Agronomijas v stis - Latvijas LauksaimniecÄ«bas universitÄte

Conference Organizing CommitteeChairman Dr. Kaspars Kampuss Latvia University of AgricultureVice-Chairman Dr. Mintauts Abolins Latvia University of AgricultureMembers Mg.agr. Marta Liepniece Latvia University of AgricultureBc. agr. Rudite Sausserde Latvia University of AgricultureJulija OrlovaLatvia University of AgricultureSecretary Mg.agr. Dace Sterne Latvia University of AgricultureConference Scientific CommitteeChairman Dr. Mintauts Abolins Latvia University of AgricultureVice-Chairman Dr. Aleksandrs Adamovics Latvia University of AgricultureMembers Dr. Kaspars Kampuss Latvia University of AgricultureDr. Jim HancockMichigan State University, USADr. Taimi PaalEstonian University of Life SciencesDr. Laura JaakolaUniversity of Oulu, FinlandDr. Remigijus DaubarasKaunas Botanical Garden of VytautasMagnus University, LithuaniaSponsorsState Stock Company „Latvia’s State Forests”(Latvijas Valsts meži)Kristapa street 30, Riga, LV-1046www.lvm.lv“Pure Food” Ltd.,Pure, Daigones 22, Pures pagasts, Tukuma rajons, LV-3124, Latviawww.purefood.lvThe Chemical Company BASFMazā NometĦu iela 45/53, Riga, LV-1002, Latviawww.agro.basf.lvLatvian Council of Science(Latvijas Zinātnes padome)Akadēmijas laukums 1, Rīga, LV – 1050, Latviawww.lzp.gov.lv3

ReviewersTwo independent reviewers from participating countries have reviewed every report accordinglyto the unified academic guidelines determined by the review structure of the ConferenceCommittee.4

15 Pormale J., Osvalde A. and Nollendorfs V.COMPARISON STUDY OF CULTIVATED HIGHBUSH AND WILD BLUEBERRYNUTRIENT STATUS IN PRODUCING PLANTINGS AND WOODLANDS, LATVIAKRŪMMELLEĥU UN SAVVAěAS MELLEĥU MINERĀLĀS BAROŠANĀSNODROŠINĀJUMA LĪMENIS RAŽOJOŠĀS SAIMNIECĪBĀS UN LATVIJAS MEŽOS16 Prokaj E., Medve A., Koczka N., Ombodi A. and Dimeny J.EXAMINATION OF CORNEL (CORNUS MAS L.) FRUITS IN BORSOD-ABAUJ-ZEMPLENCOUNTY (HUNGARY)KIZILA (CORNUS MAS L.) AUGěU IZVĒRTĒŠANA BORSOD-ABAUJ-ZEMPLENAPGABALĀ (UNGĀRIJA)17 Ripa A. and AudriĦa B.RABBITEYE BLUEBERRY, AMERICAN CRANBERRY AND LINGONBERRY BREEDINGIN LATVIAEŠA ZILEĥU, AMERICAS DZĒRVEĥU UN BRŪKLEĥU SELEKCIJA LATVIJĀ18 Rohloff J., Nestby R., Nes A. and Martinussen I.VOLATILE PROFILES OF EUROPEAN BLUEBERRY: FEW MAJOR PLAYERS, BUTCOMPLEX AROMA PATTERNSEIROPAS MELLEĥU AROMĀTS: DAŽAS GALVENĀS SASTĀVDAěAS, BETDAUDZVEIDĪGS KOPĒJAIS AROMĀTA SASTĀVS19 Rupasova Zh., Pavlovskij N., Kurlovich T., Pyatnitsa F., Yakovlev А., Volotovich A.,Pinchukova Yu.VARIABILITY OF THE STRUCTURE OF THE BIOCHEMICAL COMPOSITION OF THEFRUITS OF THE HIGHBUSH BLUEBERRYAUGSTKRŪMU MELLEĥU OGU BIOĖĪMISKĀ SASTĀVA MAINĪGUMS20 Sedlak J. and Paprstein F.MICROPROPAGATION OF HIGHBUSH BLUEBERRY CULTIVARSAUGSTKRŪMU MELLEĥU ŠĖIRĥU MIKROPAVAIROŠANA21 Seglina D., Krasnova I., Heidemane G. and Ruisa S.INFLUENCE OF DRYING TECHNOLOGY ON THE QUALITY OF DRIED CANDIEDCHAENOMELES JAPONICA DURING STORAGEKALTĒŠANAS TEHNOLOĂIJU IETEKME UZ CHAENOMELES JAPONICA SUKĀŽUKVALITĀTI UZGLABĀŠANAS LAIKĀ22 Smolarz K.SHORT INFORMATION ABOUT THE HISTORY OF THE COMMERCIAL CULTIVATIONHIGHBUSH BLUEBERRY IN POLANDĪSA INFORMĀCIJA PAR AUGSTKRŪMU KRŪMMELLEĥU KOMERCIĀLASAUDZĒŠANAS VĒSTURI POLIJĀ23 Uleberg E., Røthe G. and Martinussen I.CLOUDBERRY BREEDING IN NORWAYLĀCEĥU SELEKCIJA NORVĒĂIJĀ24 Vilka L., Rancane R. and Eihe M.FUNGAL DISEASES OF VACCINIUM MACROCARPON IN LATVIAVACCINIUM MACROCARPON SLIMĪBAS LATVIJĀ25 Vilka L., Rancane R. and Eihe M.STORAGE ROTS OF VACCINIUM MACROCARPON SPREAD AND DEVELOPMENT INLATVIAVACCINIUM MACROCARPON OGU PUVES IZPLATĪBA LATVIJĀ26 Yakovlev A., Rupasova Zh., Volotovich A.EXPERIMENTAL AND RESEARCH WORKS ON SOME OF VACCINIUM TAXA INBELARUSDAŽU VACCINIUM ĂINTS SUGU EKSPERIMENTĀLIE UN IZPĒTES DARBIBALTKRIEVIJĀ27 Žukauskien÷ J., Paulauskas A., Daubaras R., Česonien÷ L.PLANT MORPHOLOGY AND RAPD MARKER CHARACTERIZATIONS OF VACCINIUMOXYCOCCUS LITHUANIAN POPULATIONSVACCINIUM OXYCOCCUS LIETUVAS AUGU POPULĀCIJU MORFOLOĂIJA UNRAKSTUROJUMS AR RAPD MARĖIERIEM808793981031081131191221251331381426

CRANBERRY AND BLUEBERRY PRODUCTION IN LATVIADZĒRVEĥU UN KRŪMMELLEĥU AUDZĒŠANAS SITUĀCIJAS IZPĒTE LATVIJĀMintauts Abolins, Rudite Sausserde, Marta Liepniece and Dace SterneLatvia University of Agriculture, Institute of Agrobiotechnology,Liela street 2, Jelgava, Latvia, e-mail: Mintauts.Abolins@llu.lvAbstractLarge cranberries (Vaccinium macrocarpon Ait.) and highbush blueberries (Vacciniumcorymbosum L.) are relatively new cultures in Latvia. The first commercial cranberry plantationwas established in 1985. Nowadays, the area of cranberries is approximately 100 ha which makes itthe third largest in the world. The blueberry plantations compared to other European states are notso large - 170 ha. The largest part of the area of high moss peat bogs was drained and is now beingused as cranberry fields – a bogs area of approximately 10 000 ha. The examination of the situationof cranberry and blueberry production started in 2007/2008. Cranberry and blueberry plantationswere surveyed in all four fruit-growing areas of Latvia. The following parameters were examined:varieties and plantation sizes, the physiological status of the plants of different varieties afteroverwintering, growing technologies - characterisation of the soil or substrate, plantation location,growing distances; yield and yield quality. The most popular varieties of cranberries are ‘Stevens’,‘Bergman’, ’Ben Lear’, etc. The most popular cultivars of blueberries are ‘Bluecrop’, ‘Patriot’ and‘Northland’. The newest cultivars in the Latvian plantations are ‘Toro’, ‘Rubel’, ‘Blue Gold’,‘Hanna’, ‘Klara’, ‘Drapers’ and ‘Bonus’. Planting technologies of highbush blueberries: 1) mineralsoil (78 % of farmers – with specific preparation of the soil before planting; 2) peat – 22 % offarmers.KopsavilkumsLielogu dzērvenes (Vaccinium macrocarpon Ait.) un krūmmellenes (Vaccinium corymbosum L.) irsalīdzinoši jauni kultūraugi Latvijā. Pirmie komerciālie dzērveĦu stādījumi tika ierīkoti 1985. gadā.Šobrīd dzērveĦu stādījumu platības sasniedz jau 100 ha, ieĦemot trešo vietu pasaulē, betkrūmmelleĦu stādījumu platības ir mazākas, salīdzinoši ar citām Eiropas valstīm – 170 hektāri.Lielākā daĜa augstā sūnu kūdras purvi ir nosusināti un šobrīd tiek izmantoti kā dzērveĦu lauki -purvu platību aptuveni 10 000 ha. Pētījumi par dzērveĦu un krūmmelleĦu audzēšanu Latvijāuzsākti 2007/2008 gados. DzērveĦu un krūmmelleĦu stādījumi tika apsekoti visās četrāsaugĜkopības zonās Latvijā. Tika vērtēts: audzētās šėirnes un to audzēšanas platības, dažādu šėirĦuaugu fizioloăiskais stāvoklis pēc ziemošanas, audzēšanas tehnoloăijas – raksturojot augsni vaisubstrātu, atrašanās vieta, audzēšanas attālumi, raža un ražas kvalitāte. Visvairāk audzētākāsdzērveĦu šėirnes ‘Stevens’, ‘Bergman’, ‘Ben Lear’, u.c. Visvairāk audzētākās krūmmelleĦu šėirnes– ‘Bluecrop’, ‘Patriot’, ‘Northland’. Jaunākās krūmmelleĦu šėirnes Latvijā ir ‘Toro’, ‘Rubel’,‘Blue Gold’, ‘Hanna’, ‘Klara’, ‘Drapers’ and ‘Bonus’. KrūmmelleĦu audzēšanas tehnoloăijas:1) minerālaugsnē–78 % saimniecībās (pirms stādīšanas augsni speciāli sagatavojot); 2) kūdrā–22%saimniecībās.Key words: Vaccinium macrocarpon, Vaccinium corymbosum, cultivars, area, technologiesIntroductionCranberries, as it is with highbush blueberries, can play an important role in the economy of Latviaas these berries are sought in the world market for their medical and dietary properties and they areamong the best paying berries.The Latvia’s climate and the vast marshland area is the main reason why from ancient times theEuropean or the Latvian local (Vaccinium oxycoccus L.) cranberries are grown in the wild. Berryplantation is not mechanized due to their morphological properties. The yield was mostly used inthe local market.The American large cranberry (Vaccinium macrocarpon Ait.) and highbush blueberry (Vacciniumcorymbosum L..) are relatively new cultures in Latvia. The first commercial cranberry plantations7

were established in 1985. Nowadays, the area of the large cranberries is approximately 100 ha andthat is third place in the world. The blueberry plantations compared to other European countries arenot so large- about 170 ha (after Latvian Fruitgrowers Association data).The scientific research on the European or Latvian local cranberries was started in the 70ies of thelast century by the Department of Horticulture under the Faculty of Agriculture of the LatviaUniversity of Agriculture. It was found that the advantage of these cranberries lies in the growingseason, it is shorter- the beginning of flowering is about two weeks earlier, it starts already in themiddle of May. Besides, they require a lower sum of the effective temperatures than the large berrycranberries. The berries are better protected from autumn frosts, the berry texture is more gentle.For this species of cranberries it is not possible to use mechanization in harvesting (Abolins andGurtaja, 2006). Producing shoots are upright, unable to detach the berries from the plant and theberries develope unequally.Productive wild clones were found not only in the bogs of Latvia but also in Karelia (Russia) andEstonia using both physical and chemical mutagenesis. 172 cranberry genotypes were studied.Most of them -163 genotypes had been collected in Latvia, 7 - in Estonia, 1 - in Novosibirsk and 1 -in Petrozavodsk in Russia.The research was carried out propagating woody and softwood cuttings, investigating plantingdensity, substrates, morphological and biological characteristics of the plant and other issues(Gronskis and Liepniece, 2004.)Improving less productive areas of cranberry bogs with high-value varieties as well as onrecovering the cranberry degraded bog areas. It was decided to continue selection in cranberryclone test conditions in order to breed crops, to test their suitability in re-cultivated bogs. It wasalso decided to develop elaborated technologies for the propagation and cultivation of the specificgenotype.In the breeding work, the most valuable clones were chosen. Having analyzed the organic harvestof the best cranberry genotypes, it can be concluded that the greatest number of inflorescence - 800m -2 , the largest flower number 1420 m -2 and berry mass are calibrated with the genotype V-63583(Gronskis and Liepniece, 2004.)According to the length of the vegetation period, the best cranberry genotypes are divided into:medium early B-83 and V-63383; medium late V-63583, but the late is -V-21682. The highestbreeding ability for grass-like cuttings is noteworthy with the genotype V – 63583 and B – 83(Abolins.and Gurtaja, 2006).In the recultivated cranberry bogs areas - in total 40.8 ha of land, selected in different places ofLatvia, the most valuable genotype seeds were sown. The seed sowing was done by plane.Studying the recultivated cranberry areas it was found that cranberry growing was very uneven,averaging from 10 to 30 %. In some places it was associated with deep groundwater levels, sulphurspring diffluence and open places in the array as well they were destroyed by frost.However, in a more carefully prepared place - in the Experimental Plantation of Jelgava MRS(Jelgava Forestry), sowing the seeds in the area of 1 ha, the projective cover totaled to 100 % andplanting seedlings in the growing area of 0.8 ha also totaled to 100 %. The yield reached 1.7 – 2.1 tha -1 in the given stands.Materials and MethodsClimate in Latvia. The northern part of Latvia is outside the fruit zone of the temperate climate.However, nearness to the ice-free Riga Gulf Stream avoids early autumn frosts, and during thewinter becomes the reason for a relatively mild climate in the western part of Latvia. The averageprecipitation is 560 – 850 mm, the monthly average winter temperature (January) is -2.6 to – -6.6o C and the summer temperature (July) is +16.8 to +17.6 o C with the day length 17 – 18 hours inJune. In the growth season, temperature over +5 0 C lasts 180 – 200 days. The sum of the activetemperatures (> 10 o C) in the vegetative period is 1800 – 2100 0 C. There are 120 – 140 activecyclones and 160 – 180 anticyclones per year.The total area of peat bogs is 6401 km 2 or 9.9 % of all the land area of Latvia, which makesrecourses of peat – 1.7 - milliard t. There are more than 5000 peat deposits: 7 exceeding an area of5000 ha; 87 with the area of 1001- 5000 ha; 109 with the area of 501 – 1000 ha. Included are 49.3% of low grass peat bogs; 41.7 % of high moss peat bogs, 9 % others. The average depth of8

deposits is 2 – 5 m, but the maximum - up to 15 metres. The largest part of the area of high mosspeat bogs is drained and used for establishing cranberry fields- could in the future beapproximately 10 000 ha (Abolins and Gurtaja, 2006).Despite the greatly varying climatic conditions, the winter of 2007/2008 was more favorable for thegrowth and development of large cranberries and highbush blueberries, rouhg the highbushplantings could suffer from spring frosts when temperatures fell below 0ºC. In such cases, the frostdid not damage plantings where surface irrigation was available. The physiological observationscarried out in 2008 showed that only single twigs of highbush blueberries were frost damaged. Theafter effect of the damage caused by the winter of 2006/2007 caused the delayed development ofsingle twigs that later led to the twigs dying.An evaluation of the situation of cranberry and blueberry production started in the season of2007/2008 within the framework of Project No. 04.1 – 25/5. Cranberry and blueberry plantationswere surveyed in all fruit-growing zones of Latvia – Central, Eastern, Southern and Western zones.The following parameters were examined: the used varieties and plantation sizes, growingtechnologies - characterisation of soil or substrate, plantation location, growing distances, thephysiological status of plants of different varieties after overwintering, yield and yield quality.In collaboration with the researchers of the Laboratory of Plant Mineral Feeding under the Instituteof Biology of the Latvia University, in the farm „Strelnieki” (Riga District, in the mineral soil, thevariety ‘Patriot’) and Lienama Ltd (Aluksne District, in the peat bog, the variety ‘Northblue’). Thetesting of fertilizer systems: option 1 - the basic fertilizer NPK + Mg + S, a supplementary fertilizerN; Ca; S;option 2- leaf fertilizer Vito Silva; option 3- leaf fertilizer Vito Silva + B; Cu; Mo; option4–leaf fertilizer Vito Silva + B; Cu; Mo + Caltrac.The research on the development of plant phenology, the physiological status of plants, yield andits quality indicators is simultaneously carried out by the Training and Research Farm of theInstitute of Agrobiotechnology under the Faculty of Agriculture of the Latvia University ofAgriculture (www.llu.lv) in the City of Jelgava. On this base farm, the high bush blueberry andcranberry collection is structured. The high bush blueberries are planted in the mineral soil in peatcushions distributed in furrows. The plant age-7 years, biennial seedlings were planted. Cranberriesare planted in peat providing the necessary growth conditions.Results and DiscussionThe large cranberries (Vaccinium macrocarpon Ait.). The most popular varieties of cranberriesare ‘Stevens’, ‘Bergman’, ’Ben Lear’ and others, but less popular are ‘Franklin’, ‘Pilgrim’,‘Hoves’ and ‘Lemynion’ (Figure 1).‘Early Black’3,81 %‘Hoves’2,82 %Others16,93 %‘Ben Lear’19,89 %‘Lemynion’0,56 %‘Pilgrim’4,09 %‘Franklin’4,23 %‘Bergman’19,80 %‘Stevens’27,87 %Figure 1. The division of cranberry varieties in farms, %Due to the high costs involved to establish cranberry fields, most (65 %) of the farms are small -with 0.1 – 5.0 ha of land, but 21 % of farms – with 5.1 – 10.7 % with 10.1 – 15.0 and 7 % - with15.1 – 20.0 ha of land (Figure 2).9

10,1-15 ha7%15,1-20 ha7%< 20,1 ha0%5,1-10 ha21%0,1-5 ha65%Figure 2. Farm division by cranberry plantation areas, %There are three planting technologies for large cranberries used in Latvia.First - in the prepared field, cranberry tendrils are evenly spread on the soil surface and imbeddedin peat with a disc harrow. Cranberries are planted or seeded in straight lines. It is a widely usedmethod with sand substrate abroad. The drawback of the method- all tendrils are not deeply thesoil.Second - imbedding tendrils by power harrow. In order not to damage the plants the speed of thepower harrow should be reduced. One should not imbed them too deep or too shallow.Third - planting by hand using a planting stick. The drawback - a laborious process, but it can beused for planting a small area.The large cranberries unlike the local ones, start blooming later-in mid-June/early July (the localcranberries – in May/June). Thus, the harvest of the large cranberries is only affected by the latespring frosts (sprinkling should be provided). On the surveyed farms, in May maintenance workwas started in the cranberry plantations-combing, tendril cutting, and in the end of June, cranberryplantations were at the flowering stage.On the whole, in terms of weather conditions, in the second decade of September in Latvia it wascold but dry in 2008. The average air temperature of the decade was 3.7 degrees below the norm.The cold weather lasted the whole decade. During the coldest night of the decade (16 September),the first frosts were recorded -2 ºC. During the decade there was frost almost every night, duringthe coldest periods falling to -5 – -6 ºC.The highest yield of 2007 was 4500 g m -2 . In 2008 there was a harvest of 3900 g m -2 . Also, 100-berry mass was higher in 2007 by an average of 180 g and in 2008. - 150 g. In the middle andeastern areas rainfall was lower.Weed control as an essential part of cranberry management was done in two ways - by hand and byusing glyphosate pesticides as a replant treatment by weed wipers on bicycle wheels.Sand, used as a mulching material, was spread with a specially constructed spreader but there wereproblems with sand pH and weeds. Therefore, sanding is not popular. The farmers use high mosspeat, but some -sawdust or sawdust mixed with peat especially those without a sprinkler irrigationsystem.The highest cranberry harvest in the year 2007 was - 4500 g m -2 , but in the year 2008 – 3900 g m -2(Figure 3). The large berries of cranberries (above 19 mm), were on average 1 – 2,5 %, but themain harvest was produced large berries (15-16 mm), 43 – 62 % in the year 2007 and 35 – 59 %in the year 2008, accordingly.10

g m -25000,04500,04000,03500,03000,02500,02000,01500,01000,0500,00,01 farm 2 farm 3 farm 4 farm 1 farm 2 farm 3 farm 4 farm2007 2008200,0180,0160,0140,0120,0100,080,060,040,020,00,0gYield, g m-2100 berries mass, gFigure 3. The average yield of the cultivar ‘Stevens’, g m -2 , and the mass of 100 berries, g (on threefarms in the years 2007 and 2008).The analysed late cranberries varieties in the autumn frost, up to 10 % of the harvest was lost inautumn frosts on the farms of the western area of Latvia.Blueberries (Vaccinium corymbosum L.). The most popular varieties of blueberries in Latvia arethe northern highbush varieties adapted to quite cold mid-winter temperatures below -20 ºC‘Bluecrop’, ‘Patriot’ and ‘Northland’ (Hancock, 2006). The newest varieties are ‘Toro’, ‘Rubel’,‘Blue Gold’.In the collections of some farms, such varieties as ‘Hanna’, ‘Klara’, ‘Drapers’, ‘Bonus’ are startingto appear but are not popular in Latvia yet (Figure 4).Others43,9 %Northland5,6 % Patriot10,7 %Bluecrop15,6 %Stanley0,3 % Woodart0,4 %Polaris0,2 % Brigita2,2 %Northcauntry3,1 %Duke6,9 %Early Blue0,2 %Blue Ray7,5 %Northblue3,3 %Figure 4. The division of highbush blueberry cultivars on farms, %Like it was with cranberries, most blueberry farms - 80 % are small with 0.1 – 5.0 ha of land, but11 % of the farms have 5.1 – 10 ha of land (Figure 5). The main reason why small farms are somany, is the high start up cost and lack of experience in highbush blueberries cultivation.On the surveyed farms, the highbush blueberries are chosen for cultivation in one of the followingways: in mineral soil (78 % of the breeders) – the soil before planting is adequately prepared: theplants are planted in cushion peat pits (50 x 50 x 50 cm), which are filled with acidic high mosspeat, some of the farmers fill the furrows with peat and chippings or sawdust (1:1).The second option-in the peat bog (22 % of the farmers) they choose to breed highbush blueberriesin worked out peat-moss bogs, planting plants on a level field or in the beds.11

In less than half the surveyed farms, the highbush blueberry plantations were irrigated againstfrosts. Therefore, the spring frost damage was very slight. In determining soil acidity pH/KCl , in allthe surveyed farms it was within the norm from 4.3 to 5.15 (Nollendorfs, 2003).Spring maintenance includes: fertilizing, the tree crown formation, excision of the dead twigs, bedmaintenance (weeding, mulching) and irrigation. As a mulching material, moss bog peat is used aswell as chipping mulch. The research carried out in the year 2007 shows that incompletely mulchedplantations are destroyed by frosts.10,1-15 ha3%20,1-25 ha3%< 25,1-30 ha3%5,1-10 ha11%0,1-5 ha80%Figure 5. Farm division by the highbush blueberry plantation areas, %Irrigation. The farms use both surface (sprinkling) and drip irrigation. Some farm irrigation is notyet available and this condition highly affects the further growth, development and production ofhighbush blueberries.Fertilizers. Most farmers use soil analysis and adjust the required amount of fertilizers accordingly.Yield, harvest and quality. The highbush blueberry varieties ‘Northland’ – 4.12 and ‘Patriot’ – 4.06kg from a bush had the highest yields, the lowest the variety ‘Chandlers’ – 0.32 (Figure 6).kg per bush4,504,003,504,084,123,683,002,502,442,001,501,441,581,211,341,000,700,700,500,320,00PatriotPolarisNorthblueNorthlandChippewaBluecropBlueraySpartanBluejayChandlerJerseycultivarsFigure 6. The average yield of highbush blueberry cultivars per bush, kg.(Training and Research Farm of the Faculty of Agriculture under the LLU in Jelgava)The largest 100 berry mass was achieved by the variety ‘Northblue’, using the supplementaryfertilizer Vito Silva + B Cu Mo + Caltrac. For the variety ‘Patriot’, the effects of ferilizers are notessential. Depending on the fertilizer option, the variety ‘Northblue’ had the largest berries – theproportion of the very big berries was 2 – 7 % and of the big berries – 29 – 40 %. For the variety‘Patriot’, proportion of the very big berries was on average - 5 %, but of the big berries on average– 50 % of the total number of berries. The fertilizers used did not essentially affect the berry size.12

References1. Abolins M., Gurtaja L. (2006). Vaccinium spp. Production Techniques in Latvia. Proc. of VIIIth Int.Symp. on Vaccinium Culture, ISHS Acta Hort. No. 715, p. 185-190.2. Hancock J. (2006). Northern Highbush Blueberry Breeding. Proc. of VIIIth Int. Symp. on VacciniumCulture, ISHS Acta Hort. No. 715, p. 37-40.3. Gronskis I., Liepniece M. (2004). Atskaite par zinātniski pētniecisko darbu „Degradēto purvu platīburekultivācija dzērveĦu audzēšanai” (1991-1993). (Report on the scientific research work „Area ofdegraded bogs recultivation for cranberry cultivation”), grant No. 241, Jelgava.4. Nollendorfs V. (2003). Kanādas pieredze krūmmelleĦu audzēšanā (Experience of highbush blueberriesProduction in Canada). Darzs un Drava, No.6, p. 13 – 15.SELECTION OF THE EUROPEAN CRANBERRY IN LITHUANIAEIROPAS DZĒRVEĥU SELEKCIJA LIETUVĀLaima Česonien÷ 1 , Remigijus Daubaras 1 , Ina Jasutien÷ 21 Kaunas Botanical Garden of Vytautas Magnus University,Z.E.Zilibero 6, LT-46324, Kaunas, Lithuania, e-mail: l.cesoniene@bs.vdu.lt,2 Food Institute of Kaunas University of Technology, e-mail: ina.jasutiene@lmai.ltAbstractA detailed evaluation of the morphological diversity and economical properties of the Europeancranberry was carried out in 2000 – 2008 in the Kaunas Botanical Garden of Vytautas MagnusUniversity with the purpose of ascertaining the most valuable clones. The European cranberryclones were selected from the strictly protected areas of Čepkeliai, Žuvintas, and Kamanos in 1995– 1999. These clones with distinctive morphological peculiarities were propagated and planted intoacid peat (pH 3.5 – 4.0) in the field collection of the Kaunas Botanical Garden for furtherinvestigations. The results of the comprehensive evaluations of the phenotypic and geneticdiversity of the European cranberry were crowned with the selection of the most valuable clones.These clones were compared with Estonian and Russian cultivars. Berry size, yield, and the amountof biologically active substances were evaluated. The selected clones are characteristic ofexceptional biological and biochemical peculiarities. The five Lithuanian clones ‘Amalva‘,‘Žuvinta‘, ‘Vaiva‘, ‘Vita, and ‘Reda‘ were proposed to recieve the cultivar status.KopsavilkumsNovērojumi par Eiropas dzērveĦu morfoloăisko daudzveidību un ekonomiskajām īpašībām tikaveikti no 2000. – 2008. gadam Vitautas Magnus universitātes KauĦas botāniskajā dārzā ar mērėinoskaidrot vērtīgākos klonus. Eiropas dzērveĦu kloni tika izraudzīti no īpaši aizsargājamāmteritorijām Čapkeliai, Žuvintas un Kamanos, laikā no 1995. – 1999. gadam. Šie morfoloăiskiatšėirīgie kloni tika pavairoti un iestādīti skābā kūdrā (pH 3.5 – 4.0) tālākai izpētei unnovērojumiem kolekcijas stādījumos KauĦas botāniskajā dārzā. Pēc vispusīgiem pētījumiem parEiropas dzērveĦu fenotipisko un genotipisko daudzveidību rezultāti ir vainagojušies ar vērtīgākoklonu izlasi. Šie kloni tika salīdzināti ar Igaunijas un Krievijas dzērveĦu šėirnēm. Tika vērtēts ogulielums, raža un bioloăiski aktīvās vielas. Izvēlētajiem kloniem bija īpaši vērtīgas bioloăiskās unbioėīmiskās īpatnības. Pieci Lietuvas kloni ‘Amalva’, ‘Žuvinta’, ‘Vaiva’, ‘Vita’ and ‘Reda’ irpieteiktas, lai saĦemtu šėirnes statusu.Key words: anthocyanins, cultivar, morphological diversity, phenolics, selectionIntroductionThe European cranberry (Vaccinium oxycoccos L., syn. Oxycoccus palustris Pers.) belongs to themost valuable berry plants, whose natural habitats are found in moist boreal forests, ombrothrophicshagnum bogs and minerotrophic fens. This species has been intensively researched for its healthproperties in recent years. The cultivars of other species American cranberry (Vacciniummacrocarpon Aiton, syn. Oxycoccus macrocarpus (Aiton) Purs.) are known for their exceptional13

economical and biochemical characteristics. This species has been fully introduced in Lithuania.The productive cultivars ‘Ben Lear‘, ‘Early Black, ‘Stevens, and ‘Pilgrim‘ were recommended forberry growers (Budriūnien÷, 1998). The European cranberry is well adapted to Lithuanian climateconditions. The obviuos desirable traits of the European cranberry are productivity, fruit size andquality as well as disease resistance. The breeding of new cultivars set a task to enhance the levelsof the before mentioned traits. As with most crops, productivity is a main factor for selection toculture.At present the investigations on the introduction of the European cranberry are being carried out inRussia and other countries of Europe (Gorbunov, 1993).The selection of economically valuableclones of the European cranberry were carried out in the Kostroma Forest Research Station. Thelong-term research resulted in the selection of six V. oxyccocos clones. These clones wereregistered by the State Commission of the Russian Federation for Testing and Protection ofSelection Achievements as the first Russian cultivars ‘Alaya Zapovednaya’, ‘Krasa Severa’,‘Sazonovskaja’, ‘Severyanka’, ‘Sominskaja’, and ‘Khotavetskaya’ (Cherkasov et al., 1998,Makeev et al., 2000). Six cultivars suitable for garden cultivation have been selected from the wildclones in Estonia as well: ‘Kuresoo’, ’Nigula’, ‘Soontagana’, ‘Maima’, ‘Virussaare’, and ‘Tartu’.In distinguishing the different cultivars special characteristics including berry size and shape havebeen used (Ruus & Vilbaste, 1968).The evalution of the European cranberry genetic resources started in Lithuania in 1995. About 120different cranberry clones were selected from the strictly protected areas of Čepkeliai, Žuvintas,and Kamanos as well as in the raised bogs of the Ignalina, Jurbarkas and Švenčionys districts in1995 – 1999. The clones of wild cranberry with different morphological properties of leaves,stems, and berries were described. The collected cranberry shoots were used for vegetativepropagation. The collection of selected clones was planted in the Kaunas Botanical Garden ofVMU. Investigations of seasonal development, morphological and genetic diversity as well asfruiting characteristics were ascertained (Daubaras et al., 2006; Areškevičiūt÷ et al., 2006). Fiveclones of the European cranberry were selected in consideration of productivity, berry size, diseaseresistance and were approved as suitable to cultivars.Aim of this study was to evaluate the peculiarities of the five selected Lithuanian clones andcompare them with the Russian and Estonian cultivars of the European cranberry.Material and MethodsFive European cranberry clones as well as the Russian and Estonian cultivars were selected forevaluation. The clones 96-Ž-10, 96-Ž-11, and 99-Ž-10 were selected in the strictly protectedreserve Žuvintas, whereas the clones 95-A-05 and 95-A-09 were collected in different mesotrophicbogs in the East Lithuania in 1995, where great morphological variation was noticed. These cloneswere named, respectively: ‘Vita‘, ‘Amalva‘, ‘Žuvinta‘, ‘Vaiva‘ and ‘Reda‘.The collected cuttingsof these clones were planted into acid peat (pH 4.0 – 5.0) beds in the field collection of the KaunasBotanical Garden of VMU.A detailed evaluation of the morphological diversity of these clones was carried out in 2000 –2008. For the morphological characterization berry properties per clone were used: berry size,shape, colour, and waxy layer intensity. The average weight of a berry was calculated by weighing50 berries in three replications. A 1 – 5 point scale was used for berry weight estimation where 1stands for very small (1.5 g). The yield production of the clones was estimated. Thedistribution of fungal diseases were observed during the vegetation period.Samples of berries for the estimation of total phenolics compounds and antocyanins amounts weregathered during the stage of berry mass ripening.The amount of the total phenolics in the cranberry extracts was determined with the Folin-Ciocalteu reagent according to the method of Slinkard and Singleton (1977) using gallic acid as astandard. The reagent was prepared by diluting a stock solution (Sigma-Aldrich Chemie GmbH,Steinheim, Germany) with distilled water (1:10, v/v). Samples (1.0 ml, two replicates) wereintroduced into test cuvettes, and then 5.0 ml of Folin-Ciocalteu’s phenol reagent, and 4.0 ml ofNa 2 CO 3 (7.5 %) was added. The absorbance of all samples was measured at 765 nm using theGenesys10 UV/Vis spectrophotometer (Thermo Spectronic, Rochester, USA) after incubating at14

Table 1. Morphological characteristics of European cranberry cultivars and selected LithuanianclonesCultivar or Yield, kg/m 2 Berry weight, g Berry shape Berry colourclone‘Vaiva‘ 2.33 0.81 oblate red‘Reda‘ 1.34 1.13 pyriform dark red‘Vita‘ 2.06 1.03 round dark red‘Amalva‘ 1.80 1.01 oblate dark red‘Žuvinta‘ 1.65 0.97 oblate pink, unevenlycoloured‘Nigula‘ 1.49 1.07 oblong-conical red‘Kurresoo‘ 1.60 1.04 oblong-conical red‘Soontagana‘ 1.23 1.07 pyriform purple, strong waxcoating‘Maima‘ 1.16 1.1 oblong-conical light red‘Virussaare‘ 1.73 1.10 oblate pinkish, unevenlycoloured‘Krasa Severa‘ 0.86 1.30 egg-shaped pink‘Severianka‘ 0.64 0.94 egg-shaped dark red‘Dar0.58 1.85 oblate dark redKostromy‘LSD 01 0.56 0.135S x % 8.34 4.68LSD 01 - the least significant difference, P

investigated European cranberry accessions comprised from 18.8 to 53.3 % of the total phenoliccompounds.As several authors have reported cranberries and these specific biochemical components are beingassociated with human health attributes, such as maintenance of urinary tract health and antioxidantstatus (Povilaityt÷ et al., 1998; Vorsa et al., 2002). The berries of European cranberry are one of thebest sources of phenolic compounds as compared with the other berry plants, such as strawberry,black currant, raspberry etc. (Moyer et al., 2002). The American cranberry has been increasinglyresearched for its health properties in recent years as well. Significant genetic variability was foundfor total phenolics, total anthocyanins, proanthocyanidins, soluble solids, quinic acid, citric acidetc. (Vorsa et al., 2002; Zeldin et al., 2007). At present large-fruited cranberries are successfullycultivated by amateur gardeners and even some commercial plantations are being established inLithuania. Nevertheless, the wild cranberry is more suitable for cultivation on harvested peat bogsas well as for their renaturalisation.Therefore, there is an evident need to conserve wild cranberries ex situ in order to complementtheir conservation in situ and facilitate the investigation and utilisation of this wild crop relative.The unique collection of genetic resources of the European cranberry was established at the KaunasBotanical Garden of Vytautas Magnus University with the purpose to preserve the most valuableclones. In consequence of their comprehensive evaluations the selection of five prospective cloneswas achieved.ConclusionsThe estimation of the berry yield and berry size indicated statistically reliable differences amongthe selected Lithuanian clones. The clone ‘Vaiva’ was characteristic of the highest yield.The largest damage was caused by the fungal dieseases Monilinia oxycocci, Fusicoccumputrefaciens, Botrytis cinerea, and Phyllosticta elongata. Berry rot diseases damaged about 5 – 15% of the berry yield.The berries of the European cranberry accumulated from 66.64 to 244.66 mg 100 g -1 ofanthocyanins and from 309.50 to 521.74 mg 100 g -1 of the total phenolics.References1. Areškevičiūt÷ J., Paulauskas A., Česonien÷ L. and Daubaras R. (2006) Genetic characterization of wildcranberry (Vaccinium oxycoccus) collected from Čepkeliai reserve using RAPD method. Biologija, 1,pp. 5-7.2. Budriūnien÷ D. (1998) Investigation of Vaccinium macrocarpon Ait., Vaccinium oxycoccus L. varietiesin Kaunas Botanical Garden. Forestry Studies, 30, pp.23-29.3. Daubaras R., Česonien÷ L., Areškevičiūt÷ J. and Viškelis P. (2006) Evaluation of MorphologicalPeculiarities, Amount of Total Phenolics and Anthocyanins in Berries of European Cranberry(Oxycoccus Palustris). Baltic Forestry, 12(1), pp.59-63.4. Cherkasov A., Makeev V. and Makeeva G. (1998) Cultivation of Oxyccocus palustris Pers. in EuropeanRussia central regions. Forestry Studies, 30, pp.30-34.5. Gorbunov A.B. (1993) Introduction and breeding of Vacciniaceae in Siberia. Acta Horticulturae, 346,pp. 103-106.6. Makeev V.A., Cherkasov A.F. and Makeeva G.Y. (2000) Results and future outlook for Oxycoccuspalustris selection. Proceedings of the International Conference. Glubokoye-Gomel, Belarus, pp.178-180.7. Moyer R.A., Hummer K.A., Finn C.E., Frei B. and Wrolstad R.E. (2002) Anthocyanins, phenolics, andantioxidant capacity in diverse small fruits: Vaccinium, Rubus, and Ribes. Journal of Agricultural andFood Chemistry, 50, pp.519-525.8. Povilaityt÷ V., Budriūnien÷ D., Rimkien÷ S. and Viškelis P. (1998) Investigation of Vacciniummacrocarpon Ait. fruits chemical composition. Dendrologia Lithuaniae, 4, pp.55-62.9. Ruus E., Vilbaste H. (1968) Jõhvikas – põhjamaade viinamari (Cranberry as a grape from North). EestiLoodus. 8, pp.490-494. (In Estonian).10. Slinkard K. and Singleton, V.L. (1977). Total phenol analysis. Automation and comparison with manualmethods. American Journal of Enology and Viticulturae, 28, pp.49-55.11. Tarakanovas P., Raudonius S. (2003). Agronominių tyrimų duomenų statistin÷ analiz÷, taikantkompiuterines programas ANOVA, STAT, SPLIT-PLOT iš paketo SELEKCIJA ir IRRISTAT17

(Statistical analysis of agronomic evaluations by using the programmes ANOVA, STAT, SPLIT-PLOTfrom SELEKCIJA and IRRISTAT). Akademija, 56 p. (In Lithuanian).12. Vorsa N., Polashock J., Cunningham D., Roderick R. and Howell A. (2002). Evaluation of fruitchemistry in cranberry germplasm: potential for breeding varieties with enhanced health constituents.Acta Horticulturae, 574, pp.215-219.13. Zeldin E.L., McCown B.H., Krueger C.G. and Reed J.D. (2007). Biochemical characterization andbreeding of American cranberry for increased health benefits. Acta Horticulturae, 744, pp.253-258.THE EVALUATION OF EUROPEAN CRANBERRYBUSH (VIBURNUM OPULUS) FORBREEDING IN LITHUANIAIRBENES (VIBURNUM OPULUS) PIEMĒROTĪBA AUDZĒŠANAI LIETUVĀRemigijus Daubaras, Laima Česonien÷Kaunas Botanical Garden of Vytautas Magnus University,Z.E.Zilibero 6, LT-46324, Kaunas, Lithuania, e-mail: l.cesoniene@bs.vdu.lt,AbstractThe investigations were carried out at the European cranberrybush (Viburnum opulus L.) collectionat the Kaunas Botanical Garden of Vytautas Magnus University in 1999 – 2005. The biologicalpeculiarities of cranberrybush cultivars and clones were estimated according to the usual methodsof the evaluation of horticulture plants. Reliable differences were determined between accessionswith respect to productivity, the number of fruit per cluster and the mean weight of the fruit. Theinvestigations of the biochemical composition of the fruit of different cultivars and clones revealedsignificant differences in the amounts of phenolic compounds (anthocyanins and flavonols),benzoic, and ascorbic acids. The accession of Lithuanian origin P1 was typical of the largestamount of ascorbic acid (46.1 mg 100 g -1 ). The fruit of the cultivar ‘Leningradskaja Otbornaja’accumulated the largest amounts of anthocyanins. The cultivars ‘Krasnaja Grozd’ as well as theclone P2 had the largest benzoic acid amounts. The value of the most productive Europeancranberrybush accessions were determined by the number of fruit in a cluster. The results of thebiochemical investigations corroborated that the selection of European cranberrybush accessions inrespect to benzoic acid, anthocyanins and ascorbic acid amounts could be advisable.KopsavilkumsIzmēăinājumi tika veikti irbeĦu (Viburnum opulus L.) kolekcijas stādījumā Vitauta Magnusuniversitātes KauĦas botāniskajā dārzā no 1999. līdz 2005. gadam. Tika noteiktas dažādu irbeĦušėirĦu un klonu bioloăiskās īpašības. Atrastas būtiskas atšėirības starp dažādu paraugu ražību,augĜu skaitu ėekarā un augĜa vidējo masu. AugĜu bioėīmiskā sastāva pētījumi uzrādīja būtiskasatšėirības starp dažādu šėirĦu un klonu fenolu (antocianīni un flavonoli), benzoskābes unaskorbīnskābes saturu. Lietuvas izcelsmes paraugs P1 saturēja visvairāk askorbīnskābes – 46.1 mg100g -1 . Šėirnes ‘Leningradskaja Otbornaja’ augĜi saturēja visvairāk antocianīnu, bet šėirne‘Krasnaja Grozd’ un klons P2 – visvairāk benzoskābes. Produktīvāko irbenes vērtību noteica augĜuskaits ėekarā. Bioėīmiskā sastāva pētījumu rezultāti rāda, ka būtu ieteicams veikt irbeĦu selekcijuattiecībā uz askorbīnskābes, benzoskābes un antocianīnu saturu.Key words: evaluation, biochemical composition, cultivars, productivity, breeding.IntroductionThe species of genus Viburnum L. such as Viburnum opulus L. (European cranberrybush), V.trilobum Marsh. (American cranberrybush) as well as V. sargentii Koehne are widely used intraditional and folk medicine. The European cranberrybush (Viburnum opulus L.) is a native plantin Lithuania which is widely used in traditional and folk medicine. Its flowers, bark and leaves arean important medicinal raw because it possesses large amounts of the tanic substances, carotenoids,isovalerianic acid, saponines, and glycosides. The seeds contain up to 21 % of fatty oil (Bock et al.,1978). Fruit of this species accumulate significant amounts of biologically active substances. The18

fruit of V. opulus has different amounts of ascorbic acid, pectin, carotenoids, flavonols,tocopherols, anthocyanins, different polysaccharides (Евтухова et al., 2000; Евтухова et al., 2002;Лобанова et al., 1999; Оводова et al., 2000; Velioglu et al., 2006; Jordheim et al., 2007).The collection of V. opulus accessions at Kaunas Botanical Garden was started in 1998. Theevaluation of collected samples was done during 1999 – 2008. The most comprehensive researchwork on Viburnum sp. was accomplished hitherto in Russia and Ukraine. Different cultivars wereadded to the Russian official register and recommended for growing (Куденков М. andЧурканенко Н., 1998). The Ukrainian selections have small bushes, dark red fruits with a weakerbitter quality and a yield of 8 – 9 t ha -1 (Panteev et al., 1995).The investigation of productivity, fruit properties and biochemical components of V. opulusaccessions and the selection of the most valuable clones and cultivars makes it possibility to usethem for breeding.The aim of present study was to investigate the variation of yield, fruit properties and biochemicalcomponents of V. opulus accessions and select the most valuable clones and cultivars forpreservation and breeding in Lithuania.Materials and methodsThe European cranberrybush cultivars of Russian origin, the clones P1 (of Lithuanian origin) andP2 (of Ukrainian origin) were selected for evaluation. All accessions are proving easy to grow andexceptionally winter hardy. Fruit production began within two years after planting.The yield and fruit properties (yield per bush, number of fruit in a cluster and mean weight of afruit) were studied. The results were statistically analyzed and regression (R 2 ) and variation (CV)coefficients were calculated using STAT for Excel.For the average fruit weight 100 fruits were collected and examined. 20 racemes were examined toevaluate fruit number per raceme and 10 bushes were examined to evaluate the yield per bush..The investigations of the biochemical composition of the fruit were conducted in their maturestage. The amount of ascorbic acid was ascertained by the Tilmans reaction. It was titrated by asolution of 2.6-dichlorphenolindophenol sodium salt. The pigments were extracted with 95 % (v/v)grade ethanol acidified with 0.1 M HCl with the purpose to assay the total amount of anthocyanins.The total amount of anthocyanins was expressed by cyanidin 3-rutinoside. The amount of flavonolswas expressed by rutin and determined spectrophotomerically at a wavelength of 440 nm. Themethod of benzoic acid determination was based on steam distillation. The spectrophotometricanalysis was carried out by a reaction of benzoic acid with hydroxylamine-HCl and peroxide at awavelength of 315 nm (Ермаков А. and Арасимович В., 1987; Helrich, 1990).Results and DiscussionThe investigation showed that accessions differed in their yield per bush, the number of fruit in acluster and the mean weight of the fruit as well. Reliable differences between the cultivar‘Leningradskaja Otbornaja’ and other accessions were determined. A constant large average yieldwas typical of the cultivars ‘Leningradskaja Otbornaja’, P3 and ‘Zarnica’ 8.5 – 6.2 kg per bush.The cultivars ‘Zarnica-2’, ‘Souzga’, and ‘Šukšinskaja’ had the lowest yields 2.6 – 3.2 kg per bush(Table 1).The accessions of V. opulus were distinguished by significant differences in the size of a raceme.The number of fruit in a raceme varied from 28.1±5.83 (‘Upninkai’) to 51.0±6.1 (cv. ‘KrasnajaGrozd’).The cultivars ‘Upninkai’ and ‘Leningradskaja Otbornaja’ produced the largest fruit (0.74±0.09 gand 0.66±0.05 g, respectively). The cultivar ‘Šukšinskaja’ and P3 were typical of the smallest fruit,0.45±0.04 g and 0.47±0.04 g, respectively. Tge length of a cluster stalk varied from 2.9±0.10(‘Leningradskaja Otbornaja’) to 4.6±0.21 cm (P3).Statistically reliable differences were determined between the mean weight of a fruit. Estimates ofvariability indicated that the most stable trait was the mean weight of a fruit. The coefficient ofvariation did not exceed 20 %, except in the cv. ‘Krasnaja Grozd’ (22.7 %). The stability of fruitnumber in a cluster differed subject to the accession. The variation of fruit number in a cluster ofaccessions P1 and ‘Leningradskaja Otbornaja’ was low (CV < 10 %). The medium coefficient ofvariation (10 % < CV < 20 %) was determined for the clone P2 and the cultivars ‘Krasnaja Grozd’,19

‘Kijevskaja Sadovaja’, and ‘Zarnica’. The high variability of fruit number in a cluster was typicalof the cultivars ‘Souzga’ and ‘Šukšinskaja’, respectively: 30.1 % and 25.4 %.Regression analysis revealed that the response of yield per bush to the amount of fruit in a clustercould be best expressed by the exponential equation y = 0.33685 × e 0.4969x at the coefficient ofdetermination R 2 = 0.895 (Fisher’s criterion F=88.4**). The relationship between the yield andmean weight of a fruit was poor and not significant while the coefficient of regression R 2 = 0.138(Fisher’s criterion F=4.8*).On the basis of the obtained results it is possible to state that the number of fruit in a cluster is onein the most important indexes for the selection of V. opulus accessions with predictable high yield.Table 1. Comparison of V. opulus accessions fruiting characteristics, 2000 – 2007AccessionMean fruit Fruit number per Stalk length of Yield, kg/bushweight, g raceme raceme, cm‘Krasnaja Grozd’ 0.51 abcd 51.0 e 3.1 ab 5.8 abc‘Kijevskaja Sadovaja’ 0.64 efgh 34.4 abc 3.8 bcde 4.1 abc‘Leningradskaja Otbornaja’ 0.66 fgh 43.1 bcde 2.9 a 8.5 c‘Zarnica’ 0.56 cdef 35.1 abcde 3.6 abcd 6.2 abc‘Souzga’ 0.54 bcde 32.3 ab 3.9 cde 2.7 a‘Šukšinskaja’ 0.45 ab 33.8 abc 3.6 bcd 3.0 a‘Zarnica-2’ 0.51 abcd 33.8 abc 4.2 de 2.6 a‘Upninkai’ 0.74 h 28.1 a 3.9 cde 4.3 bcP1 0.58 def 37.8 abcde 4.2 de 3.2 abcP2 0.56 cdef 35.0 abcde 3.2 abc 5.9 abcP3 0.47 abc 42.5 bcde 4.6 efg 6.3 abcMeans followed by the same letter in columns are not significantly different at the P=0.05 (Duncan‘smultiple range test).The evaluation of the fruits chemical composition showed that they accumulate quite large amountsof flavonols (Figure 1).The largest amount of flavonols was found in the fruits of the cultivars ‘Krasnaja Grozd’ and P1(on average about 18 mg 100 g -1 ). The lowest content of flavonols was found in the fruits of thecultivar P2 – less than 14 mg 100 g -1 .flavonols, mg/100g20151050'Zarnica''Souzga''Šukšinskaja''Krasnajagrozd'P2accessionP1'Kijevskajasadovaja'Figure 1. Amounts of flavonols in the fruits of V. opulus, accessions, 1999 – 2002The accessions were compared according to the amounts of ascorbic acid, anthocyanins,polyphenolic compounds, and benzoic acid. Statistically reliable differences were ascertained(Table 2).The fruit of different accessions accumulated from 22.9 mg 100 g -1 (P3) to 49.9 mg 100 g -1(‘Leningradskaja Otbornaja’) of anthocyanins and from 732.5 mg 100 g -1 (‘Souzga’) to 1280.0 mg100 g -1 (P3) of polyphenolic compounds. The smallest amount of anthocyanuins was noted in thefruits of the cultivar P3 (22.9 mg 100 g -1 ) and the smallest amount of polyphenolic compounds inthe fruits of the cultivar ’Souzga’ (732.5 mg 100 g -1 ). The amount of ascorbic acid content rangedfrom 13.0 mg 100 g -1 (‘Souzga’) to 44.7 mg 100 g -1 (P3).'Leningradskaja otbornaja'20

As several authors have stated (Kozłowski et al., 2006; Рупасова and Василевская, 1999), theberries of the Ericaceae family species accumulate benzoic acid on an average of 65 mg 100 g -1 .Benzoic acid has been regarded as the preserving agent responsible for the good preservativequalities of cranberry, cowberry and products manufactured from them (Shwartz and Medrek,1968; Smolarz, 2003). The fruit of V. opulus accessions accumulated benzoic acid, with an averageof 12.4±0.90 mg 100 g -1 . The amounts of benzoic acid in different V. opulus accessions arepresented in the fig. 2. The cultivar P2 accumulated the highest amount of benzoic acid (16.3 mg100 g -1 ).benzoic acid, mg/100g181614121086420'Zarnica''Souzga''Šukšinskaja''Krasnajagrozd'P2accessionP1'Kijevskajasadovaja''Leningradskajaotbornaja'Figure 2. Amounts of benzoic acid in the fruits of V. opulus, accessions, 1999 – 2002The statistical analysis of the biochemical components of the fruit revealed a high variabilitybetween the accessions investigated. The most stable biochemical characteristic was the amount offlavonols (CV=10.4 %). Very high variations in the amount of ascorbic acid, anthocyanins andbenzoic acid amounts was ascertained (45.7, 24.1 and 20.4 %, respectively).Table 2. Amount of biochemical compounds in the fruit of V. opulus accessions, 2007AccessionBiochemical components, mg 100 g -1Polyphenolic compounds Anthocyanins Ascorbic acid‘Krasnaja Grozd’ 1136.0 bc 36.8 defg 20.9 b‘Kijevskaja Sadovaja’ 1103.1 bc 35.7 def 26.6 c‘Leningradskaja Otbornaja’ 962.0 ab 49.9 i 13.8 a‘Zarnica’ 976.9 ab 39.0 fg 26.4 c‘Souzga’ 732.5 a 34.0 cd 13.0 a‘Šukšinskaja’ 1007.0 bc 31.0 bc 31.9 d‘Zarnica-2’ 1002.5 abc 38.4 def 21.2 b‘Upninkai’ 1065.6 bc 35.2 de 33.3 deP1 1157.9 bcd 40.4 f 32.2 gP2 1220.5 bcdef 37.9 cdef 35.3 deP3 1280.0 cdef 22.9 a 44.7 fMeans followed by the same letter in columns are not significantly different at the P=0.01(Duncan‘s multiplerange test).As a result of the research the most valuable accessions (‘Leningradskaja Otbornaja’, P3, ‘Zarnica’and ‘Krasnaja Grozd’) in regard to the largest productivity and significant amounts of biologicallyactive substances could be selected for the breeding.References1. Bock K, Jensen S.R, Nielsen BJ, Norn V. (1978) Iridoid allosides from Viburnum opulus.Phytochemistry, 17(4), pp.753-756.2. Helrich K . (1990) Official methods of Analysis.3. Jordheim M., Giske N.H., Andersen O.M. (2007) Anthocyanins in Caprifoliaceae. BiochemicalSystematics and Ecology, 35, pp.153-159.21

4. Kozłowski J, Buchwald W, Mścisz A et al. Uprawa borówki i Ŝurawiny (z elementami ekologii)(Growing of Blueberries and Cranberries (With Ecological Aspects), 2006, pp.229-235. (In Polish).5. Panteev A., Batchilo A., Grakovich Z. (1995) The Breeding of Chaenomeles japonica Lindl (JapaneseQuince), Cerasus tomentosa Wall. (felt cherry), and Viburnum opulus L. (Snowball tree) in the republicBelarus. Acta Horticulturae. 390, pp.133-136.6. Shwartz J., Medrek T. (1968) Antifungal Properties of Cranberry Juice. Applied Microbiology, 16(10),pp.1524-1527.7. Smolarz K. (2003) Uprawa borówki i Ŝurawiny (Growing of Bluebeeries and Cranberries), pp.182-187.(In Polish).8. Velioglu Y.S., Ekici L., Poyrazoglu E.S. (2006) Phenolic composition of European cranberrybush(Viburnum opulus L.) berries and astringency removal of its commercial juice. International Journal ofFood Science and Technology. 41, pp.1011-1015.9. Ермаков А., Арасимович В. (1987) Методы биохимических исследований растений (The Methodsof Plants Biochemical Investigation). 136 p. (In Russian).10. Куденков М., Чурканенко Н.. (1998) Varieties of guelder rose and sea buckthorn released in Russia.Садовотство и виноградовотство (Horticulture and Viticulture). 2, pp.20-21. (In Russian).11. Лобанова А., Сысолятин С., Сакович Г., Зимина И. (1999) Масло плодов Viburnum opulus L. (Oilsof Viburnum opulus L. fruits) Химия растительного сырья (Chemistry of Vegetatyve Raw Matherial).4, pp.101-103. (In Russian).12. Оводова Р., Головченко В., Попов С., Шашков А. (2000) The isolation, preliminary study of structureand physiological activity of water-soluble polysaccharides from squeezed berries of snowball treeviburnum opulus. Биоорганическая химия (Bioorganic Chemistry). 26(1), pp.61-67. (In Russian).13. Рупасова Ж., Василевская Т. (1999) Клюква крупноплодная в Беларуси (Large Fruited Cranberry inBelarus), pp. 6-31. (In Russian).PROPAGATION AND CULTIVATION OF VACCINIUM SPECIES AND LESSKNOWN SMALL FRUITSVACCINIUM ĂINTS SUGU UN MAZĀK ZINĀMO AUGěAUGUPAVAIROŠANA UN AUDZĒŠANASamir C. DebnathAtlantic Cool Climate Crop Research Centre, Agriculture and Agri-Food Canada,P.O. Box 39088, 308 Brookfield Road, St. John’s, NL A1E 5Y7, Canada,e-mail: Samir.Debnath@AGR.GC.CAAbstractThe production of Vaccinium species crops has recently been the subject of much interest globallybecause of an improved understanding of the important role of dietary fruit in maintaining humanhealth. Cloudberry (Rubus chamaemorus L., family Rosaceae), a less known small fruit ofmedicinal importance, and the Vaccinium species are genetically heterozygous and do notreproduce progeny from seed that are similar to the seed parent. Tremendous progress in planttissue culture, resulting in great advances in micropropagation, has occurred in these crops. Ofparticular significance has been the evolution of the technology permitting multiplication of theseplants through bioreactor micropropagation. The in vitro morphogenesis seems to be highlydependent on the plant growth regulators and media used for the culture, which is again genotypespecific. Although the automation of micropropagation in bioreactors has been advanced as apossible way of reducing propagation cost, optimal plant production depends upon a betterunderstanding of the physiological and biochemical responses of plants to the signals of the culturemicroenvironment and an optimization of specific physical and chemical culture conditions tocontrol the morphogenesis of berry plants in liquid culture systems. Clonal fidelity can be a seriousproblem and molecular strategies have been developed to reduce the variation to manageablelevels. The paper focuses on conventional and bioreactor systems used for the in vitro culture of theVaccinium species and cloudberry, cultivation of micropropagules and the employment ofmolecular markers in micropropagated plants for the assessment of genetic fidelity, uniformity,stability and true-to-typeness among donor plants and tissue culture regenerants.22

KopsavilkumsVaccinium ăints kultūraugu audzēšanai pēdējā laikā visā pasaulē pievērsta pastiprināta uzmanība,jo arvien labāk tiek izprasta šo augĜu diētiskā nozīme cilvēka veselības saglabāšanai. Lācenes(Rubus chamaemorus L., Rosaceae dzimta) – mazāk zināmi augĜi ar medicīnisku nozīmi unVaccinium ăints sugas ir ăenētiski heterozigoti un, pavairojot ar sēklām, nedod vecākiem līdzīguspēcnācējus. Milzīgais progress augu šūnu kultūru izpētē ir izraisījis arī lielus uzlabojumus šokultūraugu mikropavairošanā. Īpaši nozīmīga ir bijusi tādas tehnoloăijas attīstība, kas Ĝauj šo augumikropavairošanu veikt bioreaktorā. Morfoăenēze in vitro apstākĜos ir Ĝoti atkarīga no kultivēšanāizmantotajiem augu augšanas regulatoriem un barotnes, kas ir atkarīga no genotipa. Lai ganmikropavairošanas automatizācija bioreaktoros ir attīstīta kā iespējamais pavairošanas izmaksusamazināšanas ceĜš, optimāla stādu ražošana ir atkarīga no labākas izpratnes par augu bioėīmiskoun fizioloăisko reakciju uz mikrovides signāliem un specifisku fizikālu un ėīmisku kultivēšanasapstākĜu optimizācijas, lai kontrolētu ogaugu morfoăenēzi šėidrās kultivēšanas sistēmās. Klonālāmainība var būt nopietna problēma, tāpēc ir izveidotas molekulāras metodes, lai samazinātu šomainību līdz iespējami zemākam līmenim. Šajā rakstā lielākā uzmanība pievērstakonvencionālajām un bioreaktora sistēmām Vaccinium ăints kultūraugu un lāceĦu in vitropavairošanai, mikropavairoto augu kultivēšanai un molekulāro marėieru izmantošanai ăenētiskāsatbilstības, viendabīguma, stabilitātes un autentitātes novērtēšanai starp donora augiem un audukultūrā iegūtajiem stādiem.Key words: cloudberry, propagation, micropropagation, in vitro, genotype.IntroductionThe genus Vaccinium L. (family: Ericaceae) contains about 400 species, and one or more speciesare native to all continents except Antarctica and Australia (Vander Kloet, 1988; Ballington, 2001).It is typically characterized as having fleshy, more-or-less edible fruits with very high levels ofvitamin C, cellulose, pectin and anthocyanins possessing antitumor, antiulcer, antioxidant andantiinflammatory activities (Wang et al., 1999). The proanthocyanidins in cranberries have beenshown to help prevent urinary tract infections through reduced adhesion of uropathogenicEscherichia coli (Howell et al., 2005). Lingonberry fruits and leaves are used to lower cholesterollevels and treat stomach disorders, rheumatic diseases, and bladder and kidney infections (Novelli,2003). Blueberry (Vaccinium spp.), cranberry (V. macrocarpon Ait.), and lingonberry (V. vitisidaeaL.) are three commercially cultivated Vaccinium fruit crops of economic importance.Although the majority of cultivated blueberry hectarage is in the United States and in Canada, theyare also grown commercially in Europe, Asia, Africa, Australia, New Zealand and South America(Lehnert, 2008). While the leading countries in cranberry production are the United States, Canada,Latvia and Poland; its culture has also shown promise in Austria, Germany and Russia(http:////aesop.rutgers.edu/~bluecran/cranberrypage.htm).Commercial lingonberry production primarily involves the harvesting of berries from wildpopulations in northern Europe, Asia and North America, with cultivated production still in itsinfancy compared with cranberries and blueberries (Ballington, 2001). The cloudberry (Rubuschamaemorus L., family Rosaceae), a less known small fruit crop, is a boreal circumpolar,rhizomatous dioecious perennial herb common to bogs. The berries and leaves of cloudberry arerich in vitamin C and tannins, and possess high ellagic acid content (Amakura et al., 2000).Cloudberries are used medicinally to treat scurvey and diarrhea in traditional medicine (Thiem,2003).Although conventional vegetative propagation methods by cuttings or rhizome divisions aresuccessful in these species, the micropropagation of selected germplasm can potentially multiplyplants more rapidly than traditional propagation methods. Various culture conditions, basal mediaand growth regulators have been investigated for the micropropagation of these crops on semisolidgelled media (for review, please see Debnath, 2003a, 2006a, 2007a).However, these techniques are difficult to automate and the production cost is high. Automatedbioreactors for large scale production of micropropagated plants are important for themicropropagation industry. Bioreactors are self-contained, sterile environments which capitalize onliquid nutrient or liquid/air inflow and outflow systems, designed for intensive culture and control23

over microenvironmental conditions – aeration, agitation, dissolved oxygen, etc. (Paek et al.,2005). This review provides an overview of in vitro culture and the production of micropropagatedplants of blueberry, cranberry, lingonberry and cloudberry, and also highlights the research effortsof our programme at the Atlantic Cool Climate Crop Research Centre of Agriculture and Agri-Food Canada in St. John’s, Newfoundland and Labrador.Blueberry. There are five major groups of blueberry species which are commercially-grown:1) lowbush (V. angustifolium Ait., V. myrtilloides Michx., V. boreale Hall and Aald.), 2) highbush(V. corymbosum L.), 3) half-high, which are hybrid or backcross derivatives of highbush-lowbushhybridizations; 4) southern highbush, which were developed from the hybridization of V.corymbosum with one or more species (mainly V. darrowi Camp and V. ashei Reade); and 5)rabbiteye (V. ashei). Micropropagation techniques using gelled media for axillary shoot productionhave been developed for lowbush (Debnath, 2004, 2007b), highbush (Gajdošová et al., 2006;Litwińczuk and Wadas, 2008; Tetsumura et al., 2008) and rabbiteye (Lyrene, 1980) blueberries.Shoot cultures can be initiated from nodal segments or from shoot tips. Media with low ionicconcentrations are suitable for Vaccinium culture (Debnath and McRae, 2001a). While the woodyplant medium (WPM) (Lloyd and McCown, 1980) was the best for highbush blueberrymicropropagation (Sedlak and Paprstein 2009), Debnath (2004, 2007b) established in vitrolowbush blueberry cultures on a modified cranberry tissue culture medium (BM-C) (Debnath andMcRae, 2001a).Tetsumura et al. (2008) observed a mixture of equal parts of Murashige and Skoog (1962) (MS)and WPM containing 20 µM zeatin was the best for in vitro shoot proliferation of highbushblueberry cultivars. Zeatin was effective for shoot initiation and proliferation of lowbushblueberries (Debnath, 2004) although Gonzalez et al. (2000) observed the best shoot multiplicationof highbush blueberry with 25 µM N6-[2-isopentenyl] adenine (2iP) in the culture medium.A low concentration of an auxin [5.7 µM 3-indolyl-acetic acid (IAA)] is beneficial when added tothe induction medium (Morrison et al., 2000). However, using low levels of zeatin (2-4 µM) andsucrose (20 g l -1 ), Debnath (2004) reported an increased the in vitro-shoot multiplication rate of thelowbush blueberry by about 50 to 100-fold over a 12-week interval when shoots were exposed tolower irradiance (15 µmol m -2 s -1 ). A major problem in blueberry micropropagation is theformation of unwanted callus at the base of the explants and the occurrence of spontaneousadventitious shoots (Litwińczuk and Wadas, 2008).Cao et al. (2002) reported shoot regeneration in the highbush blueberry based on a two-step pretreatmentand regeneration on TDZ medium. Explants of 2-week-old shoot cultures were incubatedthe following regime: pretreatment medium # 1 containing 5 µM TDZ and 2.6 µM naphthaleneacetic acid (NAA) for 4 days, pretreatment medium #2 containing 7 µM zeatin riboside and 2.6 µMNAA for 3 days, regeneration medium containing 1 µM TDZ for 6 weeks, and last on a mediumwithout growth regulators for 10 days. Debnath (2009a) developed a two-step shoot regenerationprotocol in lowbush blueberry where leaf cultures produced multiple buds and shoots on 2.3–4.5 µM TDZ within 6 wk of culture initiation. The greatest shoot regeneration came from youngexpanding basal leaf segments positioned with the adaxial side touching the culture medium andmaintained for 2 weeks in darkness. TDZ-initiated cultures were transferred to a mediumcontaining 2.3–4.6 µM zeatin and produced usable shoots after one additional subculture.Application of bioreactor micropropagation in Vaccinium crops is still at the infancy stage. Aprotocol for Vaccinium micropropagation using a temporary immersion bioreactor (TIB) system ina liquid medium combined with a in vitro culture on a semi-solid gelled medium has beendeveloped in the author’s laboratory. Successful shoot regeneration and proliferation have beenobtained in the lowbush blueberry (Figure 1.), cranberry and lingonberry (S. C. Debnath,unpublished).In vitro-derived shoots are rooted either in vitro (Litwińczuk and Wadas, 2008; Tetsumura et al.,2008) or, most frequently, in ex vitro conditions on an acidic substrate such as 1 peat : 1 perlite(v/v) (Gonzalez et al., 2000) and 4 peat : 2 vermiculite : 1 perlite (v/v/v) (Morison et al., 2000)without an auxin-pretreatment. An auxin-pretreatment was unnecessary for the ex vitro rooting ofblueberries (Gonzalez et al., 2000) although Debnath (2009a) found 80 % to 90 % rooting inlowbush blueberries when microshoots were dipped in 4.9 mM 3-indolebutyric acid (IBA) beforeplanting in 3 peat : 2 perlite (v/v) medium. For ex vitro rooting, the microcuttings are generally24

maintained in a mist chamber with very high relative humidity (95 %) and then transferred to agreenhouse (85 % relative humidity, RH) for acclimatization. In vitro rooting can be induced in theshoot proliferation medium containing 1-2 µM zeatin (S.C Debnath, personal communication) orwithout plant growth regulators (PGR) (Tetsumura et al., 2008).Figure 1. Shoot proliferation of wild lowbush blueberry 12 weeks after transfer to a bioreactorsystem containing liquid medium supplemented with 2 µM zeatin.Cranberry. Marcotrigiano and McGlew (1991) and Smagula and Harker (1997) recommend a high2iP concentration along with an auxin (IAA or IBA) in the culture media to increase cranberryshoot proliferation. Debnath and McRae (2001b) established in vitro cranberry cultures andmaintained them in a medium containing low levels of cytokinin to avoid excessive callusformation at the base of explants and the formation of somaclonal variants.Shoot organogenesis from cranberry explants has been reviewed by McCown and Zeldin (2005). Anumber of factors such as genotype, culture medium (including growth regulators and theircombinations), the physical environment, the explant development stage, etc. can affectadventitious shoot regeneration. Qu et al. (2000) regenerated shoots from cranberry leaves byculturing on a basal medium supplemented with 10 µM TDZ + 5 µM 2iP. Elongation ofadventitious shoots began 2 weeks after transfer to the basal medium without growth regulators.Both in vitro and ex vitro methods have successfully been used to root and acclimatizemicropropagated cranberry shoots (Qu et al., 2000; Debnath and McRae, 2001b). For in vitrorooting, shoots are cut at the base and then placed onto an auxin-free medium (Qu et al., 2000;Debnath and McRae, 2001b, 2005). In vitro-derived shoots (>1.5 cm long) can also be rooted exvitro in shredded sphagnum moss in pots (Qu et al., 2000).Debnath and McRae (2005) developed a protocol that enables cranberry multiplication in one step,i.e. multiplying shoots and having them rooted in the same culture medium containing 2-4 µMzeatin. The main advantage of this protocol is that all the shoot tips of the in vitro-grown plantletscan be used for shoot proliferation and rooting, whereas basal rooted nodal segments can betransferred to the peat-perlite medium and acclimatized in the greenhouse (Debnath, 2008).Lingonberry. Lingonberries grow wild in diverse habitats, ranging from lowland to upland andmountain areas, in largely acid soils to pure peat bogs (Gustavsson, 1997). Two subspecies of V.vitis-idaea have been recognized: the larger lowland race as V. vitis-idaea ssp. vitis-idaea (L.)Britton and the dwarf arctic-montane race as V. vitis-idaea ssp. minus (Lodd.) Hult. (Hulten, 1949).Various culture conditions, basal media, and growth regulators have been investigated for axillaryshoot proliferation of the lingonberry (Debnath and McRae, 2001a; Jaakola et al., 2001; Debnath,2005a, b). Debnath and McRae (2001a) compared four different media for the shoot proliferationof lingonberry cultivars: ‘Regal’, ‘Splendor’ and ‘Erntedank’, and found that a reasonable balanceof shoot multiplication rate and desirable growth characteristics was attained in a new medium25

(BM-C) formulated in the author’s laboratory (Debnath and McRae, 2001a). Debnath (2005a)observed that TDZ supported shoot proliferation in lingonberries at low concentrations (0.1 to 1µM) but inhibited shoot elongation. However, usable shoots were obtained within 4 weeks bytransferring shoot clusters to the culture medium containing 1µM zeatin. In the lingonberry, shootproliferation is greatly influenced by explant orientation, changing the orientation of explants fromvertically upright to horizontal increases the axillary shoot number, but decreases shoot height andleaf number per shoot (Debnath, 2005a). Debnath (2005b) observed that the best response wasafforded by sucrose at 20 g l -1 both in terms of explant response and shoot development potential,although glucose supported shoot growth equally well, and in a wild clone at 10 g l -1 it resulted inbetter in vitro growth than sucrose.The first adventitious shoot regeneration from lingonberry leaves was described by Debnath andMcRae (2002). Later, the regeneration efficiency has been much improved by Debnath (2005c)where leaf explants were cultured on the 1-5 µM TDZ-containing a nutrient medium for 8 weeksfor bud and shoot regeneration followed by transferring on to the medium containing 1-2 µM zeatinfor shoot elongation. Adventitious shoots have also been regenerated from hypocotyl segments ofseedlings from open-pollinated seeds of lingonberry cultivars and a wild clone (Debnath, 2003b).Multiple bud and shoot regeneration can be obtained using apical segments of the hypocotyl fromin vitro-grown lingonberry seedlings by incorporating 5-10 µM TDZ in the regeneration medium.Such TDZ-induced buds can be proliferated and elongated on a shoot proliferation mediumcontaining 1-2 µM zeatin and 20 g l -1 sucrose. Callus, bud, and shoot regeneration frequency, callusgrowth, and the number of buds and shoots per regenerating explant depend not only on thespecific segment of the hypocotyl, but also on the parental genotype (Debnath, 2003b).For rooting, 3 to 4 cm long in vitro-derived shoots are excised just above the original explant,dipped in 39.4 mM IBA powder, planted in a 2 peat : 1 perlite (v/v) medium and maintained in ahumidity chamber [(22 ∀ 2ΕC, 95 % RH, 16 h photoperiod, 55 µmol m -2 s -1 photosynthetic photonflux (PPF)]. In vitro proliferated shoots root easily within 4 weeks. Plantlets can be acclimatizedby gradually lowering the humidity over 2 to 3 weeks and hardened-off plants can be maintained inthe greenhouse at 20 ∀ 2ΕC, 85 % RH, and 16 h photoperiod at a maximum PPF of 90 µmol m -2 s -1(Debnath and McRae, 2001a, 2002; Debnath, 2003a, b; 2005a, b, c).Cloudberry. In vitro propagation of cloudberry has been reported in a gelled medium throughaxillary shoot initiation from seedling explants (Thiem, 2001) and through meristem cultures(Martinussen et al., 2004). When meristem cultures were sub-cultured from clusters of 3 – 5shoots, approximately 70 and 50 shoots were produced per cluster within 6 weeks at 8.9 µM BAPfor the female cv. ‘Fjellgull’ and the male cv. ‘Apollen’, respectively. The addition of 5.5 µMgibberellic acid (GA3) reduced the number of shoots. Auxins (IBA, NAA) promoted rootdevelopment in vitro, but inhibited the formation of new shoots (Martinussen et al., 2004).Debnath (2007c) established a protocol for the in vitro culture of wild cloudberry clones using abioreactor system combined with a gelled medium. Cultures were established on a modifiedcranberry (V. macrocarpon Ait.) tissue culture medium containing 8.9 µM BAP. The addition of5.8 µM GA3 in 8.9 µM BAP-contained medium improved shoot proliferation. TDZ supportedrapid shoot proliferation at low concentration (1.1 µM) but induced a 20 to 30 % hyperhydricity ina plastic airlift bioreactor system containing a liquid medium. The bioreactor-multipliedhyperhydric shoots were transferred to a gelled medium containing 8.9 µM BAP and 5.8 µM GA3and produced normal shoots within 4 weeks of culture. Proliferated shoots were rooted on a pottingmedium with a 65 % to 75 % survivability rate of rooted plants. Growth and morphology ofmicropropagated plants. Increased branching and vigorous vegetative growth are often noted inplants produced through in vitro culture. Morrison et al. (2000) observed that micropropagatedlowbush blueberry plants from shoots that passed through several subcultures produced ten-foldmore rhizomes than those of stem cuttings. The micropropagated lowbush blueberry plantsproduced longer and more stems with more leaves per stem than the conventional cuttings(Debnath, 2007b).Softwood cutting-derived ‘Herbert’ highbush plants grew more slowly and produced less andshorter shoots than micropropagated ones, although the majority of cutting-propagated plantsdeveloped flowers earlier, flowered more abundantly and bore larger berries than those of tissueculture plants (Litwiczuk et al., 2005). Micropropagated cranberry plants have an excellent juvenile26

period and produce vigorous vegetative growth (mostly runners) during their first season but do notproduce flowers until their third growing season (Serres and McCown, 1994). Micropropagated‘Bergman’, ‘Pilgrim’ and ‘Stevens’ plants produced more runners and uprights with more leavesper upright than the conventional cuttings (Debnath, 2008).Debnath (2005d, 2006b) observed that the in vitro-derived lingonberry plants produced morestems, leaves and rhizomes than the conventional cuttings. Under field condition, rhizomeproduction and total plant weight were greater for tissue culture plants than for stem cuttings in thelingonberry cultivar, ‘Sanna’ (Gustavsson and Stanys, 2000). After 4 years of growth, the tissueculture plants of ‘Splendor’ and ‘Erntedank’ lingonberries produced berries with more antioxidantactivity, although the berry diameter, number and yield per plant were higher in the stem cuttingplants (Foley and Debnath, 2007).ConclusionsThe commercial propagation of the Vaccinium species and cloudberry by tissue culture isbecoming increasingly common as it is a reliable and efficient method, especially for the rapidintroduction of new cultivars. In breeding programs, the technique can provide advantages in: (i)the mass production of elite selections and for analysis in a replicated trial of new releases, (ii)germplasm conservation, (iii) accelerating the breeding process by in vitro selection.Large-scale liquid cultures combined with automated bioreactors can eliminate most manualhandling in micropropagation and decrease production costs significantly. Cultures in liquidmedium are advantageous for several plant species but may limit the gas exchange of the plantmaterials and often cause asphyxia and hyperhydricity, resulting in malformed plants and loss ofmaterial.True-to-type propagules and genetic stability are prerequisites for the application ofmicropropagation. Molecular markers are powerful tools in the genetic identification of clonalfidelity. Special classes of markers including restriction fragment length polymorphism (RFLP),random-amplified polymorphic DNA (RAPD), arbitrary primed PCR (AP-PCR), DNA amplifiedfingerprinting (DAF), simple (short) sequence repeat (SSR), short tandem repeat (STR), sequencecharacterized amplified region (SCAR), sequence-tagged sites (STSs), amplified fragment lengthpolymorphism (AFLP) and inter simple sequence repeat (ISSR) are appropriate for geneticanalysis of tissue culture-raised plants. RAPD and ISSR marker analyses have been developed inthe author’s laboratory to identify genetic diversity in the Vaccinium species (Debnath, 2007d,2009b) and in cloudberry germplasm (Debnath, 2007e), and can be used to study the clonal fidelityof the micropropagated plants of these species.References1. Amakura Y., Okada M., Tsuji S. and Tonogai Y. (2000) High-performance liquid chromatographicdetermination with photodiode array detection of ellagic acid in fresh and processed fruits. Journal ofChromatography A, 896, pp.87-93.2. Ballington J.R. (2001) Collection, utilization, and preservation of genetic resources in Vaccinium.HortScience, 36, pp.213-220.3. Cao X., Hammerschlag F.A. and Douglass L (2002) A two-step pretreatment significantly enhances shootorganogenesis from leaf explants of highbush blueberry cv. Bluecrop. HortScience, 37, pp.819-821.4. Debnath S.C. (2003a) Micropropagation of Small Fruits. In: Jain S.M. and Ishii K. (eds) Micropropagation ofWoody Trees and Fruits, Kluwer Academic Publications, Dordrecht, Germany, pp.465-506.5. Debnath S.C. (2003b) Improved shoot organogenesis from hypocotyl segments of lingonberry (Vacciniumvitis-idaea L.). In Vitro Cellular and Developmental Biology – Plant, 39, pp.490-495.6. Debnath, S.C. (2004) In vitro culture of lowbush blueberry (Vaccinium angustifolium Ait.). Small FruitsReview, 3, pp.393-408.7. Debnath S.C. (2005a) Micropropagation of lingonberry: influence of genotype, explant orientation, andovercoming TDZ-induced inhibition of shoot elongation using zeatin. HortScience, 40, pp.185-188.8. Debnath S.C. (2005b) Effects of carbon source and concentration on development of lingonberry (Vacciniumvitis-idaea L.) shoots cultivated in vitro from nodal explants. In Vitro Cellular and Developmental Biology –Plant, 41, pp.145-150.9. Debnath S.C. (2005c) A two-step procedure for adventitious shoot regeneration from in vitro-derivedlingonberry leaves: shoot induction with TDZ and shoot elongation using zeatin. HortScience, 40, pp.189-192.10. Debnath S.C. (2005d) Morphological development of lingonberry as affected by in vitro and ex vitropropagation methods and source propagule. HortScience, 40, pp.760-763.27

11. Debnath S.C. (2006a) Propagation of Vaccinium in vitro: a review. International Journal of Fruit Science, 6,pp.47-71.12. Debnath S.C. (2006b) Influence of propagation method and indole-3-butyric acid on growth and developmentof in vitro- and ex vitro-derived lingonberry plants. Canadian Journal of Plant Science, 86, pp.235-243.13. Debnath S.C. (2007a) Strategies to propagate Vaccinium nuclear stocks for the Canadian berry industry.Canadian Journal of Plant Science, 87, pp.911-922.14. Debnath S.C. (2007b) Influence of indole-3-butyric acid and propagation method on growth and developmentof in vitro- and ex vitro-derived lowbush blueberry plants. Plant Growth Regulation, 51, pp.245-253.15. Debnath, S.C. (2007c). A two-step procedure for in vitro multiplication of cloudberry (Rubus chamaemorusL.) shoots using bioreactor. Plant Cell, Tissue and Organ Culture, 88, pp.185-191.16. Debnath S.C. (2007d) An assessment of the genetic diversity within a collection of wild cranberry(Vaccinium macrocarpon Ait.) clones with RAPD-PCR. Genetic Resources and Crop Evolution, 54, pp.509-517.17. Debnath S.C. (2007e). Inter-simple sequence repeat (ISSR)-PCR analysis to assess genetic diversity in acollection of wild cloudberry (Rubus chamaemorus L.) clones. Journal of Horticultural Science andBiotechnology, 82, pp.727-732.18. Debnath S.C. (2008). Zeatin-induced one-step in vitro cloning affects the vegetative growth of cranberry(Vaccinium macrocarpon Ait.) micropropagules over stem cuttings. Plant Cell, Tissue and Organ Culture, 93,pp.231-240.19. Debnath S.C. (2009a) A two-step procedure for adventitious shoot regeneration on excised leaves of lowbushblueberry. In Vitro Cellular and Developmental Biology – Plant, 45, pp.122-128.20. Debnath S.C. (2009b) Development of ISSR markers for genetic diversity studies in Vaccinium angustifolium.Nordic Journal of Botany, 27, pp.141-148.21. Debnath S.C. and McRae K.B. (2001a) In vitro culture of lingonberry (Vaccinium vitis-idaea L.): theinfluence of cytokinins and media types on propagation. Small Fruits Review, 1, pp.3-19.22. Debnath S.C. and McRae K.B. (2001b) An efficient in vitro shoot propagation of cranberry (Vacciniummacrocarpon Ait.) by axillary bud proliferation. In Vitro Cellular and Developmental Biology – Plant, 37,pp.243-249.23. Debnath S.C. and McRae, K.B. (2002) An efficient adventitious shoot regeneration system on excised leavesof micropropagated lingonberry (Vaccinium vitis-idaea L.). Journal of Horticultural Science anddBiotechnology, 77, pp.744-752.24. Debnath S.C. and McRae K.B. (2005) A one-step in vitro cloning procedure for cranberry (Vacciniummacrocarpon Ait.): the influence of cytokinins on shoot proliferation and rooting. Small Fruits Review, 4,pp.57-75.25. Foley S.L. and Debnath S.C. (2007) Influence of in vitro and ex vitro propagation on anthocyanin content andanti-oxidant activity of lingonberries. Journal of Horticultural Science andd Biotechnology, 82, pp.114-118.26. Gajdošová A., Ostrolucká M.G., Libiaková G., Ondrušková E. and Šimala D. (2006) Microclonal propagationof Vaccinium sp. and Rubus sp. and detection of genetic variability in culture in vitro. Journal of Fruit andOrnamental Plant Research, 14, pp.103-118.27. Gonzalez M.V., Lopez M., Valdes A.E. and Ordas R.J. (2000) Micropropagation of three berry fruit speciesusing nodal segments from field-grown plants. Annals of Applied Biology, 137, pp.73-78.28. Gustavsson B.A. (1997) Breeding strategies in lingonberry culture (Vaccinium vitis-idaea). ActaHorticulturae, 446, pp.29-137.29. Gustavsson B.A. and Stanys V. (2000) Field performance of ‘Sanna’ lingonberry derived by micropropagationvs. stem cuttings. HortScience, 35, pp.742-744.30. Howell A.B., Reed J.D., Krueger C.G., Winterbottom R., Cunningham D.G. and Leahy M. (2005) A-typecranberry proanthocyanidins and uropathogenic bacterial anti-adhesion activity. Phytochemistry, 66, pp.2281-2291.31. Hulten E. (1949) On the races in the Scandinavian flora. Svensk Botanisk Tidskrift Bd, 43, pp.383-406.32. Jaakola L., Tolvanen A., Laine K. and Hohtola A. (2001) Effect of N 6 -isopentenyladenine concentration ongrowth initiation in vitro and rooting of bilberry and lingonberry microshoots. Plant Cell, Tissue and OrganCulture, 66, pp.73-77.33. Lehnert D. (2008) Blueberry production is skyrocketing worldwide. − The Fruit Growers News, GreatAmerican Publishing, USA. Available at: http://www.fruitgrowersnews.com/pages/arts.php?ns=908.34. Litwińczuk W. and Wadas M. (2008) Auxin-dependent development and habituation of highbush blueberry(Vaccinium × covilleanum But. Et Pl.) 'Herbert' in vitro shoot cultures. Scientia Horticulturae, 119, pp.41-48.35. Litwi⏐czuk W., Szczerba G., and Wrona, D. (2005) Field performance of highbush blueberries (Vaccinium Ηcorymbosum L.) cv. >Herbert= propagated by cuttings and tissue culture. Scientia Horticulturae, 106,1pp.62-169.36. Lloyd G. and McCown B. (1980) Commercially feasible micropropagation of mountain laurel, Kalmialatifolia, by use of shoot tip culture. Combined Proceedings International Plant Propagators' Society, 30,pp.421-427.37. Lyrene P.M. (1980) Micropropagation of rabbiteye blueberries. HortScience, 15, pp.80-81.28

38. Marcotrigiano M. and McGlew S.P. (1991) A two-stage micropropagation system for cranberries. Journal ofthe American Society for Horticultural Science, 116, pp.911-916.39. Martinussen I., Nilsen G., Svenson L., Junttila O. and Rapp K. (2004) In vitro propagation of cloudberry(Rubus chamaemorus). Plant Cell, Tissue and Organ Culture, 78, pp.43-49.40. McCown B.H. and Zeldin E.L. (2005) Vaccinium spp. Cranberry. In: R. E. Litz R.E. (ed) Biotechnology ofFruit and Nut Crops, Biotechnology in Agriculture Series No. 29, CAB International, Wallingford, UK,pp.247-261.41. Morrison S., Smagula J.M. and Litten W. (2000) Morphology, growth, and rhizome development ofVaccinium angustifolium Ait. seedlings, rooted softwood cuttings, and micropropagated plantlets.HortScience, 35, pp.738-741.42. Murashige T. and Skoog F. (1962) A revised medium for rapid growth and bioassays with tobacco tissuecultures. Physiologia Plantarum,15, pp.473-497.43. Novelli S. (2003) Developments in berry production and use. Bi-weekly Bulletin, Agriculture and Agri-FoodCanada, Vol. 16, No. 21, pp.5-6.44. Paek K.Y., Chakrabarty D. and Hahn E.J. (2005) Application of bioreactor systems for large scale productionof horticultural and medicinal plants. Plant Cell, Tissue and Organ Culture, 81, pp.287-300.45. Qu L., Polashock J. and Vorsa N. (2000) A high efficient in vitro cranberry regeneration system using leafexplants. HortScience, 35, pp.948-952.46. Serres R. and B. McCown. (1994) Rapid flowering of microcultured cranberry plants. HortScience, 29,pp.159-161.47. Sedlak J. and Paprstein F. (2009) In vitro multiplication of highbush blueberry (Vaccinium corymbosum L.)cultivars. Acta Horticulturae, 810, pp.575-580.48. Smagula J.M. and Harker J. (1997) Cranberry micropropagation using a lowbush blueberry medium. ActaHorticulturae, 44, pp.343-347.49. Tetsumura T., Matsumoto Y., Sato M., Honsho C., Yamashita K., Komatsu H., Sugimoto Y. and Kunitake H.(2008) Evaluation of basal media for micropropagation of four highbush blueberry cultivars. ScientiaHorticulturae, 119, pp.72-74.50. Thiem B (2001) Micropropagation of cloudberry (Rubus chamaemorus L.) by initiation of axillary shoots.Acta Societatis Botanicorum Poloniae, 70, pp.11-1651. Thiem B. (2003) Rubus chamaemorus L. − a boreal plant rich in biologically active metabolites: a review.Biology Letters, 40, pp.3-13.52. Vander Kloet S.P. (1988) The genus Vaccinium in North America, Agriculture Canada Publication, 1828,Ottawa, Canada, 201 p.53. Wang H., Nair M.G., Strasburg M., Chang Y.C., Booren A.M., Gray J.I. and DeWitt D.L. (1999) Antioxidantand antiinflammatory activities of anthocyanins and their aglycon, cyanidin, from tart cherries. Journal ofNatural Products, 62, pp.294-296.CLIMATICALLY DETERMINATE PROJECTIONS OF RESOURCES OFVACCINIUM SPECIES IN BELARUS TO 2050KLIMATISKI NOTEIKTA PROGNOZE VACCINIUM ĂINTS SUGĀM BALTKRIEVIJĀLĪDZ 2050. GADAMValery GrimashevichForest Institute of the National Academy of Sciences of Belarus,Gomel, 220012 Minsk str. Surganova, 2v, Belarus, e-mail: grimashevich@gmail.comAbstractBased on the predicted dynamics of the forest fund in terms of global climate change we havemade a climatically determinate projection of the resources of major species of wild berry plants,namely, cowberry (Vaccinium vitis-idaea L.), bog blueberry (Vaccinium uliginosum L.), Europeancranberry (Oxycoccus palustris Pers.) and bilberry (Vaccinium myrtillus L.), in Belarus to the year2050 at 5-year intervals. The amounts of European cranberries and bilberries may increase, whilethose of cowberries and bog blueberries may decrease towards the end of the forecast period. Onthe whole the amounts of all the principal wild berry plants may increase by 11 percent.29

KopsavilkumsBalsoties uz prognozēto meža fonda dinamiku sakarā ar globālajām klimata pārmaiĦām, tika veiktaklimatiski noteikta prognoze galveno savvaĜas ogu sugu resursiem, tai skaitā brūklenēm(Vaccinium vitis-idaea L.), savvaĜas zilenēm (Vaccinium uliginosum L.), Eiropas dzērvenēm(Oxycoccus palustris Pers.) un mellenēm (Vaccinium myrtillus L.) Baltkrievijā līdz 2050. gadam arlaika intervālu 5 gadi. Eiropas dzērveĦu un melleĦu ražas var palielināties, bet brūkleĦu unsavvaĜas zileĦu samazināties aprakstītā perioda gaitā. Kopumā apskatīto svarīgāko meža ogu ražasvarētu palielināties par 11 procentiem.Key words: Vaccinium vitis-idaea L., Vaccinium uliginosum L., Vaccinium myrtillus L.,Oxycoccus palustris Pers., forecast.IntroductionNowadays forecasting is used on all levels of the national economy. Of prime importance is theenvironmental forecast. This is required to plan harmonious exploitation and protection of plantresources on the basis of scientific anticipation of environmental changes.Recent trends in Belarus are towards reorientation of the environmental policy to suit requirementsfor steady development. In this regard, therefore, major problems are those associated withforecasts of environmental changes. The most intricate ecological forecasts are required to foreseechanges in forest plant resources, including wild berry plant resources. In specific cases predictedenvironmental impact assessments may serve to devise scenarios of changes in plant resources indifferent regions of the country.The last few years have witnessed a decline in the resource potential of wild berry plants due to theincreasing scale of society’s impact on the forest ecosystems. Cuttings, reforestation, forest firesare very detrimental to the berry fields. Also, this decline is associated with radioactivecontamination, digression of forest phytocoenoses, drainage reclamation, violation of harvestingregulations and wanton consumption of berries. Management for timber production alone cannotpromote reproduction of minor forest products (Grimashevich, 2002, 2005).Forecasts for minor forest produce resources and their relationship with the dynamics of foreststands might ensure the rational use and protection of side products. A perfect forecast of thedynamics of forest stands, including berry fields, offers a means of carrying out silviculturalpractices to prevent negative trends.We recognise the following forecasts in relation to the forecast duration: seasonal (up to 1 year),short-range (1 to 5 years), extended-range (5 – 15 years), long-range (15 years to several decades)and very-long-range (several hundreds of years) (Loginov, 2004). Forty-two years being left to theyear 2050, our investigation was based on the long-range forecast.Materials and methodsUsing data from forest surveys of all the stands of forest-forming species occurring in Belarus andeach of the state production forestry associations (SPFA) and taking into consideration thegeobotanical subdivision, we systematised the data obtained and analysed the condition andstructures of the stands assayed. Principal parameter values and pre-existing dynamics of resourcesof the berry plants assayed were obtained on the basis of our earlier investigations and regularitiesrevealed in the current research (Grimashevich, 2002, 2005).Estimates of climate changes that may occur in Belarus over the period of the first fifty years of the21 st century were made on the basis of the results obtained through the use of the had CM2atmospheric circulation model (Great Britain). The years 1960 – 1990 were taken as the baseperiod. Climate changes, essential to forestry, according to this model, are a monthly rise inaverage temperature by 0.6 °С to 2.9 °С, a decrease in the transpiration rate and an insignificantincrease of rainfall in winter months when its role as a source of moisture for the current-yearvegetation period is slight (Loginov, 2004).In forecasting changes in the resources of wild berry plants at 5-year intervals we used verified dataobtained by us in the years 2003 – 2004; the data on variations in the typological structure of foreststands obtained by Lazareva (2007) in 2006 – 2007 being used as a basis for the data. Our forecast30

of changes in the typological structure of forest stands is in close agreement with that made by theRussian investigator Minin (2000). Hence our forecast of changes in the resources of wild berryplants in Belarus correlates with the dynamics of areas of forest stands and swamps by forest typeto the year 2050.To develop the climatically determinate dynamics of pineries of different forest types Lazareva(2007) used two main principles of theoretical representation of typological relations, namely, theSukachev net of edapho-phytocoenotic series of forest types and the Pogrebniak edaphic net offorest site types. The principle of the typological continuum of forest formation was used. Theoverlapping of some forest types points to the most commonly encountered edaphicallyconditioned associations of these types, which makes it possible to make a prediction about theirdynamics in theory (Loginov, 2003; Alexandrovich, 2003). Each forest type is represented by anumber of associations, of which the native one has the most significant place.With the model of changes of climate parameter values for each of the geobotanical districts to theyear 2050 and the relative values for ‘shifts’ in the air temperature change of 1 °С Lazareva (2007)succeeded in forecasting changes in the forest vegetation of the republic to the above mentionedyear.It is found that aridization may cause colonisation of mires by woody vegetation as a result ofwhich over the forecast period sedge-grass downy birch forests (P.-Betuleta caricosa), sedge-grassbogmoss downy birch forests (P.-Betuleta caricoso-sphagnosa), black alder forests (G.-Alneta),pineries (Pineta) and willow woods (Saliceta) may cover 40,000 to 45,000 hectares. Young standsof small-leaved trees and bushes may cover 11,000 to 12,000 ha of idle hay fields and pastures bythe year 2010 and 20,000 ha by 2020.By the year 2010, about 30,000 ha of cutover peatlands and 1,300 to 1,400 ha of quarries will beturned over to the forest fund. Also, about 20,000 ha of used peatbogs and about 1,500 ha ofquarries will be turned over to the forest fund in 2011-2020. It is expected that no less than 65percent of cutover peatlands and no less than 80 percent of quarries will be afforested while theremainder will be transformed into wetlands (Loginov, 2004). Large unproductive agriculturallands have already been turned over to the forest fund. To date, most of these lands have beenafforested.As a consequence of the reforestation of unproductive forestry lands and the colonisation of forestfreeareas by the year 2020 the forest cover percentages in Belarus will most likely be as high as 41– 42 percent. In other words, the forest cover percentage may approach that recorded in Belarus inthe latter half of the 19 th century.With respect to the stock and forest exploitation it is anticipated that future changes will bepositive. By the year 2020, for instance, more than 1 million ha of forest stands will mature and thearea of maturity stands will be almost doubled, which will account for 13.0 – 13.5 % of stockedforestlands. By 2015, young stands of the 1 st age class will most probably cover 1.2 million ha,which will account for 15 % of stocked forestlands. It is anticipated that the years 2025-2030 willsee the equalisation of the age structure (Loginov, 2004).In all likelihood, the prescribed cut will increase by 70 – 75 % by the year 2020, principal fellingvolume will rise to 11.8 million m 3 , felling volume of coniferous timber will increase by a factor of2.2 and account for 6.2 million m 3 a year. By the year 2020 the percentage of environment-orientedpartial clear cuttings will most likely run to 25 % of their total volume, which will amount to asmuch as 3.0 million m 3 a year (Loginov, 2004).The above changes formed the basis of the climatically determinate projections of wild berry plantresources in Belarussian forests to the year 2050 (Grimashevich, 2001, 2002, 2005, 2008;Lazareva, 2007; National strategy of steady development of the Republic of Belarus, 1997;Strategic plan of development of the forestry in Belarus, 1997).Results and DiscussionThe most detailed forecast of environmental changes in Belarus for 2010 – 2020 was made in 2004(Loginov, 2004). To forecast it was necessary to:- develop appropriate procedures;- reveal possible environmental changes;31

- substantiate a complex of measures to prevent and minimise negative environmentalchanges.We summed up and analysed the findings of researches on probable changes in plant resources topromote the development of a system of measures to be taken to prevent the decline in the resourcepotential of wild berry plants in Belarus.It is speculated that the global threshold level of consumption of primary produce has alreadyincreased many-fold, which generated the biosphere depletion and global ecological crisis(Loginov, 2004). The data obtained are indicative of the distinct relationship between globalchanges in the biosphere and anthropogenous factors (Loginov, 2003; Alexandrovich, 2003).About 11,500 plant species, including 2,100 higher plants and 9,000 to 9,400 lower plants, amongwhich are algae, lichens and fungi, are found naturally in Belarus (Loginov, 2004). Forestvegetation covers more than 38 percent of the territory of the republic.Belarus is located at the interface between two vegetation zones, namely, the boreal zone in whichconiferous forests predominate and the nemoral one in which deciduous summer broadleavedforests predominate. Here, therefore, three geobotanical subzones are recognised, namely, thesubzone of oak-dark coniferous southern taiga forests, the subzone of hornbeam-oak-darkconiferous sub-taiga forests and the subzone of mixed broadleaved-pine forests (Loginov, 2004).The last few years have been characterised by a large-scale dieback of spruce, oak and ash standsbrought on by droughts and heavy pest infestations. Forest fires, infectious tree diseases, windfalls,windbreaks, flooding and underflooding also cause grave damage to crops. Drainage reclamationthat brought on aridity in Polesye was one of the main factors extremely detrimental to theecosystems in Belarus. To date, the percentage of lichen, heath and bog moss forests has increasedby 1.5 – 2.0 percent. And this process will build up in the future (Loginov, 2004).In the context of global warming the monthly rise in temperature, the decrease in the depth of snowcover and freezing, and the increase in the probability of droughts are of paramount importance forforestry (Loginov, 2003; Alexandrovich, 2003). The increments therewith will most likely increaseby 5 % by 2020 and 10 % by 2050, fruits and seeds of forest trees and wild berries will presumablyripen 7 to 10 days and over earlier, and the silvicultural season will apparently begin 10 to 15 daysearlier (Loginov, 2004). Among the negative consequences of global warming are marked changesin the stand structure caused by the shift of the boundaries of the geographical ranges of spruce(Picea), hornbeam (Carpinus) and grey alder (Alnus incana), a longer fire danger period,favourable conditions for the reproduction of forest pests, the increase in probability of springfrosts, unfavourable growing conditions of stands owing to the decline of water table, higher ratesof evaporation and transpiration and unfavourable wintering conditions for plants due to unstablesnow cover. In Brest and Gomel Polesye the negative consequences will most likely be much morepronounced (Loginov, 2003; Loginov, 2004; Alexandrovich, 2003).Within the past fifteen years the percentage of pine formations dominated by the Vaccinium specieshas decreased by 6.4 percent. According to the data of scientific projections, global warming mayresult in an expansion of the distribution of mixed and broadleaved forests and forest-steppe and areduction in the area of coniferous stands.Forecasts of changes in the composition, productivity and resource potential of vegetation cover arerequired to take timely measures to adapt forestry and related branches of the economy of Belarusto the projected climate changes.The results of the 2006 – 2007 research done within the project on the climatically determinateforecasting of the typological structure of pineries suggest that by 2050 the transformation will verylikely bring about a decline in productivity of about 800,000 ha of pine forests, mainly at the costof upland pineries (Lazareva, 2007). More than 300,000 ha of mossy pine forests (Pinetapleuroziosa) will most likely grade into cowberry ones (Pineta vacciniosa); in this case the yieldclass may drop from 2 to 3. Around 700,000 ha of fresh mossy pine forests may grade into dryones; in this case the yield class may drop from 1 to 2. Also, the research points towards a probabledecline in productivity of around 40,000 hectares of bracken pineries (Pineta pteridiosa),particularly in the northern and central parts of the republic.The yield class for about 15,000 ha of wood sorrel pine forests (Pineta oxalidosa) in the northernand central parts of the republic may drop moderately from 1a to 1; in this case increasing aridity isnot a limiting factor for the growth of the pine stands. The yield class for cowberry pineries may32

fall from 2 to 3 and that for heath pine forests (Pineta callunosa) may drop from 3 to 4 (Lazareva,2007). The data of the forecast therefore indicate that productivity of around 200,000 ha of polytric(Pineta polytrichosa), bilberry (Pineta myrtillosa) and paludal pine forests, including sedge-grass(Pineta caricosa), sedge-grass-bog moss (Pineta caricoso-sphagnosa) and bog moss (Pinetasphagnosa) pineries will very likely increase with the increasing aridity (Lazareva, 2007).The forecast suggests that warming may cause a shift of the southern limit of the spruce continuosdistribution and replacement of the species by other pine forest-forming trees. In wood sorrel,bracken, bilberry and most mossy pineries spruce may be replaced by hornbeam and other types ofthe nemoral flora. Hence we predicted probable changes in the resources of wild berry plants fromchanges in the area of the pineries.The data obtained indicate that by 2050 the amounts of cowberry may decrease by 3.3 % (from2,528 to 2,444 tons). The decrease may be a maximum in the Brest (18 %), Gomel (15 %) andMogilev (10 %) SPFAs. In the Grodno and Minsk SPFAs the amount of cowberry may decrease by4 % and 2 %, respectively. The amounts in the Vitebsk SPFA may remain constant. Beginning in2030, the cowberry amount may stabilise in all the SPFAs as the result of equalisation of the forestecosystems.By the year 2050 the amount of bog blueberry may decrease by 5.3 % (from 1,132 to 1,072 tons).In view of aridization in Polesye the decrease may be at a maximum in the Gomel (11 %), Mogilev(8 %), Grodno (7 %) and Brest (5 %) SPFAs. The amount of bog blueberry may decrease by 4 % inthe Minsk SPFA and increase by 3 % in the Vitebsk SPFA. By the same reasoning as for cowberry,beginning in 2030 the bog blueberry amounts may stabilise in all the SPFAs.Table 1. Forecast of climatically determinate dynamics of the berry resources of all the principalwild berry plants in BelarusSPFABrestDynamics of wild berry resources by year in the form of tons : percent2008 2010 2015 2020 2025 2030 2035 2040 2045 20505,8161005,8101005,739995,672985,599965,533955,533955,533955,533955,53395Vitebsk14,56010014,68910115,29810515,96411016,74011517,66112117,66112117,66112117,66112117,661121Gomel8,0621008,0451007,987997,819977,700967,763957,763957,763957,763957,76395Grodno4,1441004,1801014,2791034,3771064,4381074,5401094,5401094,5401094,5401094,540109Minsk13,60210013,71810114,17210414,84810915,40711315,96811715,96811715,96811715,96811715,968117Mogilev2,8061002,8051002,8501022,8701022,9461053,0221073,0221073,0221073,0221073,022107TOTAL48,99010049,24710150,32510351,55010552,83010854,38711154,38711154,38711154,38711154,387111By the year 2050 the amount of the European cranberry in the republic may increase by 0.4 %(from 7,552 to 7,583 tons). In view of aridization in Polesye, however, the decrease in theEuropean cranberry amount may reach a maximum in the Gomel (12 %), Brest (11 %), Grodno (8%), Mogilev (7 %) and Minsk (3 %) SPFAs. In the Vitebsk SPFA the European cranberry amountsmay increase by 9 percent. Beginning in 2030, the cowberry amounts may stabilise in all theSPFAs.By the year 2050 the resources of bilberry in the republic may increase by 15 % (from 37,778 to43,288 tons). In view of aridization in Polesye, however, the bilberry resources may decrease by 4% and 3 % in the Brest and Gomel SPFAs, respectively. The forecast suggests that the resources of33

ilberry may increase by 30 % in the Vitebsk, 21 % in the Minsk, 15 % in the Mogilev and 12 % inthe Grodno SPFAs. Beginning in 2030, the cowberry resources may stabilise in all the SPFAs.According to the projections, by 2050 the amounts of all the principal wild berry plants in Belarusmay increase by 11 % (from 48,990 to 54,387 tons; Table 1.). In view of aridization in Polesye,however, the decrease in the amounts may be a maximum in the Brest (5 %) and Gomel (5 %)SPFAs. In the Vitebsk, Minsk, Grodno and Mogilev SPFAs the berry amounts may increase by 21percent, 17 %, 9 % and 7 %, respectively. Beginning in 2030, the amounts of the wild berry plantsmay stabilise in all the SPFAs.The predicted stabilisation of berry resources by 2030 will be due not only to the levelling andimprovement of the age and species structures of the forest fund but also to the measures taken tostabilise the general environmental situation in the republic. Among these are reswamping, repairsto drainage systems, the establishment of forest strips and the certification of harvesting berries.ConclusionsBased on the principal parameter values for principal wild berry plant resources for 2008 andfactors that govern their climatically determinate dynamics, we have forecasted the dynamics of theresources to the year 2050 at 5-year intervals.The amounts of European cranberries and bilberries may increase, while those of cowberries andbog blueberries may decrease towards the end of the forecast period. On the whole the amounts ofall the principal wild berry plants may increase by 11 percent.A complex of silvicultural and other measures should be worked out to conserve and increaseminor forest product resources. Silvicultural and special-purpose practices are being developed toovercome negative berry plant productivity trends.References1. Alexandrovich Y.M., Kochanovsky C.B. and Shershen L.I. (2003) Report. Project Forecasting ofenvironmental changes and working out measures to ensure environmental safety of the Republic ofBelarus for 2010-2020, Economic Research Institute of Ministry of Economic Affairs of Belarus, Minsk,Belarus. (In Russian) .2. Grimashevich V.V. (2001) Effects of spring frosts on the bearing of bilberries in Polesye. In: Questionsof Forest Ecology and Forest Exploitation in Ukrainian Polesye, issue 2 (8), Volyn, Zhitomir, theUkraine, pp. 152-154. (In Russian)3. Grimashevich V.V. (2002) The rational use of minor forest produce resources in Belarus, ForestInstitute of NAS of Belarus, Gomel, Belarus, 261p. (In Russian)4. Grimashevich V.V. (2005) The efficient use of resources of wild berry plants and edible mushrooms inBelarus in the conditions of market-oriented economy In: Transactions of the Forest Institute of NAS ofBelarus, issue 64, Forest Institute of NAS of Belarus, Gomel, Belarus, pp. 421-430. (In Russian)5. Grimashevich V.V. (2008) Substantiation of methods for a climatically determinate forecast of resourcesof wild berry plants and edible mushrooms. In: Kovalevich A.I. (ed.) Theoretical and Applied Aspects ofRational Use and Reproduction of Minor Forest Products. The International Practical-ScientificConference, Forest Institute of NAS of Belarus, Gomel, Belarus, pp. 28-32. (In Russian)6. Lazareva M.S. (2007) Report. Project 1.04/1 A climatically determinate forecast of the typologicalstructure of pineries and resources of minor forest products in Belarus to the year 2050. Phase 1.04/1.02A climatically determinate forecast of the typological structure of pineries in Belarus to the year 2050(state registration No 20065447), Forest Institute of NAS of Belarus, Gomel, Belarus, 61 p. (In Russian)7. Loginov V.F. (ed.) (2004) A forecast of environmental changes in Belarus for 2010-2020,Minsktipproekt, Minsk, Belarus, 180 p. (In Russian)8. Loginov V.F., Sachek G.I. and Mikutsky V.S. (2003) Climate changes and their consequences inBelarus, TONPIK Additional Liability Co, Minsk, Belarus, 330 p. (In Russian)9. Minin A.A. (2000) Phehology of Russian Plain: data and general conclusions, Minsk: ABF PublishingCo, 160 p. (In Russian)10. National strategy of steady development of the Republic of Belarus (1997) Minsk, Belarus (In Russian)11. Strategic plan of development of the forestry in Belarus (1997) Ministry of Forestry of Belarus, Minsk,Belarus178 p. (In Russian)34

HIGHBUSH BLUEBERRY BREEDINGAUGSTKRŪMU MELLEĥU SELEKCIJAJim HancockMichigan State University, East Lansing, USA, email: hancock@msu.eduAbstractMost of the blueberry cultivars now grown in the world came from the breeding programs ofFrederick Coville (1908 – 1937), George Darrow (1938-1958) and Arlen Draper (1965 - present) ofthe United States Department of Agriculture, and Paul Lyrene (1978 - present) of the University ofFlorida. Until about 30 years ago, highbush blueberry cultivation was restricted to cold climates, asthe original cultivated species, Vaccinium corymbosum, required about 800 – 1000 chilling hoursbelow 7 0 C for normal floral development. To expand the range of highbush blueberry cultivationinto the southern USA, Ralph Sharp of the University of Florida and Darrow hybridized theoriginal northern highbush types with native southern species. There are now three types ofhighbush blueberry varieties grown worldwide - Northern, Southern and Intermediate. These varyin the number of chilling hours they require for normal floral development and their level oftolerance to winter cold. The primary goals of today’s southern and Intermediate highbush breedersare to obtain early ripening types with high plant vigor, disease resistance and a later bloom(particularly in Florida). Northern highbush breeders are concentrating on flavor, longer storingfruit, expanded harvest dates, disease and pest resistance and machine harvestability. Current trendsin highbush blueberry breeding include speeding the cultivar release process, expanding thegermplasm base of varieties and licensing.KopsavilkumsLielākā daĜa no krūmmelleĦu šėirnēm, kas pašreiz tiek audzētas pasaulē ir izveidojuši FrederickCoville (1908 – 1937), George Darrow (1938 – 1958), Arlen Draper (1965 – pašlaik) un PaulLyrene (1978 – pašlaik). Apmēram pirms 30 gadiem augstkrūmu melleĦu audzēšanu ierobežojaaukstais klimats, jo kultivētām Vaccinium corymbosum sugām, lai ziedkopa normāli attīstītos,nepieciešamas 800 – 1000 aukstuma stundas zem + 7 0 C. Lai paplašinātu diapazonu augstkrūmumelleĦu audzēšanā ASV dienvidu daĜā, R. Šarps no Floridas universitātes un G.Darovs krustojavietējās ziemeĜu augstkrūmu mellenes ar vietējām dienvidu sugām.Šobrīd ir trīs veidu augstkrūmu melleĦu šėirnes, ko audzē visā pasaulē – ziemeĜu, dienvidu unstarpformu. Tās atšėiras ar nepieciešamo aukstuma stundu skaitu, lai attīstītos normālas ziedkopasun krūmi būtu aukstumizturīgi. Galvenais selekcijas mērėis dienvidu un starpformu krūmmellenēmir iegūt agrīnas, ar lielu augšanas spēku, pret slimībām izturīgas un vēlu ziedošas (īpaši Floridā)krūmmellenes. ZiemeĜu augstkrūmu melleĦu selekcionāri koncentrē uzmanību uz aromātisku oguieguvi, ilgāku augĜu uzglabāšanos, ienākšanās laika pagarināšanu, slimību un kaitēkĜu izturību unizturību pret mehanizētu vākšanu. Nozīmīgi augstkrūmu melleĦu selekcijā ir virzība uz ātrākušėirnes atzīšanas procesu, šėirĦu gēnu banku paplašināšanu un licences izsniegšanu.Key words: Vaccinium corymbosum, varieties, cultivarsIntroductionThere are now three types of highbush blueberry varieties grown - Northern, Southern andIntermediate. These vary in the number of chilling hours they require for normal floraldevelopment and their level of tolerance to winter cold. Northern highbush varieties are adapted toquite cold mid-winter temperatures below -20 0 C, but grow well anywhere there are 800 – 1000hours of chilling. These are grown primarily in Australia, France, Germany, Michigan, New Jersey,New Zealand, the Pacific Northwest, Poland and Chile. Southern highbush varieties do not toleratewinter temperatures much below freezing and require chilling hours under about 350 hours. Theyare grown primarily in Australia, Argentina, California, Florida, Chile and southern Spain.Intermediate highbush varieties have a wide range in chilling requirements from 400 – 800 hours.They generally fail in the colder climates because they bloom too early and are too slow to harden35

in the fall, resulting in freeze damage to the flower buds. The Intermediate highbush types aregrown primarily in Arkansas, Chile, North Carolina and the Pacific Northwest.History of blueberry breeding.Blueberry breeding is a very recent development (Hancock, 2006a; Lyrene, 1998). Highbushbreeding began in the early 1900s in New Jersey, with the first hybrid being released in 1908 byFrederick Coville of the United States Department of Agriculture (USDA). He conducted thefundamental life history studies of the blueberry that served as the basis of cultivation such as soilpH requirements, cold and day-length control of development, pruning strategies and modes ofpropagation. Working with Elizabeth White and others, he collected several outstanding wildclones of V. corymbosum and V. angustifolium, which he subsequently used in breeding improvedtypes. Over 75 % of the current blueberry acreage is still composed of his hybrids, most notably‘Bluecrop’, ‘Jersey’, ‘Weymouth’, ‘Croatan’, ‘Blueray’, ‘Rubel’ and ‘Berkeley’ (Mainland, 1998).George Darrow took over the USDA program after Coville died in 1937 and made importantcontributions on the crossibility and phylogeny of the native Vaccinium species working with thetaxonomist W. H. Camp (Hancock, 2006b). He formed a large collaborative testing network thatencompassed both private growers and Agricultural Experiment Station Scientists (AES) scientistsin Connecticut, Florida, Georgia, Maine, Massachusetts, Michigan, New Jersey and North Carolina.From 1945 to 1961, he sent out almost 200,000 hybrids to his cooperators for evaluation. ArlenDraper followed Darrow and focused on mixing the genes of most wild Vaccinium species into thecultivated highbush background. He maintained and strengthened Darrow’s collaborative networkand released a prodigious number of southern, intermediate and northern highbush cultivars, withimproved fruit color and firmness, smaller pedicle scars and higher productivity (Hancock andGalletta, 1995). His Northern highbush ‘Duke’ and ‘Elliott’ have been major successes, along withhis newer Intermediate release ‘Legacy’. Mark Ehlenfedlt took over the USDA program in 1998.Ralph Sharp began working in the 1950s in Florida on the development of Southern highbush typesin collaboration with Darrow (Sharp and Darrow, 1959; Lyrene, 1998). To expand the range ofhighbush blueberry cultivation into the southern USA, they hybridized the original northernhighbush types with native southern species. Sharp was the first collector of V. darrowii forbreeding, and until very recently, all southern highbush cultivars contained genes from his wildclones. Sharp developed a number of successful cultivars, including ‘Sharpblue’, which was growncommercially until very recently. Paul Lyrene took over the breeding work in Florida in the late1970s.Stanley Johnson at Michigan State University spent a considerable amount of time in the 1950s and1960s improving the cold tolerance of highbush by crossing it with V. angustifolium. Out of thiswork came the “half-high” cultivar Northland and the mostly pure Northern highbush type‘Bluejay’, which was released by his successor Jim Moulton. The program was abandoned in1978, but was renewed in 1990 by Jim Hancock.Joseph Eberhart, in Washington released three Northern highbush cultivars, Pacific, Olympia, andWashington in the 1920s and 1930s. ‘Olympia’ is still grown today in the Pacific Northwest, butnot planted.Outside of the USA, blueberry breeding work was conducted in Australia, Germany and NewZealand. Johnston sent open pollinated seed to D. Jones and Ridley Bell in Australia in the 1960sthat generated the important Northern highbush cultivar ‘Brigitta Blue’ along with several others.Narandra Patel at HortResearch in New Zealand released the Northern highbush cultivars Nui, Puruand Reka from breeding material initially provided by the University of Arkansas and the USDA atBeltsville in the 1960 and 1970s. Walter Heermann in Germany, working with seed provided byFrederick Coville, released several Northern highbush varieties in the 1940s and 1950s including‘Blauweiss-Goldtraube’, ‘Blauweiss-Zukertraube’, ‘Heerma’, ‘Rekord’, ‘Ama’ and ‘Gretha’.Current Breeding Goals. The current goals of Southern and Intermediate highbush breeders are toobtain early ripening types with high plant vigor, disease resistance and a later bloom (particularlyin Florida). Established breeding lines are being used for this purpose, along with hybrids derivedfrom native V. ashei, V. elliottii and V. darrowii. There is also growing interest in developing verylow chill, evergreen types that fruit in both the summer and fall (Lyrene, 2007; Darnell andWilliamson, 1997).36

Southern and Intermediate highbush cultivars are being developed at several locations, includingArkansas, Australia, California, Florida, Georgia, Mississippi, Chile and Spain. Paul Lyrene at theUniversity of Florida has the most active program dealing with very low chill genotypes and hasreleased many high impact cultivars including ‘Emerald’, ‘Jewel’, ‘Misty’ and ‘Star’. JimBallington in North Carolina has the most significant program operating at the interface betweenNorthern and Southern highbush types, and has generated a number of important cultivarsincluding ‘Lenore’, ‘New Hanover’, ‘O’Neal’, ‘Reveille’ and ‘Sampson’. O’Neal is a very lowchill type, while the rest are intermediate. Jim Moore and now John Clark at the University ofArkansas have focused on mixing southern wild species with northern types and released‘Ozarkblue,’ a late Intermediate type. Scott NeSmith at the University of Georgia has generatedseveral new early Intermediate varieties including ‘Rebel’, ‘Camelia’ and ‘Palmetto’. SteveStringer, Arlen Draper and Jim Spears at the USDA in Mississippi have developed a number ofIntermediate highbush types including ‘Biloxi’, ‘Gupton’ and ‘Magnolia’. Several private breedingprograms have also emerged that are developing Southern and Intermediate highbush typesincluding Atlantic Blue in Spain, Berry Blue in Michigan and Chile, Driscoll Associates inCalifornia, Mountain Blue Orchard in Australia and Vital Berry in Chile.Northern highbush breeders are concentrating on flavor, longer storing fruit, expanded harvestdates, disease and pest resistance and machine harvestability. Established breeding lines are beingused in these efforts, along with complex hybrids made up of V. darrowi, V. angustifolium, V.constablei and most of the other wild species. Even though it has limited winter hardiness, V.darrowii has proven to be an interesting parent in colder climates, because it passes on apowderblue color, firmness, high flavor, heat tolerance and potential upland adaptations.Northern highbush blueberries are currently being bred in New Jersey, Michigan, Oregon andChile. Jim Hancock at Michigan State University is focusing on late maturing, long storinggenotypes and has released three new Northern highbush cultivars that show high promise,‘Aurora’, ‘Draper’ and ‘Liberty’. Mark Ehlenfeldt of the USDA program in New Jersey is focusingon identifying genotypes with high disease resistance and tolerance to winter cold, and has releasedseveral cultivars including ‘Chanticleer’ and ‘Hannah’s Choice’. Nicholi Vorza at the Cranberryand Blueberry Research Station of Rutgers University has begun a program in New Jersey todevelop locally adapted highbush cultivars with machine harvestability and high fruit quality. ChadFinn of the USDA in Oregon is active in identifying genotypes that are well suited to the PacificNorthwest. The HortResearch program has recently changed hands to Dave Brazelton and FallCreek Nursery in Oregon. Other worldwide northern highbush breeding projects include ‘BerryBlue’ in Michigan and Chile, Driscoll Associates in California, the University of Talca and VitalBerry in Chile.Recent trends in highbush breeding. There has been a recent trend to speed up the cultivar releaseprocess. While it used to take up to 25 years from the original cross to the farm, there is now a pushto go from seed to release in 10 – 12 years, with 8 years being thought possible. In the early days, aplant was often evaluated for 6 – 12 years before selection and then the elites were evaluated foranother 6 – 12 years in replicated trials before release. A good example is ‘Elliott,’ which wasreleased 25 years after the cross. Today, the primary selection and replicated trial stages have eachbeen reduced to 3 to 4 years in some programs. ‘Draper’ was released 12 years after the originalcross, and ‘Aurora’ and ‘Liberty’ were evaluated only 10 years.The rapid expansion of the industry and the need for new improved types has stimulated thisacceleration, along with a need to keep evaluation costs low. The key to the success of thisapproach is to make sure that the replicated trials are conducted across a wide range ofenvironments, so that the full potential of the selections is recognized as soon as possible. Still,today’s growers must be more prepared to abandon a cultivar if it fails to live up to expectations ora better one appears.Another trend which is to produce varieties with a blend of species genes. In the early days,virtually all the cultivars were pure derivatives of V. corymbosum, with at little bit of V.angustifolium. The southern breeders changed all of this when they used V. darrowii and to someextend V. ashei to reduce the chilling requirement of southern highbush. With increasingfrequency, native species genes are finding their way into the blood of northern types. Forexample, Legacy is 25 % V. darrowii and 2 % V. angustifolium; ‘Sierra’ is 20 % V. darrowii, 15 %37

V. ashei, 13 % V. constablei and 2 % V. angustifolium. Breeders are finding that the use of V.darrowii has dramatic impacts on fruit quality, and it only takes two or three generations to restorewinter hardiness (Hancock et al, 1995). The complex genetic background of modern breedingpopulations also makes testing of superior genotypes across broad climatic zones imperative tofinding their optimal adaptive zone, particularly for selections of southern and intermediatehighbush families. For example, a few years ago we split our breeding families between Oregonand Michigan, and evaluated them independently at each location (Finn et al., 2003). We used adiverse array of families with varying amounts of southern species blood in their heritage. The elitefamilies that emerged in Michigan where also elite in Oregon, but there were a number of elitefamilies in Oregon that proved poorly adapted to the heat and cold in Michigan. Had we relied onMichigan screens, we would have disregarded some important families.One other important change in highbush breeding is the move towards patenting and licensingblueberry varieties. Today, only the USDA breeding program does not license their varieties, andthey are headed in this direction. This move has come primarily as a means to support furtherbreeding work, as State resources dwindle. Licensing may save some public programs fromextinction due to diminishing state support, but it will also restrict the availability of new varieties.References1. Darnell, R.L., Williamson J.G. (1997) Feasibility of blueberry production in warm climates. Acta Hort.,446, pp. 251-256.2. Finn C.E., Hancock J.F., Mackey T., Serce S. (2003) Genotype x environment interactions in highbushblueberry (Vaccinium sp. L.) families grown in Michigan and Oregon. J. Amer. Soc. Hort. Sci., 128, pp.196-200.3. Hancock J.F. (2006a) Northern highbush breeding. Acta Hort., 715, pp. 37-40.4. Hancock J.F. (2006b) Highbush blueberry breeders. HortScience, 41, pp. 20-21.5. Hancock J.F., Galletta GJ (1995) Dedication: Arlen D. Draper: Blueberry Wizard. Plant BreedingReviews, 13, pp. 1-10.6. Lyrene P.M. (1998) Ralph Sharpe and the Florida blueberry breeding program. In: Cline, WO,Ballington J.R. (eds.). Proceedings of the 8 th North American Blueberry Research and ExtensionWorkers Conf. North Carolina State University, Raleigh. pp. 1-7.7. Lyrene P.M. (2007) Breeding southern highbush blueberries. Plant Breeding Reviews (In press).8. Mainland C.M. (1998) Frederick Coville’s pioneering contributions to blueberry culture and breeding.Proc N Amer Blueberry Workers Conf, Wilmington, NC.9. Sharp R.H., Darrow G.M. (1959) Breeding blueberries for the Florida climate. Proc. Fla. State Hort.Soc., 72, pp. 308-311.FRUIT DEVELOPMENT IN VACCINIUM SPECIESVACCINIUM SUGU AUGěU ATTĪSTĪBALaura JaakolaDepartment of Biology, University of Oulu, POB 3000, FIN-90014 Oulu, Finland,e-mail: laura.jaakola@oulu.fiAbstractFruit development and ripening represent one of the most complex developmental processes inplants. Functionally, the role of fruits is to cover the developing seeds and promote the dispersal ofmature seeds through the production of attractive colour, flavour and aroma compounds. Afterfertilisation, the first phases of fruit development include the division and the expansion of thecells. The ripening phase is initiated after the completed seed maturation. Tissue softening andaccumulation of flavour compounds, aromatic volatiles and pigments occurs during the ripeningphase. The quality of fruits is determined by the different developmental steps via the signallingcascade that is responsible for the metabolic and structural changes during the ripeningphenomenon. Genus Vaccinium is widespread over the world and it includes many economicallyimportant cultivated and wild berry species. Fruits of the Vaccinium species are non-climacteric38

and anatomically they belong to false berries, many-seeded fleshy fruits in which the inferior ovaryalong with floral tube ripens into an edible pericarp. Vaccinium berries are especially rich withphenolic compounds that are known to possess antioxidative properties. This review focuses oncharacteristics of fruit development in both cultivated and the wild Vaccinium species.KopsavilkumsAugĜa attīstība un nogatavošanās ir viens no sarežăītākajiem attīstības procesiem augos.Funkcionāli augĜa uzdevums ir pasargāt jaunattīstītās sēklas un veicināt to nogatavošanos, reizēveicinot arī to iekrāsošanos, atbilstošai garšai un aromātam. Pēc apaugĜošanās, pirmajās augĜaattīstības fāzēs, tiek veicināta šūnu dalīšanās un augšana. AugĜa nogatavošanās fāze sākas pēc tam,kad beidzas sēklu nobriešana. AugĜa audi paliek mīkstāki un garšas un aromātu veidojošosavienojumu un pigmentu uzkrāšanās notiek nogatavošanās fāzes laikā. AugĜa kvalitāti nosakadažādi attīstībā izmantotie signāli, kas atbild par vielmaiĦas un strukturālām pārmaiĦāmnogatavošanās laikā. Vaccinium ăints ir plaši izplatīta visā pasaulē un tajā iekĜautas daudzasekonomiski svarīgas kultivētas un savvaĜas sugas. To augĜi anatomiski pieder pie neīstām ogām –daudzsēklu mīkstajiem augĜiem. Ogas ir īpaši bagātas ar fenola savienojumiem, kam, kā zināms,piemīt antioksidantu īpašības. Šajā rakstā dots augĜa attīstības raksturojums gan kultivētām, gansavvaĜas Vaccinium sugām.Key words: berries, fruit development, Vaccinium.IntroductionFruit development and ripening, typically preceded by successful flower pollination, represent oneof the most complex and important developmental processes in plants. Functionally, the role offruits is to cover the developing seeds and promote the dispersal of mature seeds by frugivoreanimals through the production of attractive colour, flavour and aroma compounds in addition tonutritional value. After fertilisation, the first phases of fruit development include the division andthe expansion of the cells. The ripening phase is initiated after seed maturation has been completed.Tissue softening and accumulation of flavour compounds, aromatic volatiles and pigments occursduring the ripening phase (Brady 1987, Giovannoni 2001, 2004).Fruits can be classified into climacteric and non-climacteric fruits according to the differences inthe respiration rate and the production of the plant hormone ethylene during the ripening phase.According to the structure of the pericarp, fruits are classified as non-dehiscent (fleshy) ordehiscent (dry) fruits. Most fruits develop from a gynoecium that contains one or more carpels. Inpseudocarpic fruits, organs other than the gynoecium (eg. receptacle bracts, floral tube, or theenlarged axis of the inflorescence) participate in the formation of the fruit (Gillaspy et al. 1993,Giovannoni 2004). The genus Vaccinium is widespread over the world with about 450 species ofevergreen and deciduous woody plants varying from dwarf shrubs to trees. Most Vaccinium speciesoriginate from the cooler areas of the Northern hemisphere, although tropical species also exists.Well-known members of the genus are cultivated northern or southern highbush blueberries (V.corymbosum hybrids), lowbush (V. angustifolium) or rabbiteye blueberries (V. ashei Reade) andcranberries (V. macrocarpon), in addition to commercially utilized wild bilberries (V. myrtillus)and lingonberries (V. vitis-idaea). Other better known wild species are European cranberries (V.oxycoccos, V. microcarpum), odon (V. uliginosum) and numerous other especially American wildVaccinium species. The Vaccinium species require soil with low pH and they grow mostabundantly in heaths, bogs and acidic woodlands.Pollination. Most northern highbush blueberries are self-pollinated, meaning that the pollen of thesame individual plant can lead in successful fruit development. However, for many cultivars crosspollination produces higher fruit set and larger fruit. Southern highbush blueberries are onlypartially self-pollinated (Krebs and Hancock 1990). Hokanson and Hancock (2000) tested selffertilityin controlled hand self- and cross-pollinations with individual V. corymbosum, V.angustifolium and V. myrtilloides plants and detected that all three species showed a significantreduction in self fruit set and in the proportion of fertilized ovules that developed into mature seedin self compared to outcross fruit.39

Rabbiteye and lowbush blueberries are also largely self-infertile and therefore cross pollination isrequired for the good fruit set and berry size. Bumble bees and wild bees pollinate blueberryflowers naturally. Cranberries are self-pollinated but insect or wind disturbance is needed for thepollen release (Rieger). Most wild Vaccinium species are cross pollinated by insects. Selfpollinationis possible in bilberry and lingonberry but it reduces seed production (Nuortila et al.2002). Fruit development. The fruit of the Vaccinium species is fleshy, round or oval by shape, theycontain several to many seeds and belong in the category of berries. More strictly, the Vacciniumfruits are classified as false or epigynous berries, in which the fruit is formed from an inferiorovary, but the floral tube ripens along with the ovary. In true berries the entire ovary wall ripensinto a soft pericarp (Fahn 1990). The fruits can grow clustered like blueberries and lingonberries orsingly in branch or leaf axis like bilberries or cranberries.Most Vaccinium species growing in the Northern hemisphere flower from May to June and produceberries from July to October. The fruit development of blueberries takes 45 to 90 days, and varies alot between different species, cultivars, and growth locations. The growth of most blueberries,especially later ripening cultivars, has been found to exhibit a double sigmoid pattern (Godoy et al.2008). Cranberry fruits mature in 60-120 days after fertilization, depending on the cultivar andweather. Among the cultivated blueberries highbush blueberries reach maturity faster (typically 45-75 days) compared to lowbush or rabbiteye blueberries (Rieger).The development of bilberries from flower to ripe fruit lasts usually 55 to 70 days, varying betweendifferent years. The different phases of bilberry fruit development and ripening are presented infigure 1. Typically, the beginning of fruit development involves divisions and expansions of thecells (stages 2 – 3). During the ripening (stages 4 – 6) the accumulation of anthocyanins, and othersecondary metabolites and sugars as well as the softening of the cell walls occur. Compared to thebilberry, lingonberry flowers and fruits ripen later during the season, approximately 78 – 84 daysafter full blossom (Gustavsson 2001).Phenolic compounds. The berries of the Vaccinium species are known especially for their highcontent of phenolic compounds that are strong antioxidants and possess health beneficialproperties. Vaccinium berries are among the best sources of anthocyanins and proanthocyanidins(Ovaskainen et al. 2008). Therefore, the accumulation of phenolic compounds has been one of thefocus areas in the study on the fruit development of the Vaccinium species. The composition offlavonoids and other phenolic compounds in fruit development and ripening has been analysed inbilberries, cranberries, and blueberries (Jaakola et al. 2002, Vvedenskaya and Vorsa 2004,Castrejon et al. 2008, Celik et al. 2008). The overall profile of phenolic compounds at theirdifferent stages of development shares similarities between the examined Vaccinium species.Figure 1. Fruit development and the ripening of bilberry from pollination to ripe fruit.At the beginning of fruit development proanthocyanidins and flavonols are the main flavonols inVaccinium fruits. At the onset of the ripening, the content of proanthocyanidins decreases at thesame time anthocyanins begin to accumulate. A high content of proanthocyanidins at the earlyphases of fruit development has also been detected from fruits other than the Vaccinium species.Proanthocyanidins have been suggested to provide protection against fungal pathogens and thepredation of unripe fruits (Harborne 1997).40

The levels of flavonols are more constant over the period of fruit development, although especiallyqurcetin glycosides are found to be in slightly higher level at the beginning of the fruitdevelopment. In addition to quercetin glycosides, many Vaccinium berries contain myricetinglycosides (Määttä-Riihinen et al. 2004). In the bilberry, myricetin glycosides were found toaccumulate during the ripening phase, along with anthocyanins (Jaakola et al. 2002). Of thephenolic compounds, in addition to flavonoids Vaccinium berries contain hydroxycinnamic acids.Blueberries contain high levels of caffeic acids (Määttä-Riihinen et al. 2004). The antioxidantactivity during fruit development has been shown to be associated with the total phenolic content atthe different developmental stages (Castrejon et al. 2008, Celic et al. 2008).Other nutraceuticals. There is a lot of information on the nutritional value of Vaccinium berries, butit is mainly focused on ripe fruits. Only a limited number of studies report the contents of thenutritional compounds during fruit development. Cano-Merdano and Darnell (1997) analysed sugaraccumulation during the development of rabbiteye blueberries. Glucose and fructose are the mainsugars in Vaccinium fruits, whereas the contents of sucrose are low (Cano-Merdano and Darnell1997, Viljakainen et al. 2002). According to studies of rabbiteye blueberries, the levels of sugarswere at their lowest about 20 days after bloom, after which the contents increased up to ten fold.The maximum levels were reached between 60 and 90 days after bloom, reaching the maximum inthe ripe fruits.Celic et al. (2008) analysed the content of organic acids at four different maturity stages ofcranberries. The most abundant organic acid in cranberries was citric acid (73 %) followed bymalic and ascorbic acid. The overall concentration of citric and malic acid increased over theripening, whereas the ascorbic acid concentration decreased. Regulation of fruit development.Recent discoveries have begun to reveal the developmental cues that are responsible for primingand initiating the ripening in fruit bearing species (Giovannoni 2004, 2007). The tomato (Solanumlycopersicum) has been the model system of choice due to its genetic resources and the knowledgethat researchers have of its ripening physiology and biochemistry of ripening (Seymour et al.,2008). However, key information has also been obtained from the model plant Arabidopsis, and toa lesser extent from strawberries (Fragaria spp).The current state of the art is that a number of ripening related genes are known from these speciesincluding the ripening inhibitor (rin) and the Colourless non-ripening (Cnr) (Giovannoni, 2007).The gene at the tomato ripening inhibitor (rin) locus is a member of the MADS-box SEPALLATA(SEP) sub-family; LeMADS-RIN (Vrebalov et al., 2002). MADS-box genes were previouslyassociated with floral development, but LeMADS-RIN is necessary for ripening. The gene at theCnr locus encodes an SBP-box transcription factor (Manning et al., 2006), that is likely to interactwith the promoters of the SQUAMOSA (SQUA) sub-family of MADS-box genes (Lännenpää et al.,2004). These include TDR4, which shows enhanced expression during tomato fruit ripening(Eriksson et al., 2004), but has yet to be assigned a function. This regulatory network appears to beconserved across fruit bearing species.Nonetheless, links between the regulatory factors and the down stream effectors are poorlyunderstood and thus far the studies are limited almost exclusively to the tomato, despite the diverseripening behaviour of other important fruiting species.Studies clarifying the molecular basis of fruit development in the Vaccinium species have, untilrecently, been limited and nothing is known about the genes controlling the fruit ripening in theVaccinium species. Some structural genes encoding the key enzymes of the flavonoid biosyntheticpathway have been characterised from cranberry (Polaschock et al., 2002) and bilberry fruits(Jaakola et al., 2002). Just recently, two SQUA sub-family of MADS-box genes have been clonedfrom the bilberry (Jaakola et al., unpublished). The other of the two cloned transcription factorgenes was related by sequence homology with tomato TDR4 and Arabidopsis FRUITFULL (FUL)genes. The functional analyses revealed a hitherto unsuspected link between the SQUA MADS-boxgene and the production of secondary metabolites.ConclusionsThe genus Vaccinium includes several economically important berry species. Therefore, the studyon the quality characteristics over the development and ripening of the fruits can open newcommercially exploitable applications in the future. Understanding better the gene x environment41

interaction during the ripening process is an important part of fruit quality research. Fruitdevelopment in various Vaccinium species shares several similarities, despite the different growthhabits of the plants (e.g. highbush blueberry vs. cranberry). Even though there are differences in thephenolic profiles between the Vaccinium species, the trend in the accumulation of the compoundgroups is the same.The development of new analytical methods has allowed for the accumulation of the newinformation in the entire area of plant science. However, most information to date has beengathered from the model species and the most economically important crop species. Along withnew efficient sequencing technologies, we will soon have vastly more gene level informationavailable for studying the gene x environment interaction in the various Vaccinium berries.AcknowledgementsThe two referees of the manuscript are thanked for the improvements in the paper.References1. Brady C.J. (1987) Fruit ripening. Annual Review of Plant Physiology, 38, pp. 155-178.2. Cano-Medrano R. and Darnell R.L. (1997) Sucrose metabolism and fruit growth in partenocarpic vsseeded blueberry (Vaccinium ashei) fruits. Physiologia Plantarum, 99, pp. 439-446.3. Castrejon A.D.R., Eicholz I., Rohn S., Kroh L.W. and Huyskens-Keil S. (2008) Food Chemistry, 109,pp. 564-572.4. Celik H., Özgem M., Serce S. and Kaya C. (2008) Phytochemical accumulation and antioxidant capacityat four maturity stages of cranberry fruit. Scientia Horticulturae, 117, pp. 345-348.5. Gillaspy G., Ben-David H. and Gruissem W. (1993) Fruits: a developmental perspective. Plant Cell, 5,pp. 1449-1451.6. Giovannoni J. (2001) Molecular biology of fruit maturation and ripening. Annual Review of PlantPhysiology and Plant Molecular Biology, 52, pp. 725-49.7. Giovannoni J.J. (2004) Genetic regulation of fruit development and ripening. Plant Cell, 16, pp. 170-180.8. Giovannoni J.J. (2007) Fruit ripening mutants yield insights into ripening control. Current Opinion inPlant Biology, 10, pp. 283-289.9. Godoy G., Monterubbianesi G. and Tognetti J. (2008) Analysis of highbush blueberry (Vacciniumcorymbosum L.) fruit growth with exponential mixed models. Scientia Horticulturae, 115, pp. 368-376.10. Gustavsson B.A. (2001) Genetic variation in horticulturally important traits of fifteen wild lingonberryVaccinium vitis-idaea populations. Euphytica, 120, pp. 173-182.11. Eriksson E., Bovy A., Manning K., Harrison L., Andrews J., De Silva J., Tucker G. and Seymour G(2004) Effect of the Colorless non-ripening mutation on cell wall biochemistry and gene expressionduring tomato fruit development and ripening. Plant Physiology, 136, pp. 4184-4197.12. Fahn A. Plant Anatomy. 1990 (4 th ed.) Pergamon press plc. Oxford, UK. p.489.13. Harborne J. (1997) Phytochemistry of fruits and vegetables: an ecological overview. In F Tomas-Barberan, ed, Phytochemistry of fruits and vegetables. Oxford University press, New York, pp. 335-367.14. Hokanson K., and Hancock J. (2000) Early-acting inbreeding depression in three species of Vaccinium(Ericaceae). Sexual Plant Reproduction, 13, pp.145-150.15. Jaakola L., Määttä K., Pirttilä A. M., Törrönen R., Kärenlampi S. and Hohtola A. (2002) Expression ofstructural genes involved in anthocyanin biosynthesis in relation to anthocyanin and flavonoid levelsduring bilberry (Vaccinium myrtillus L.) fruit development. Plant Physiology, 130, pp. 729-739.16. Krebs S.L. and Hancock J.F. (1990) Early-acting inbreeding depression and reproductive success in thehighbush blueberry, Vaccinium corymbosum L. Theoretical and Applied Genetics, 79, pp. 825-832.17. Lännenpää M., Jänönen I., Hölttä-Vuori M., Gardemeister M., Porali I., Sopanen T. (2004) A new SBPboxgene BpSPL1 in silver birch (Betula pendula) Physiologia Plantarum, 120, pp. 491-500.18. Manning K., Tör M., Poole M., Hong Y., Thompson A.J., King G.J., Giovanonni J.J. and Seymour G.B.(2006). A naturally occurring epigenetic mutation in an SBP-box transcription factor inhibits tomatofruit ripening. Nature Genetics, 38, pp. 948-952.19. Määttä-Riihinen K.R., Kamal-Eldin A., Mattila P.H., Gonzales-Paramas A.M. and Törrönen R. (2004)Distribution and contents of phenolic compounds in eighteen Scandinavian berry species. Journal ofAgricultural and Food Chemistry, 52, pp. 4477-4486.20. Nuortila C., Tuomi J. and Laine K. (2002) Inter-parent distance affects reproductive success in twoclonal dwarf shrubs, Vaccinium myrtillus and Vaccinium vitis-idaea (Ericaceae). Canadian Journal ofBotany, 80, pp. 875-884.42

21. Polashock J.J., Griesbach R.J., Sullivan R.F. and Vorsa N. (2002) Cloning of a cDNA encoding thecranberry dihydroflavonol-4-reductase (DFR) and expression in transgenic tobacco. Plant Science, 163,pp. 241-251.22. Seymour G., Poole M., Manning K., King G. (2008) Genetics and epigenetics of fruit development andripening. Current Opinion in Plant Biology, 11, pp. 58-63.23. Ovaskainen M. L., Törrönen R., Koponen J.M., Sinkko H., Hellström J., Reinivuo H. and mattila P.(2008) Dietary intake and major food sources of polyphenols in Finnish adults. Journal of Nutrition,138, pp. 562-566.24. Rieger, M. http://www.uga.edu/fruit/bluberi.html25. Viljakainen S., Visti A. and Laakso S. (2002) Concentrations of organic acids and soluble sugars injuices from Nordic berries. Acta Agriculturae Scandinavica, 52, pp. 101-109.26. Vrebalov J., Ruezinsky D., Padmanabhan V., White R., Medrano D., Drake R., Schuch W., GiovannoniJ. (2002) A MADS-box gene necessary for fruit ripening at the tomato ripening-inhibitor (Rin) locus.Science, 296, pp. 343-346.27. Vvedenskaya I.O. and Vorsa N. (2004) Flavonoid composition over fruit development and maturation inAmerican cranberry, Vaccinium macrocarpon Ait. Plant Science, 167, pp. 1043-1054.NORDIC BILBERRY PROJECTZIEMEěVALSTU MELLEĥU PROJEKTSLaura Jaakola 1 , Mika Paassilta 1,5 , Marko Suokas 1 , Inger Martinussen 2 , Kristiina Antonius 3 ,Andreas Åkerström 4 , Asdis Helga Bjarnadottir 5 , Úlfur Óskarsson 5 , Anja Hohtola 1 , AnnaMaria Pirttilä 1 , Simo Moisio 6 , Olavi Junttila 7 , Ulla Bång 4 and Hely Häggman 11 Department of Biology, University of Oulu, POB 3000, FIN-90014 Oulu, Finland,2 Bioforsk, P.O.B.6232, N-9292 Tromso, Norway,3 Nordic Gene Bank (NordGen), P.O.B. 41, SE-23053 Alnarp, Sweden,4 SLU, Department of Agricultural Research for Northern Sweden, P.O.B. 4097, SE-90403 Umeå, Sweden,5 Agricultural University of Iceland, Hvanneyri, 311 Borgarnes, Iceland,6 Arctic Flavours Association, Kauppakatu 20, FI-89600 Suomussalmi, Finland,7 University of Tromsø, Department of Biology, Dramsveien 201, N-9037 Tromsø , Norway,e-mail: laura.jaakola@oulu.fiAbstractWild berries are a characteristic part of Northern nature and a particular speciality of Nordiccountries. Wild berries are also a rich and valuable resource that has not yet been exploited in asatisfactory level. Approximately 90 – 95 % of the whole wild berry crop yield is left unpicked inthe Nordic forests every year. The challenges of wild berry utilization are similar in Nordiccountries - the logistics of berry picking including traceability, fragmented sector structure as wellas the high share of unprocessed raw material in export. The Nordic project focusing on bilberry(Vaccinium myrtillus) “Bilberry: Towards functional food markets” (2007 – 2009) is a part of theNew Nordic Food programme funded by the Nordic Innovation Centre. The programme aims toenhance cooperation and innovation among companies that utilize the natural resources of theNordic countries. The aim of the project is to improve wild berry production and utilization in theNordic and global market. To achieve this goal a network between the Nordic experts presentingthe different fields of the wild berry sector has been established. The project has focused onmarketing research, quality issues, biodiversity and the traceability of wild berries; especially thebilberry. The results of the marketing survey were published in November 2008. The aim of thesurvey was to generate an overall picture of the companies working with wild berries in Nordiccountries and to gather information on the existence and willingness of the berry companies tocooperate in wild berry supply, logistics, marketing and research and development. According tothe results, a general agreement for the need of increased cooperation at the Nordic level washighlighted.43

KopsavilkumsSavvaĜas ogas ir Skandināvijas dabas raksturīga sastāvdaĜa un īpaša ZiemeĜvalstu dabas vērtība.SavvaĜas ogas ir arī bagāts un vērtīgs resurss, kas vēl nav izmantots apmierinošā līmenī. Aptuveni90 – 95 % no visas savvaĜas ogu ražas ZiemeĜvalstu mežos katru gadu paliek nenovāktas. SavvaĜasogu izmantošanas problēmas Skandināvijas valstīs ir līdzīgas - ogu savākšanas loăistika, ietverotizpķti, sadrumstaloto sektora struktūru, kā arī lielu daĜu neapstrādātās izejvielas eksportā.ZiemeĜvalstu projekts koncentrējas uz mellenēm (Vaccinium myrtillus) „Mellenes: ceĜā uzfunkcionālās pārtikas tirgu” (2007 – 2009) un ir daĜa no jaunās ZiemeĜvalstu pārtikas programmas,ko finansē ZiemeĜvalstu inovācijas centrs.Programmas mērėis ir veicināt sadarbību un inovāciju starp uzĦēmumiem, kuri izmanto dabasresursus ZiemeĜvalstīs, kā arī uzlabot savvaĜas ogu ražošanu un izmantošanu ZiemeĜvalstīs unPasaules tirgū. Lai sasniegtu šo mērėi, jākooperējas starp ZiemeĜvalstu ekspertiem, kas nodarbojasar dažādiem pētījumu virzieniem par savvaĜas ogām. Projekts ir vērsts uz tirgus pētījumiem,kvalitātes jautājumiem, bioloăisko daudzveidību un savvaĜas ogu, it sevišėi, melleĦu izpēti.Mārketinga aptaujas rezultāti tika publicēti 2008. gada novembrī. Aptaujas mērėis bija iegūtvispārēju priekšstatu par kompānijām, kas strādā ar savvaĜas ogām ZiemeĜvalstīs un apkopotinformāciju par uzĦēmumu gatavību sadarboties savvaĜas ogu piegādē, loăistikā, tirdzniecībā,pētniecībā un attīstībā. Balstoties uz iegūtajiem rezultātiem, tika panākta vispārēja vienošanās parstiprākas kooperācijas nepieciešamību ZiemeĜvalstu līmenī.Key words: bilberry, quality, marketing survey, new nordic foodIntroductionBilberry (Vaccinium myrtillus L.) belongs to the most important wild berries in northern Europeand is recognized for its bioactive properties (Lau et al. 2005, Canter & Ernst 2004). Wild berriesare a valuable part of European nature and tradition. In the northern and eastern parts of Europewild berries grow abundantly and in these areas the picking of wild berries and mushrooms is animportant recreation for people. About half of the wild berries are picked for personal consumptionand the remainder are used for commercial utilization. In these areas wild berries are a speciality,which could be utilised and marketed notably better. The average bilberry yield in Scandinavia hasbeen estimated to be over 500 million kg per year, from which only 5 – 8 % is used (Salo 1995).Wild berries are an excellent source for functional food that should demand a higher valuation andproduct development in Europe. Nowadays, the biggest part of wild berries picked commercially inNorth Europe is exported as frozen unprocessed raw material to East Asia or the Central-Europeanfood industry. China and Japan are the biggest buyers of European wild berries, a market of whichis increasingly focused on health products. The wild berry industry in Europe is typically fairlysmall and fragmented. One problem is that the annual wild berry crop yields vary markedly indifferent areas and the yield estimates of the crop have been inaccurate with the present methods.In addition, to better utilise the valuable raw material more knowledge on the uniform quality ofwild berries growing in different areas is needed. By characterising the attributes of the growthareas of the best wild berry crops, would provide a sustainable and natural ways to improve thefuture prospects of wild berry production in a changing climate. Moreover, sustainable methods forimproving the logistics of wild berry picking would be needed. A Nordic project focusing on thebilberry (Vaccinium myrtillus) “Bilberry: Towards functional food markets” was initiated in 2007.The project is a part of the New Nordic Food programme funded by the Nordic Innovation Centre.The programme aims to enhance cooperation and innovation among companies that utilize thenatural resources of the Nordic countries. The aim of the project is to improve wild berryproduction and utilization in the Nordic and global market. Three work packages includingnetworking, marketing research and quality aspects have been the basis of the project to achieve thestated goal. The general scheme of the project is presented in figure 1.44

Figure 1. A general scheme of the Nordic bilberry project.Networking. The bilberry project is co-ordinated by the University of Oulu, Finland, and the otherparticipants are the Arctic Flavours Association and MTT Agrifood Research Jokioinen fromFinland, the Nordic Gene Bank (NordGen), the Swedish University of Agricultural Sciences(SLU), Bioforsk, the University of Tromsø and Biolink from Norway and the AgriculturalUniversity of Iceland. There are also some companies and other institutions as network partners ofthe project. During the project, information gathered on companies dealing with wild berries inNordic countries was listed.The bilberry project organized the Nordic wild berry seminar at the University of Oulu, Finland on6 th -7 th November 2008. In the seminar, there were altogether 50 participants from Finland, Sweden,Norway, UK, Canada and Japan. The participants were representatives of research institutes, wildberry companies and other related organizations. The two-day program consisted of presentationson health and quality issues, marketing and the product development of Nordic wild berries. Apanel discussion with the title: “Wild berry production in the northern areas – Guidelines for thefuture” was held at the end of the seminar. In the discussions, increased co-operation between allinterested parties in the wild berry sector was emphasised. The general consensus was that in thefuture, there would be a need for a joint organisation (e.g. Nordic Wild Berry Association) tomaintain the established networking and productive discussions via the general meetings andmailing lists.Marketing survey. The aim of the marketing survey was first of all to generate an overall picture ofthe rather heterogeneous and large group of Nordic companies working with wild berries –especially bilberries. Another aim was to gather information on how the companies feel aboutcertain issues related to the wild berry sector, e.g. if companies have problems in gaining enoughwild berries. A special emphasis was given to cooperative actions between the companies. The aimwas to find out whether, and to what extent, the companies are willing to cooperate in order toreach some of their common goals and what are the areas of business that they consider worthwhileto cooperate in. The common goals include wild berry supply, logistics, marketing and researchand development. The survey was carried out in 2007 – 2008. First task was to gather informationfrom the Nordic companies dealing with wild berries. The eight page questionnaire was deliveredto 1300 companies, 200 of which were Finnish, 750 Swedish and 350 Norwegian. The results wereanalysed, compiled and first published in the Nordic Wild Berry Seminar at Oulu, Finland inNovember 2008 and afterwards also as a printed report (Paassilta et. al., 2009). The results of thesurvey shown that Nordic wild berry companies are for increased co-operation in Nordic levelconcerning several common issues. For instance, creating a uniform traceability system for allNordic countries in wild berry picking was supported. Most of companies also supported thedevelopment of common Nordic wild berry brand in the future.45

Quality research. The most important issue for berries as a raw material for the functional foodmarket is the quality characteristics of the fruit. Fruit quality is a consequence of proper fruitdevelopment, which is a complicated biochemical process and, to a great extent, geneticallyregulated. However, also environmental factors such as light conditions and temperature affect theripening process, and the yearly fluctuations can influence the content of secondary metabolites inripening fruits (Åkerstöm et al. 2009). In terms of bilberry production, more knowledge on thefactors affecting the ripening process and quality of the fruit is needed. In the Nordic Bilberryproject, quality research is going on in several areas. The effect of the growth conditions (daylength and temperature) has been studied in a controlled experiment in a phytotrone using clonalmaterial (Martinussen et al. unpublished). Additionally, bilberry samples from various altitudeshave been collected as well as berry samples from trials in controlled environments. Also bilberriesfrom different latitudes that have been growing in the same growth conditions for several yearshave been analysed (Åkerstrom et al. unpublished). Moreover, molecular level study on theregulation of bilberry fruit development has revealed new information on transcription factors thatare necessary for fruit ripening and also about the accumulation of anthocyanins - the importantpigments and antioxidants (Jaakola et al. unpublished).In the project, bilberry clones have been collected from all Nordic countries. The samples arepresently in the tissue culture, and they will be planted in the test field of the University of Oulu forfurther extend the clone collection for future research purposes. One task of the project was tooptimise a method for bilberry diversity analysis. The retrotransposon based method has shown thatgenetic diversity exists between and among the bilberry populations from different regions andorigins (Antonius et al. unpublished).At present, a poor knowledge of the genome of the bilberry or other Vaccinium species is thelimiting factor for many applications. A better knowledge of the bilberry genome could be utilisedin determining the origins of small fruits for marketing purposes. Increasing berry imports andexports have demonstrated the need for new methods to confirm the origin of the raw material.Moreover, one major problem in the global wild berry markets is that the final products may alsocontain other berry or plant species than what is mentioned on the product label. In the Nordicbilberry project a bilberry fruit specific EST-library is under construction, with 454-sequencingtechnology (Roche Diagnostics). Moreover, a new DNA level method for authenticity analyses ofwild berry species has been developed (Jaakola et al. unpublished).ConclusionsThe Nordic bilberry project has reached most of the results that were set at the beginning of theproject. The project has shown that with co-operation and concerned and active participants it iseasier to achieve the desired goals. The marketing survey among the companies dealing with wildberries gives a fresh overview on the prospects of the Nordic wild berry business. The scientificefforts in the project are shedding light on the gene x environment interaction related to the qualityissues of the bilberry. These results are to some extent applicable to other wild berries also.Moreover, the networking and discussions between the interested participants of the whole wildberry sector have emphasised the need for increased international co-operation and created newideas for future activities. However, the long term challenges in the wild berry sector still needadditional brainstorming, research work, product development, customer surveys and action.Acknowledgements‘The Bilberry – towards functional food markets’ project is funded by Nordic Innovations Centre.References1. Canter P.H. and Ernst E. (2004) Anthocyanosides of Vaccinium myrtillus (bilberry) for night vision –systematic review of placebo-controlled trials. Survey of Opthalmology, 49, pp. 38-50.2. Lau F.C., Shukitt-Hale B. and Joseph J.A. (2005) The beneficial effects of fruit polyphenols on brainaging. Neurobiology of Ageing, 26S, pp. 128-132.3. Paassilta M., Moisio S., Jaakola L. and Häggman H. (2009) Voice of the Nordic wild berry industry. Asurvey among the companies. Oulu University press, Oulu, 84 p.4. Salo K. (1995) Non-timber forest products and their utilization. In: Hytönen M. (eds) Multiple-useForestry in the Nordic Countries. Gummerrus Press, Jyväskylä, pp. 117-155.46

5. Åkerström A., Forsum Å., Rumpunen K., Jäderlund A. and Bång U. (2009) Effects of sampling time andnitrogen fertilization on anthocyanidin levels in Vaccinium myrtillus fruits. Journal of Agriculture andFood Chemistry, 57, pp. 3340-3345DOMINANT PHYTOPHAGES OF EDIBLE HONEYSUCKLE (LONICERA EDULIS Turcz.ex Freyn) IN BELARUS AND THE EFFICIENCY OF BIOLOGICAL PREPARATIONSAPPLICATION AGAINST THEMDOMINĒJOŠIE ĒDAMĀ SAUSSERŽA (LONICERA EDULIS Turc. Ex Freyn) KAITĒKěIBALTKRIEVIJĀ UN BIOPREPARĀTU EFEKTIVITĀTE TO KONTROLEINatallia Kaltun, Svetlana Yartchakovskaya, Ryta MikhnevichInstitute of Plant Protection, p. Priluki, 223011 Mira Str.2., Мinsk region, Belaruse-mail: belizr@tut.byAbstractThe objective of our research was the study of the specific and structural diversity of phytophagesin honeysuckle plantations and the generalization of optimization system of their phytosanitarycondition. The phonological observations on the development of the host plant and phytophageswere carried out in 2005 – 2008 in plantations of the Institute of fruit growing Minsk region. Thepest records were kept every 10 days, starting with «buds breaking» on not less than 10 bushes ofevery variety. The experiments on the evaluation of the efficiency the biological preparationsagainst the main honeysuckle phytophages were accomplished in 4 times repetition (3 bushes perrepetition). The biological preparations against the dominant pest species were applied during themost vulnerable development periods for the noxious organisms.It has been determined that the main pests both by occurrence and number in the honey suckleplantations of Belarus are honeysuckle – cereal aphid - Rhopalomyzus lonicerae Siebold, roseleafroller - Archips rosana L., bud codling moth - Spilonota ocellana Den. et Schiff. Sporodicdamage is caused by spider mite - Tetranychus urticae Koch. and the European fruit lecanium -Parthеnolecanium corni corni Bouch. Among three being studied (‘Goluboe vereteno’,‘Vasilievskaya’ and ‘Lakomka’) the variety ‘Lakomka’ is less damaged by pest infestation. Alsothis variety was not damaged by the specialized honeysuckle pest – honeysuckle - cereal aphid.It has been determined that the biological preparation baciturine, ps, titre 45 – 60 mlrd spores g -1manufactured based on Bacillus thuringiensis, var. darmstadiensis, strain № 24 – 91 at the Instituteof Microbiology National Academy of Sciences of Belarus and the Institute of plant protection atthe rate of application 6 l ha -1 decreases the rose leafroller caterpillars number by about 42 – 55 %.The efficiency of the experimental batch of the biological preparation lecanicil application alsoproduced in Belarus based on the fungus Lecanicillium (Verticillium) lecanii (Zimm.) Zare et W.strain BL-1 6 l ha -1 against honeysuckle-cereal aphid eventes a decrease of 22 – l42 %.KopsavilkumsŠo pētījumu mērėis bija izpētīt ēdamā sausserža kaitēkĜu daudzveidību plantācijās un apkopot datuspar to fitosanitārās situācijas optimizācijas iespējām. Saimniekauga un kaitēkĜu fenoloăiskienovērojumi tika veikti no 2005. līdz 2008. gadam AugĜkopības Institūta plantācijās Minskasapgabalā. KaitēkĜi tika uzskaitīti ik pēc 10 dienām, sākot no pumpuru plaukšanas fāzes ne mazākkā 10 krūmiem no šėirnes. Biopreparātu efektivitātes pētījumi pret nozīmīgākajiem ēdamāsausserža kaitēkĜiem tika veikti četros atkārtojumos pa 3 krūmiem katrā atkārtojumā. Biopreparātitika pielietoti kaitīgā organisma visjutīgākajā attīstības fāzē.Skaita un izplatības ziĦā nozīmīgākie ēdamā sausserža kaitēkĜi Baltkrievijā bija graudaugu laputs -Rhopalomyzus lonicerae Siebold, rožu lapu tinējs - Archips rosana L. un pumpuru kode - Spilonotaocellana Den. et Schiff. Atsevišėos gadījumos postīga bija arī tīklērce Tetranychus urticae Koch.un Parthеnolecanium corni corni Bouch. Starp trīs pētītajām šėirnēm (‘Goluboe vereteno’,‘Vasilievskaya’ un ‘Lakomka’), vismazāk kaitēkĜu bojājumu bija šėirnei ‘Lakomka’. Šo šėirninebojāja arī specifisks sausseržu kaitēklis – sausseržu-graudaugu laputs.47

Tika noteikts, ka biopreparāts baciturīns (ps, titrs 45 – 60 mljr. sporu g -1 , ražots uz Bacillusthuringiensis, var. darmstadiensis celma Nr. 24 – 91 bāzes Baltkrievijas Nacionālās zinātĦuakadēmijas Mikrobioloăijas institūtā un Augu aizsardzības institūtā) devā 6 l ha -1 samazina rožulapu tinēja kāpuru skaitu par 42 – 55 %. Eksperimentālā preparāta lecanicila (ražots Baltkrievijā uzsēnes Lecanicillium (Verticillium) lecanii (Zimm.) Zare et W. celma BL-1 bāzes) pielietošanasamazināja sausseržu-graudaugu laputu skaitu par 22 – 42 %.Key words: entomocoenosis, phytophage, edible honeysuckle, biological preparations, efficiencyIntroductionThe honeysuckle has appeared in amateur orchards of Belarus rather recently. In the eighties of theXX-th century, however, it quickly gained an increasing distribution as it is early ripening andfrost-resistant and can grow on poor soils at minimum expense (Panteev A.V., 1997). Fructificationstarts 3 – 4 years after planting. In the conditions of Belarus the edible honeysuckle startsblossoming at the second or third decade of April and the flowers resist temperature decrease up to-8 ºС. The berries of early varieties start ripening at the end of May-beginning of June, 7 – 10 daysearlier than garden strawberries. Honeysuckle berries are a pantry of vitamins and biologicallyactive substances, have a pleasant taste similar to the blueberry and render curative and preventiveproperties.The average edible honeysuckle productivity in the conditions of Belarus varies from 1.5 to 2.5 kgper bush, however, it is not always stable and also often low, which in many respects is determinedby losses caused by noxious organism damages though until recently there was an opinion that darkblue or edible honeysuckle as well as the majority of ornamental honeysuckles practically were notdamaged by noxious organisms and not infected by diseases (http:luzhok.ru/encyclop/garden/treesbrush/art489.html). However, it is possible to say with confidence that the longer the bush iscultivated, more it suffers from these or other pests. V.P. Vasiliev (1975) points out that in theUkraine the honeysuckle is damaged by 27 insect species, representatives of 7 orders and 1 mitespecies. Based on Z.S. Babenko’s (1982) data in a taiga zone of western Siberia 37 honeysucklepest insect species of the mainly leaf-eating kind are registered. In a non-chernozem zone of Russiathe honeysuckle damage by the following pests are noted: rose tortrix moth (Archips rosana L.),black currant tortrix moth (Pandemis ribeana Hb.), honeysuckle plume moth (Platyptiliacalodactyla Den. & Schiff.), honeysuckle aphid (Semiaphis lonicerae Shap.), willow scale(Chionaspis salicis L.) (Plekhanova M. N., 1990). In the middle zone of Russia the dangeroushoneysuckle pests are: European fruit lecanium, willow scale, honeysuckle-cereal aphid,honeysuckle whitefly, rose, omnivorous and currant tortrix moths, honeysuckle hooktip moth,honeysuckle plume moth (Naumova L.V., 2002). In Latvia the following pests are registered forthe honeysuckle: red spider mite – Tetranychus urticae Koch, honeysuckle apical aphid –Semiaphis tataricae Aizneberg., honeysuckle-spruce aphid – Prociphilus xylostei de Geer,honeysuckle-cereal leaf roller – Rhopalomyzus lonicerae Siebold, frost leafroller – Exapatecongelatella Clerck, rose tortrix moth – Archips rosana Linnaeus, honeysuckle striped sawfly –Zaraea fasciata Linnaeus., honeysuckle miner – Phytogromyza xylostei Robineau-Desvoidy(Rupice A.A., 1981).The research dealing with honeysuckle phytophages indicates the necessity of carrying outprotective measures against pests under conditions of their mass development. There is informationon the efficiency of preparations such as actellic, rogor, confidor, inta-vir, fufanon, decis againstpest insects; against mites – the acaricides: omite, mavrik; against aphids – garlic, tobacco, pepperinfusions (http:flower.onego.ru/kustar/lonice_v.html). As honeysuckle is an early ripening crop andit takes not more than two months from the beginning of bud breaking to berry maturation, it isnecessary to spray against the phytophages only after the fruit harvest.Till 2005 there were no purposeful researches on studying the specific and structural variety ofedible honeysuckle phytophages, terms definition, expediency and the efficiency carrying out ofprotective measures against them.In this connection the objective of the present research was the edible honeysuckle specific pestcomposition determination in Belarus, discovering the most widespread and harmful phytophage48

species and studying the efficiency of the biological plant protection products available againstthem.Materials and MethodsThe stationary phytosanitary edible honeysuckle inspections, the experiences on studying thebioecological features and dynamics of pests development, an estimation of the phytophageharmfulness degree were carried out in the plantations of the Institute of Fruit Growing in Minsk ona total area of 2 hectares.The estimate of the phytosanitary condition of the plantings was done using the general methods(Alekhin V.T. et al, 1988; Green N. et al., 1996).Records of the phytophage number were carried out every decade starting from the phenophase of‘buds breaking’ not less than on 10 bushes of every variety. The leaf-eating phytophages numberwas determined by calculating per 2 m of branches taken in regular intervals from 4 bush sides.The aphids were recorded by calculating the colonies number per 100 leaves from each modelingbush. The mites number was determined by binocular viewing calculating both pest imago andlarvae number per 100 leaves from each modeling bush. Scale and soft scale records were done bylarvae and shields records per 2 m of branches from 4 bush sides. Honeysuckle plume moth recordswere determined by opening 100 berries from each registration bush, starting from the end of theirgrowth period with 5 days periodicity.The experiments concerning the efficiency of biological preparations against the main honeysucklephytophages were done in 4 repetitions (3 bushes repetition). The biological preparations againstthe dominant pest species were applied during the most vulnerable for noxious organismdevelopment periods: against honeysuckle-cereal aphid – during the pest number increase period;rose leaf roller – II-instar larvae stage.The systematization, generalization and statistical processing of the collected material was donebased on dispersive, correlation and regression analysis methods (Zar H.J., 1996; Ulanova E.S. andZabelin V.N., 1990).Results and DiscussionAs a result of the research it was determined that during the period from honeysuckle budding tillthe berries maturation the edible honeysuckle is damaged by leaf-eating butterflies (rose tortrixmoth - Archips rosana L., bud moth - Spilonota ocellana Den. et Schiff., winter moth -Operophthera brumata L.), the number of which during the years of observations (2005 – 2008)varied from 2.1 (2007) to 4.5 (2008) caterpillars, on the average, per 2 m of branches (Table 1).In all the years of the research among leaf-eating pests the greatest threat was represented by therose tortrix moth. This pest caterpillar number varied from 1.4 to 3.9 per 2 m of branches. Thehoneysuckle bud moth in honeysuckle plantings also caused its annual damage, however, itsnumber was much lower and did not exceed 1.2 caterpillars per record unit. Winter moth wasobserved in honeysuckle plantations only in 2005 and in 2007 and the caterpillar number did notincrease 0.2 per 2 m of branches.A complex of sucking pests in honeysuckle plantings are the following insect and mite species:honeysuckle-cereal aphid (Rhopalomyzus lonicerae Siebold), European fruit lecanium(Parthеnolecanium corni corni Bouch.), red spider mite (Tetranychus urticae Koch.). Thedominating rolе in the complex of the sucking phytophages belongs to the honeysuckle-cerealaphid. The pest eggs number for years of inspection fluctuated from 0.3 to 2.4 per 2 m of branchesand the maximum phytophage number in the maturing berries was 6.6 – 8.9 colonies (1 colony upto 20 individuals) per 100 leaves (Table 1). The red spider mite number years of research except2008, also was high and after harvest had reached 3.7 – 6.8 individuals per leaf. The European fruitlecanium was noticed in honeysuckle plantations only during the last two years of researches andthe wintered larvae number during bud breaking was 3.0 – 5.9 individuals per 2 m of branches.Thus, it has been determined that the main pests both by occurrence and number in honeysuckleplantings in Belarus are honeysuckle-cereal aphid - Rhopalomyzus lonicerae Siebold, rose tortrixleaf roller - Archips rosana L., bud moth - Spilonota ocellana Den. et Schiff. Sporadic damage iscaused by the red spider mite - Tetranychus urticae Koch. and European fruit lecanium -Parthеnolecanium corni corni Bouch.49

Table 1. Specific composition and honeysuckle phytophage number (Institute of Fruit Growing,Minsk region, 2005 – 2008)Phytophage species, record unit 2005 2006 2007 2008Archips rosana , average larvae number per 2 m of 2.6 1.4 1.5 3.9branchesSpilonota ocellana, average number of larvae per 2 1.1 1.2 0.4 0.6m of branchesOperophthera brumata , average number of larvae 0.1 - 0.2 -per 2 m of branchesTotal number of leaf-eating butterflies 3.8 2.6 2.1 4.5Rhopalomyzus lonicerae, colonies per 100 leaves 8.9 6.6 7.3 6.8Tetranychus urticae,, average individuals number per 6.8 4.4 3.7 -leafParthеnolecanium corni corni , larvae number per 2m of branches- - 5.9 3.0Since 2006 research was started on the evaluation of the damageability by the dominantphytophages of honeysuckle cultivars regionalized in Belarus. The most spread cultivars:‘Lakomka’, ‘Goluboe vereteno’ and ‘Vasilievskaya’ were under study. As a result of three yearsrecords and observations it has been determined that a variety ‘Lakomka’ is damaged by a complexof phytophages to a lesser degree than the varieties ‘Vasilievskaya’ and ‘Goluboe vereteno’(Figure 1).Colonies,per 100 leaves1210864Caterpillars, per 2 mof branches1210864LakomkaGoluboe veretenoVasilievskaya2202006 2007 200802006 2007 2008Rhopalomyzus loniceraeArchips rosana12121010Larvae, per 2 mof branches864Individuals,per leaf864202007 2008Parthenolecanium corni corni202006 2007Tetranychus urticaeFigure 1. Regionalized edible honeysuckle cultivars colonization by main phytophages(Samokhvalovichi, Мinsk region, 2006-2008)On the cultivar ‘Lakomka’ for all years of inspections there was no honeythuckle-cereal aphiddamage, while on the cultivars ‘Vasilievskaya’ and ‘Goluboe vereteno’ the pest number reached 6– 8 colonies per 100 leaves. The rose tortrix moth number in cv. ‘Lakomka’ bushes for all years ofinspection did not increase 2.6 caterpillars per 2 m of branches in comparison with the cultivars‘Vasilievskaya’ and ‘Goluboe vereteno’ where there were 4 – 5 caterpillars. It was noticed that theEuropean fruit lecanium and red spider mite also to a lesser degree colonized cv. Lakomka. Therewere no essential differences between the cultivars‘Goluboe vereteno’ and ‘Vasilievskaya’ byphytophages damage.50

Thus, it has been determined that the cv. ‘Lakomka’ in the conditions of Belarus is the leastdamaged by main phytophages and not damaged by the honeythuckle-cereal aphid.For the sake of extending the spectrum of preparations allowed for application on ediblehoneythuckle, in 2008 a series of experiments were started on studying the efficiency of biologicalplant protection products against sucking and leaf-eating phytophages. Studying the efficiency andpossibility of biological preparation application on the crops where the chemical means ofprotection is limited and is authorized only after harvesting is rather actual and a perspectivedirection for research. For studying, the biological product baciturine, ps. is taken, titer 45 – 60milliard viable spores g -1 (spore-crystal complex and exotoxin Bacillus thuringiensis, var.darmstadiensis, strain № 24 – 91), developed at the Microbiology Institute of the NationalAcademy of Sciences of Belarus and at the Institute of Plant Protection. Also the antagonisticactivity of the experimental sample of the biological preparation lecanicil developed in Belarus isbased on a high-active strain of the fungus Lecanicillium (Verticillium) lecanii Zimm. Zare et W.Gams strain BL-1.The spraying of bushes by a 1 % working solution of a biological product baciturine against rosetortrix moth caterpillars was carried out on April 30, at the end of honeysuckle blossoming, when90 % of pest caterpillars had reached the II-instar larvae stage. The experimental sample oflecanicil was tested in a 1 % concentration against the honeysuckle-cereal aphid at the beginning ofberry coloring the pest number increase. The records of the phytophage number were noted justbefore harvest and on the 3 rd and 7 th day after spraying. Repetition – 3 bushes taken at random byone block in a row.Table 2. Efficiency of the biological preparation baciturine against the rose tortrix moth in ediblehoneythuckle (Cultivar-‘Goluboe vereteno’, p. Samokhvalovichi, Minsk r., 2008)Number ofcaterpillars per 2 m BiologicalDate ofof branches after efficiency, %Varianttreatment, croptreatmentphenophaseBaciturin, ps., titer 45-60mlrd. spores g -1 (6 l hа -1 )Number ofcaterpillarsper 2 m ofbranchesbeforetreatment4.330.04 end ofhoneythuckleblossomingon the 3 rddayon the 7 thdayon the 3 rddayon the7 th day1.9 2.4 41.6 55.0Control (without treatment) 4.5 - 4.4 4.3 - -As a result of the trials it was determined that the application of the biological preparationbaciturine, ps., titer 45 – 60 mlrd. spores g -1 at the rate of preparation application and workingsolution use 600 l ha -1 caused a 41,6 % kill of rose tortrix moth on the 3 rd day after treatment and 55% on the 7 th day after the product application (Table 2).Таble 3. Efficiency of the experimental sample of the biological preparation lecanicil against thehoneythuckle-cereal aphid in edible honeythuckle (cv. ‘Vasilievskaya’, p. Samokhvalovichi, Мinskregion, 2008)VariantNumber ofindividualsper 100leavesbeforetreatmentLecanicil, 6 l ha -1 132.8Control (withouttreatment)Treatment date, cropphenophase2.06, start ofhoneythuckleberries coloringNumber of individualsper 100 leaves aftertreatmenton the 3 rddayon the 7 thdayBiologicalefficiency, %on the 3 rddayon the 7 thday126.1 120.2 22.3 41.8133.3 - 134.5 135.3 - -51

The application of an experimental sample of the preparation lecanicil based on the fungusLecanicillium lecanii strain BL-1, in a 1 % concentration in the beginning of berry coloring againstthe honeythuckle-cereal aphid caused a pest number decrease on the 7 th day after treatment of41.8% (Table 3).ConclusionsIt was determined that the main pests both by occurrence and number in honeythuckle plantationsin Belarus are the honeythuckle-cereal aphid - Rhopalomyzus lonicerae Siebold, the rose tortrixmoth - Archips rosana L. and the bud moth - Spilonota ocellana Den. et Schiff. Considerably lessdamage is caused by – the red spider mite - Tetranychus urticae Koch. and the European fruitlecanium - Parthеnolecanium corni corni Bouch.It was revealed, that cv ‘Lakomka’ in the conditions of Belarus is not damaged by thehoneythuckle-cereal aphid and to a lesser degree, in comparison with the cultivars ‘Vasilievskaya’and ‘Goluboe vereteno’ is colonized by rose tortrix moth, the European fruit lecanium and the redspider mite.The biological preparation baciturine, ps., titer 45 – 60 mlrd viable spores g -1 (spore-ctystalcomplex and the exotoxin Bacillus thuringiensis, var. darmstadiensis, strain № 24 – 91) at the rateof preparation application 6 l hа -1 and at the working solution rate of 600 l hа -1 has caused 41.6 %death rate of rose tortrix moth caterpillars on the 3 rd day after treatment and 55 % - on the 7 th dayafter preparation application. The efficiency of application of an experimental sample of thepreparation lecanicil produced based on the fungus Lecanicillium (Verticillium) lecanii Zimm.Zare et W. Gams strain BL-1. 6 l hа -1 at the beginning of berry coloring against honeythucklecerealaphid was 41.8 % effective on the 7 th day after treatment.References1. Zar H.J.(1996) Biostatistical analysis, Prentice-Hall Int., London, 662 p.2. Alekhin V.T., Ermakov A., Cherkashin V.I. (1988) Kontrol phytosanitarnogo sostoyaniya sadov ivinogradnikov (Control of phytosanitary orchard and vineyard condition) Zatschita i karantin rastenij,2, pp. 54-57. (In Russian).3. Green N., Staut U., Talor D. (1996) Biologiya. Kolichestvennaya ekologiya (Biology. Quantitativeecology), Moskva, 2, pp. 127-150. (In Russian).4. Babenko Z.S. (1982) Nasekomye-phytophagi plodovykh i yagodnykh rastenij lesnoy zony Priobiya(Insects–phytophages of fruit and berry plants of the forest Priob zone), Tomsk: Izdatelstvo TomskogoUniversiteta, 27 p. (In Russian).5. Vasiliev V.P. (1975) Vrediteli selskokhozyajstvennykh kultur i lesnykh nasazhdenij (Pests of agriculturalcrops and forest plantations), Kiev: Urozhaj, 3, pp. 364-365. (In Russian).6. Lonicera. Available at: http: flower.onego.ru/kustar/lonice_v.html, 15.01.2009.7. Lonicera. Available at: http:luzhok.ru/encyclop/garden/treesbrush/art489.html., 15.01.2009.8. Naumova L.V. (2002) Vrediteli zhimolosti sjedobnoy (Edible honeysuckle pests), Zatschita i karantinrastenij, 5, 57 p. (In Russian).9. Panteev A.V. (1997) Yagody na dache (Berries in private plot), Minsk, Sovremennoe slovo, 320 p. (InRussian).10. Plekhanova M.N. (1990) Aktinidiya, limonnik, zhimolost (Aktinidia, magnolia vine, honeythuckle),Leningrad: Agropromizdat, 87 p. (In Russian).11. Rupice A.A. (1981) Vrediteli dereviev i kustarnikov v zelenykh nasazhdeniyakh Latvii (Pests of trees andbushes in green plantations of Latvia), Riga, Zinatne, 264 p. (In Russian).12. Ulanova E.S., Zabelin V.N. (1990) Metody korrelyatsionnogo i regressionnogo analiza vagrometeorologii (Methods of correlation and regression analysis in agrometeorology), Leningrad, 201p. (In Russian).52

CHEMICAL COMPOSITION OF HIGHBUSH BLUEBERRY CULTIVARSKRŪMMELLEĥU ŠĖIRĥU ĖĪMISKAIS SASTĀVSSolvita Kampuse 1 , Elga Šnē 1 , Dace Šterne 2 , Inta Krasnova 31 Faculty of Food Technology, Latvia University of Agriculture (LLU), Liela Street 2, Jelgava, Latvia,e-mail: skampuse@inbox.lv2 Faculty of Agriculture, LLU, Jelgava, Latvia3 The Latvia State Fruit Growing Institute, Graudu 1, Dobele, LatviaAbstractThe breeding of blueberries has expanded so far that now the number of the highbush blueberrycultivars has reached several hundreds, and sixty of them are particularly widespread. The chemicalcomposition and suitability for the processing of highbush blueberries has been researched quiteextensively, mostly in the USA, but there are no such studies and data for blueberries grown inLatvia. Therefore the aim of this investigation was to compare the biochemical composition ofdifferent highbush blueberry cultivars grown in Latvia.The experiments were done at the Faculty of Food Technology, Latvia University of Agriculture(LLU), Jelgava, and at the Latvia State Fruit Growing Institute, Dobele during the year 2008. Thesamples of highbush blueberry cultivars were collected from the blueberry collection of Institute ofAgrobiotechnology (LLU). The contents of titratable acids, soluble solids, ascorbic acid, totalphenols, and anthocyanins of frozen fruits of the highbush blueberry cultivars 'Northland', 'Spartan','Barkeley', Duke', 'Chippewa', 'Bluecrop', 'Jersey', 'Blueray', 'Chandler', and 'Bluejay' wereanalysed.The cultivars ‘Northland’ and 'Chippewa' had the highest anthocyanin content (on average 297.59and 288.83 mg 100 g -1 , respectively), the highest phenol content was detected in highbushblueberry cultivar 'Spartan' (on average 381.14 mg 100 g -1 ), the highest content of ascorbic acidwas in cultivar ‘Bluejay’ (on average 11.8 mg 100 g -1 ), the highest titratable acids content in theberries of the cultivar ‘Chandler’ (1.35 %), and the cultivar with the highest soluble solids contentwas ‘Duke’ (on average 12.99 % soluble solids).KopsavilkumsMelleĦu audzēšana jau ir izplatījusies tādā apmērā, ka uz doto brīdi krūmmelleĦu šėirĦu skaits jauir sasniedzis vairākus simtus un sešdesmit no tām ir plaši izplatītas. KrūmmelleĦu ėīmiskais sastāvsun piemērotība pārstrādei tiek intensīvi pētīta – visvairāk ASV, bet pagaidām nav tādu pētījumu undatu par Latvijā audzētajām krūmmellenēm. Pētījuma mērėis bija salīdzināt dažādu Latvijā audzētokrūmmelleĦu šėirĦu bioėīmisko sastāvu.Eksperimenti tika veikti Latvijas Lauksaimniecības universitātes Pārtikas tehnoloăijas fakultātē unLatvijas Valsts AugĜkopības institūtā 2008. gadā. KrūmmelleĦu šėirĦu paraugi tika ievākti noAgrobiotehnoloăijas institūta (LLU) kolekcijas. ŠėirĦu 'Northland', 'Spartan', 'Barkeley', Duke','Chippewa', 'Bluecrop', 'Jersey', 'Blueray', 'Chandler' un 'Bluejay' paraugiem tika analizētastitrējamās skābes, šėīstošā sausna, askorbīnskābe, kopējie fenoli un antociāni.ŠėirĦu ‘Northland’ un ‘Chippewa’ paraugiem tika konstatēts augstākais antociānu saturs (vidēji297.59 un 288.83 mg 100 g -1 ). Augstākais kopējo fenolu saturs noteikts šėirnes 'Spartan' paraugam(vidēji 381.14 mg 100 g -1 ), augstākais askorbīnskābes saturs bija škirnei ‘Bluejay’ (vidēji 11.8 mg100 g -1 ). Augstākais titrējamo skābju saturs bija šėirnes ‘Chandler’ (1.35 %) paraugam, un šėirne araugstāko šėīstošās sausnas saturu bija ‘Duke’ (vidēji 12.99 % šėīstošās sausnas).Key words: Vaccinium corymbosym, ascorbic acid, soluble solids, phenols, anthocyanins, titratableacidsIntroductionThe highbush blueberries (family Ericaea, genus Vaccinium corymbosym) are a relatively novelshrub berry crop. In the early 1900’s two enthusiasts - Elizabeth White and Dr. Frederick Coville -aspired to domesticate the wild blueberries. Their efforts and selection work by plant breeders andand pathologists has created nowadays plump, juicy, sweet and easy-to-pick cultivated blueberries.53

The major growing areas of highbush blueberries are in the United States (in more than 38 states)and in some provinces in Canada; gradually they are being propagated all around the world - SouthAmerica, Australia and New Zealand, and Europe (US Highbush Blueberry Council, 2009).The popularity and demand of blueberries is growing increasingly due to the demand for theirbioactive compounds (polyphenolics, pectic acids, ascorbic acid, carotenes and others) and theirantioxidant activity (Sinelli et.al., 2008).Blueberries contain polyphenols in high quantities: the antocyanins (cyanidin, delphinidin,malvidin, peonidin, petunidin), flavonoids (cathechin and epicatechin, myricetin, quercetin andkaempferol), phenolic acids (ellagic acid, benzoic and cinnamic acid) and others. The content andcomposition of polyphenolics depends on the growing conditions, cultivating methods, maturity atharvest and other conditions (Giovanelli et.al. 2009; Giovanelli et.al. 2002; US HighbushBlueberry Council, 2009). Giovanella et.al. (2009) reported concentrations of total phenolics andtotal antocyanins, accordingly from 250 to 310 mg 100g -1 and from 92 to 126 mg 100 g -1 . Thesedata are similar with those reported Prior et.al. (1998): total phenolics from 233 to 273 mg *100 g -1and antocyanins from 62 to 157 mg 100 g -1 .Antioxidants help to protect the body against free radicals caused damage, diseases and acceleratedaging. The USDA Human Nutrition Research Center on Aging (Boston, MA) has stated thatblueberries have the highest antioxidant activity among fruits. They used a test called ORAC(oxygen radical absorbance capacity), which determines the antioxidant capacity of foods, andshowed that fresh blueberries the ORAC is 2400 per 100 grams and that is more than other freshfruits and vegetables (for example, the blackberry ORAC is 2000 to 100 g -1 , strawberry - 1500 to100 g -1 , apple 400 to 100 g -1 (Prior et.al., 1998; Cao et.al., 1998).Beaudry (1992) has suggested highbush blueberries quality standards: >10 % soluble solids content(SSC), 0.3 – 1.3 % titratable acidity (TA), pH between 2.25 and 4.25 and SSC/TA ratio between 10and 33.Saftner et. al. (2008) explored the 11 most popular varieties of highbush blueberries (‘Chanticleer’,‘Duke’, ‘Hannah’s Choice’, ‘Weymouth’, ‘Berkeley’, ‘Bluecrop’, ‘Bluegold’, ‘Coville’, ‘Elliott’un ’Lateblue’) and concluded that their soluble solids content (SSC) differs from 10.6 – 13.2 %;varieties which SSC is the lowest are ‘Lateblue’, ‘Coville’ and ‘Duke’ (accordingly 10.6; 10.8 and10.9), but highest are - ‘Chanticleer’ (13.0) and ‘Bluegold’ (13.2). The difference in titratableacidity (expressed as citric acid) among cultivars is large: ‘Coastal’ had only 0.35 % of the acid,whereas ‘Lateblue’ and ‘Elliott’ has a two times larger amount 1.22 – 1.27 %. Fruit pH rangedbetween 2.5 to 3.4 where the two last of mentioned varieties had a pH 2.5, but the ‘Bluegold’,‘Bluecrop’, ‘Berkeley’, ‘Hannah’s Choice’ and ‘Chanticleer’ had the highest pH 3.1 – 3.4. Allcultivars have these above mentioned Beaudry (1992) standards excepting three - ‘Coastal’,‘Elliott’ and ‘Lateblue’ (Saftner et.al., 2008).Another autor (Giovanelli et.al., 2009) studied the blueberries cultivars ‘Goldtraube’, ‘Patriot’,Bluecrop’ and ‘Darrow’. These varieties agree with Beaudry (1992) suggested standards in regardto pH which is 2.90 – 3.15, but the titratable acidity (expressed as citric acid) is higher – from 1.15(‘Goldtraube’) to 1.47 % (‘Bluecrop’).Due to their chemical content, blueberries are acknowledged as very healthy food and are sold notonly fresh but also in jams, as blueberry juice and they can also be canned, frozen and dried, andproducers are still looking for the new processing techniques.The breeding of blueberries has expanded so far that now the number of the highbush blueberrycultivars has reached several hundreds, and sixty of them are particularly widespread. (AugĜi unogas Latvijā mūsdienu augĜu dārzā, 2008).The chemical composition and suitability for processing for highbush blueberries has beenresearched quite extensively, mostly in USA, but there are no similar studies and data ofblueberries grown in Latvia.Therefore the aim of this investigation was to compare the biochemical composition of differenthighbush blueberry cultivars grown in Latvia.Materials and methodsThe experiments were done at the Faculty of Food Technology, Latvia University of Agriculture(LLU), Jelgava, and at the Latvia State Fruit Growing Institute, Dobele. The samples of highbush54

lueberry cultivars were collected from the blueberry collection (7 years old bushes) of the Instituteof Agrobiotechnology (LLU), Jelgava.The samples were analyzed after freezing. After harvesting the blueberries were sorted, frozen inthe freezer PORKKA BF 710 at a temperature of -25±2 °C, then packaged and stored for onemonth in the freezer chamber VTK 201 U at a temperature of -20±2 °C.The contents of titratable acids, soluble solids, ascorbic acid, the total phenols, and anthocyanins offrozen fruits of highbush blueberry cultivars 'Northland', 'Spartan', 'Barkeley', Duke', 'Chippewa','Bluecrop', 'Jersey', 'Blueray', 'Chandler', and 'Bluejay' were analysed.The content of ascorbic acid was determined by titration with a 0.05-M iodine solution (Moor etal., 2005). 25 g of berries were doused with a 100 ml of 6 % solution of oxalic acid andhomogenized for 1 minute. Then the sample was filtered. 2 ml of 1 % solution of starch was addedto10 ml of filtrate and the filtrate was titrated until a change of colour, which does not disappearduring 30 seconds. The content of ascorbic acid mg per 100 g of berries was calculated from thefollowing equation [1]:CVsample= 400 ⋅ , [1]Vstan dardwhere V sample – volume of the iodine solution titrated in a sample, ml;V standard – volume of the iodine solution titrated in a standard solution, ml.Total titratable acids were determined by titration with 0.1 N NaOH (ISO 750:1998) in fresh andfrozen berries.The contents of the soluble solids were determined by refractometer (ISO 2173:2003) in fresh andfrozen berries.Total phenol content was determined by the photometric method with Folin-Ciocalteau reagent(Singleton et al., 1999). For analyses of phenols the Folin-Ciocalteau reagent and 4 ml 7.5 %sodium carbonate was used. After 30 minutes the samples were analyzed with a spectrophotometerat a wave length of 765 nm. As a control solution 1 ml water with 5 ml Folin-Ciocalteau reagentand 4 ml 7.5 % sodium carbonate solution was used. The content of phenols was calculated fromformula [2]:CX = a ⋅10 , [2]where C – content of phenols, mg 100 g -1 ;a – the amount of analyzed sample, g.The results of all analyses were recalculated to 100 g of dry weight.Total anthocyanins were determined by the spectrophotometric method (Moor et al., 2005).Initially 50 g of berries were homogenized. Then 20 g of this volume was doused with 40 g ofethanol and 1.5 M HCl solution (85:15 by volume) and homogenized for 1 minute. Then thesample was filtered, and light absorption at 535 nm was detected with a spectrophotometer. Thesample was diluted until the absorption coefficient was between 0.6 and 0.8. The content mg per100 g was calculated with the equation [3]:whereA⋅v⋅d⋅1000 C =; [3]980⋅mA – absorption coefficient;v – volume of the extraction (90);d – dilution;m – sample weight in g.Results and DiscussionThe total anthocyanins of highbush blueberry cultivars differed between 59 and 119 mg 100 g -1 offresh weight (Figure 1).55

Total anthocyanins, mg 100ḡ 13002702402101801501209060300a* ab**NorthlandChippewabcBerkeleycdDukecd d dBluejayBluecropSpartaneJerseyeBluerayfChandlerFigure 1. Total anthocyanin content in highbush blueberry cultivars.*Values, marked with the same letter, are not significantly different at p=0.05.** Bars corresponds the standard error of the mean of the cultivar.The cultivars ‘Northland’ and 'Chippewa' had the highest anthocyanin content (on average 297.59and 288.83 mg 100 g -1 , respectively).The total phenol content of highbush blueberries differed between 226 and 381 mg 100 g -1 of freshweight (Figure 2). This is even higher than reported in the literature (Giovanella et.al., 2009; Prioret.al, 1998).400350a***bcdTotal phenols, mg 100g -1300250200150100500efghiiSpartanBluecropBluerayChandlerNorthlandJerseyBerkeleyBluejayChippewaDukeFigure 2. Total phenol content in highbush blueberry cultivars.*Values, marked with the same letter, are not significantly different at p=0.05.** Bars corresponds the standard error of the mean of the cultivar.The highest phenol content was measured in the highbush blueberry cultivar 'Spartan' (on average381.14 mg 100 g -1 ).The ascorbic acid content in the highbush blueberry cultivars was low compared to the other berrycrops (currants, strawberries, etc. (AugĜi un ogas Latvijā mūsdienu augĜu dārzā, 2008; Moor et al.,2005): from 6.9 to 11.8 mg 100 g -1 (Figure 3).56

Ascorbic acid, mg 100ḡ 112108642a* b**bcddddeeff0BluejayJerseyChandlerBluerayChippewaBluecropBerkeleySpartanNorthlandDukeFigure 3. Ascorbic acid content in highbush blueberry cultivars.*Values, marked with the same letter, are not significantly different at p=0.05.** Bars corresponds the standard error of the mean of the cultivar.The cultivar ‘Bluejay’ had the highest content of ascorbic acid (in average 11.8 mg 100 g -1 ).The titratable acids content in highbush blueberries differed between 0.5 and 1.4 %, similarly tothat reported in the literature (Beaudry, 1992; Saftner et.al., 2008). There were high differencesbetween the cultivars in titratable acid content (Figure 4).1.61.4a*Titratable acids, %1.21.00.80.60.40.2**bcdefgggg0.0ChandlerJerseyBluerayBluecropSpartanBluejayBerkeleyDukeChippewaNorthlandFigure 4. Titratable acid content in highbush blueberry cultivars.*Values, marked with the same letter, are not significantly different at p=0.05.** Bars corresponds the standard error of the mean of the cultivar.The titratable acid content in the berries of the cultivar ‘Chandler’ (1.35 %) were significantlyhigher than in all other evaluated cultivars. The titratable acids content in most of the cultivars didnot exceed 1 % and is low compared to other berry cultivars. It is important for processing to findblueberry cultivars with the higher acidity therefore the cultivar ‘Chandler’ could be more suitablefor the production of juice and other preserves.57

Soluble solids, %1413121110987a***b b bcddefg6DukeJerseySpartanBluecropChippewaBluerayBluejayChandlerNorthlandBerkeleyFigure 5. Soluble solids content in highbush blueberry cultivars.*Values, marked with the same letter, are not significantly different at p=0.05.** Bars corresponds the standard error of the mean of the cultivar.The soluble solids content in the evaluated highbush blueberry cultivars differed between 8.5 and13 % (Figure 5). The cultivar ‘Duke’ had the highest soluble solids content (on average 12.99 %soluble solids). The soluble solids content in some blueberry cultivars grown in Latvia could belower than in other growing regions due to more rainfall, less sunshine, and a colder climate. Forexample, the soluble solids content in the berries of the cultivar ‘Berkeley’ were only on average8.5 %, which is significantly lower than that mentioned in the literature (Saftner et. al., 2008).ConclusionsThe cultivars ‘Northland’ and 'Chippewa' had the highest anthocyanin content (on average 297.59and 288.83 mg 100 g -1 , respectively), the highest phenol content was measured in the highbushblueberry cultivar 'Spartan' (on average 381.14 mg 100 g -1 ), the highest content of ascorbic acidwas in the cultivar ‘Bluejay’ (on average 11.8 mg 100 g -1 ), the highest titratable acids content in theberries of cultivar ‘Chandler’ (1.35 %), and the cultivar with the highest soluble solids content was‘Duke’ (in average 12.99 % soluble solids).In total, the chemical composition of the evaluated highbush blueberry cultivars grown in Latviawere similar to the literature, but there was tendency that blueberries in Latvia had higher phenolcontent and lower soluble solids content compared to those grown in the other growing regions.References1. Beaudry R. (1992) Blueberry quality characteristics and how they can be optimized. In: Annnual Reportof Michigan State Horticultural Society (122 nd ). Michigan State Horticultural society, Morrice, MI,USA, pp. 140-145.2. Cao G., Booth SL., Sadowski JA, Prior RL. (1998) Increases in human plasma antioxidant capacity afterconsumption of controlled diets high in fruits and vegetables. A.M., J. Clin. Nutr., 68, pp. 1081-1087.3. Giovanelli G., Buratti S. (2009) Comparison of polyphenolic composition and antioxidant activity ofwild Italian blueberries and some cultivated varieties. Food Chemistry, 112, pp. 903-908.4. Giovanella G., Sellappan S., Akoh CC., Krewer G. (2002) Phenolic compounds and antioxidant capacityof Georgia- grown blueberries and blackberries. Journal of Agricultural and Food Chemistry, 50, pp.2432-2438.5. Prior RL., Cao G., Martin A., Sofic E., McEwen J., O’Brien C. Lischner N., Ehlenfeldt M., Kalt W.,Krewer G., Mainland CM. (1998) Antioxidant capacity a influenced by total phenolic and anthocyanincontent, maturity, and variety of Vaccinium species. Journal of Agricultural and Food Chemistry, 46,pp. 2686-2693.58

6. Saftner R., Polashock J., Ehlenfeldt M., Vinyard B. (2008) Instrumental and sensory qualitycharacteristics of blueberry fruit from twelve cultivars. Postharvest Biology and Technology, 49, pp. 19-26.7. Sinelli N., Spinardi A., Di Egidio V., Mignani I., Casiraghi E., 2008. Evaluation of quality andnutraceutical content of blueberries (Vaccinium corumbosym L.) by near and mid- infrared spectroscopy.Postharvest Biology and Technology, 50, pp. 31-36.8. US Highbush Blueberry Council (2009) Available at: www.blueberry.org/blueberries.htm (accessedAugust 25, 2009).9. 400 AugĜi un ogas Latvijā mūsdienu augĜu dārzā. (2008) Sast. Birulis I. A/S “Lauku Avīze”, p. 102.10. Singleton V. L., Orthofer R. M., Lamuela-Raventos R. M. (1999) Analysis of total phenols and otheroxidation substrates and antioxidants by means of Folin-Ciocalteu reagent: Methods in Enzymology, No299, pp. 152–178.11. Moor U., Karp K., Põldma P.,Pae A. (2005) Cultural systems affect content of anthocyanins and vitaminC in strawberry fruits. European Journal of Horticultural Science, 70 (4), pp. 195–201.BIOCHEMICAL COMPOSITION AND ANTIRADICAL ACTIVITY OF ROWANBERRY(SORBUS L.) CULTIVARS AND HYBRIDS WITH DIFFERENT ROSACEAE L.CULTIVARSPĪLĀDŽU (SORBUS L.) ŠĖIRĥU UN TO HIBRĪDU AR CITIEM ROSACEAE L.AUGěAUGIEM ANTIOKSIDATĪVĀ AKTIVITĀTE UN BIOĖĪMISKAIS SASTĀVSKaspars Kampuss 1 , Solvita Kampuse 2 , Elga BerĦa 2 , Zanda Krūma 2 , Inta Krasnova 3 , IneseDrudze 41 Faculty of Agriculture, Latvia University of Agriculture (LLU), Liela street 2, Jelgava, Latvia2 Faculty of Food Technology, LLU, e-mail: skampuse@inbox.lv3 The Latvia State Fruit Growing Institute, Dobele, Latvia4 Pure Horticultural Research Centre, Abavas 2, Pure, LV-3124, LatviaAbstractRowanberry (Sorbus aucuparia L.) is a common yellowish, wild berry that grows in the Northernpart of Europe. Rowan has been described as an important source of flavonoids and theirantioxidant activity affects reactive oxygen species and lipid peroxidation. The aim of this studywas to evaluate the biochemical composition of 8 rowanberry cultivar fruits and the fruits of theirhybrids with Rosaceae L. cultivars and to establish the correlation of their biochemical compositionwith antiradical activity. The experiments were done at the Faculty of Food Technology, LatviaUniversity of Agriculture (LLU) and in the Latvia State Fruit Growing institute, Dobele. Thecontent of ascorbic acid, the total phenols, anthocyanins, carotenoids, soluble solids, titrable acids,tannins and the antiradical activity of fresh and frozen rowanberry×hawthorn 'Granatnaya',rowanberry×chokeberry 'Likiornaya', rowanberry (Sorbus aucuparia) 'Rosina', rowanberry (Sorbusaucuparia) 'Zholtaya', wild rowanberry (Sorbus aucuparia), rowanberry×pear 'Alaya Krupnaya',rowanberry (Sorbus aucuparia) 'Rosina Variegata', rowanberry (Sorbus aucuparia) 'KrasnayaKrupnaya' were analysed. There were no significant differences between the chemical compositionof fresh and frozen rowanberry samples. The highest content of ascorbic acid was in fruits of therowanberry ‘Rosina’, 'Rosina Variegata', 'Krasnaya Krupnaya', and 'Zholtaya' (49 – 53 mg 100 g -1 ).The highest content of carotenoids were detected in rowanberry×hawthorn 'Granatnaya‘ (13.04 mg100 g -1 ), but the highest phenol content was detected in the rowanberry and chokeberry hybrid'Likiornaya' (484.9 mg 100 g -1 ). 'Likiornaya' showed also the highest antiradical activity (11.2 g ofberries per 1g of DPPH radical).KopsavilkumsPīlādži (Sorbus aucuparia L.) ir izplatīts savaĜas augĜaugs, kas aug Eiropas ziemeĜu daĜā. Pīlādži irraksturoti kā nozīmīgs flavonoīdu avots un to antioksidatīvā aktivitāte ietekmē reaktīvo skābekli unlipīdu peroksidāciju. Pētījuma mērėis bija izvērtēt bioėīmisko sastāvu 8 pīlādžu šėirĦu un to59

hibrīdu ar citiem Rosaceae L. augĜaugiem, noteikt korelāciju starp to antioksidatīvo aktivitāti unbioėīmisko sastāvu.Eksperimenti tika veikti Latvijas Lauksaimniecības universitātes Pārtikas tehnoloăijas fakultātē unLatvijas Valsts AugĜkopības institūtā 2008. gadā. Pīlādžu šėirĦu un hibrīdu paraugi tika ievākti noPūres Dārzkopības izmēăinājumu stacijas kolekcijas. Analizēts tika svaigu un saldētu pīlādža ×vilkābeles 'Granatnaya', pīlādža × aronijas 'Likiornaya', pīlādža (Sorbus aucuparia) 'Rosina',pīlādža (Sorbus aucuparia) 'Zholtaya', savaĜas pīlādža (Sorbus aucuparia), pīlādža × bumbiera'Alaya Krupnaya', pīlādža (Sorbus aucuparia) 'Rosina Variegata', pīlādža (Sorbus aucuparia)'Krasnaya Krupnaya' paraugu askorbīnskābes, kopējo fenolu, karotinoīdu, šėīstošās sausnas,miecvielu saturs un antioksidatīvā aktivitāte.Starp svaigiem un saldētiem pīlādžu un to hibrīdu paraugiem netika konstatētas būtiskas atšėirības.Augstākais askorbīnskābes saturs bija ‘Rosina’, 'Rosina Variegata', 'Krasnaya Krupnaya', un'Zholtaya' paraugiem (49 – 53 mg 100 g -1 ). Augstākais kopējo karotinoīdu saturs bija pīlādža ×vilkābeles 'Granatnaya‘ augĜiem (13.04 mg 100 g -1 ), bet augstākais kopējo fenolu saturs bijapīlādža un aronijas hibrīdam 'Likiornaya' (484.9 mg 100 g -1 ). 'Likiornaya' uzrādīja arī augstākoantioksidatīvo aktivitāti (11.2 g ogu uz 1 g of DFPH radikāĜa).Key words: ascorbic acid, phenols, antiradical activity (DPPH), frozen, freshIntroductionRowanberry (Sorbus aucuparia L.) is a common yellowish, wild berry that grows in the northernpart of Europe. They have been described as an important source of flavonoids and theirantioxidant activity affects reactive oxygen species and lipid peroxidation (Gil-Izquierdo andMellenthin, 2001).The fruits of rowan (Sorbus aucuparia L.) have been traditionally used for jellies and jams, buttheir wider use as food ingredients has been less popular because of their bitter taste. The firstsweet rowanberry clones were selected in the Sudety mountain area, in the current Czech Republicarea in the 19 th century. A breeding program for sweet rowanberries was started by Michurin inRussia at the beginning of 20 th century, resulting in interesting hybrids of the rowanberry (Sorbusaucuparia L.) with the Aronia, Malus, Mespilus, or Pyrus species. Sweet rowanberries have beenbred particularly for northern conditions and have shown excellent winter-hardiness in Russia andFinland. The taste of these berries is less astringent than that of wild rowanberries, and the berriesare often larger. The total phenolic content can vary greatly among the sweet rowanberry cultivarsranging from 550 – 1014 mg 100 g -1 of fresh weight of berries. A high correlation between theantioxidant capacity and phenolic contents of sweet rowanberries was established (Hukkanen et al.,2006). Including different types of berries other Finnish authors found that the phenoliccomposition data showed no remarkable correlation between antioxidant activity and totalphenolics. A statistically significant correlation was observed between flavonol content andantioxidant activity, and between hydroxycinnamic acid and antioxidant activity (Kähkönen et al.,2001).Rowanberries contain also carotenoids, vitamin E, and vitamin C, which might also contribute totheir antioxidant capacity. According to Piir and Niiberg (2003) carotenoid levels in sweetrowanberries are as high as those in carrots, and levels of vitamin C are close to those ofstrawberries, varying from 12-21 mg 100 g -1 (‘Granatnaya’) to 86 mg 100 g -1 (‘Zholtaya’) (Piir andNiiberg, 2003; Häkkinen et al., 1999). Also higher vitamin C content is found in the rowanberryvarieties (S. aucuparia) than in the hybrid cultivars.The Pure Horticultural Research Centre has a large collection with sweet rowanberry cultivars andtheir hybrids with Rosaceae L. genus cultivars collected from Russia and other countries. But thereis still little information about the biochemical composition and the nutritional value of the fruits ofthese cultivars.Therefore the aim of this study was to evaluate the biochemical composition and nutritional valueof 8 rowanberry cultivars and hybrids with other Rosaceae L. genus cultivars and to findcorrelations between of the biochemical composition and the antiradical activity.60

Materials and methodsThe experiments were done at the Faculty of Food Technology, Latvia University of Agriculture(LLU), Jelgava, and at the Latvia State Fruit Growing Institute, Dobele. The samples of rowancultivars and their hybrids were collected from the Pure Horticultural Research Centre collection ofgenetic resources.The samples were analyzed fresh and after freezing. After harvesting the rowanberries were sorted,frozen in a freezer PORKKA BF 710 at the temperature of -25±2 °C, then packaged and stored forone month in the freezer chamber VTK 201 U at a temperature of -20±2 °C.Contents of titratable acids, soluble solids, ascorbic acid, total phenols, carotenoids, and theantiroxidant activity (DPPH) of the fresh and frozen fruits of wild rowanberry (Sorbus aucuparia),rowanberry×hawthorn 'Granatnaya', rowanberry×chokeberry 'Likiornaya', rowanberry (Sorbusaucuparia) 'Rosina', rowanberry (Sorbus aucuparia) 'Zholtaya', rowanberry×pear 'AlayaKrupnaya', rowanberry (Sorbus aucuparia) 'Rosina Variegata', and rowanberry (Sorbus aucuparia)'Krasnaya Krupnaya' were analysed.The content of the ascorbic acid was determined by titration with a 0.05-M iodine solution (Mooret al., 2005). 25 g of berries were doused with 100 ml of 6 % solution of oxalic acid andhomogenized for 1 minute. Then the sample was filtered. 2 ml of 1 % solution of starch was addedto10 ml of filtrate and the filtrate was titrated until change of colour, which does not disappearduring 30 seconds. The content of ascorbic acid mg per 100 g of berries was calculated from thefollowing equation [1]:CVsample= 400 ⋅ , [1]Vstan dardwhere V sample – volume of the iodine solution titrated in a sample, ml;V standard – volume of the iodine solution titrated in a standard solution, ml.The total titrable acids were determined by titration with 0.1 N NaOH (ISO 750:1998) in fresh andfrozen berries.The contents of soluble solids were determined by a refractometer (ISO 2173:2003) in fresh andfrozen berries.The total phenol content was determined by the photometric method with a Folin-Ciocalteaureagent (Singleton et al., 1999). For analyses of phenols the Folin-Ciocalteau reagent and 4 ml7.5% sodium carbonate was used. After 30 minutes the samples were analyzed with aspectrophotometer at the wave length of 765 nm. As a control solution 1 ml water with 5 ml Folin-Ciocalteau reagent and a 4 ml 7.5 % sodium carbonate solution was used. The content of phenols iscalculated from formula [2]:CX = a ⋅10 , [2]where C – content of phenols, mg 100 g -1 ;a – the amount of analyzed sample, g.The results of all analyses were recalculated to 100 g of dry weight.Carotenoids were analyzed by the spectrophotometric method at a wave length of 440 nm(Ермаков , 1987). One to two grams of homogenized berries were placed in a 100 ml conic retortand 20 ml 96 % ethanol was added. The sample was stirred by a magnetic stirrer for 15 min then 25ml petrol ether was added and stirring was continued for one more hour. After 3 – 4 hours whenboth layers were completely separated, the top (yellow) layer was used for the further detection ofcarotenoids at a wave length of 440 nm. The carotene equivalent (KE) was found, using agraduation curve with K 2 Cr 2 O 7 . The content of carotenoids (mg 100 g -1 ) was calculated by equation4:0 .208⋅25⋅100⋅KEX =, [4]36⋅awhere 0,208 and 36 coefficients for the relationship between K 2 Cr 2 O 7 and carotenoids;61

25 – dilution coefficient;KE – carotene equivalent from the graduation curve;a – sample weight, g.The antiradical activity (ARA) of frozen berries was analyzed by the spectrophotometric methodwith the N,N-diphenil-N’-picrilhydrazil (DPPH) reagent at a wave length of 517 nm (Milauskas etal., 2004). 50 ml of ethanol was added to 10 g of the homogenized sample; the glasses were closedby Parafilm and stirred for 2 hours for extraction. After 2 hours top clear layer was decanted, 50 mlethanol added to the sample and the extraction was repeated for 2 hours. 2.9 ml 1·10 -4 M DPPH wasfilled in the cuvette and 100 µl of extract added. The sample was stirred and placed in the dark for30 min. Then the absorption was measured at 517 nm (spectrophotomter UV – 1650 PC). ARA wascalculated by equation 5:ADFPH− AsampleARA =⋅100% , [5]ADFPHwhere: A DPPH – absorption of DPPH reactive;A sample - absorption of DPPH after addition of fruit extract.Tannins. The total content of tannins was detected using the traditional method by titration with 0.1n KMnO 4 (Шмыд, 1960). The content of tannins (x) was calculated from formula 6:where( v − v )1 2⋅ 0.04157 ⋅100X = , [6]av 1 - the amount of 0.1 n KMnO 4 used in the first titration, ml;v 2 – the amount of 0.1 n KMnO 4 used in the second titration, ml;a – the amount of 0.1 n KMnO 4 used for oxidizing of 10 ml of 0.1 oxalic acid, ml.Results and DiscussionThe evaluated components of chemical composition did not significantly (p=0.13-0.59) differbetween the fresh and frozen samples that demonstarte the possibility of frozen rowanberry usagesimilar to the fresh ones.The ascorbic acid content of rowanberry cultivars and their hybrids is similar to many other fruitspecies and did not exceed 53 mg 100 g -1 , which is similar to that mentioned in literature (Gil-Izquierdo Mellenthin., 2001). The highest content of ascorbic acid was detected in the rowanberry‘Rosina’, 'Rosina Variegata', 'Krasnaya Krupnaya', and 'Zholtaya' (Figure 1). The wild rowanberryand hybrids with different Rosaceae L. genus cultivars had the significantly lower content ofvitamin C.ascorbic acid, mg 100g -15040302010a a aabcdd0`Rosina``RosinaVariegata``KrasnayaKrupnaya``Zholtaya`Wildrowanberry`AlayaKrupnaya``Granatnaya``Likiornaya`Figure 1. Ascorbic acid content in rowanberries and their hybrids.*Values, marked with the same letter, are not significantly different at p=0.0562

The titrable acids content of the evaluated rowanberry cultivars and the hybrids was 1.6 – 2.8 %(Table 1). The highest acidity was found in the wild rowanberry, but the lowest titrable acidscontent were in the hybrids rowanberry×hawthorn 'Granatnaya', and rowanberry×chokeberry'Likiornaya'.Table 1. Biochemical composition of rowanberries and their hybridsCultivarTitrable acids, % Carotenoids, mg/100g Tannins, %Rowanberry×pear 'Alaya Krupnaya' 2,61 ± 0,10 ab 9,24 ± 0,16 c 0,33 ± 0,02 bRowanberry×hawthorn 'Granatnaya' 1,73 ± 0,04 d 13,04 ± 0,68 a 0,37 ± 0,01 abRowanberry (Sorbus aucuparia ) 'Krasnaya Krupnaya' 2,65 ± 0,12 ab 7,25 ± 0,24 d 0,13 ± 0,02 cRowanberry×chokeberry 'Likiornaya' 1,62 ± 0,02 d 10,48 ± 0,05 b 0,39 ± 0,02 abRowanberry 'Rosina Variegata' 2,67 ± 0,02 ab 8,08 ± 0,14 d -Rowanberry (Sorbus aucuparia ) 'Rosina' 2,48 ± 0,09 b 10,04 ± 0,27 bc 0,38 ± 0,00 abWild rowanberry (Sorbus aucuparia ) 2,80 ± 0,03 a 9,52 ± 0,31 bc 0,42 ± 0,02 aRowanberry (Sorbus aucuparia ) 'Zholtaya' 2,09 ± 0,03 c 9,53 ± 0,36 bc 0,32 ± 0,04 bThe soluble solids content in rowanberries varied between 11 and 18 % (Figure 2). An interestingfact was that the highest soluble solid content was in the wild rowanberry (Sorbus aucuparia) (18.0%, in average). All other rowanberry cultivars and hybrids had a significantly lower soluble solidcontent than the wild rowanberry and did not reach even 16 % though in literature the content ofsoluble solids in fruits of cultivars 'Rosina' and 'Zholtaya' were mentioned as being above 19 %(Hukkanen et al., 2006). This means that the soluble solids of rowanberries can greatly fluctuate indifferent regions and during different years.Soluble solids, %181614abcc12ddde10Wildrowanberry`Zholtaya``Granatnaya``Rosina``Likiornaya``AlayaKrupnaya``KrasnayaKrupnaya`Figure 2. Content of soluble solids in rowanberries and their hybrids.*Values, marked with the same letter, are not significantly different at p=0.05`RosinaVariegata`The total phenol content of rowanberries and their hybrids differed between 162 and 485 mg 100g -1of fresh weight (Figure 3). It is lower than reported in literature (Hukkanen et al., 2006). Thehighest phenol content was detected in the rowanberry and the chokeberry hybrid 'Likiornaya' (inaverage 484.9 mg 100 g -1 ).The differences in the total carotenoid content of eight rowanberry cultivars and hybrids werebetween 7 and 13 mg 100 g -1 of fresh berry weight (Table 1). The rowanberry and hawthorn hybrid'Granatnaya' had the highest carotenoid content (13.04 mg 100 g -1 , in average).63

The total tannin content of the evaluated samples was between 0.3 and 0.4 %, and only rowanberrycultivar 'Krasnaya Krupnaya' had a significantly lower content of total tannins (0.13 % on average)(Table 1). The wild rowanberry had the highest content of tannins (0.42 % in average) whichexplains the most astringent flavour of these berries compared to other rowanberry cultivars andhybrids.The DPPH radical scavenging activity of the evaluated samples ranged from 2.5 to 11.2 g of berriesper g of DPPH radical (Figure 3). The highest antiradical activity (DPPH) was found in therowanberry and chokeberry hybrid 'Likiornaya' (11.2 g of berries per g of DPPH radical).There was a significant (p=0.01) correlation between the antiradical activity and total phenoliccontent (r=0.960) (Figure 3).1210Cultivar8ZholtayaWild rowanberryAntiradical activity (DPPH)642100200300400500RosinaLikiornayaKrasnaja KrupnaGranatnayaAlaya KrupnayaTotal phenols, mg/100gFigure 3. Pearson correlation plot between the antiradical activity (DPPH) and total phenolics ofrowanberries and their hybrids.The correlation between antiradical activity and phenolic content was found also in otherinvestigations with berries (Hukkanen et al., 2006; Gil et.al., 2002). There was no statisticallysignificant correlation between vitamin C content and antiradical capacity and also betweencarotenoids and antiradical capacity although these bioactive compounds are mentioned as strongantioxidants (Puupponen-Pimiä et al., 2001; Sergio et al., 1999; Sies and Stahl, 1995). This couldbe explained by the similar amounts of vitamin C and carotenoids in several rowanberry cultivarsand hybrids while the phenolic content was different for the three better cultivars. It was also foundthat a significant negative correlation exists between the antiradical activity and the titrable acidcontent (p=0.008, r=-0.886). It is difficult to find any explanation for this correlation and it seemsthat it is specific only to the evaluated rowanberry cultivars.ConclusionsThere were no signifficant diferences between the chemical composition of the fresh and frozenrowanberry samples.The highest content of ascorbic acid was found in rowanberries ‘Rosina’, 'Rosina Variegata','Krasnaya Krupnaya', and 'Zholtaya' (49 – 53 mg 100 g -1 ). The highest content of carotenoids wasdetected in the rowanberry×hawthorn 'Granatnaya‘ (13.04 mg 100 g -1 ), but the highest phenolcontent was detected in the rowanberry and chokeberry hybrid 'Likiornaya' (484.9 mg 100 g -1 ).'Likiornaya' also had the highest antiradical activity (11.2 g of berries per 1g of DPPH radical).64

There was a significant correlation established only between the antioxidant activity and the totalphenolic content (r=0.886).References1. Gil-izquierdo, A.; Mellethin, A. (2001) Identification and quantitation of flavonols in rowanberry(Sorbus aucuparia L.) juice. Eur. Food Res. Technol., 213, pp. 12-17.2. Gil M. I., Tomás-Barberán F. A., Hess-Pierce B., Kader A. A. (2002) Antioxidant Capacities, PhenolicCompounds, Carotenoids, and Vitamin C Contents of Nectarine, Peach, and Plum Cultivars fromCalifornia. J. Agric. Food Chem., 50 (17), pp. 4976–4982.3. Häkkinen S. H., Kärenlampi S. O., Heinonen I. M., Mykkanen H. M., Törrönen A. R. (1999) Content ofthe Flavonols Quercetin, Myricetin, and Kaempferol in 25 edible Berries. J. Agric. Food Chem., 47, pp.2274-2279.4. Hukkanen A. T., Pölönen S. S., Kärenlampi S. O., Kokko H. I. (2006) Antioxidant Capacity andPhenolic Content of Sweet Rowanberries. J. Agric. Food Chem., 54, pp. 112-119.5. Kähkönen M. P., Hopia A. I., Heinonen M. (2001) Berry Phenolics and their Atioxidant Activity. J.Agric. Food Chem., 49, pp. 4076-4082.6. Miliauskas G., Venskutonis P.R., van Beek T.A. (2004) Sreening of radical scavenging activity of somemedicinal and aromatic plant extracts. Food Chemistry, 85, pp. 231-237.7. Moor U., Karp K., Põldma P.,Pae A. (2005) Cultural systems affect content of anthocyanins and vitaminC in strawberry fruits. European Journal of Horticultural Science, 70 (4), pp. 195–201.8. Piir R.; Niiberg T. (2003) Rowan in garden and kitchen Pihlakas aias ja köögis; Maahele Raamat: Tallin,Estonia.9. Puupponen-Pimiä R., Nohynek L., Meier C., Kähkönen M., Heinonen M., Hopia A., Oksman-CaldenteyK.M. (2001) Antimicrobial properties of phenolic compounds from berries. J. of Applied Microbiology,90, pp. 494–507.10. Sergio A. R., Paiva M.D., Russell R. M. (1999) MD ß-Carotene and Other Carotenoids as Antioxidants.Journal of the American College of Nutrition, 18 (5), pp. 426-433.11. Sies H.,Stahl W. (1995) Vitamins E and C, beta-carotene, and other carotenoids as antioxidants.American Journal of Clinical Nutrition, 62, pp. 1315-1321.12. Singleton V. L., Orthofer R. M., Lamuela-Raventos R. M. (1999) Analysis of total phenols and otheroxidation substrates and antioxidants by means of Folin-Ciocalteu reagent: Methods in Enzymology, No299, pp. 152–178.13. Методы биохимического исследования растений (1987) Под ред. А. И. Ермакова, Ленинград,Агропромиздат. (Methods for plant biochemical analysis, Leningrad, Agropromizda), pp. 112 – 113.(in Russian).14. Шмыд О.И. (1960) Природные дубильные вещества. // Биохимические методы анализараастений.) (Natural tannins. In: Bichemical methods of plant analysis), 239 p. ( (In Russian).RESEARCH ON THE MINERAL COMPOSITION OF AMERICAN CRANBERRIESAND WILD CRANBERRIES IN LATVIAAMERIKAS LIELOGU UN SAVVAěAS DZĒRVEĥU OGU MINERĀLĀ SASTĀVASALĪDZINOŠS IZVĒRTĒJUMSAndis Karlsons, Anita Osvalde, Vilnis NollendorfsInstitute of Biology, University of Latvia, Miera Street 3, Salaspils LV-2169, Latvia,e-mail: augi@email.lubi.edu.lvAbstractWild cranberry (Vaccinium oxycoccus L.) is one of the small fruit species of commercialimportance in Latvia, traditionally used in folk-medicine and food. Typically, there are widefluctuations in yield annually and between different parts of the country. The commercialcultivation of American cranberry (Vaccinium macrocarpon Ait.) was successfully started duringlast 15 years and today Latvia is the fourth major cranberries producing country. With the increasein consumption of cranberries, widely considered being as one of the healthiest foods, it becomesimportant to have detailed information on the nutritional content of cranberries. The aim of thisstudy was to compare the contents of twelve biologically essential elements (N, P, K, Ca, Mg, S,65

Fe, Mn, Zn, Cu, Mo, B) in berries of two Vaccinium species: V. oxycoccus and V. macrocarpon.Together 78 (leaf and berry) samples were collected from 4 main cranberry producing sites and 3native bogs during autumn 2004.The present study revealed significant differences in the mineral composition of V. oxycoccus andV. macrocarpon. Cultivated cranberry fruits had a higher content of P and Fe while wildcranberries showed higher levels of Ca, Mg Mn, Zn, Cu and B. Plant leaf analysis revealed organspecificdistribution of mineral elements in both cranberries studied. The present study shows thatfruits of both V. macrocarpon and V. oxycoccus are a valuable source of the microelements: Mn,Fe, Cu, Mo, and B in human nutrition. Since the wild cranberry has a especially highconcentrations of Mn, 100 g of fresh berries could supply 121% of the adult daily requirement.KopsavilkumsSavvaĜas dzērvenes (Vaccinium oxiccocus L.) ir Latvijā augoša ogu suga, kuru tradicionāli izmantopārtikā un tautas medicīnā. Raksturīgi, ka savvaĜas dzērveĦu ražas lielums ievērojami atšėiras gaduno gada, kā arī variē dažādās Latvijas vietās. Pēdējos 15 gados uzsākta Latvijā Amerikas lielogudzērveĦu (Vaccinium macrocarpon Ait.) kultivēšana un šobrīd Latvija ir ceturtā lielākā Amerikaslielogu dzērveĦu ražojošā valsts pasaulē. Palielinoties dzērveĦu, kā īpaši veselīga produkta,patēriĦam ir svarīgi gūt pēc iespējas pilnīgāku priekšstatu par šo ogu minerālo sastāvu. Šī pētījumamērėis bija salīdzināt divas Vaccinium ăints sugas V. oxycoccus un V. macrocarpon pēc 12 biogēnoelementu (N, P, K, Ca, Mg, S, Fe, Mn, Zn, Cu, Mo, B) satura ogās. 2004 gada rudenī tika ievākti78 lapu un ogu paraugi četrās ražojošās saimniecībās un trīs dabiskos purvos Latvijā.Pētījumā tika konstatētas ievērojamas atšėirības savvaĜas un Amerikas lielogu dzērveĦu oguminerālajās sastāvā. Kultivēto dzērveĦu ogās bija augstāks P un Fe, bet savvaĜas dzērvenēsaugstāks Ca, Mg, Mn, Zn, Cu un B līmenis. Pētījums apstiprina, ka V. oxicoccus un V.macrocarpon ogas ir vērtīgs mikroelementu Mn, Fe, Cu, Mo un B avots cilvēka pārtikā. Jāatzīmēīpaši augsts mangāna saturs, 100 g svaigas ogas nodrošina 121 % no pieauguša cilvēkanepieciešamās Mn diennakts devas.Key words: Vaccinium oxycoccus L., Vaccinium macrocarpon Ait., fruit mineral compositionIntroductionWild cranberry (Vaccinium oxycoccus L.) is one of the small fruit species of commercialimportance in Latvia, traditionally used in folk-medicine and food. Typically, there are widefluctuations in yield annually and between different parts of the country (Ripa 1988; Yudina 1988).The yield of V. oxycoccus ranges from a few kilograms to 1000 kg ha -1 (from 5 to 100 berries m 2 )(Cherkasov, 1988; Ripa, 1988; Yudina, 1988). Vaccinium oxycoccus usually is found on acid soilswith humus type peat, dismoder or mor, and all the soils are poor in nutrients (Stewart and Nilsen,1993; Schaminee et. al., 1995).The commercial cultivation of the American cranberry (Vaccinium macrocarpon Ait.) was startedonly during last 15 years and is one of the youngest branches of agriculture in Latvia with a highpotential in country’s economical and ecological future. Today with more than 100 ha ofcommercial plantings Latvia is the fourth major cranberry producing country in the world.American cranberry producing plantings are mostly developed in high bog territories. V.macrocarpon, fruit indigenous to North America, are characterized as high yielding crop withsignificantly higher productivity (to 40 t ha -1 ) in comparison with the wild cranberry (Karlsons andOsvalde, 2007).Cranberries are valued for their fresh taste, high dietary and health values, as well as their greatpotential for being processed. Cranberry juice, sugared fruits, raisins, and other products havebecome increasingly popular in Latvia. A growing body of research suggests that the cranberry is arelatively unique fruit which accumulates one of the highest concentrations of phenolic compoundsamong fruit species with demonstrable human health benefits (Vinson et al., 2001; Leahy et al.,2002; Howell et al., 2005). Cranberries are a good source of anthocyanins and their antioxidants,which plays a vital role in the prevention of neuronal and cardiovascular illnesses, cancer anddiabetes (Konczak and Zhang, 2004). There are several reports focused on the effect ofanthocyanins in cancer treatments (Castaneda-Ovando et al., 2008; Lule and Xia, 2005;66

Nichenametla et al., 2006), human nutrition (Stintzing and Carle, 2004), and its biological activity(Kong et. al., 2003).Increased consumption of fruits and vegetables can help replace foods high in saturated fats, sugarand salt and thus improve the intake of most micronutrients and dietary fibre (Ekholm et al., 2007).Daily consumption of fresh fruits and vegetables (>400 g·d -1 ) is recommended to help preventmajor non-communicable diseases such as cardiovascular diseases and certain cancers (WHO,2003). Thus the chemical composition of Vaccinium spp. has important implications for humanhealth. Many minerals are essential for normal metabolic functions and are required components ina balanced diet (Grusak and DellaPenna, 1999).The mineral element contents of plants are known to be affected by the species and cultivar of theplant, soil conditions, weather conditions, the use of fertilizers and the state of the plants maturityat harvest (Pietola and Salo, 2000; Bálint et al., 2001).The aim of this study was to compare the contents of twelve biologically essential elements (N, P,K, Ca, Mg, S, Fe, Mn, Zn, Cu, Mo, B) in berries of two Vaccinium species: V. oxycoccus and V.macrocarpon.Materials and methodsThe study was carried out on the wild cranberry (V. oxycoccus) and the American cranberry (V.macrocarpon) crops in different regions of Latvia. Together 78 (leaf and berry) samples werecollected from 4 main cranberry producing sites (Aluksne, Talsu, Madonas and Riga district) and 3native bogs (Riga, Jelgava and Saldus district) during autumn 2004.For each leaf sample 200 current season upright tips and for the berry sample about 400g ofcranberry berries were collected from locations representative of the planting. The leaf and berrymaterial was oven-dried at 60 o C and ground. Then the samples were dry-ashed in concentratedHNO 3 vapours and re-dissolved in HCl solution (HCl - distilled water mixture 3:100) (Rinkis et al.,1987).Concentrations of 12 biogenous elements (N, P, K, Ca, Mg, S, Fe, Mn, Zn, Cu, Mo, B) weredetermined in all berry and leaf samples. The levels of Ca, Mg, Fe, Cu, Zn, and Mn were estimatedby atomic absorption spectrophotometer (Perkin Elmer AAnalyst 700, acetylene-air flame)), thoseof N, P, Mo, B by colorimetry, S by turbidimetry, and K by flame photometer (Jenwey PFP7, airpropanebutane flame).The mineral element content in the berries was expressed as mg·100g -1 fresh fruit. All chemicalanalyses were done in the Laboratory of plant mineral nutrition of the Institute of Biology,University of Latvia. The levels of statistical significance were determined with MS Excel 2003. T-test “Two-Sample Assuming Unequal Variances” (p

Table 1. Nutrient concentrations in cranberry berry samples from producing plantings (Aluksne,Talsu, Madonas and Riga district) and woodlands (Riga, Jelgava and Saldus district) in Latvia.V.macrocarponV. oxycoccosElementRange Mean ± SE Range Mean ± SEMacroelements (mg·100g -1 fresh berries)N 10.4 – 65.0 40.3 ± 3.38 a 1 13.0 – 78.0 35.1 ± 9.36 aP 6.5 – 11.7 9.1 ± 0.26 a 6.5 – 7.8 6.5 ± 0.13 bK 52.0 – 98.8 79.3 ± 2.08 a 78.0 – 93.6 81.9 ± 1.82 aCa 7.8 – 14.3 10.4 ± 0.39 a 9.1 – 18.2 13.0 ± 1.30 bMg 5.2 – 9.1 6.5 ± 0.13 a 7.8 – 9.1 7.8 ± 0.13 bS 5.2 – 14.3 9.1 ± 0.39 a 6.5 – 18.2 11.7 ± 1.43 aMicroelements (mg·100g -1 fresh berries)Fe 0.22 – 1.17 0.51 ± 0.049 a 0.33 – 0.42 0.36 ± 0.010 bMn 0.06 – 0.57 0,21 ± 0.005 a 2.18 – 3.95 2.78 ± 0.194 bZn 0.07 – 0.17 0,12 ± 0.005 a 0.14 – 0.19 0.16 ± 0.007 bCu 0.04 – 0.08 0.05 ± 0.002 a 0.06 – 0.08 0.07 ± 0.002bMo 0.01 – 0.02 0.01 ± 0.001 a 0.01 – 0.02 0.01 ± 0.001 aB 0.03 – 0.12 0.07 ± 0.005 a 0.10 – 0.17 0.13 ± 0.008 b1 Means with different letters in a row were significantly different (t-Test, p < 0.05)Statistically significant differences (p

The contribution of V. macrocarpon and V. oxycoccus fruits as a dietary source of mineral elementswas estimated from our study results. The potential contribution of 100 g of cultivated and wildcranberries to the Recommended Dietary Allowances (RDA) (USDA RDA chart, 2004) for mineralelements is presented in Table 3.Table 3. Contribution of 100 g of cranberries to the Recommended Dietary Allowance (RDA) foradults per day.RDA*,Element concentrationmg 100 g -1 g fresh fruit% of RDA supplied by 100 gcranberriesElement mg V.macrocarpon V.oxycoccos V.macrocarpon V. oxycoccosP 700 9.1 6.5 1.32 0.97K 2500 79.3 81.9 3.16 3.28Ca 1000 10.4 13.0 1.07 1.31Mg 420 6.5 7.8 1.64 1.93S 850 9.1 11.7 1.06 1.39Fe 8 0.51 0.36 6.33 4.46Mn 2.3 0.21 2.78 9.26 120.96Zn 11 0.12 0.16 1.11 1.49Cu 0.9 0.05 0.07 6.00 7.67Mo 0.045 0.01 0.01 8.89 8.89B 1.5 0.07 0.13 4.73 8.73* USDA RDA chart (2004)Wild and cultivated cranberries supply 9.26 % and even 120.96 %, respectively, of the adult dailyrequirement for Mn. The content of Fe, Cu, Mo, and B in 100 g fresh fruits of both cranberriesstudied also contributes from 4.46 % to 8.89 % of the daily micronutrient requirement. Frommacronutrients only K in V. macrocarpon and V. oxycoccus fruits was found in appreciableamounts (3.16 and 3.28 %, respectively of the RDA). One hundred grams of fresh wild andcultivated cranberries may supply also a few percent of RDA for P, Ca, Mg, S and Zn.The relationship between food and health becomes increasingly significant as consumers nowdemand healthy, tasty and natural foods that have been grown in uncontaminated environments.Numerous studies have shown that among horticultural crops fruits are an important source ofdietary nutrients, especially with respect to minerals (Grusak and DellaPenna, 1999). Among berryfruits, cranberries are considered to be not only an excellent source of phenolic compounds andvitamins, but also as valuable source of mineral nutrients (Ekholm et. al., 2007).Our research revealed statistically significant differences between V. macrocarpon and V.oxycoccus results for P, Ca, Mg, Fe, Mn, Zn, Cu and B in fruit samples and P, K, Ca, Mg, S, Fe,Mn, Zn and B in leaf samples. Nitrogen is the controlling element for American cranberry nutritionand adequate fertilization, in general, is used to maintain renewal growth, crop production, andflower bud development for the next crop (DeMoranville, 1997). However surprisingly similar Nconcentrations were found in the cultivated American cranberry and wild cranberry leaf samples. Itshould be stressed that V. oxycoccos growing medium – sphagnum peat are especially N poor(Stewart and Nilsen 1993). Our research revealed a considerably higher content of Mn in V.oxycoccus leaves (1583.0 mg kg -1 ) in comparison to American cranberry leaves (282.9 mg kg -1 ).This phenomenon could be explained by the different pH of the growing substrata for both speciesstudied. Significantly lower pH and consequentially higher availability of Mn in natural high bogswas found in our previous studies (Karlsons and Osvalde, 2007). Both cranberry fruit Mn contentssupported these differences.Cultivated cranberry fruits had higher content of P and Fe while wild cranberry fruits showed thehighest levels of Ca, Mg, Mn, Zn, Cu and B. The sequences with regard to the content of macroandmicroelements in V. macrocarpon fruits were as K > N > Ca > P = S > Mg and Fe > Mn > Zn69

B > Cu > Mo, respectively. The order of macro- and micronutrient concentrations in V.oxycoccos was N > K > Ca = S > Mg > P and Mn > Fe > Zn > B > Cu > Mo.In general, the fruit mineral nutrient concentrations found in the studied species in Latvia weresimilar or considerably higher (Ca, Mg, Fe, Zn and Cu) than values reported for the Americancranberry (USDA, 2008). Reported mean values for Fe content (0.25 mg·100g -1 fresh fruit) in V.macrocarpon fruits (USDA, 2008) were almost twice lower than our Fe results for Americancranberries. To the author’s knowledge, there is little comparable data in the literature which showthe detailed mineral content of V. oxycoccus. However the macronutrient content in wildcranberries analyzed was lower (except Ca), but Mn concentrations significantly higher than thereported values for V. oxycoccos in Finland (Ekholm et. al., 2007)The nutritional significance of fruits as a dietary source of minerals is related to the contributionthey make to the Recommended Dietary Allowance (RDA). The present study shows that fruits ofboth V. macrocarpon and V. oxycoccus are a ood sources of Mn (9.26 % and 120.96 % of therecommended daily dose, accordingly) in human nutrition. The content of Fe, Cu, Mo, and B in100 g of fresh fruits of both cranberries studied also contributes from 4.46 % to 8.89 % of the dailymicronutrient requirement.All macronutrients, except K, in both species of cranberries were stated in relatively lowconcentrations. Only K in V. oxycoccus berries was found in appreciable amounts (3.28 % of theRDA). One hundred grams of fresh wild and cultivated cranberries may supply also a few percentof the RDA for P, Ca, Mg, S and Zn. The availability of Ca in the body to great extent depends onthe calcium to phosphorous ratio. The recommended optimal Ca : P ratio in the diets is 1.0 to 1.3(Calvo & Park, 1996). In our study such a Ca : P ratio was characteristic only for Americancranberries.ConclusionsThe present study revealed significant differences in the mineral composition of V. oxycoccus andV. macrocarpon. Cultivated cranberry fruits had higher content of P and Fe while wild cranberrieshad higher levels of Ca, Mg Mn, Zn, Cu and B. Plant leaf analysis revealed organ-specificdistribution of mineral elements in both species of studied cranberries.The present study shows that fruits of both V. macrocarpon and V. oxycoccus are a valuable sourceof microelements: Fe, Cu, Mo, B and especially Mn, in human nutrition.References1. Bálint A.F., Kovacs G. and Erdei L.J. (2001) Comparison of the Cu, Zn, Fe, Ca and Mg contents of thegrains of wild, ancient and cultivated wheat species. Cereal Research Communications, 29, pp. 375–382.2. Calvo M.S. and Park Y.K. (1996) Changing phosphorus content of the U.S. diet: potential for adverseeffects on bone. J. Nutr., 126, pp. 1168–1180.3. Castaneda - Ovando A., Pacheco-Hernandez L., Paez-Hernandez E., Rodriguez J.A. and Galan-VidalC.A. (2008) Chemical studies of anthocyanins: A review. Food Chemistry, 113, pp. 859–871.4. Cherkasov A.P. (1988) The cranberry yields in the USSR. Acta Botanica Fennica, 136, pp. 65 – 68.5. DeMoranville C.J. (1997) Cranberry nutrition and fertilizers. In: H.A. Sandler (ed), CranberryProduction: a Guide for Massachusetts, Univ. of MA Extensions, pp. 81-85.6. Ekholm P., Reinivuo H., Mattila P., Pakkala H., Koponen J., Happonen A., Hellström J. and Marja-Leena Ovaskainen M.L. (2007) Changes in the mineral and trace element contents of cereals, fruits andvegetables in Finland. Journal of Food Composition and Analysis, 20, pp. 487–495.7. Grusak M.A. and DellaPenna D. (1999) Improving the nutrient composition of plants to enhance humannutrition and health. Annu. Rev. Plant Physiol. Plant Mol. Biol., 50, pp. 133-161.8. Howell A.B., Reed J.D., Krueger C.G., Winterbottom R., Cunningham D.G. and Leahy M. (2005) A-type cranberry proanthocyanidins and uropathogenic bacterial anti-adhesion activity. Phytochemistry,66, pp. 2281-2291.9. Karlsons A. and Osvalde A. (2007) Characterarion of the American cranberry and wild cranberrynutrient status in cultivated plantings and natural bogs of Latvia. Proc. International Conf. “Vacciniumspp. and less known small fruits: cultivation and health benefit”, Nitra, Slovak Republic, pp. 78-79.10. Konczak I. and Zhang W. (2004) Anthocyanins-more than nature´s colours. Journal of Biomedicine andBiotechnology, 5, pp. 239–240.70

11. Kong J. M., Chia L. S., Goh N.K., Chia T. F. and Brouillard R. (2003) Analysis and biological activitiesof anthocyanins. Phytochemistry, 64 (5), pp. 923–933.12. Leahy M., Speroni J. and Starr M. (2002) Latest development in cranberry health research. Pharm. Biol.,40, pp. 50-54.13. Lule S.U. and Xia W. (2005) Food phenolics, pros and cons: A review. Food Reviews International, 21(4), pp. 367–388.14. Nichenametla S.N., Taruscio T.G., Barney D.L. and Exon J.H. (2006) A review of the effects andmechanisms of polyphenolics in cancer. Critical Reviews in Food Science and Nutrition, 46 (2), pp.161–183.15. Pietola L. and Salo T. (2000) Response of P, K, Mg and NO3-N contents of carrots to irrigation, soilcompaction, and nitrogen fertilization. Agricultural and Food Science in Finland, 9, pp. 319–331.16. Rinkis G., Ramane H. and Kunicka T. (1987) Methods of soil and plant analysis. Zinatne, Riga, 200 p.(In Russian).17. Ripa A. (1988) Rational use, reproduction and protection of cranberries in the Latvian SSR. ActaBotanica Fennica, 136, pp. 69 – 72.18. Stewart C.N. and Nilsen E.T. (1993) Association of edaphic factors and vegetation in several isolatedAppalachian peat bogs. Bulletin of the Torrey Botanical Club, 120, pp. 128 – 135.19. Stintzing F.C. and Carle R. (2004) Functional properties of anthocyanins and betalains in plants, food,and in human nutrition. Trends in Food Science and Technology, 15 (1), pp. 19–38.20. Vinson J.A., Su X., Zubik L. and Bose P. (2001) Phenol antioxidant quantity and quality in foods: fruit.J. Agric. Food Chem., 49, pp. 5315-5321.21. WHO. (2003) Diet, nutrition and the prevention of chronic diseases. WHO, Geneva22. Yudina V.F. (1988) On the European cranberry in Soviet Karelia. Acta Botanica Fennica, 136, pp. 73 –76.23. USDA National Nutrient Database for Standard Reference.(2008) www.nal.usda.gov/24. USDA RDA chart. (2004) www.iom.eduCLIMATIC EFFECTS ON THE PRODUCTION AND QUALITY OF BILBERRIES(VACCINIUM MYRTILLUS)KLIMATA IETEKME UZ MELLEĥU (VACCINIUM MYRTILLUS)RAŽU UN KVALITĀTIInger Martinussen 1 , Jens Rohloff 2 , Eivind Uleberg 1 , Olavi Junttila 3 , Anja Hohtola 4 , LauraJaakola 4 and Hely Häggman 41 Norwegian Institute for Agricultural and Environmental Research, Bioforsk Nord Holt, Box 2284, N-9269Tromsø, Norway, e-mail: inger.martinussen@bioforsk.no;2 Department of Biology, NTNU, N-7491 Trondheim, Norway;3 Institute of Biology, University of Tromsø, N-9037 Tromsø, Norway;4 University of Oulu, Department of Biology, P.O. Box 3000, FIN-90014 Oulu, Finland.AbstractThe bilberry (Vaccinium myrtillus), also called European blueberry, is one of the most significantwild berries in the Nordic countries. The berries are recognized for their bioactive properties anddistinctive aroma and flavour. The effects of climate on the production and the quality of bilberrieshave been studied in a controlled experiment in a phytotrone using clonal material. In theexperiment individual plants from two Northern clones and two Southern clones of bilberry weregrown at 12° and 18 °C. At each temperature 2 different light treatments were tested; 1) 24 hour (h)natural light (long day) and 2) 12 h natural light (short day). All plants were kept outside duringflowering to ensure pollination by insects. Berries were sampled when ripe, weighed and stored at -80°C for later analysis.KopsavilkumsMellenes (Vaccinium myrtillus), sauktas arī Eiropas mellenes, ir Ĝoti nozīmīgas savvaĜas ogasZiemeĜu valstīs. Ogas ir atzītas to bioloăiski aktīvo vielu satura un īpašās smažas un garšas dēĜ.Klimata ietekme uz ražu un tās kvalitāti tika pētīta kontrolējamos eksperimenta apstākĜos,71

izmantojot klonu materiālu. Atsevisėi augi no diviem ZiemeĜu melleĦu kloniem un diviemDienvidu melleĦu kloniem tika audzēti 12° un 18 °C temperatūrā. Katras temperatūras apstākĜiemtika izmantoti divu veidu apgaismojumi: 1) 24 stundas dabīgs apgaismojums (garā diena) un 2) 12stundas dabīgs apgaismojums (īsā diena). Lai nodrošinātu apputeksnēšanos ar kukaiĦu palīdzību,visi augi ziedēšanas laikā tika turēti āra apstākĜos. Ogu nogatavošanās laikā tika ievākti oguparaugi, nosvērti un uzglabāti – 80 °C vēlākām analīzēm.Key words: bilberry, clonal differences, European blueberry, quality, temperature effectsIntroductionThe bilberry (Vaccinium myrtillus L.), also called the European blueberry, is a long-livedericaceous dwarf shrub that grows wild in Europe and Asia, and most abundantly in the northernand eastern parts of Europe. The berries are recognized for their distinctive aroma and flavor andtheir high concentrations of phenolic compounds such as anthocyanins (Lätti et al., 2008). Obviousdifferences between the European blueberry and other blueberry species like V. angustifolium, V.corymbosum (highbush), V. ashei Reade (rabbiteye) and V. myrtilloides Minchx (sour-top lowbush)are the blackish-blue colour of the flesh compared to the whitish fruit flesh of the others. Resultsfrom Giovanelli and Buratti (2009) show that total phenolics and total anthocyanin concentrationswere, respectively, two fold and three fold higher in V. myrtillus than in V. corymbosum. Thebilberries also had a higher anthocyanin-to-total phenolic ratio. In a study from Finland (Lätti et al.,2008) it was found that the berries of the southern regions had a significantly lower content of totalanthocyanins compared with the central and northern regions. The aim of this study was to studythe role of the genotype and the environment (temperature and daylength) under controlledconditions in a phytotrone on the contents of carbohydrate, acids and phenols in the bilberry. Suchinformation is important for the development of the European blueberry as a commercial crop.Material and MethodsMaterial and experimental design. Individual plants of four clones derived from the southern (S1and S2) and northern (N1 and N2) part of Finland were grown in phytotron in Tromsø, Norway at12° and 18 °C. At each temperature 2 different light treatments were tested; 1) 24 hour (h) naturallight (long day) and 2) 12 h natural light (short day). All plants were kept outside during floweringto ensure pollination by insects. Berries were sampled when ripe, weighed and stored at -80°C forlater.Bilberry Extraction Procedure. Frozen bilberries (3-6 berries) were sliced with a scalpel, and 320mg f.w. of each sample was transferred to a round-bottom shaped microtube (2 ml). Pre-cooled(-20°C) methanol (400 µl) containing ribitol as the internal standard (25 µg/ ml), was added to eachtube and vortexed for 5 seconds (s). Sample tubes were treated for 1 hour (h) at 60°C in anultrasonic bath, and cooled down to room temperature before the next step. 200µl chloroform wasadded, and tubes vortexed for 5 s to remove the lipids. Additional 400 µl H 2 O were added andtubes vortexed for 10 s. Samples were centrifuged at 13,000 rpm and 4°C for 10 min. An aliquot of300 µl from the clear supernatant was transferred into a V-shaped 1.5 ml microtube for GC-MSanalysis. Yet another 300 µl aliquot was stored in a 1.5 ml microtube at -20 °C for later phenolanalyses. The GC-MS samples were dried in a speed vac overnight without heating and stored at -80°C prior to further processing. For the derivatization of extracted sugars, acids and secondarymetabolites, sample residues were re-dissolved in 80 µl of 20 mg ml -1 methoxyamine hydrochloridein pyridine, and treated at 30°C for 90 min in an incubator (200 rpm). Finally, samples were treatedwith 80 µl of MSTFA (N-Methyl-N-(trimethylsilyl)trifluoroacetamide) at 37°C for 30 min in anincubator (200 rpm). Samples were transferred to 1.5 ml autosampler vials with glass inserts, andstored at -20 °C prior to GC-MS analysis.Gas Chromatography–Mass Spectrometry analysis (GC-MS). An Agilent 6890/5975 GC-MS wasused for all analyses. Sample volumes of 1 µl were injected with a split ratio of 25:1. GCseparations were carried out on a HP-5MS capillary column (30 m × 0.25 mm i.d., film thickness0.25 µm). The injection temperature was 230 °C, and the interface was set to 250 °C. The carriergas was helium at a constant flow rate of 1 ml min -1 The GC temperature program was held72

isothermically at 70°C for 5 min, ramped from 70 °C to 310 °C at a rate of 5 °C min, and finallyheld at 310 °C for 7 min (analysis time: 60 min). The MS source was adjusted to 230 °C and amass range of m/z 50-700 was recorded. All mass spectra were acquired in EI mode.Chromatogram visualization and peak integration was carried out using the Agilent ChemStationsoftware. For mass spectra evaluation and peak identification, the AMDIS software (v. 2.64) wasused in combination with the following mass spectral libraries: NIST05 database and a target TMSdatabase (Max-Planck Insitute for Molecular Plant Physiology, Golm, Germany). All compoundswere quantified based on the internal standard ribitol and expressed as mg/100 g f.w.Total Phenolics. The analysis of total phenolics content was based on a modified Folin-Ciocalteumethod (Ainsworth and Gillespie, 2007). Berry extracts (see Bilberry Extraction Procedure) werediluted 1:40 before incubation at ambient temperature for 2 h. Samples (200 µl) were transferred toa clear 96-well microplate, and the absorption was measured at 750 nm on a plate reader(Labsystems Multiskan MS). Total phenolics were expressed as mg gallic acid equivalents (GAE)100 g f.w. of berries.Statistics. Statistics were done using Mintitab software. At each treatment there were twoindividual plants of each clone.Results and DiscussionAt the lowest temperature the two Northern clones produced berries earlier than the two Southernclones (Figure 1) and Southern clones produced far more berries during short days than during longdays. These results indicate that the Northern clones are able to grow rapidly at reasonably lowtemperatures when given 24 hour of light, while the Southern clones are adapted to shorter daysand are not able to use the 24 h light efficiently. At 18 °C all clones produced berries as early as 28days after pollination (Figure 2). The Northern clones produced unexpectedly more berries than theSouthern clones during both the long day and the short day treatment at 18 °C.Berries produced at 12 °C had a significantly higher % of sugars (sucrose, fructose and glucose),phenolic acids and total phenols than berries produced at 18 °C (Table 1). The increased amount oftotal phenols found at low temperatures contradicts the findings of Rieger et al. (2008) where thelevel of anthocyanins in bilberries decreased in amounts while the antioxidant level increased withrising altitudes. The study of Rieger et al. (2008) was, however, only focused on anthocyanins andwas not performed under controlled conditions. Howard et al. (2003) analyzed five commercialcultivars and 13 breeding selections of V. corymbosum L. grown at the same location for totalphenolics over two growing seasons and found that the variation between genotypes was muchgreater than that observed between growing seasons.However, several genotypes varied significantly over the two growing seasons indicating thatenvironmental growing conditions can impact the amount of phenolics in blueberries.Figure 1. Accumulated production of berries at 12°C.♦ clone S1, ■ clone S2, ▲ clone N1, × clone N273

Figure 2. Accumulated production of berries at 18°C.♦ clone S1, ■ clone S2, ▲ clone N1, × clone N2Studies on raspberries (Anttonen and Karjalainen, 2005) also indicate that growing conditions(light, temperature and soil condition) affect the phenolic content in northern latitudes; however,the variation between genotypes was considerable.Table 1. Analysis of % sugars, % acids and total phenols in bilberries grown at 12° and 18°C.Temp.°C % Sugars % Acids Total phenols mg/100 g12 13.1 3.7 583.818 9.2 2.9 556.8(The results are presented as the mean for all clones and all treatments at the given temperature.)ConclusionsThe results show that the clones of bilberries originating from northern areas are better adapted tolow temperatures and long days (24 h light) than clones originating from southern areas. Breedingshould therefore aim both for the selection of material for northern conditions and material forsouthern conditions. Low temperatures lead to a significantly higher content of sugars, acids andtotal phenols. Berries from the northern areas possess the added value of sweetness and a highercontent of the health beneficial phenols. In conclusion, breeding material should be selected basedon the evaluation of results achieved from the health benefit compounds of bilberry clones.Moreover, the environmental effects on berry quality will open the door for breeding specificclones for specific regions.AcknowledgementsThe study was financed by Norden Nordic Innovation Centre, program “New Nordic Food” and theNorwegian Research Council, program “Matprogrammet”.References1. Ainsworth E.A. and Gillespie G.M. (2007) Estimation of total phenolic content and other oxidationsubstrates in plant tissues using Folin–Ciocalteu reagent. Nature Protocols, 2(4), pp. 875-877.2. Anttonen M.J. and Karjalainen R.O. (2005) Environmental and genetic variation of phenolic compoundsin red raspberry. J. Food Comp. Analysis, 18, pp. 759-769.3. Giovanelli G. and Buratti S. (2009) Comparison of polyphenolic composition and antioxidant activity ofwild Italian bluberries and some cultivated varieties. Food Chem., 112, pp. 903-908.4. Howard, L., Clark J.R. and Brownmiller C. (2003) Antioxidant capacity and phenolic content inblueberries as affected by genotype and growing season. J. Sci. Food Agric., 83, pp. 1238-1247.5. Lätti A.K., Riihinen K.R. and Kainulainen P.S. (2008) Analysis of anthocyanin variation in wildpopulations of bilberry (Vaccinium myrtillus L.) in Finland. J. Agric. Food Chem., 56, pp. 190-503.6. Rieger G., Müller M., Guttenberger H. and Bucar F. (2008) Influence of altitudinal variation on thecontent of phenolic compound in wild populations of Calluna vulgaris, Sambucus nigra, and Vacciniummyrtillus. J. Agric. Food Chem., 56(19), pp. 9080-9086.74

IN VITRO PROPAGATION OF SEVERAL VACCINIUM CORYMBOSUM L. ANDVACCINIUM VITIS-IDAEA L. CULTIVARSVAIRĀKU VACCINIUM CORYMBOSUM L. UN VACCINIUM VITIS-IDAEA L. ŠĖIRĥUIN VITRO PAVAIROŠANAMária Gabriela Ostrolucká 1 , Alena Gajdošová 1 , Emilia Ondrušková 2 and GabrielaLibiaková 11Institute of Plant Genetics and Biotechnology SAS, Akademická 2, P.O.Box 39A, 950 07 Nitra, SlovakRepublic; e-mail: gabriela.ostrolucka@savba.sk2Institute of Forest Ecology, Branch for Wood Plants Biology Nitra, Akademická 2, P.O.Box 39A, 950 07Nitra, Slovak Republic; e-mail: ondruskova@savzv.skAbstractIn the paper the efficient in vitro regeneration and propagation systems in several Vacciniumcorymbosum L. and Vaccinium vitis-idaea L. cultivars are described. It was found that shootregeneration ability is highly dependent on the cultivar and cytokinin applied. Zeatin showed itselfto be efficient for axillary and adventitious shoot induction in both species. A zeatin concentrationof 2 mg l -1 was the best for axillary shoot regeneration in V. corymbosum, while zeatin in aconcentration of 0.75 - 1.5 mg l -1 was suitable for V. vitis-idaea cultivars. Multiple adventitiousshoot formation on leaf explants of V. corymbosum occurred on the medium with 0.5 mg l -1 zeatin.In V. vitis-idaea indirect shoot regeneration was observed in cv. ´Red Pearl´ after transfer of thecalli on the medium with 0.5 mg l -1 zeatin. For long-term proliferation of in vitro regeneratedaxillary or adventitious shoots an AN medium with 0.5 mg l -1 zeatin was successfully used.Satisfactory rooting of isolated microshoots was achieved in vitro on an AN medium supplementedwith 0.8 mg l -1 IBA and 0.8 g l -1 charcoal.KopsavilkumsŠajā rakstā ir apskatīta efektīva in vitro reăenerācijas un pavairošanas sistēma vairākām Vacciniumcorymbosum L. un Vaccinium vitis-idaea L. šėirnēm. Dzinumu reăenerācijas spēja ir atkarīga nošėirnes un apstrādes ar citokinīniem. Zeatīns bija efektīvs, lai veicinātu sānu un adventīvo dzinumuaugšanas veidošanu. V. corymbosum sāndzinumu veidošanai piemērotākā zeatīna koncentrācija bija2 mg l -1 , bet V. vitis-idaea šėirnēm piemērotāka bija 0.75 - 1.5 mg l -1 zeatīna koncentrācija. Barotnēar 0.5 mg l -1 zeatīnu novērota masveidīga adventīvo dzinumu veidošanās V. corymbosum lapueksplantiem. Netieša dzinumu reăenerācija tika novērota V. vitis-idaea šėirnei ‘Red Pearl’ pēckallusa pārnešanas uz barotni ar 0.5 mg l -1 zeatīnu. Zeatīns 0.5 mg l -1 koncentrācijā tika sekmīgipielietots AN vidē, lai nodrošinātu ilgstošu in vitro reăenerēto sānu un adventīvo dzinumuproliferāciju. Apmierinoša izolēto mikrodzinumu apsakĦošanās tika sasniegta AN vidē, kaspapildināta ar 0.8 mg l -1 indolilsviestskābes un 0.8 g l -1 kokogĜu.Key words: Vaccinium corymbosum L., Vaccinium vitis-idaea L., axillary shoot and adventitiousshoot regenerationAbbreviations: AN – Anderson medium; IBA – indole-3-butyric acid; TDZ - thidiazuron (Nphenyl-N1-1,2,3,-thiadiazol-5-ylurea);2-iP (N6-[2-Isopentenyl]adenine)IntroductionThe Vaccinium corymbosum L. and Vacinium vitis-idaea L. represent commercially important andbiologically valuable small fruits (Song and Sing, 2004) desirable for cultivation in various parts ofthe world. Production of high quality plants in large amounts is needed for commercial plantationestablishment. In this respect, in vitro plant regeneration from dormant buds and throughadventitious organogenesis enables effective mass production of plant material and to the createnecessary conditions for the application of genetic engineering. Several papers have describedmicropropagation methods for these economically important species (Shibli and Smith, 1996;Jaakola et al., 2001; Debnath and McRae, 2002; Ostrolucká et al., 2002; Cao et al., 2003;75

Ondrušková et al., 2003; Petri and Burgos, 2005; Gajdošová et al., 2006; Gajdošová et al., 2007).However, the results show that the different cultivars within the same species can differ in theirrequirements for the optimal growth regulator concentrations therefore it is important to get exactinformation on culture conditions for different cultivars.In this paper micropropagation by axillary branching and adventitious shoot regeneration isdescribed in a wide range of Vaccinium corymbosum L. and Vaccinium vitis-idaea L. cultivars.Material and methodsAs an initial plant material for axillary shoot formation stem cuttings with dormant buds werecollected from the selected cultivars of mature plants during February and the beginning of March:Vaccinium corymbosum L. - cvs. ´Berkeley’, ´Bluecrop´, ´Blueray´, ´Duke´, ´Brigitta´ andVaccinium vitis-idaea L. - cvs. ´Red Pearl´ and ´Koralle´. Nodal segments with buds were washedunder running water for 1 h and sterilised 2 min in 70 % ethanol, 6 min in a 0.1 % solution ofmercuric chloride with 3 drops of Tween, followed by washing 3x15 min in sterile distilled water.For shoot regeneration dormant apical and axillary buds were used, from which the upper scaleswere removed after sterilisation. Anderson’s Rhododendron medium - AN (Anderson, 1980) with30 g l -1 sucrose, 8 g l -1 Phyto agar, pH adjusted to 4.8 – 5.0 and supplemented with differentcytokinin concentrations was used for cultivation. Cultures were maintained in the growth chamberat 23 ± 2 o C under 16/8 light and dark photoperiod and a light intensity of 50 µmol m -2 s -1 providedby white fluorescent lamps. In V. corymbosum cvs. 0.5 and 2.0 mg l -1 zeatin and in V. vitis-idaea L.cvs. 0.25 – 2.0 mg l -1 zeatin and 2.5 - 20.0 mg.l -1 2-iP, were tested for axillary shoot regeneration.The influence of cytokinins, zeatin and 2-iP, on shoot regeneration ability was evaluated after 5weeks. Data evaluation was performed using Statgraphic PLUS 5 for Windows.For adventitious shoot regeneration leaves from the in vitro plants of V. corymbosum L. - cvs.´Berkeley´, ´Bluecrop´ and ´Brigitta´ with cut margins were cultivated horizontally with adaxialsurface on an AN medium with 30 g l -1 sucrose, 8 g l -1 Phyto agar, pH adjusted to 4.8-5.0,supplemented with 0.5 mg l -1 zeatin and 2.2 mg l -1 TDZ. Their regeneration ability based on shootproliferation was evaluated in three subcultures, during a five week period. In V. vitis-idaea L. -cvs. ´Red Pearl´ and ´Koralle´ as primary explants stem cuttings and leaves from in vitro plantswere used for adventitious shoot induction. For each experiment 30 explants were cultivated (5explants/culture dish x 6 culture dishes). AN medium with zeatin in concentrations 2.2 and 4.4mg.l -1 or thidiazuron in concentrations 1.1 mg l -1 , 2.2 mg l -1 and 3.3 mg l -1 was used the forinduction of adventitious organogenesis. After 5 weeks of cultivation the explants were transferredinto an AN medium with 0.5 mg.l -1 zeatin for shoot multiplication. The percentages of explantsregenerating shoots or inducing callus and the number of regenerated shoots per explant after 3subcultures were recorded. The long-term shoot proliferation was performed on an AN mediumwith 0.5 mg l -1 zeatin. For the microshoot rooting an AN medium supplemented with 0.8 mg l -1IBA and 0.8 g l -1 charcoal was tested.Results and DiscussionThe results of the experiments on shoot regeneration from dormant apical and axillary buds in V.corymbosum L. showed that shoot regeneration ability is highly dependent on cultivar andcytokinin concentrations. Zeatin in a concentration of 2 mg l -1 was proved to be more effective forshoot regeneration in comparison with 0.5 mg l -1 zeatin (Figure 1).The statistically significant differences in the number of shoots per explant were obtained on amedium with 2 mg l -1 zeatin in tested cultivars with the highest regeneration ability in cvs.‘Brigitta’ and ‘Blueray’.76

16BM ean N o. of shoots/explant14121086420ABlueray Duke Berkeley Bluecrop BrigittaFigure 1. Effect of zeatin (A - 0.5 mg l -1 ; B - 2.0 mg l -1 ) on V. corymbosum L. shoot regeneration.The positive influence of zeatin on multiple shoot induction from apical and axillary buds wasconfirmed also in V. vitis-idaea cvs. Statistically significant differences in the number of shoots perexplant were obtained in cv. ‘Koralle‘ with the highest shoot formation on the zeatin concentrationof 0.75 mg l -1 . The differences between the control and the variants with different concentrations of2-iP were not statistically significant (Table 1.).Table 1. Multiple range analysis for shoot number/explant at different zeatin and 2-iPconcentrations in Vaccinium vitis-idaea L. - cvs. ‘Koralle’ and ‘Red Pearl’Zeatin Mean number of shoots/explantMean number of shoots/explant(mg l -1 2-iP (mg l -1 ))‘Koralle’ ‘Red Pearl’‘Koralle’ ‘Red Pearl’0.00 1.24 a 0.99 a 0.00 1.24 a 0.99 a0.25 3.00 ab 3.93 b 2.50 1.25 a 1.82 ab0.50 3.84 bc 5.02 bc 5.00 1.60 a 3.44 bcd0.75 5.19 c 5.55 bc 10.00 1.42 a 2.84 abc1.00 4.76 bc 6.98 c 15.00 1.27 a 6.66 e1.50 3.69 abc 6.90 c 17.50 1.16 a 5.47 de2.00 3.13 abc 6.20 bc 20.00 1.09 a 4.68 cdeIn cv. ‘Red Pearl’ the both tested cytokinins were effective in shoot regeneration. On the mediawith zeatin the highest shoot regeneration was obtained at the zeatin concentration 1.0 – 1.5 mg l -1 .The most effective 2iP concentration was 15 mg l -1 (Table 1.). Regeneration ability was differentfor each cultivar. The shoot proliferation per explant was higher in ´Red Pearl´ in comparison withcv. ´Koralle´ for both tested cytokinins.For induction of adventitious organogenesis on the leaf explants of V. corymbosum L. 0.5 mg l -1zeatin and 2.2 mg l -1 TDZ were used. On the media with TDZ callus formation was observed, butno adventitious buds were visible during the 5 weeks of cultivation. Shoot formation occurred onlyafter their transfer on medium with 0.5 mg.l -1 zeatin, which confirms the positive zeatin influenceon shoot regeneration. On the other hand, multiple adventitious shoots were formed after 5 weeksof cultivation on the leaf explants initially cultivated on an AN medium with 0.5 mg.l -1 zeatin,produced via direct regeneration. After the 3rd subculture on the same medium a high number ofvigorous shoots with good elongation growth was obtained per explant in the tested cultivars(Table 2.).77

Table 2. Adventitious shoot regeneration from leaf tissue in Vaccinium corymbosum L. with 0.5mg l -1 zeatinCultivar% of leavesTotal number of adventitious shootsMean no. ofregeneratingII.III.I. subcultivationshoots/explantshootssubcultivation subcultivation‘Berkeley’ 35 110.0 237.14 362.0 18.1‘Bluecrop’ 10 13.3 50.33 133.5 6.68‘Brigitta’ 40 129.37 257.50 380.0 19.0The regeneration ability of the cultivars was different. The highest in vitro shoot regeneration wasobtained in cv. ´Brigitta´ (19.0), which is in correlation with regeneration achieved via culture ofapical and axillary buds (Figure 1.).In the V. vitis-idaea L. an AN medium with zeatin 2.2 mg l -1 and 4.4 mg l -1 or thidiazuron 1.1 mg l -1 , 2.2 mg l -1 and 3.3 mg l -1 was used for the induction of adventitious organogenesis on the stemand leaf explants of in vitro plants. On the media with TDZ callus formation was observed, buteven after transfer on medium with zeatin no shoot formation occurred and calli necrotized. Incontrast to V. corymbosum L. no direct regeneration of adventitious buds was recorded in V. vitisidaeaL. on media with zeatin. The callus formation was observed on leaf and stem explants ofboth tested cvs. ´Red Pearl´ and ´Koralle´, with small differences in the percentage of callusformation between tested zeatin concentrations (2.2 and 4.4 mg l -1 ). However, the percentage ofcallus formation was markedly higher from stem tissue in comparison with leaf tissue. After thetransfer of callus on an AN medium with 0.5 mg l -1 zeatin adventitious buds developed on thecallus surfaces on both leaf and stem explants only in cv. ´Red Pearl´. No bud induction and shootregeneration occurred from callus in cv. ‘Koralle’. After the 3 rd subculture the number ofadventitious shoots in ‘Red Pearl’ regenerated from the leaf originated callus was significantlyhigher than from the stem callus. The total shoot number reached 1592 shoots/explant inducedfrom the leaf and 266 from the stem explants (Table 3).Table 3. Indirect regeneration of adventitious shoots in Vaccinium vitis-idaea L. on AN mediumwith zeatinShoot regeneration via callus phaseCultivars‘Red Pearl’‘Koralle’Primaryexplant2.2 mg l -1zeatin (A)4.4mg l -1zeatin (B)0.5 mg l -1 zeatinA + Bcallus % callus % total number of shootsTotal mean numberof shoots/explantLeaves 20.0 23.3 1592 53.07Stems 83.3 83.3 266 44.33Leaves 13.3 6.6 0 0Stems 16.6 16.6 0 0Our results, as well as the results of other researchers, confirmed that zeatin is the best for axillaryand adventitious shoot regeneration and proliferation in Vaccinium spp. (Chandler and Draper,1986; Rowland and Ogden, 1992; Marcotrigiano et al., 1996; Cao and Hammerschlag, 2000).Unlike who results of other researchers (Reed and Abdelnour-Esquivel, 1991; Debnath and McRae,2002; Meiners et al., 2007), the used higher zeatin concentrations (4.0 mg l -1 and more), in ourexperiments the zeatin in the lower concentrations was successfully applied for axillary (0.75 – 2.0mg l -1 ; Figure 2.) and adventitious (0.5 mg l -1 ) shoot regeneration in V. corymbosum and V. vitisidaea, which is an important factor reducing the micropropagation cost, as well as minimizingundesirable somaclonal variation, as it was confirmed in the previous paper of Ostrolucká et al.(2007).For long-term proliferation of in vitro regenerated axillary or adventitious shoots an AN mediumwith 0.5 mg l -1 zeatin can be successfully used in both Vaccinium species, on which formation ofvigorous multiple shoot cultures was observed (Figure 3). The differences in intensity of shootproliferation on an AN culture medium with the zeatin content 0.5 mg l -1 confirmed dependence ofshoot formation and multiplication on the regeneration ability of the cultivars. Similar correlationswere found also by other authors (Popowich and Filipenya, 1997).78

Figure 2. Axillary shoot regeneration in V. vitis-idaea cv.’Koralle’ on AN medium with 0.75 mg l -1zeatin (A) and cv.’Red Pearl’ on AN medium with 15 mg l -1 2-iP (B), control (C).Satisfactory rooting of isolated microshoots in V. corymbosum L. and V. vitis-idaea L. wasachieved on an AN medium supplemented with 0.8 mg l -1 IBA and 0.8 g l -1 charcoal. In V.corymbosum the percentage of rooting was 95 %, 90 % and 80 % in cv. ‘Berkeley’, ‘Bluecrop’ and‘Brigitta’, respectively. In V. vitis-idaea the rooting percentage reached 60 % and 40 % in ‘ReadPearl’ and ‘Koralle’, respectively. Rooted plantlets transplanted into peat substrate weresubsequently acclimatized under greenhouse conditions without any special light treatment, withregular irrigation, in containers at the beginning (2 weeks) covered by lid, later opened. Plantlets inlength 80 – 100 mm (after 2 months) were replanted into bigger containers (120 mm in diameter)and placed under open-air conditions. Transfer of regenerants from in vitro to ex vitro conditionsand their acclimatization was successful, as almost 80-90 % of transferred plants survived.Figure 3. Adventitious shoot proliferation in V. vitis-idaea cv.’Red Pearl’derived from leaf tissuecultivated on AN medium with 0.5 mg l -1 zeatin (A, B). High shoot proliferation in V. corymbosumcv. ‘Bluecrop’ on AN medium with 0.5 mg l -1 zeatin (C).In conclusion we can state, that efficient cloning protocols were developed for the several selectedblueberry and lingonberry cultivars, which enable large-scale propagation of high quality, true totype plants for economic and growing needs.AcknowledgementsThe work was supported by Slovak Grant Agency VEGA, project no. 2/0004/08 and MVTS-COST863 Action.References1. Anderson W.C. (1980) Tissue culture propagation of red and black raspberries, Rubus idaeus and R.occidentalus. Acta Hort. (ISHS), 112, pp. 124-132.2. Cao X. and Hammerschlag F.A. (2000) Improved shoot organogenesis from leaf explants of highbushblueberry. Hort. Science, 35, pp. 945-947.3. Cao X., Fordham I., Douglass L. and Hammerschlag F. (2003) Sucrose level influencesmicropropagation and gene delivery into leaves from in vitro propagated highbush blueberry shoots.Plant Cell Tiss. Org. Cult., 75, pp. 255-259.79

4. Debnath S.C. and McRae K.B. (2002) An efficient adventitious shoot regeneration system on excisedleaves of micropropagated lingonberry (Vaccinium vitis-idaea L.). J. Hort. Sci. and Biotech., 77, pp.744-752.5. Gajdošová A., Ostrolucká M.G., Libiaková G., Ondrušková E. and Simala D. (2006) Microclonalpropagation of Vaccinium sp. and Rubus sp. and detection of genetic variability in culture in vitro. J.Fruit and Ornamental Plant Research, 14 (Suppl.1), pp. 61-76.6. Gajdošová A., Ostrolucká M.G., Libiaková G. and Ondrušková E. (2007) Protocol for micropropagationof Vaccinium vitis-idaea L. In: Jain S.M. and Häggman H. (eds) Protocols for Micropropagation ofWoody Trees and Fruits, Springer, Berlin Heidelberg New York, pp. 447-464.7. Chandler C.K. and Draper A.D. (1986) Effect of zeatin and 2iP on shoot proliferation of three highbushblueberry clones in vitro. Hortic. Sci., 25, pp. 1065-1066.8. Jaakola L., Tolvanen A., Laine K. and Hohtola A. (2001) Effect of N6-isopentenyladenine concentrationon growth initiation in vitro and rooting of bilberry and lingonberry. Plant Cell Tiss. Org. Cult., 66, pp.73-77.9. Marcotrigiano M., McGlew S.P., Hackett G. and Chawla B. (1996) Shoot regeneration from tissueculturedleaves of the American cranberry (Vaccinium macrocarpon). Plant Cell Tiss. Org. Cult., 44, pp.195-199.10. Meiners J., Schwab M. and Szankowski I., (2007) Efficient in vitro regeneration systems for Vacciniumspecies. Plant Cell Tiss Organ Cult., 89, pp. 169-176.11. Ondrušková E., Ostrolucká M.G. and Gajdošová A. (2003) Cytokinin influence on Vaccinium vitis-idaeaL. regeneration in vitro. Folia Oecologica, 30, pp. 99-105.12. Ostrolucka M.G., Gajdosova A. and Libiakova G. (2002) Influence of zeatin on microclonal propagationof Vaccinium corymbosum L. Propagation of Ornamental Plants, 2, pp. 14-18.13. Ostrolucká M.G., Gajdošová A., Libiaková G., Hrubiková K. and Bežo M. (2007) Protocol formicropropagation of selected Vaccinium spp. In: Jain S.M. and Häggman H. (eds) Protocols forMicropropagation of Woody Trees and Fruits, Springer, Berlin Heidelberg New York, pp. 445-455.14. Petri C. and Burgos L. (2005) Transformation of fruit trees. Useful breeding tool or continued futureprospect? Transgenic Research, 14, pp. 15-26.15. Popowich E.A. and Filipenya V.L. (1997) Effect of exogenous cytokinin on viability of Vacciniumcorymbosum explants in vitro. Russian J. of Plant Physiology, 44, pp. 104-107.16. Reed B.M. and Abdelnour-Esquivel A. (1991) The use of zeatin to initiate in vitro cultures of Vacciniumspecies and cultivars. HortScience, 26, pp. 1320-1322.17. Rowland L.J. and Ogden E.L. (1992) Use of a cytokinin conjugate for efficient shoot regeneration fromleaf sections of highbush blueberry. Hort. Science, 27, pp. 1127-1129.18. Shibli R.A. and Smith M.A.L. (1996) Direct shoot regeneration from Vaccinium pahlae (Othelo) andV.myrtillus (bilberry) leaf explants. Hort. Science, 31, pp. 1225-1228.19. Song G.Q. and Sing K.C. (2004) Agrobacterium tumefaciens-mediated transformation of blueberry(Vaccinium corymbosum L.). Plant Cell Rep., 23, pp. 475-484.COMPARISON STUDY OF CULTIVATED HIGHBUSH AND WILD BLUEBERRYNUTRIENT STATUS IN PRODUCING PLANTINGS AND WOODLANDS, LATVIAKRŪMMELLEĥU UN SAVVAěAS MELLEĥU MINERĀLĀS BAROŠANĀSNODROŠINĀJUMA LĪMENIS RAŽOJOŠĀS SAIMNIECĪBĀS UN LATVIJAS MEŽOSJolanta Pormale, Anita Osvalde, Vilnis NollendorfsInstitute of Biology, University of Latvia, Miera iela 3, Salaspils, Latvija, e-mail: augi@email.lubi.edu.lvAbstractHighbush blueberries (Vaccinium corymbosum L.) grown on more than 170 ha have become animportant horticultural commodity in Latvia with a high market demand. To realize the fullpotential of this modern high yielding crop, balanced plant nutrition is vitally important to ensureadequate growth and fruit production. The main aim of this study was to find out the actual mineralnutrition status of cultivated highbush and wild blueberries (Vaccinium myrtillus L.) in Latvia aswell as to evaluate the peculiarities of blueberry mineral nutrition in producing plantings and nativewoodlands. About 100 (soil and plant) samples were collected from different blueberry producingsites and 5 woodlands during the summer of 2008. Plant leaf analysis and soil testing were used to80

evaluate the blueberries supply with all of the biogenous elements (N, P, K, Ca, Mg, S, Fe, Mn, Zn,Cu, Mo, B). Our results suggest that only about 40 % of the plant leaf nutrient indices and soil testswere in the optimal range. Insufficient levels of N, P, Ca, Mo, B and increased concentrations ofMn in highbush blueberry leaves were the main problems. In spite of the high heterogeneity of theelement concentrations in soils, deficiency of N, Zn, Mo, B was found in the vast majority ofsamples analyzed. Although forest soils were characterized as an acid nutrient poor environment,wild blueberries showed especially high efficiency of K, Ca, Mg, Fe, and Mn accumulation in theirleaves. The present investigation forms the basis for the development of scientific knowledge basedblueberry fertilization practices in LatviaKopsavilkumsPēdējos gados Latvijā strauji attīstās jaunas netradicionālas ogu kultūras - augstkrūmmu melleĦu(Vaccinium corymbosum) audzēšana. KrūmmelleĦu stādījumu kopplatība Latvijā 2008. gadā jausasniedza 170 ha. Darba mērėis bija noskaidrot un salīdzinoši izvērtēt krūmmelleĦu un savvaĜasmelleĦu (Vaccinium myrtillus) minerālās barošanās stāvokli Latvijā, kā arī novērtēt minerālāsbarošanās īpatnības ražojošu saimniecību un meža augsnēs. 2008. gadā no piecām Latvijaslielākajām krūmmelleĦu saimniecībām un piecām dabisko mežu audzēm ievāca 100 (augsnes unlapu) paraugus. Noteica 12 augiem nepieciešamos barības elementus (N, P, K, Ca, Mg, S, Fe, Mn,Zn, Cu, Mo, B), augsnes pH un kopējo sāĜu koncentrāciju (EC). No visiem iegūtajiem augsnes unlapu analīžu rezultātiem tikai 40 % rādītāju bija optimālā līmenī. KrūmmelleĦu lapās kopumāraksturīgs – N, P, Ca, Mo un B deficīts, kā arī paaugstinātas Mn koncentrācijas. Neskatoties uzelementu koncentrāciju augsto heterogenitāti, N, Zn, Mo un B deficītu konstatēja vairākumamizanalizēto augšĦu paraugu. Lai arī meža augsnes raksturojas ar īpaši skābu un barības elementiemnabadzīgu vidi, savvaĜas mellenes uzrāda augstu K, Ca, Mg, Fe, Mn un Zn uzĦemšanasefektivitāti. Pētījumā gūtās atziĦas papildina zināšanas un izpratni par krūmmelleĦu mēslošanaslikumsakarībām Latvijā.Key words: Vaccinium corymbosum L., Vaccinium myrtillus L., mineral nutrition, plant analysis,soil testingIntroductionThe wild blueberry (Vaccinium myrtillus L.) is one of the most popular wild-harvested fruit inLatvia, traditionally used fresh and processed as a health food. The leaves and fruits of the wildblueberry are dried for teas and used in folk-medicine. Unfortunately berry production of the wildblueberry fluctuates annually with weather conditions – spring frosts and summer droughts cangreatly decrease yields (Kuchko, 1988). Latvia, where wild blueberries have been picked forcenturies, is now replacing them with cultivated North American highbush varieties.The commercial cultivation of the highbush blueberry (Vaccinium corymbosum L.) in Latvia wassuccessfully started during last 10 years. Highbush blueberries grown on more than 170 ha havebecome an important horticultural commodity in Latvia with high market demand. Cultivatedblueberry fruits and juice have become increasingly popular due to their excellent taste, highdietary and health values. Among berry fruits, blueberries are considered to be not only anexcellent source of phenolic compounds and vitamins, but also a valuable source of minerals anddietary fibre (Ehlenfeldt and Prior, 2001; Trehane, 2004; USDA National Nutrient Database forStandard Reference, 2006).Many external factors are important in producing high blueberry yields including soil type, light,temperature, water availability and quality, as well as management practices. Highbush blueberriesevolved in low pH (optimum between pH 4.3 and 4.8) soils that were poor in nutrients and rich inorganic matter are adapted specifically to these soil conditions and are generally regarded as verysensitive to excessive fertility (Smolarz and Mercik, 1989; Hanson and Hancock, 1996; Hanson,2006). Although the fertilization requirements of cultivated blueberries are relatively smaller thanfor other berry crops, investigations indicate that balanced and precise fertilizer applications canimprove the nutrient status, growth, development and yield of blueberries (Percival et al., 2003).81

While the nutrient status of highbush blueberries in the United States and Canada (the mainblueberry production countries in the world) has been studied in considerable detail (Eck, 1988;Strik et al., 1993; Hart et al., 2006), investigations on optimal cultivation technologies of theblueberry crop in Latvia are in their very beginning stages. At present high bush blueberries inLatvia are cultivated on light, acid mineral soils rich in organic matter as well as on high bogs afterpeat extraction. As highbush blueberries are grown in many non-native regions all over the world(Strik, 2005) and often on soils that have been differently modified by acidification, liming ororganic matter incorporation, direct application of nutrient recommendations and managementpractices from North America are limited in their practicality. Therefore intensive scientificresearch on soil, climatic requirements and the mineral nutrition regime of highbush blueberries aswell as wild blueberries in Latvia are very important.The main aim of this study was to find out the actual mineral nutrition status of the cultivatedhighbush (Vaccinium corymbosum L. ) and wild blueberries (Vaccinium myrtillus L.) in Latvia aswell as to evaluate the peculiarities of blueberry mineral nutrition in producing plantings and nativewoodlands.Materials and methodsAbout 100 (soil and plant) samples were collected from different blueberry producing sites(Aluksne, Jelgava, Riga, Valmiera and other districts) and 5 woodlands (Cesis, Saldus, Tukums,Valka districts and forest territory of Jurmala city) in Latvia during summer 2008.The soil samples were taken with a soil probe to a depth of 20 cm. For each sample, five to eightsubsamples were obtained and thoroughly mixed to form one sample. The soil samples were airdried;plant roots and all particles, mineral and organic with a diameter larger than 2 mm wereremoved by sieving through a 2-mm sieve. To determine the plant available amounts of 12biogenous elements (N, P, K, Ca, Mg, S, Fe, Mn, Zn, Cu, Mo, B) the soil samples were extractedwith 1 M HCl (soil - extractant volume ratio 1:5) (Rinkis et al., 1987).For each highbush blueberry plant sample 50-100 of the most recently fully expanded leaves fromcurrent season shoots were collected from locations representative of the planting. Wild blueberryleaf materials were collected at each site as a composite sample from an area of about 10x10 m.The plant material was dried at 60 º C and ground. Then the plant samples were dry-ashed withHNO 3 vapours and re-dissolved in HCl solution (HCl - distilled water mixture 3:100) (Rinkis et al.,1987).Concentrations of 12 biogenous elements (N, P, K, Ca, Mg, S, Fe, Mn, Zn, Cu, Mo, B) weredetermined in all soil and leaf samples. The levels of Ca, Mg, Fe, Cu, Zn, and Mn were estimatedby an atomic absorption spectrophotometer (Perkin Elmer AAnalyst 700, acetylene-air flame),those of N, P, Mo, B by colorimetry, S by turbidimetry, and K by flame photometer (Jenwey PFP7,air-propane butane flame). Soil pH was determined in 1 M KCl (soil - extractant volume ratio1:2.5) potentiometrically by pHmeter Sartorius PB-20 (Rinkis et al., 1987). Soil electricalconductivity (EC, mS cm -1 ) was determined in distilled water extract (soil - distilled water volumeratio 1:5) by the conductometer Hanna EC 215. Analytical replication was performed at least threetimes. The levels of statistical significance were determined using MS Excel 2003. T-test “Two-Sample Assuming Unequal Variances” (p

espectively). It should be stressed that almost all the elements concentration range, with exceptionof Ca and Mo, in woodland soils was narrower.Table 1. Nutrient concentrations in blueberry soil samples from producing plantings and woodlandsin Latvia, 2008.Concentrations in 1M HCl extraction, mg l -1Ele-mentV. corymbosum L. V. myrtillus L.optimal inhighbushrange mean ± SE range mean ± SEblueberry soilsN 15-245 70.59±8.30a 1 13-32 22.87±1.61b 120 - 150P 34-1908 334.92±56.17a 27-47 35.27±1.43b 150 - 180K 19-500 150.43±18.48a 30-130 70.07±6.27b 100 - 140Ca288-16900 2388.46±572.16a 7-1400 588.73±84.57b 700 - 1200Mg 100-3875 383.92±101.51a 22-350 109.33±18.78b 120 - 250S 7.0-825 79.54±26.12a 7-11 8.67±0.33b 30 - 50Fe 24-9750 1115.62±275.72a 90-380 177.93±23.87b 600 - 15000.10-15 - 25Mn 185.00 47.31±7.87a 0.70-24.00 10.19±2.42b1.35-8 - 20Zn 20.00 5.77±0.69a 1.60-12.50 5.77±0.72a0.45-2.5 – 4.0Cu 17.50 3.61±0.57a 0.10-2.00 0.51±0.13bMo 0.01-0.14 0.04±0.005a 0.01-0.41 0.06±0.03a 0.06 – 0.20B 0.10-1.40 0.48±0.06a 0.10-1.00 0.42±0.08a 0.6 – 1.2pH KCl 2.76-7.20 4.30±0.16a 2.59-4.01 3.06±0.10b 4.2 - 4.8EC,mS cm -1 0.15-5.39 1.05±0.19a 0.20-0.37 0.28±0.01b 0.8 – 1.21 Means with different letters in a row were significantly different (t-Test, p < 0.05)Significantly (p

concentration of Mn (814.4 mg kg -1 ) was found in wild blueberry leaves, which exceeded the valuefor cultivated blueberries 4.3 times.Table 2. Mineral element concentrations in V. corymbosum L. and V. myrtillus L. leaf samples inLatvia, 2008ElementConcentrations in dried tissueoptimal levels inhighbushblueberry leavesV. corymbosum L. V. myrtillus L.range mean ± SE range mean ± SEMacronutrients (%)N 0.63-4.6 1.36±0.13a 1 0.80 – 1.5 1.08 ± 0.05b 1,70 – 2,00P 0.07-0.33 0.16±0.01a 0.08 – 0.17 0.12 ± 0.01b 0,20 – 0,30K 0.36-1.70 0.62±0.04a 0.64 – 1.0 0.77 ± 0.03b 0,45 - 0,70Ca 0.23-0.80 0.40±0.02a 0.56 – 1.12 0.84 ± 0.05b 0,50 - 0,80Mg 0.12-0.31 0.18±0.01a 0.17 – 0.54 0.32 ± 0.03b 0,15 - 0,30S 0.08-0.46 0.18±0.01a 0.12 – 0.18 0.15 ± 0.01b 0,15 - 0,25Micronutrients (mg kg -1 )Fe 26.0-362.0 76.4±11.2a 54.0 – 128.0 81.87 ± 6.76a80 - 150Mn 24.00-880.00 189±29.4a 130.0 – 1720.0 814.40 ± 146.42b 40 - 100Zn 8.8-76.0 19.3±2.4a 14.0 – 26.0 18.60 ± 0.96a 20 - 60Cu 0.8-362.0 19.8±11.0a 3.6 – 6.4 4.97 ± 0.24b 8 - 12Mo 0.10-2.49 0.46±0.09a 0.2 – 0.3 0.23 ± 0.01b 1 - 5B 7.0-58.0 24.1±2.2a 15.0 –30.0 22.73 ± 1.21a 30 - 601 Means with different letters in a row were significantly different (t-Test, p < 0.05)The results obtained on the percentage of highbush blueberry plant and soil samples in low,optimum and excessive nutrient supply levels are shown in Figure 1. Almost 60 % of the plantsamples were completely sufficient in macronutrients. The worse situation was found for themicronutrient supply – more than 55 % of indices were in the deficient range, optimal supply wasprovided only for 31 % of leaf samples. In general, equal distribution (39 %) between all indices inlow and optimal levels was found for highbush blueberry soil samples. Unlike blueberry leaves, abetter situation was found for the micronutrient supply in cultivated blueberry soils.P ercen ta g e of sa m p les in d ifferen tn u trien t su p p ly levels, %706050403020100Macronutrients Micronutrients All indicesALeavesLowOptimalExcessive50454035302520151050Macronutrients Micronutrients All indicesBSoilsLowOptimalExcessiveFigure 1. Distribution of highbush blueberry leaf (A) and soil (B) samples in different nutrientsupply levels in Latvia, 2008DiscussionIncreased consumer demand for blueberries is being driven by the health benefits associated withfunctional food characteristics. Wild blueberry have historically been used to help improveeyesight, treat diarrhoea, improve blood sugar levels in diabetics, strengthen blood vessels andimprove circulation (Trehane, 2004). The powerful antioxidant properties of the highbush84

lueberry appear to play a conclusive role in the prevention and delay of certain diseases such ascancer, cardiovascular issues, and the aging process (Ehlenfeldt and Prior, 2001).To realize the full potential of modern high yielding crop, balanced plant nutrition is vitallyimportant to ensure adequate growth and fruit production. A deficiency in any of the essentialnutrients will disrupt either the vegetative or reproductive growth cycles in plants (Marshner, 1995;Fuqua et al, 2005).The results obtained on the nutrition status of the highbush blueberry revealed the main problemsin plants supply with essential mineral elements in Latvia. In general, our results suggest that onlyabout 40 % of plant tissue nutrient indices were within the sufficient range. Mean macronutrientconcentrations, with the exception of low N and slightly decreased P and Ca could be characterizedas optimal. Nitrogen is the controlling element in blueberry nutrition and adequate fertilization isnecessary to maintain renewal growth, crop production, and flower bud development (Strik andHart, 1997). Our study suggests that optimal N levels were provided by only 20 % of blueberry leafsamples in Latvia. It can be mentioned that deficiencies of N are the most frequently encounteredproblems in growing blueberries in North America also (Pritts, 2000; Fuqua et al 2005).The main tendencies in mean micronutrient supply were also detected – deficiency of Mo, slightlydecreased B, optimal levels of Fe and Zn, as well as increased concentrations of Mn and Cu in theplant tissue. While Mn concentrations in highbush blueberry leaves were rather high, in most ofcases they could not be considered as toxic, with the exception of the highest concentrations: 450mg kg -1 to 880 mg kg -1 . As the cultivation environment influences the chemical composition ofplant leaves, our study suggests that the increased Mn concentrations in highbush blueberriesreflect high Mn availability in low-pH soils, excessive soil Mn, and sometimes the mistakenapplication of fertilizers containing Mn. According to recommendations based on the first studieson blueberry nutrition in Latvia the use of the foliar micronutrient (Cu, Zn, B, Mo) fertilization wasstarted during recent years to correct the specific deficiencies stated in the tissue tests.Consequently, excessive Cu concentrations could be caused by the adhesion properties of foliarfertilizers and the use of fungicides.Our results revealed statistically significant differences between wild blueberry and highbushblueberriy results for N, P, K, Ca, Mg, S, Mn, Cu, Mo in leaf samples. Cultivated highbushblueberry leaves had higher mean concentrations of N, P, S, Cu, and Mo while wild blueberryleaves showed higher levels of K, Ca, Mg, and Mn. It is interesting that there were no significantdifferences in leaf Fe contents between wild blueberry and highbush blueberry growing incultivated and forest soils with completely different Fe concentrations: 1115.6 mg Fe l -1 and 177.9mg Fe l -1 , respectively. Although wild blueberry leaves were comparatively high in Mn content (onaverage, 814.4 mg kg -1 ), reported mean values for Mn (1900 mg kg -1 ) in wild blueberry leaves fromNorthern Europe (Reumann et al., 2002) and Poland - 2758 mg kg -1 (Kozanecka et al., 2002) weretwice to three times higher than our Mn results for wild blueberries in Latvia.Our previous studies suggest that, in general, blueberry fruit chemical analysis confirmed mineralelement differences in wild blueberry and highbush blueberry leaves. A comparison of the twospecies studied showed that the highest mean K, Ca, and Mn concentrations were alsocharacteristic for wild blueberry fruits (Pormale et al, 2009).Several investigations have shown that leaf analysis provides a picture of the nutrient status of acrop at a particular point in time resulting from all factors affecting plant growth and is an excellentmethod to be used on established plantings (Strik et al., 1993; Marschner, 1995). But thisdiagnostic method cannot detect the character of the nutrient deficiencies or toxicities and the soilcontent of the nutrients. Therefore soil testing is very important in determining the ability of thesoil to supply the nutrients needed for optimum plant growth.Although a particularly broad range of element concentrations in highbush blueberry soil sampleswas found, in general, soil tests revealed nutrient deficiencies in the cultivated blueberries.Deficiencies of N, Zn, Mo, and B as well as increased Mn levels were the main problems found.Our results suggest that only 39 % of all soil indices in blueberry producing plantings in Latviawere in the optimal range. It should be noted that the lowest macro- and microelementconcentration levels in cultivated blueberry soils are highly comparable with elementconcentrations in nutrient poor forest soils, thus indicating insufficient fertilization. This could85

seriously limit the highbush blueberry yield in Latvia. Although mean levels of soil pH/ KCl and ECmeet requirements of soil standards, the samples with marginal values did not correspond to theoptimum for highbush blueberry growth and development.The results obtained on nutrient concentrations in wild blueberry soils revealed significantly lowerN, P, K, Ca, Mg, S, Fe, Mn and Cu levels compared with highbush blueberry soils. Particularly lowconcentrations were found for N and S in the woodland soils. This is not surprisingly becausenutrients in anion form are more leachable from light acid forest soils, as well as during the last 20years industrial emissions have been seriously decreased in Latvia (ěulko et al., 2008).Although both wild blueberries and highbush blueberries can be classified as typical calcifuges,since they thrive in nutrient poor soils with low pH (Korcak, 1989), wild blueberries showed anespecially high efficiency rate of K, Ca, Mg, Fe, Mn, accumulation in their leaves. Thisphenomenon can be considered as a potential wild plant physiological adaptation mechanism toinfertile soils.ConclusionsThe results obtained on the nutrition status of the highbush blueberry revealed the main problemsin plant supply with essential mineral elements in Latvia. Insufficient levels of N, P, Ca, Mo, B andincreased concentrations of Mn in highbush blueberry leaves were found to be the main problems.In spite of the high heterogeneity of the element concentrations in soils, the deficiency of N, Zn,Mo, B was found in a vast majority of samples analyzed. In general, our results suggest that onlyabout 40 % of plant leaf nutrient indices and soil tests were in the optimal range. Although forestsoils were characterized as acidic and a nutrient poor environment, wild blueberries showed anespecially high efficiency of K, Ca, Mg, Fe, Mn accumulation in their leaves. The presentinvestigation forms the basis for the development of ongoing scientific research and knowledgeabout blueberry fertilization practices in Latvia.References1. Eck P. (1988) Blueberry science. Rutgers University Press, N.J., 284 p.2. Ehlenfeldt M.K and Prior R.L. (2001) Oxygen radical absorbance capacity (ORAC) and phenolic andanthocyanin concentrations in fruit and leaf tissues of highbush blueberry. Journal of Agricultural andFood Chemistry, 49, pp. 2222-2227.3. Fuqua B., Byers P., Kaps M., Kovacs L., Waldstein D. (2005) Growing blueberries in Missuri. MissuriState University Bulletin, 44, 47 p.4. Hanson E. and Hancock J. (1996) Managing the nutrition of highbush blueberries. Michigan StateUniversity, Extension Bulletin E-2011, 46 p.5. Hanson E.J. (2006) Nitrogen fertilization of highbush blueberry. Acta Horticulturae, 715, pp. 347-351.6. Hart J., Strik B., White L., Yang W. (2006) Nutrient management for blueberries in Oregon. OregonState University Extension service EM 8918, 14 p.7. Kozanecka T., Chojnicki J., Kwasowski W. (2002) Content of heavy metals in plant from pollution-freeregions. Polish Journal of Environmental Studies, 11, pp. 395-399.8. Kuchko A.A. (1988) Bilberry and cowberry yields and the factors controlling them in the forests ofKarelia, U.S.S.R. Acta Botanica. Fennica, 136, pp. 23-25.9. ěulko I., Dubakova I., Frolova M., KovaĜevska A. (2008) Atmosfēras gaisa piesārĦojuma pārrobežupārneses ietekme uz Latvijas ekosistēmu (Transboundary air pollution effects on ecosystem of Latvia),Latvijas Vides, ăeoloăijas un meteoroloăijas aăentūra, Rīga, 43 p.10. Marschner H. (1995) Mineral nutrition in higher plants, Academic Press, London, 889 p.11. Nollendorfs V. (2004) Augsto krūmmelleĦu prasības pēc augsnes un mēslojuma (Highbush blueberrysoil and fertilization demands). Agropols, 12, Ražība, 6, pp. 8-10. (In Latvian).12. Percival C.D., Janes D.E., Stevens D.E., Sanderson K. (2003) Impact of multiple fertilizer applicationson plant growth, development, and yield of wild lowbush blueberry (Vaccinium augustifolium Ait.). ActaHorticulturae, 626, pp. 415-421.13. Pormale J., Osvalde A., Karlsons A. (2009) Investigation on the essential mineral element contents ofcultivated and wild blueberry fruits in Latvia. In Proc. of the International conference: Environmentallyfriendly and safe technologies for quality of fruits and vegetables, 14-16 January, Faro, Portugal (inpress).14. Pritts M. (2000) Blueberry nutrition on upland soils. New York Fruit Quarterly, 8, pp. 14-20.86

15. Reimann C., Kollen F., Frengstad B., Kashulina G., Niskavaara H., Englmaier P. (2001) Comparison ofthe element composition in several plant species and their substrate from a 1 500 000 km 2 area inNorthen Europe. The Science of the Total Environment, 278, pp. 87-112.16. Rinkis G., Ramane H., Kunicka, T. (1987) Methods of soil and plant analysis. Zinatne, Riga (inRussian).17. Smolarz K. and Mercik, S. (1989) Growth and yield of highbush blueberry Bluecrop cv. (VacciniumCorymbosum L.) in relation to the level of nitrogen fertilizer. Acta Horticulturae. 241, pp. 171-174.18. Strik B., Brun C., Ahmedullah M., Antonelli A., Askham L., Barney D., Bristow P., Fisher G., Hart J.,Havens D., Ingham R., Kaufman D., Penhallegon R., Pscheidt J., Scheer B., Shanks C., William R..(1993) Highbush blueberry production. Oregon State University Extension Service PNW 215, 80 p.19. Strik B. and Hart J. (1997) Blueberry fertilizer guide. Oregon State University Extension FG 78, 4 p.20. Strik B. (2005) Blueberry – an expanding world berry crop. Chronica Horticulturae, 45, pp. 7-12.21. USDA National Nutrient Database for Standard Reference (2006) Available at: www.nal.usda.gov/,04.03.2009.22. Trehane J. (2004) Blueberries, cranberries and other vacciniums, Timber Press, Cambridge, 256 p.EXAMINATION OF CORNEL (CORNUS MAS L.) FRUITS IN BORSOD-ABAUJ-ZEMPLEN COUNTY (HUNGARY)KIZILA (CORNUS MAS L.) AUGěU IZVĒRTĒŠANA BORSOD-ABAUJ-ZEMPLENAPGABALĀ (UNGĀRIJA)Eniko Prokaj, Anita Medve, Noemi Koczka, Attila Ombodi, Judit DimenySzent Istvan University, Faculty of Agricultural and Environmental Sciences, Institut of HorticulturalTechnology, Godollo, Pater Karoly ut 1. 2103. Hungary, e-mail: Prokaj.Eniko@mkk.szie.huAbstractThe domestication of cornel has already begun in the neighbour countries by the selection ofvaluable individuals. The spontaneously growing North-Hungarian specimens could be a goodmaterial for similar selection and domestication because foreign cultivars may not adapt to the dryclimate of Hungary. Fruit collectors are making from cornel berries delicious palinka (a kind ofspirit) and jam. The needs of the processing industry and the market is larger than the gathering canprovide, therefore the culturing of new cultivars would be favorable. Our purpose was to showthere are several valuable individuals in the Hungarian cornel population, which have outstandingqualities. We observed individuals growing in a natural habitat in 2006, measured the weight offruit, seeds, and the parameters of fruit length and width. The 19 samples were collected in theAggtelek Karsts and the southern slopes of Bükk hill. The measured fruits weighed between the 1.2– 2.85gramm, the seeds were 0.27 – 0.41gramm, so the calculated flesh ratios were average 72.93 –86.66 %. The average lengths of fruits were between 15.43mm and 20.02 mm, and the averagewidths of them were between 10.41 – 14.67 mm. Even in a small sample there can be foundvaluable fruit types for further examination, although it is necessary to observe a wider range ofsamples to find a good cultivar which is adapted to the local climate.KopsavilkumsKizila kultivēšana kaimiĦu valstīs ir sākusies ar vērtīgāko indivīdu atlasi. ZiemeĜungārijā spontāniaugošie eksemplāri varētu būt labs materiāls līdzīgai atlasei un kultivēšanai, jo ievestās šėirnesvarētu nebūt piemērotas sausajam Ungārijas klimatam. AugĜu vācēji no kizila ogām gatavo garšīgu“palinku”(alkoholiskā dzēriena veids) un ievārījumu. Pārstrādes rūpniecības vajadzības un tirgus irlielāks kā ogu vācēji var nodrošināt, tādēĜ jaunu šėirĦu kultivēšana varētu būt izdevīga. Mūsunolūks bija parādīt dažas vērtīgas ungāru kizila populācijas ar izcilu kvalitāti. 2006. gadā mēsnovērojām eksemplāru augšanu dabiskā vidē, nosakot augĜu un sēklu svaru un augĜu garuma unplatuma parametrus. Aggtelek Karsts un Bükk kalna nogāzēs tika savākti 19 paraugi. AugĜu masabija starp 1.2 – 2.85 g, sēklu masa 0.27 – 0.41 g, aprēėinātā augĜa mīkstuma masas attiecība pretsēklu masu bija vidēji 72.93 – 86.66 %. Vidējais augĜa garums bija starp 15.43 un 20.02 mm unvidējais platums bija 10.41 – 14.67 mm. Pat mazā paraugā varēja atrast vērtīgus augĜu veidus87

tālākai pārbaudei, tomēr ir nepieciešams novērot daudz vairāk paraugu, lai atrastu labu šėirni, kurair piemērota vietējiem klimatiskajiem apstākĜiem.Key words: cornel, fruit parameters, flesh ratioIntroductionThe cornel (European cornel, cornelian cherry, Cornus mas L.) is a medium to large deciduousshrub or small tree growing to 5 – 12 m tall. The fruit is an oblong red drupe containing a singleseed. The fruit is edible, it has an acidic flavour which is best described as a mixture of cranberryand sour cherry; it is mainly used for making jam, makes an excellent sauce and also can be eatendried. The cornel is a relatively new species in the circle of the fruit breeders and growers. In thosecountries, where cornel has native populations with high variability and valuable genetic sources,with the mapping and selection of precious specimens, the cultivar breeding has already begun(Priszter, 1990). In the Ukraine, Bulgaria, Slovakia, Austria, France, Germany, Poland and Turkeya systematic collecting, selecting and breeding program has started in the last years (Klimenko,2004). In Bulgaria, Poland, and Romania there are several selected cultivars and in Slovakia evenbreeded cultivars are available as for instance ’Santana’, ’Titus,’ ’Ovidus’, etc... (Pirc, 1992;Porpaczy, 1997; Sipos, 2002; Zeitlhöfer, 2002; Porpáczy, 2004; Klimenko, 2004). In the KievBotanical Garden were bred several cultivars which became popular in the USA named ’Pioneer’,’Elegant’, ’Red-star’ (Zeitlhöfler, 2002). According to Reich’s (2004) study, the cultivars bred bySvetlana Klimenko are very delicious (’Elegant’, ’Pioneer’, ’Red Star’, ’Siretski’, ’Vavilov’). Themain cultivars of the Russian cornel plantations are ’Ispolinskij’, ’Karazogal’, ’Kyrymzy-zogal’,’Gjul-zogal’ and since 1990, two Danish cultivars were introduced ’Macrocarpa’ and ’ Cormas’(Zeitlhöfer, 2002). In Hungary between 1972 and 1981 cornel collecting and processing was on top(Szepesi, 1983), but in the near future, large-scale production is expected with the promotion ofcultivar evaluation and wider cultivation, because cornel grows well in relatively dry soils and iseasy to harvest by shaking (Sipos, 2002). However, cornel can be collected but is also valuable forbio production, because they are not damaged by dangerous pests. 50 years ago, Nyékes (1953)described a Hungarian form of Cornus mas called forma csaszloiensis. It has scarlet red drupes,weight is about 2.7 – 4 g, and the size is about 21 – 27 x 12 – 17 mm. There is only one Hungariancornel cultivar in ornamental use, called ’Autumn Fire’. Its fruits are pear shaped and 4 – 5 g heavy(Priszter, 1990). Priszter (1962) described 11 types of shape and size of the cornel fruits. The mostwell spread types are the forma mas, the f. macrocarpa, and the f. sphaerocarpa. Szepesi (1983)pressed for plantations of wild trees, but because of the modified lifestyle and customs, thecollecting does not ensure enough fruits for industry. Plantations could be established on suchareas, where wood production is not economic. Generally cornel and the other (so-called) forestfruits are pioneer plants. These could be a kind of „fruit bearing forests” near human settlementswith a double interest: 1. providing a concentrated fruit collecting area; and 2. using the soil andenvironment protecting function of the shrubs. Cornel is prolific but teems unevenly. To turnproductive it takes 5 – 6 years, then bears generally (30 kg plant -1 ) 7 – 11 t ha -1 . The fruits can beharvested by hand or shakers for tart cherry. (Porpáczy, 1999; Gilbert and Lorraine, 2003).Propagation is difficult by seeds; they need cold stratification (Priszter, 1990). However, seedlingsare different from mother plants, for the preservation of the attributes of the mother plant it is betterto propagate by suckers, division, cuttings or grafting (Priszter, 1990; Pirc, 1992; Schmidt et al.,1996). Fruit collectors are making from cornel berries delicious palinka (a kind of spirit) and jam.The needs of the processing industry and the market is larger than the collecting can provide,therefore the culturing of new cultivars would be advisable (Makai and Balázs, 2002). Our purposein this study was to show that there are several valuable and variable individuals in the Hungariancornel population, which have outstanding qualities. This might be the first step for furtherexamination and probably the start of the breeding work.Materials and MethodsWe examined the drupes of wild cornel genotypes, collecting samples from the area of ourhometown (the Karsts of Aggtelek and the Bükk-hill southern slopes). In both area the collectingand the use of the fruits in daily cuisine is remarkable. Our study is useful for the evaluation of88

each type, and for comparison samples to each other, but not suitable to give a general synopsis ofcornel populations in the observed area, because the number of samples was very small. Thelocation of the sampled bushes are far from each other (a few 10 km-s) and the local climaticfactors in some cases were quite different for characterization of the locus, but the average of allsample is eligible for comparing to the data of studies in the surrounding countries. The samplename is the name of the town near the sampling place. At each place we took sample from morethan one bush, because the fruit size and colour is very various even on the plants that are growingat the same location, these were marked with a number of the order in the sample name. Thecollection places were near to the following 7 towns, (the number of observed bushes is written inbrackets): Szögliget (3), Szinpetri (2), Varbóc (3), Tornaszentandrás (2), Kács (2), Kisgyır (4),Felsıtárkány (3); all together there are 19 cornel bushes. The samples were harvested between 9 thand 24 th of September in 2006. Only the fully ripe, easily separable, healthy, good quality fruitswere collected. In the measurement only 20 pieces of randomly selected fruits were examined.Each of the 20 fruits was measured one by one within 24hrs of the harvest. The measured attributeswere the followings: weight of fruit in grams, weight of seed (g), length and width of fruit(millimetres). Then the next attributes were calculated: flesh weight (g), proportion of flesh(percentage) and profile index. For weight measuring, we used an OHAUS EXPLORER balance totwo places of decimals. The width and length were measured by calliper square (1/20) to one placeof decimals. The flesh weight was calculated with this formula: fruit weight minus seed weightequal to flesh weight. The flesh ratio (%) equal to ((fruit weight minus seed weight) divided byfruit weight) multiplied by 100 and the profile index is equal to the fruit length devided by thefruit width. For data registration Microsoft Excel was used, analysis of one-factor variance andanalysis of regression was used for the evaluation. The significant difference was calculated at 5%of error.Results and DiscussionTable 1 shows all the measured and calculated data of samples. The mean of the weight of allsamples is 1.95g, comparing to it, 10 samples are not significantly different, 4 samples arepositively significant. The Szinpetri 2 sample has an outstandingly high value (2.85 g) in a positivedirection. The Kács 2 sample shows the lowest average weight (1.2 g), being the negativesignificance peak. According to the data of references, the natural cornel populations have a fruitweight between 1.5 – 4.12 g (Demir et al., 2003), 1.46 – 3.81 g (Ercisli et al., 2006), 0.55 – 3.44 g(Brindza et al., 2007). Priszter (1990) found that forma mas type has a weight of about 1.4 g.Karadeniz (2002) reported that the fruit weight of selected types was 3.08 – 3.71 g. In anotherstudy Pirlak et al. (2003) measured 2.9 – 5.2 g. Our data comparing the references is similar, butthere was no outstandingly heavy weight. The order of the weight of the seed does not follow theorder of the fruit weight; the correlation between them is not significant (Figure 1).Weight of seed (g)0.50.450.40.350.30.25y = 0.04x + 0.25r = 0.360.21 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8 3Weight of fruit (g)szögliget1szögliget2szögliget3színpetri1színpetri2varbóc1varbóc2varbóc3tornaszentadrás1tornaszentadrás2kács1kács2kisgyır1kisgyır2kisgyır3kisgyır4felsıtárkány1felsıtárkány2felsıtárkány3Figure 1. Correlation of fruit weight and seed weight of cornel samples89

Felsıtárkány 1 has the heaviest seed: 0.41 g, but it has only 1.89 g fruit weight. The Kisgyır 1,Szinpetri 2, Felsıtárkány 3, Kisgyır 3 and Szinpetri 1 samples have similar values, but theSzinpetri 1 and Szinpetri 2 samples have the fruit weigh on the 1 st and 3 rd place. The lightest seedbelonged to the Szögliget 1 sample (0.27 g) and the Varbóc 3, Kisgyır 2, Varbóc 2,Tornaszentandrás 1 and Szögliget 3 are significantly not different. Priszter (1990) described thetypic (f. mas) cornel seed weight about 0.2 g. Demir et al. (2003) examined 6 types and found theseed weight between 0.14 – 0.37 g; and Brindza et al. (2007) reported 0.11 – 0.55 g seed weight ofcornel. We measured the cornel seed weight between 0.27 – 0.41 g similar to the references. Theresults of flesh weight measurement gave nearly the same order of samples as the fruit weight did.Between the two attributes there is a significant correlation illustrated in figure 2. The mean of theflesh weight of samples is 1.61 g; the means of 9 samples are not significantly different from thisvalue. Five-five samples are positively and negatively different from this value in a significant way.The heaviest flesh weight belonged to Szinpetri 2 sample (2.45 g) and Kács 1 sample is similar toit. The Kács 2 sample had the lightest flesh weight 0.88 g, significantly different from all samples.The mean of flesh proportion of the 19 samples was 81.93 %, but the values of each sample werescattered on a wide range from a 72.93 % to 86.66 %. The data, found in the references, are alsovaried on similar wide range as Ercisli et al. (2006) recorded in natural populations 79 – 88 % andBrindza et al. (2007) reported between 80 and 87 %. Our 18 samples were similar to these results,but Kács 2 with 72.93 % flesh ratio was remarkably low. This could be due to the flesh stackedinseparably to the seed. The highest flesh ratio belonged to the Kács 1 sample. This fact proves thatcornels even growing on the same locus can show great variability.Table 1. The results of measurement combined with the results of ANOVAFruit SeedFruitName ofFlesh ProportionFruit widthweight weightlengthsamplesweight (g) of flesh (%)(mm)(g) (g)(mm)Profile indexFelsıtárkány 1 1.89 fgh 0.41 a 1.47 ghi 78.05 jk 16.31 ef 13.64 b 1.2Felsıtárkány 2 1.97 ef 0.37 bcde 1.6 efg 81.4 fg 18.64 bc 13.01 cde 1.43Felsıtárkány 3 1.71 hij 0.4 ab 1.31 ijk 76.59 k 18.47 c 11.9 i 1.55Kács 1 2.6 b 0.34 defg 2.26 a 86.66 a 19.37 ab 14.67 a 1.32Kács2 1.15 k 0.32 fgh 0.83 l 63.95 l 16.81 de 10.41 j 1.62Kisgyır 1 1.97 ef 0.41 a 1.56f g 78.86 ij 20.02 a 12.34 fghi 1.62Kisgyır 2 2.05 def 0.3 ghij 1.75 cde 85.18 abc 16.93 de 13.57 bc 1.25Kisgyır 3 1.9 fgh 0.38 abc 1.52 fgh 79.71 hi 18.36 c 12.6 efg 1.46Kisgyır 4 2.15 de 0.31 fghi 1.84 bcd 85.46 abc 18.57 c 13.59 b 1.37Szinpetri 1 2.4 bc 0.38 abcd 2.03 b 84.35 cd 18.64 bc 13.58 b 1.38Szinpetri 2 2.85 a 0.4 ab 2.45 a 85.82 abc 19.85 a 14.58 a 1.37Szögliget 1 1.74 ghi 0.27 j 1.48 ghi 84.41 cd 15.54 g 12.49 efgh 1.24Szögliget 2 2.21 cd 0.33 efgh 1.89 bc 85.13 bc 17.38 d 12.66 efg 1.38Szögliget 3 1.97 ef 0.27 ij 1.69 def 86.04 ab 17.24 d 13.27 bcd 1.3Tornaszentandrás1 1.73 gh i 0.29 hij 1.44 ghij 83.11 de 17 de 12.57 efg 1.35Tornaszentandrás2 1.95 efg 0.35 cdef 1.6 efg 81.73 ef 18.32 c 12.83 def 1.43Varbóc 1 1.68 hij 0.32 fgh 1.36 hijk 80.68 fgh 15.72 fg 12.1 ghi 1.3Varbóc 2 1.5 j 0.29 hij 1.21 k 80.37 fgh 15.43 g 11.98 hi 1.29Varbóc 3 1.56 ij 0.3 ghij 1.26 jk 80.17 ghi 17.08 d 10.65 j 1.61mean 1.95 0.34 1.61 81.93 17.67 12.76 1.4p-value5.5E-29 2.9E-28 2.2E-33 2.6E-56 8.9E-18 2.1E-12 3.6E-48SD5% 0.22 0.04 0.19 1.5 0.75 0.56 0.04The mean of the fruit length of all samples was 17.67 mm. Seven samples were not significantlydifferent, seven samples were different in a positive direction and five were negative compared to90

the mean. Kisgyır 1 sample has the longest fruits average of 20.02 mm, Szinpetri 2 and Kács 1samples were similar to it. The shortest fruits belonged to the Varbóc 2 samples (15.43 mm).Weight of flesh (g)2.42.221.81.61.41.210.8y = 0.9565x - 0.2549r = 0.991 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8 3Weight of fruit (g)Figure 2. Correlation of fruit weight and flesh weight of cornel samplesszögliget1szögliget2szögliget3színpetri1színpetri2varbóc1varbóc2varbóc3tornaszentadrás1tornaszentadrás2kács1kács2kisgyır1kisgyır2kisgyır3kisgyır4felsıtárkány1felsıtárkány2felsıtárkány3Demir et al. (2003) examined 6 types and found the fruit length between 15.95 – 20.77 mm.Brindza et al. (2007) reported 12.55 – 19.55 mm fruit length of cornel. Our results were similar tothe references. The results of fruit weights gave a mean of 12.76 mm, eight samples were notsignificantly different from it. Six samples were significantly different in a positive direction – fiveof them had the highest fruit weight also – and five samples were in negative significancesaccompanied with the lowest fruit weight ranking. Kács 1 sample was 14.67 mm wide, and Kács 2was 10.41 mm. Our data was very similar to data mentioned in the references: 10.91 – 16.4 mm(Demir et al., 2003) and 7.43 – 15.22 mm (Brindza et al., 2007).Width of fruit (mm)191817161514131211109y = 2.51x + 7.88r = 0.891 1.5 2 2.5 3Weight of fruit (g)szögliget1szögliget2szögliget3színpetri1színpetri2varbóc1varbóc2varbóc3tornaszentadrás1tornaszentadrás2kács1kács2kisgyır1kisgyır2kisgyır3kisgyır4Figure 3. Correlation of fruit weight and fruit width of cornel samplesfelsıtárkány1felsıtárkány2felsıtárkány3The correlation of fruit weight and fruit width is strong, illustrated in Figure 3. The width of fruit isgrowing together with the fruit weight, but the length of the fruit does not follow this tendency.91

Table 2. Groups of cornel samples and their attributesCornus mas forma Sample’s name Profile index Shape, colour andweight of fruitsforma mas (basic type)Felsıtárkány 2, 3Kács 2Szögliget 3Tornaszentandrás 1, 2Varbóc 1, 2, 3Kisgyır 1Kisgyır 3f. mas or costataforma macrocarpa Kács 1Kisgyır 4Szinpetri 1, 2Szögliget 2forma sphaerocarpa Felsıtárkány 1Kisgyır 2Szögliget 11.43; 1.551.621.31.35; 1.431.3; 1.29; 1.611.621.461.321.371.38; 1.371.381.21.251.24red, cylindrical-ellipticless than 2gstriate, ovoiddark red, elliptic,heavier than 2 gdark red, roundishand nearly 2gThe length and the width of fruits show a great variance even among the fruits of one sample, butthe calculated profile index is a reliable number to compare the samples and describe the shape offruits. Using the measured length, width, weight and the calculated profile index data, the samplescan be divided in groups of fruit forms. The base of the fruit form groups were the works ofPriszter (1962) and Fintha and Szabó (2005). We classified the samples in the following groupsshowed in table 2.ConclusionsThe results of our experiments correspond to the data of similar studies in neighbour countries.Observing a small number of specimens, we found valuable types for further investigation likeSzinpetri 2 and Kács 1 with heavy fruits and outstanding flesh proportion, and Kisgyır 1 and 3with dark red colour and easy to core attributes. The possibility of finding valuable specimens ofcourse is higher when the sample amount is larger, and this increases the chance of finding andselecting several well-adapted local cultivars.References1. Brindza J., Brindza P., Grigorieva O., Klimenko S.V., Tóth D. (2007) Slovakian Cornelian Cherry(Cornus mas L.): Potential for Cultivation, Acta Horticulturae, 760, pp. 433 – 436.2. Demir F., Kalyoncu I. (2003) Some nutritional, pomological and physical properties of cornelian cherry(Cornus mas L.), Journal of Food Engineering, 60 (3), pp. 335-341.3. Ercisli S., Orhan E., Esitken A. (2006) Genetic diversity in fruit quality traits in cornelian cherry (Cornusmas L.), Asian Journal of Chemistry, 18 (1), pp. 650-654.4. Fintha I., Szabó A. (2005) Vizsgálatok az ÉK-Alföld somfáinak termésein (Cornus mas L. 1753)különös tekintettel a „császlói” formára, Botanikai Közlemények, 92 (1-2), pp. 159-165.5. Gilbert J., Lorraine G. (2003) Commercial Potential and Development of New Berry Crops: AnOverview, Acta Horticulturae, 626, pp. 380.6. Karadeniz T. (2002) Selection of native Cornelian cherries grown in Turkey, Journal AmericanPomological Society, 56 (3), pp. 164-167.7. Klimenko S.V. (2004) The cornelian cherry (Cornus mas L.): Collection, preservation and utilization ofgenetic resources, Journal of Fruit and Ornamental Plant Research Special ed., 12, pp. 93-98.8. Makai P., Balázs G. (2002) Sompálinka. (Cornel brandy) - In: Farnadi É. (szerk.). Hagyományok ÍzekRégiók. (Traditions, taste, regions) FVM AMC, Budapest, pp. 356-357. (in Hungarian).9. Nyékes I. (1953) A császlói som. (Cornel of Császló), A Kertészeti és Szılészeti Fıiskola Évkönyve,(Yearbook of University of Horticulture), pp. 169-173. (in Hungarian).10. Pirc H. (1992) A húsos som Ausztriából. (Cornus mas L. from Australia) (Translated by Koczor L.),Kertészet és Szılészet (Horiculture and Viticulture), 51 (49), 9. (in Hungarian).11. Pirlak L., Güleyrüz M., Bolat I. (2003) Promising cornelian cherries (Cornus mas L.) from theNortheastern Anatolia region of Turkey, Journal American Pomological Society, 57 (1), pp. 14-18.12. Porpáczy A. (1997) Húsos som. (Cornus mas L.). In: Soltész M. (szerk.). Integrált gyümölcstermesztés.Integrated fruit production. Mezıgazda Kiadó, Budapest, pp. 794-795. (in Hungarian).92

13. Porpáczy A. (1999) A húsos som termesztése. Production of cornel. In.: Papp J., Porpáczy A., (szerk).Szeder, ribiszke, köszméte, különleges gyümölcsök. Bogyósgyümölcsőek II. (Blackberry, currants,gooseberry, special fruits. Soft fruits II.) Mezıgazda Kiadó, Budapest, pp. 225-226. (in Hungarian).14. Porpáczy A. (2004) Húsos som. (Cornus mas L.). In: Pap J. (szerk). A gyümölcsök termesztése. (Fruitproduction), Mezıgazda Kiadó, Budapest, pp. 510 -511. (in Hungarian).15. Priszter Sz. (1962) A húsos som (Cornus mas L.) terméseinek változatossága. (Variability of cornelfruits), Botanikai Közlemények (Botanical Reports), 49 (3-4), pp. 276-279. (in Hungarian).16. Priszter Sz. (1990) A húsos som. (Cornus mas L.), Akadémiai Kiadó, Budapest (in Hungarian).17. Reich L. (2004) Uncommon fruits for every garden. Timber Press, Portland, Cambridge, pp. 169-177.18. Schmidt G., Tóth I., Maráczi L. (1996) Cornus. – In: Schmidt G., Tóth I. (szerk). Díszfaiskola.(Ornamental plant nursery), Mezıgazda Kiadó, Budapest, pp. 526-528. (in Hungarian).19. Sipos B.Z. (2002) Elfelejtett növényeink. (Our forgotten plants), Kertészet és Szılészet (Horticultureand Viticulture), 51 (35), pp. 12-14. (in Hungarian).20. Szepesi J. (1983) Erdei gyümölcsök. (Forest fruits). In: Bondor A. (szerk.). Erdei termékek győjtése ésfeldolgozása. (Collection and processing of forest fruits), Mezıgazdasági Kiadó, Budapest, pp. 23-57.(in Hungarian).21. Zeitlhöfler A. (2002). Die obstbauliche Nutzung von Wildobstgehölzen. (Using wild fruits for fruitproduction), Diplomarbeit, Fachhochschule Weihenstephan, Freising. (in German).RABBITEYE BLUEBERRY, AMERICAN CRANBERRY AND LINGONBERRYBREEDING IN LATVIAEŠA ZILEĥU, AMERIKAS DZĒRVEĥU UN BRŪKLEĥU SELEKCIJA LATVIJĀAlfreds Ripa, Biruta AudriĦaNational Botanic Garden of Latvia, e-mail: informacija@nbd.gov.lvAbstractThe purpose of the breeding work was to create adapted to the climatic conditions of Latvia anddisease resistant cultivars of rabbiteye blueberry (Vaccinium ashei Reade), American cranberry(Vaccinium macrocarpon Ait.) and lingonberry (Vaccinium vitis-idaea L.). The main task was thebreeding of cultivars with large berries, high productivity and different ripening time. Breedingresearch has been carried out in National Botanic Garden of Latvia since 1980. In the work, classicplant breeding, interspecific breeding and polyploidy breeding have been employed, and wildlingonberry clones were used. The rabbiteye blueberry cultivar ‘Salaspils Izturīgā’ was selectedfrom seedlings of open pollination of the cultivar ‘Tifblue’ in 1993 and the cultivar ‘Lielogu’ wasselected from the hybrid (‘Delite’ x ‘Woodart’) seedlings in 1995. From the hybrid (cranberrycultivar ‘Franklin’ x lingonberry) seedlings were selected the cultivar ‘Dižbrūklene’ in 1997, thecultivar ‘Salaspils Agrās’ in 1996 and the cultivar ‘Tīna’ in 2006. The lingonberry cultivar‘Salaspils Ražīgā’ origin of the wild lingonberry clone was selected as cultivar in 1993; the cultivar‘Rubīna Lāse’ was selected in 1988 from open pollination seedlings of the cultivar ‘SalaspilsRažīgā’, but the cultivar ‘Jūlija’ was selected in 1995 from open pollination seedlings of the clone‘Krasnojarska’.KopsavilkumsSelekcijas mērėis bija radīt Eša zileĦu Vaccinium ashei Reade, Americas lielogu dzērveĦu(Vaccinium macrocarpon Ait.) un brūkleĦu (Vaccinium vitis-idaea L.) šėirnes, kas būtu piemērotasLatvijas klimatiskajiem apstākĜiem un slimību izturīgas. Galvenie uzdevumi bija izveidot šėirnes arlielām ogām, ražīgas un ar dažādiem ienākšanās laikiem. Selekcija tika veikta NacionālajāBotāniskajā dārzā Latvijā kopš 1980. gada. Darbā tika izmantota klasiskā selekcija, starpsuguselekcija un poliploīdija, kā arī izmantoti savvaĜas brūkleĦu kloni. Eša zilenes šėirne ‘SalaspilsIzturīgā’ tika izveidota no brīvas apputes ‘Tifblue’ sēklaudžiem 1993. gadā, bet šėirne ‘Lielogu’tika atlasīta no hibrīda (‘Delite’ x ‘Woodart’) sēklaudžiem 1995. gadā. No hibrīda (dzērveĦu šėirne‘Franklin’ x brūklene) sēklaudžiem tika atlasīta šėirne ‘Dižbrūklene’ (1997.), ‘Salaspils Agrās’(1996.) un ‘Tīna’ (2006.). BrūkleĦu šėirne ‘Salaspils Ražīgā’ tika atlasīta no savvaĜas klona 1993.gadā. Šėirne ‘Rubīna Lāse’ tika atlasīta 1988. gadā no šėirnes ‘Salaspils Ražīgā’ brīvas apputes93

sēklaudžiem, bet šėirne ‘Jūlija’ tika atlasīta 1958. gadā no klona ‘Krasnojarska’ brīvas apputessēklaudžiem.Key words: clones, clusters, hybrid, phenological phases, pollination, resistance, upright, vine,yield.IntroductionThe abundant yields and medicinal properties of the berries of rabbiteye blueberries, Americancranberries and lingonberries contribute to the popularity of these cultures. The fresh berries ofrabbiteye blueberries contain 16.6 g kg -1 of soluble dry matter, 7.8 g kg -1 of glucose and fructose, to1.4 g kg -1 of titrable acid, 0.001 g kg -1 amino acid. The rabbiteye blueberry berries are a valuablesource of physiologically active and mineral substances (Ripa 1992, Ripa 1998). Americancranberries are characterized by a considerable amount of biologically active substances. Thecontent of anthocyans in fresh berries constitutes 0.604 – 0.186 g kg -1 and catechines 0.152 – 0.126g kg -1 , and vitamin C 0.062 – 0.068 g kg -1 . The fresh berries contain 10.3 – 11.6 g kg -1 of solubledry matter, 7.2 – 8.2 g kg -1 glucose and fructose, to 3.0 g kg -1 of titrable acid. The berries are avaluable source of pectin and mineral substances (Ripa 1980, Ripa 1996). The berries of thelingonberry contain 17.7 g kg -1 of soluble dry matter, 10.37 g kg -1 of glucose and fructose, to 2.11 gkg -1 of titrable acid. The lingonberry berries are a valuable source of physiologically activesubstances and mineral substances (Ripa 1981, Ripa et al. 1992).The purpose of the breeding work was to create adapted to climatic conditions of Latvia anddisease resistant cultivars of the rabbiteye blueberry (Vaccinium ashei Reade), the Americancranberry (Vaccinium macrocarpon Ait.) and the lingonberry (Vaccinium vitis-idaea L.). The maintask was the breeding of cultivars with large berries, high productivity and different ripening times.Materials and methodsIn 1983 the ripe berries of the following open pollination clones of lingonberries were gathered:‘Salaspils Ražīgā’, ‘Krasnojarska’, and in 1986 the ripe berries of the following open pollinationcultivars of rabbiteye blueberries were gathered: ‘Delite’, ‘Tifblue’. In 1988 – 1989 the selfpollinationand cross-pollination of rabbiteye blueberry cultivars was carried out. During crosspollinationthe cultivar ‘Woodart’ was used as the pollinator. In 1987 – 1990 the cross-pollinationof cranberry cultivar ‘Franklin’ with pollen of lingonberry clones was carried out. The lingonberryclones from the wild were distributed in 1980.During cross-pollination the flower buds were isolated by gauze sacs. In each cultivar 30 bloomswere isolated. The blooms were pollinated two days after their opening. Pollen was collected fromunfolded blooms. The germination capacity of the pollen was determined in a medium consistingof 1.0 g kg -1 agar and 15.0 g kg -1 saccharose. The berries obtained from open-pollination and crosspollinationwere stored in a fridge and stratified for six months at +4 °C. The seeds of hybrid(cranberry cultivar ‘Franklin’ x lingonberry), before sown, were soaked for 24 hours in a 0.5 g kg -1solution of colchicine. The seeds were sown in acid peat (pH 3.5 – 4.5) 2 – 3 mm deep and coveredwith sand. In the seedling boxes the peat was constantly kept humid at an air temperature of 22 °C.A month after shoot emergence the seedlings of rabbiteye blueberries, lingonberries and hybrids(cranberry x lingonberry) were planted in acid peat at a distance of 5 x 10 cm and later replanted ina permanent place at a distance of 2 x 1 m.The soil for planting the rabbiteye blueberry seedlings of open- and cross-pollination was sandyclay, soil reaction neutral to slightly acid (pH KCl – 6.1). The soil had a high level of organicmatter (11.3 g kg -1 ), and total nitrogen (0.3 g kg -1 ) - the level easily absorbed potassium was medium(0.01 g kg -1 ) and that of phosphorus was high (0.024 g kg -1 soil), the level of replaceable calciumwas 0.279 g kg -1 soil. The level of trace elements (copper, boron, iron) in the soil was sufficient, i.e.0.0003, 0.0001 and 0.0040 g kg -1 soil, respectively. Since 1992 each spring (in May)supplementary fertilizers have been cultivated around the seedlings superphosphate 70.0 – 80.0 kgha -1 , potassium sulphate 20.0 – 30.0 kg ha -1 . The soil for planting the lingonberry and hybrid(cranberry x lingonberry) seedlings was 20 – 30 cm thick acid sphagnum peat (pH KCl 2.3 – 2.6).The peat had a high level of organic matter (87.5 – 94.9 g kg -1 ), and total nitrogen (1.1–1.5 g kg -1 ),the level of easily absorbed potassium was high (0.060–0.148 g kg -1 soil) and that of phosphorus94

was medium (0.014 – 0.017 g kg -1 soil), the level of replaceable calcium was 0.089 – 0.290 g kg -1soil. The level of trace elements (copper, boron, iron) in the peat was sufficient, i.e. 0.0003, 0.0004and 0.0016-0.0080 g kg -1 substrate, respectively. The lingonberry and hybrid (cranberry xlingonberry) plants had been grown using organic methods.During the growing period the following qualities were determined: growth rhythm (phenologicalphases), the colour, shape, and quality of the berries, the yield of berries, the weight of berries, thefrost and disease resistance of plants.Results and DiscussionThe rabbiteye blueberry cultivar ‘Salaspils Izturīgā’ was selected from seedlings of the openpollination of the cultivar ‘Tifblue’ in 1993.‘Salaspils Izturīgā’. Shrub: upright, vigorous, the ten years old shrub is about 1.5 m high and 1.4 mwide, open spreading, tolerates -36 °C, productive, the average yield of berries in 14 years was 3.06kg per shrub. The leaves are small (the length 5.5 – 6.1 cm, width 2.4 – 2.7 cm), bright green,elliptic. The raceme is loose cluster, in the cluster are 7 – 37 flowers. The flowers are small, whitewith medium anthocyans paint. The berries are medium (the length 1.0 – 1.3 cm, diameter 1.3-1.6cm, the weight of one berry 1.2 – 1.8 g), dark blue, good quality, round-oblate, and ripens about 22July.Figure 1. Rabbiteye blueberry cultivar ‘Salaspils Izturīgā’The rabbiteye blueberry cultivar ‘Lielogu’ was selected from hybrid (‘Delite’ x ‘Woodart’)seedlings in 1995.‘Lielogu’. Shrub: upright, vigorous, the 10 years old shrub is 1.8 m high and 1.75 m wide, veryproductive, the average yield of berries in 9 years was 6.05 kg per shrub, tolerates -34 °C. Theleaves are medium (the length 6.6 – 7.1 cm, width 3.2 – 3.6 cm), bright green, elliptic. The flowersare medium size, white with medium anthocyans paint. The raceme is loose cluster, in the clusterare till 39 berries. The berries are large (the length 1.0 – 1.2 cm, diameter 1.5 – 1.6 cm, the weightof one berry 1.7 – 2.0 g.), aromatic, light blue, good quality, ripens about 20 July.The vegetation period for the rabbiteye blueberry cultivars ‘Salaspils Izturīgā’ and ‘Lielogu’ starteddepending on the year’s climatic conditions from April 14 th to 28 th , when the average air dailytemperature was above 4.7 °C, bud swelling continued a week, after that the buds began to dehisceand green leaves appeared. In the beginning of May, when the average air daily temperature isabove 10 °C and the positive temperature sum is 126 – 220 °C, the flower buds begin to swell.Depending on the vegetation period and the average air daily temperature the rabbiteye blueberrycultivars ‘Salaspils Izturīgā’, ‘Lielogu’ begin to flower from the 17 th to 23 rd of May and lasts 22 –27 days. During the period of flowering the sum of positive temperatures is 220 – 550 °C. The firstberries of the cultivars ‘Salaspils Izturīgā’ and ‘Lielogu’ ripen from 20 th to 22 nd July.95

From the hybrid (cranberry cultivar ‘Franklin’ x lingonberry) seedlings the cultivars ‘SalaspilsAgrās’ (in 1996), ‘Dižbrūklene’ (in 1997), and ‘Tīna’ (in 2006) were selected.‘Salaspils Agrās’. This cultivar produces large to medium berries (the length 1.5 – 1.8 cm, thediameter 1.4 – 1.8 cm, the weight of one berry – 1.2 – 2.0 g). The berries are round, dark red, ripenvery early. Fine textures vines produce short uprights that are capable of good production, theaverage yield of berries was in 6 years 3650 kg ha -1 . The elliptical leaves are medium large (thelength – 9 – 12 mm, the width – 3 – 5 mm), bright green. The buds of the top of uprights arevegetative-generative. The raceme is intercalary cluster, in the cluster are 1 – 9 flowers. Theflowers are light red. The berries cluster is loose, in the cluster are 1 – 5 berries.‘Dižbrūklene’. The berries are exceptionally large (the length – 1.8 – 2.1 cm, the diameter – 1.4 –2.0 cm, the weight of one berry – 1.7 – 2.4 g) blackish red, round to oval. The berries ripen veryearly. This productive cultivar produces coarse vines with medium high uprights, the average yieldof berries in 6 years was 2810 kg ha -1 . The elliptical leaves are medium large (the length 10 – 11mm, the width – 4 – 5 mm), bright green. The buds on the top of uprights are vegetative –generative. The raceme is intercalary cluster, in the cluster are 1 – 8 flowers. The flowers are lightred. The berries cluster is loose; in the cluster are 1 – 6 berries.‘Tīna’. This cultivar produces red, round exceptionally large berries (the length – 1.6 – 1.8 cm, thediameter – 1.3 – 1.6 cm, the weight of one berry – 1.4 – 2.2 g). The berries ripen early. Themoderately vigorous vines produce short uprights that have consistently large yield, in 6 years theaverage yield of berries was 4530 kg ha -1 . The elliptical leaves are medium large (the length – 10-11 mm, the width – 4 – 5 mm), bright green. The buds on the top of uprights are vegetativegenerative.The raceme is intercalary cluster, in the cluster are 1 – 8 flowers. The flowers are lightred. The berries cluster is loose; in the cluster are 1 – 6 berries.Figure 2. Hybrid cranberry x lingonberry ‘Tīna’The vegetation period of the hybrid cultivars ‘Salaspils Agrās’, ‘Dižbrūklene’, and ‘Tīna’ startedfrom 28-th April to 18-th May, when the average daily air temperature was 4.4 °C. The growth ofshoots began from 11-th to 28-th of May. Buds forming the uprights were the first ones to grow.After opening of the buds the first thirty five days the uprights growth was intensive. During thattime their length reached 3.0-7.0 cm.At the end of May, when the average daily air temperature was over 10 °C and sum of positivetemperatures exceeded 412 °C, the buds of the uprights began to swell and the flower budsemerged. Hybrid cultivars under trial began to flower from 14 th June to 6 th July and continued for30 – 35 days. The flowering period of each flower lasted for 15 – 17 days.The ripening of the berries depends on cultivar properties and the average daily air temperatureduring the vegetation period. The first berries of the very early hybrid cultivars ‘Salaspils Agrās’and ‘Dižbrūklene’ ripened from September 5 th to 11 th , and of the early hybrid cultivar ‘Tīna’ – fromSeptember 8 th to 15 th .The lingonberry cultivar ‘Salaspils Ražīgā’ originated from a clone found in PĜavu forestry (Talsiregion), selected as cultivar in 1993 (AudriĦa, 1996).96

‘Salaspils Ražīgā’. Vigorously growing, upright, compact shrub (mature plant height 16 – 35 cm),with moderate plant spread (rhizome production). Leaves are dark green, medium size (length 1.6 –2.2 cm, width 0.7 – 1.1 cm).This cultivar has only one pronounced blooming period (middle of May – beginning of June), thesecond blossoming is sparse. Flowers are set in medium size clusters (2 – 12 flowers) or alone.Flowers are white or white with anthocyan paint. Berries ripen in the end of August – beginning ofSeptember. Fruit clusters are medium size (2 – 10 berries). Berries are deep red, roundish, mediumsize (0.21 – 0.31 g), length 0.7 – 1.1 cm, diameter 0.7 – 1.0 cm. The cultivar is productive – fruityield of mature plants – 90 – 180 g per plant. The cultivar is winter hardy and showed relativelyhigh resistance to “little leaf disease”.Figure 3. Lingonberry cultivar ‘Salaspils Ražīgā’The lingonberry cultivar ‘Rubīna Lāse’ was selected in 1988 from open pollination seedlings of‘Salaspils Ražīga’.‘Rubīna Lāse’. It is vigorous, upright, compact shrub with moderate plant spread. Mature plantheight 20 – 30 cm. Leaves are light green, medium size (length 1.8 – 2.6 cm, width 0.8 – 1.4 cm).This cultivar has the ability to bloom twice in a season, but the second blossoming is sparse. Moreintensive second blossoming was observed in the years when the first blooms were damaged byfrosts. The average first blossoming period is in the middle of May – beginning of June, but thesecond – end of July – beginning of August. Flowers are set in medium size clusters or alone.Flowers are white with anthocyan paint, larger then flowers of other cultivars. The first berry cropripens in the beginning to middle of August, but the second crop – in the end of September –beginning of October. Fruit clusters are medium size (2 – 9 berries). Berries are very deep red,drop-like, medium size (0.30 – 0.42 g), length 1.0 – 1.3 cm, and diameter 0.8 – 1.0 cm. Berriesyield 35 – 200 g per plant. The characteristic distinctive feature of ‘Rubīna Lāse’ from otherlingonberry cultivars is their relatively light colour of leaves contrasting with very dark berries, andan unusual crop-like form of berries. The cultivar is winter hardy and showed high resistance to“little leaf disease” (AudriĦa, 2004).The lingonberry cultivar ‘Jūlija’ was selected in 1995 from open pollination seedlings of the clone‘Krasnojarska’.‘Jūlija’. It has compact, 13 – 20 cm high shrub with moderate plant spread. Leaves are dark green,medium size (length 1.7 – 2.1 cm, width 0.6 – 1.2 cm). The cultivar has only one pronouncedblossoming period (middle of may-beginning of June). Flowers are set in medium size clusters (2 –13 flowers) or alone. Flowers are white with anthocyan paint. ‘Jūlija’ is an early ripening cultivar –in the end of July to beginning of August. Berries are dark red, roundish, medium size (0.20-0.39g), length 0.6 – 1.0 cm, and diameter 0.7 – 1.0 cm. Fruit yield 21 – 92 g per plant. This cultivar alsois winter hardy, and showed high resistance to “little leaf disease’ (AudriĦa, 2004).97

ConclusionsThe highly productive and cold resistant cultivars of rabbiteye blueberries ‘Salaspils Izturīgā’ and‘Lielogu’ were selected.The highly productive hybrid (cranberry cultivar ‘Franklin’ x lingonberry) cultivars ‘SalaspilsAgrās’, ‘Dižbrūklene’, and ‘Tīna’ were selected. These very early and early ripening cultivars havelarge and very large berries.The highly productive and disease resistant cultivars of lingonberry ‘Salaspils Ražīgā’, ‘RubīnaLāse’ and ‘Jūlija’ were selected.References1. AudriĦa B. (1996) The first results of cowberry breeding in Latvia. In: Baltic Botanic Gardens in 1994-1995. Estonia, Latvia, Lithuania. Salaspils, pp. 48-56.2. AudriĦa B. (2004) New lingonberry cultivars and their disease resistance. In: Baltic Botanic Gardens in2002-2003. Estonia, Latvia, Lithuania. University press, Tartu, pp. 83-89.3. Ripa A. (1980) Dzērvenes (The cranberries). Avots, Rīga, 98 p. (in Latvian).4. Ripa A. (1981) Dzērvenes, brūklenes, mellenes un augstās zilenes mūsu galdam (The cranberries,lingonberries, bilberries and higbush blueberries for our table). Zinību biedrība, Rīga, 25 p. (in Latvian).5. Ripa A. (1992) Dzērvenes, zilenes, brūklenes dārzā (The cranberries, blueberries, lingonberries in thegarden). Avots, Rīga, 104 p. (in Latvian).6. Ripa A., Kolomijceva V., AudriĦa B. (1992) KĜukva krupnoplodnaja, golubika visokaja, brusnika. (Thelarge-fruit cranberry, highbush blueberry, lingonberry). Zinātne, Rīga, 215 p. (in Russian).7. Ripa A. (1996) Amerikas lielogu dzērvene (The American large-fruit cranberry). Zinību biedrība, Rīga,75 p. (in Latvian).8. Ripa A. (1998) Augstās zilenes (The higbush blueberries). Zinību biedrība, Rīga, 21 p. (in Latvian).VOLATILE PROFILES OF EUROPEAN BLUEBERRY: FEW MAJOR PLAYERS, BUTCOMPLEX AROMA PATTERNSEIROPAS MELLEĥU AROMĀTS: DAŽAS GALVENĀS SASTĀVDAěAS, BETDAUDZVEIDĪGS KOPĒJAIS AROMĀTA SASTĀVSJens Rohloff 1 , Rolf Nestby 2 , Arnfinn Nes 3 , and Inger Martinussen 41 The Plant Biocentre, Department of Biology, Norwegian University of Science and Technology (NTNU), N-7491 Trondheim, Norway, e-mail: jens.rohloff@bio.ntnu.no;2 Bioforsk Grassland and Landscape Division, Norwegian Institute for Agricultural and EnvironmentalResearch (NIAER), N-7500 Stjørdal, Norway;3 Bioforsk Arable Crops Division, NIAER, N-2849 Kapp, Norway;4 Bioforsk Arctic Agriculture and Land Use Division, NIAER, N-9269 Tromsø, NorwayAbstractAs part of a project on cultivation and industrial exploitation of European Blueberry (Vacciniummyrtillus ) started in 2008, berries from different wild populations from South, Mid and NorthNorway were investigated. One aspect of fruit quality analyses was to identify and describeblueberry aroma profiles. Volatiles were extracted by headspace solid-phase microextraction (HS-SPME) and analysed by gas chromatography/ mass spectrometry (GC/MS). A total of 132 potentialaroma volatiles could be detected, of which 99 structures were identified based on MS databasesearch and retention indices, also comprising aroma impact compounds not being described inblueberries earlier. Detected aliphatic and aromatic structures belonged to different chemicalgroups such as alkanes, acids, alcohols, aldehydes, esters, ketones and mono- and sesquiterpenes.Ten major compounds (mostly C 4 -, C 6 - and C 9 -structures) accounted for averagely 65–75 %relative amount of all detected peaks. However, HS-SPME analyses revealed complex volatileprofiles including terpenes (23 compounds, e.g. p-cymene, 1,8-cineole, linalool) and aromaticstructures (10 compounds, e.g. benzaldehyde, ethyl benzoate, 2-phenylethyl acetate, benzylbenzoate), which contribute to the characteristic and flavourful blueberry aroma.98

KopsavilkumsEiropas melleĦu (Vaccinium myrtillus) ogas no dienvidu, vidus un ziemeĜu Norvēăijas savvaĜasaudzēm tika pētītas kā daĜa no 2008. gadā uzsāktā projekta par šo ogu kultivēšanu unekspluatēšanu. DaĜa no augĜu kvalitātes analīžu uzdevumiem bija identificēt un aprakstīt melleĦuaromātu. Gaistošās vielas tika izdalītas ar HS-SPME metodi un analizētas ar gāzu hromatogrāfijas– masu spektrometrijas (GC/MS) metodi. Kopumā tika noteiktas 132 aromātu veidojošas gaistošāsvielas, no kurām 99 sastāvdaĜu struktūras tika identificētas, balstoties uz spektra datubāzesmeklējumiem un izdalīšanās laiku, tai skaitā atrastas sastāvdaĜas, kas iepriekš mellenēs navaprakstītas. Noteiktās alifātiskās un aromātiskās struktūras vielas piederēja dažādām ėīmiskajāmgrupām ,tādām kā alkāni, skābes, spirti, aldehīdi, ēteri, ketoni, terpēni. Desmit galvenās sastāvdaĜas(galvenokārt C 4 -, C 6 - un C 9 - struktūras) sastādīja, vidēji 65 – 75 % no relatīvā visu noteiktosastāvdaĜu daudzuma. Tomēr HS-SPME analīze uzrādīja Ĝoti daudzveidīgu gaistošo vielu sastāvu,ieskaitot terpēnus (23 sastāvdaĜas, piemēram, p-cimēns, 1,8-cineols, linalols) un aromātiskasstruktūras (10 sastāvdaĜas, piemēram, benzaldehīds, etilbenzoāts, 2-feniletilacetāts,benzilbenzoāts), kas nosaka raksturīgo un bagātīgo melleĦu aromātu.Key words: Vaccinium myrtillus L., headspace (HS), solid-phase microextraction (SPME), gaschromatography/mass spectrometry (GC/MS), aroma, quality.IntroductionEuropean blueberry (Vaccinium myrtillus L.), also called bilberry, is a perennial dwarf shrub in theEricaceae family, being native to northern and eastern parts of Europe and Asia. Compared to otherspecies in the same genus e.g. highbush blueberry (V. corymbosum L.), the wild blueberriesproduce fruits with a higher content of desirable polyphenols and other health-beneficialcompounds (Giovanelli and Buratti, 2009). Furthermore, the characteristic and pleasant flavour ofberries from wild-growing plants is very complex compared to cultivated highbush blueberries(Parliment and Kolor, 1975; Hirvi and Honkanen, 1983a) and rabbiteye blueberries (Horvat et al.,1996). Already in 1969, Von Sydow and Anjou published results about the vast variety of 109aroma volatiles found in V. myrtillus, and described 19 aliphatic alcohols, 24 aliphatic aldehydesand ketones, 26 terpene derivatives, 24 aromatic compounds, and 16 other chemical structures.Berry samples from putative progenitor species of cultivated highbush, rabbiteye and lowbushblueberries have been shown to contain many of the same aroma volatiles (Baloga et al., 1995),and later reports underscored the complexity of aroma patterns also of cultivated Vaccinium speciesthrough the identification of new potential key aroma volatiles such as sulphur-compounds(Hanoglu and Pucarelli, 2007) and other chemical structures (Di Cesare et al., 1999). Major goalsof our preliminary study on aroma volatile composition of berrries from wildgrowing V. myrtillusplants in Norway were the (1) Identification of aroma-impact compounds, (2) Influence ofmaturation stage on aroma patterns, and (3) Potential effect of location on berry aroma , in order tocharacterize the significance of different factors affecting the flavour properties of blueberries.Materials and MethodsPlant Material. Blueberry samples from different wild populations from South, Mid and NorthNorway where harvested at maturation stage in August-September 2008 and stored at -20°C priorto extraction and analysis.Aroma Volatile Analysis. Frozen berries were cut into halves and a total of 3 g from single halvesof 10 – 15 fruits were placed in a 15 ml headspace vial (Supelco Inc.). After adding 5 ml H 2 O and 1g NaCl, the vial was closed with a screw cap with Teflon-coated septum, and the sample wasconstantly agitated on a magnetic stirrer during extraction (45 min). Headspace solid-phasemicroextraction (HS-SPME) was applied for isolation and concentration of volatile aromacompounds by using a manual SPME holder (Supelco Inc.) with a PDMS/DVB-coated 65 µm fibreexposed to the atmosphere in the sample vial (Rohloff, 2004). HS-SPME sampled volatiles weredesorbed in the the injection port of a gas chromatograph (GC) for 3 min. Aroma volatiles wereanalysed using a VARIAN Star 3400 CX GC coupled with a Saturn 3 mass spectrometer (GC/MS).GC separations were carried out on a HP-5MS capillary column (30 m × 0.25 mm i.d., filmthickness 0.25 µm). Injection temperature was 220 °C, and the interface was set to 220 °C. The99

carrier gas was helium, the GC temperature program was held isothermically at 40°C for 1 min(splitless injection mode), ramped from 40° to 220 °C at a rate of 4.5 °C min, and finally held at220 °C for 4 min (analysis time: 45 min). The MS source was adjusted to 220 °C and a mass rangeof m/z 40 – 300 was recorded. Compounds were tentatively identified based on mass spectraldatabase search using the NIST05 MS Database, a customized in-house fruit flavour MS database,and retention indices from literature.Statistic Analysis. GC/MS data (TIC) were analysed using AMDIS deconvolution software (v.2.64). The raw data sets of 32 samples were aligned using the on-line service ‘SpectConnect’ atMassachusetts Institute of Technology (MIT, 2009). Chemometric Principal Component Analysis(PCA) was carried out with the statistical software Minitab (v. 15.1.30).Results and DiscussionThe application of HS-SPME coupled with GC/MS revealed 132 volatile aroma compounds in 32different samples of blueberries harvested at different loactions in Norway. A total of 99compounds could be tentatively identified using mass spectral database search. The most abundantcompounds were related to the group of C 6 -structures or so-called ‘green leaf volatiles’ (Figure 1).In addition, impact-aroma compounds belonging to the chemical group of monoterpenes, couldalso be extracted from all samples, e.g. 1,8-cineole, linalool, and linalyl acetate.Figure 1. GC/MS chromatogram of a headspace-SPME profile of blueberry aroma volatiles.Important compounds such as C 6 -structures or ‘green leaf volatiles’ (hexyl- and hexenyl-derivatives),terpenes (1,8-cineole, linalool) and aldehydes (nonanal, decanal) are indicated in the figure.Based on chemometric PCA of all data sets from GC/MS analyses, blueberry samples could begrouped into different clusters depending on from which location the blueberries were harvested(Figure 2). Samples from South Norway clustered in an own group, while the samples from MidandNorth-Norway were overlapping. Regarding differences in the maturation stage, not-fullyripened blueberries were clearly separated from fully-ripened fruits, thus indicating significantdifferences in their aroma profiles.100

Figure 2. Chemometric PCA of 32 blueberry samples from different locations in Norway.Five berry samples from South Norway clearly clusterd in an own group (right side of the figure with dashdotline); these were not fully-ripened fruits in contrast to all other berry samples harvested at full maturationstage.All identified aroma volatiles are presented in Table 1, showing the vast diversity of chemicalstructures found in berry samples from wild-growing Vaccinium myrtillus.Table 1. Identified aroma volatiles in 32 blueberry samples from Norwegian Vaccinium myrtilluspopulations.Aliphatic Estersnonanolethyl acetate2-butyl 1-octanolethyl 2-methylpropanoate2-ethyl-1-decanolmethyl 3-methylbutanoatehexyl octanolethyl 2-methylbutanoateAliphatic Ketonesethyl 3-methylbutanoate2-hexanone3-methylbutyl acetate4-hepten-2-onemethyl 2-hydroxy-3-methylbutanoate4-methyl 2-heptanone3-methyl-2-butenylacetate4-octen-3-onepentyl acetate6-methyl-5-hepten-2-onemethyl hexanoate2-dodecanoneethyl 3-hydroxy-3-methylbutanoategeranyl acetoneethyl 2-hydroxy-3-methylbutanoateβ-iononeethyl hexanoateAliphatic Acids(Z)-3-hexenyl acetatenonanoic acidhexyl acetatedecanoic acid(E)-2-hexenyl acetateundecanoic acid(Z)-2-hexenyl acetatehexadecanoic acid(E,E)-2,4-hexadienyl acetateMonoterpenes and Sesquiterpenes*hexyl propanoatecumene2-ethylhexanoatetricyclene(Z)-3-hexenyl butanoateα-pinene(E)-2-hexenyl butanoatesabineneethyl octanoateβ-myrcene(Z)-3-hexenyl 3-methylbutanoateα-terpinenehexyl-3-methylbutanoatep-cymenehexyl-2-methylbutanoate1,8-cineoleoctyl 2-methylpropanoatelimonenehexyl hexanoate(Z)-ocimeneisopropyl myristate(E)-ocimeneAliphatic Aldehydesγ-terpinene101

2-methyl butanal(Z)-3-hexenalhexanal(E)-2-hexenalheptanal(Z)-2-heptenal2,4-heptadien-1-aloctanal(E)-2-octenalnonanal(E,Z)-2,6-nonadienal(E)-2-nonenaldecanaldodecanaltetradecanalAliphatic Alcohols2,4-hexadien-1-ol(Z)-3-hexenol(E)-2-hexenolhexanol1-octen-3-oloctanolterpinolenelinaloolcitronellalα-terpineolcyclocitrallinalyl acetategeranialbornyl acetate(E)-anetholeβ-caryophyllene*(E,E)-farnesyl acetate*Aromaticstoluenestyrenebenzaldehydepropyl benzeneacetophenone5-ethyl-m-xylenep-methyl benzaldehydeethyl benzoate2-phenylethyl acetatebenzyl benzoateMajor chemical groups being represented comprise a total of 66 aliphatic hydrocarbons (29 esters,15 aldehydes, 10 alcohols, 8 ketones, 4 acids), 23 terpenes (21 mono- and 2 sesquiterpenes), and 10aromatic structures. The abundance of esterified aroma volatiles contribute to the overall fruityflavour of the European blueberry, though also many of the other reported aliphatic structures havesimilar aroma properties. Except for 1,8-cineole (mint-spicy note), most of the identified terpeneshave to be considered as minor constituents. However, due to their low olfactory threshold,characteristic flavour notes are added to blueberry aroma such as α-terpinene, p-cymene, limonene,geranial (citrus-lemon-like), β-myrcene, (Z)- and (E)-ocimene, γ-terpinene, terpinolene (herb-spicynotes) and the aroma-impact compound linalool with its acetate (flowery-fruity notes). In addition,many of the detected aromatic structures have also strong aroma potential and supplement thecharacteristic blueberry aroma with their spicy, flowery, and fruity notes. In general, the presentedresults underscore the suitabilty and sensitivity of HS-SPME for the fast and reliable description ofaroma volatiles from plant samples (Rohloff, 1999, 2002; Rohloff et al. 2004), also with regard tothe detection of low-abundance compounds (Rohloff, 2004; Rohloff and Bones, 2005). Aromavolatile patterns found in our study, are in accordance with earlier results from V. myrtillus (VonSydow and Anjou, 1969). Furthermore, many of the described structures have also been reportedfrom other cultivated Vaccinium species (Parliment and Kolor, 1975; Hirvi and Honkanen,1983a,b; Baloga i, 1995; Horvat et al., 1996; Di Cesare et al., 1999; Polashock et al., 2007;Hanoglu and Pucarelli, 2007).ConclusionsResults from our study show the complexity but also homogeneity of aroma compounds beingdetected in blueberries (Vaccinium myrtillus) from Norwegian populations, not least becausealmost 90 % of the identified structures were found in all samples. However, aroma patternsdiffered with regard to location and maturation stage, and underline the significance of bothenvironmental, genetical and ontogenetical factors and thus, potential effects on blueberry aromaand quality. These questions will be further adressesd in our blueberry project as a continuation ofthe preliminary study.AcknowledgementsFinancial funding from the Research Council of Norway (RCN) through grant no. 184797 isgreatly acknowledged.102

References1. Baloga D.W., Vorsa N., and Lawter L. (1995) Dynamic headspace gas chromatography-massspectrometry analysis of volatile flavor compounds from wild diploid blueberry species. In: RousseffR.L. and Leahy M.M. (eds.), Fruit Flavors: Biogenesis, Characterization and Authentication, ACSSymposium Series 596, Oxford University Press, USA, pp. 235-47.2. Di Cesare L.F., Nani R., Proietti M., and Giombelli R. (1999) Volatile composition of the fruit and juiceof blueberry cultivars grown in Italy. Industrie Alimentari, 38, pp. 277-282.3. Giovanelli G. and Buratti S. (2009) Comparison of polyphenolic composition and antioxidant activity ofwild Italian bluberries and some cultivated varieties. Food Chemistry, 112, pp. 903-908.4. Hanoglu A. and Pucarelli F. (2007) Determination of key aroma-active compounds by GC-O AromaExtract Dilution Analysis (AEDA) in cranberry and blueberry fruits. In: Hofmann T., Meyerhof W., andSchieberle P. (eds) Recent Highlights in Flavor Chemistry & Biology, Proceedings of the 8 th WartburgSymposium on Flavor Chemistry and Biology, Eisenach, Deutsche Forschungsanstalt fürLebensmittelchemie, Garching, Germany, pp. 269-275.5. Hirvi T. and Honkanen E. (1983a) The aroma of some hybrids between high-bush blueberry (Vacciniumcorymbosum L.) and bog blueberry (Vaccinium uliginosum L.). Zeitschrift für Lebensmittel-Untersuchung und -Forschung, 176, pp. 346-349.6. Hirvi T. and Honkanen E. (1983b) The aroma of blueberries. Journal of the Science of Food andAgriculture, 34, pp. 992-996.7. Horvat R.J., Schlotzhauer W.S., Chortyk O.T., Nottingham S.F. and Payne J.A. (1996) Comparison ofvolatile compounds from rabbit eye blueberry (Vaccinium ashei) and Deerberry (V. stamineum) duringmaturation. Journal of Essential Oil Research, 8, pp. 645-648.8. MIT (2009) SpectConnect. Massachusetts Institute of Technology (MIT), http://spectconnect.mit.edu.9. Parliment T.H. and Kolor M.G. (1975). Identification of the major volatile components of blueberry.Journal of Food Science, 40, pp. 762-763.10. Polashock J.J., Saftner R.A. and Kramer M. (2007) Postharvest Highbush blueberry fruit antimicrobialvolatile profiles in relation to anthracnose fruit rot resistance. Journal of the American Society forHorticultural Science, 132, pp. 859-868.11. Rohloff J. (1999) Monoterpene composition of essential oil from peppermint (Mentha × piperita L.) withregard to leaf position using solid-phase microextraction and gas chromatography/mass spectrometryanalysis. Journal of Agricultural and Food Chemistry, 47, pp. 3782-3786.12. Rohloff J. (2002) Volatiles from rhizomes of Rhodiola rosea L. Phytochemistry, 59, pp. 655-661.13. Rohloff J. (2004) Essential Oil Drugs – Terpene Composition of Aromatic Herbs. In: Dris R. and JainS.M. (eds.) Production Practices and Quality Assessment of Food Crops. Vol. 3: Quality Handling andEvaluation, Kluwer Academic Publishers, Dordrecht, The Netherlands, pp. 73-128.14. Rohloff J., Nestby R., Folkestad J.A. and Iversen T.-H. (2004) Influence of rain cover cultivation ontaste and aroma quality of strawberries (Fragaria ananassa Duch.). Journal of Food, Agriculture &Environment, 2, pp. 74-82.15. Rohloff J. and Bones A.M. (2005) Volatile profiling of Arabidopsis thaliana - Putative olfactorycompounds in plant communication. Phytochemistry, 66, pp. 1941-1955.16. Von Sydow, E. and Anjou, K. (1969) Aroma of bilberries (Vaccinium myrtillus). I. Identification ofvolatile compounds. Lebensmittel-Wissenschaft und-Technologie, 2, pp. 78-81.VARIABILITY OF THE STRUCTURE OF THE BIOCHEMICAL COMPOSITION OFTHE FRUITS OF THE HIGHBUSH BLUEBERRYAUGSTKRŪMU MELLEĥU OGU BIOĖĪMISKĀ SASTĀVA MAINĪGUMSRupasova Zh. 1 , Pavlovskij N. 1 , Kurlovich T. 1 , Pyatnitsa F. 1 , Yakovlev А. 1 , Volotovich A. 2 ,Pinchukova Yu. 31 Central Botanical Garden of the NAS of Belarus,220012 Minsk str. Surganova, 2v, Belarus, e-mail: rupasova@basnet.by2 Polessky State University, e-mail: volant777@tut.by3 Mogilev State University of food technologies, e-mail: mti@mogilev.byAbstractIn this article the values of the coefficients of variation of 30 indicators of the biochemicalcomposition of the fruits of the blueberry in a three-year cycle of supervision are presented. Signs103

with resistance against the complex influence of meteorological factors based on the level of thevariability of traits are designated. On the basis of the comparative analysis of the averagedvariation coefficients calculated for a varietal row of Vaccinium corymbosum L. during a long-termcycle of observation it has been established that the parameters of accumulation of solids, fructose,total soluble sugars, flavonols, total bioflavonols, benzoic acid, nitrogen, potassium, calcium andmagnesium in fruits can be characterized by the smallest degree of genotypic variability and,consequently, of intervarietal distinctions, whereas the content of free organic acids, anthocyaninsproper and the values of a sugar-acid index in fruits are characterized by the highest degree ofgenotypic variability.KopsavilkumsŠajā rakstā ātspoguĜoti 30 melleĦu ogu bioėīmiskā sastāva indikatoru variācijas koeficienti trīsgadu izmēăinājumu ciklā. Salīdzinošai analīzei ir aprēėināts vidējais variācijas koeficientsVaccinium corymbosum L. šėirnu rindai ilgtermiĦā ir novērots, ka sausnes, fruktozes, kopējošėīstošo cukuru, flavonolu, kopējo bioflavonolu, benzoskābes, slāpekĜa, kālija, kalcija un magnijauzkrāšanās ogās var raksturot ar mazāku genotipisko mainību un, tātad arī mazākām atšėirībāmstarp šėirnēm. Turpretim brīvo organisko skābju saturu, antociānu saturu un cukuru-skābjuattiecības indeksu raksturo augstāka genotipiskā mainība.Key words: Vaccinium corymbosum L., cultivars, biochemical composition, fruitsIntroductionOne major aspect of the introductory research examining berry plant cultivars is an integratedassessment of the biochemical structure of fruits which can prove that the latter contain a widespectrum of wholesome substances. At the same time it is well-known that the quantity of thesesubstances in a fruit is cultivar dependant. In recent years the collection stock of the CentralBotanical Garden of Belarus’ NAS has been replenished with some new taxons of Vacciniumcorymbosum L. that have different ripening times under the weather conditions in Belarus. Amongthem are such early-ripening varieties as Bluetta, Northblue, Weymouth, Duke, Reka, Earliblue,Spartan, Puru, Nui; such mid-ripening varieties as Bluecrop, Northland, Patriot, Toro, Jersey andsuch late-ripening varieties as Elizabeth and Coville. This replenishment gives additionalopportunities to expand the assortment of the varieties offered for regionalization and selectionwhich is done by choosing the most promising ones according not only to their crop andbioproductional characteristics, but also to the nutritive and vitamin value of berries that dependson their biochemical composition.At the same time it seems fair to suppose that there exist some distinctions among the cultivarsaccording to the genotypic variability of the parameters that influence the accumulation of certaincompounds in fruits. The distinctions can indirectly specify the degree of the genetic determinancyof the parameters and make it possible to bring out the characteristics that are more or less stablewhen breeding new cultivars.Materials and methodsIn 2006 – 2008 research was conducted into the biochemical composition of the above-listedcultivars of Vaccinium corymbosum L. examining a wide spectrum of indicators relating todifferent classes of active substances. Fresh averaged samples of vegetable material were taken todetermine the content of: solid matter – in accordance with GOST (State Standard) 8756.2 – 82(1982); ascorbic acid (vitamin C) – using the standard indophenol method (Ermakov, etc., 1987);titratable acids (total acidity) – using the volumetric method (Ermakov, etc., 1987). Dried at 65degrees Celsius averaged samples of fruits were used to determine the content of such chemicalelements as nitrogen, phosphorus, potassium using the method of K.P. Fomenko and N.N. Nesterov(1971); calcium and magnesium – using the complexometric method (Ermakov, etc., 1987);glucose, fructose, sucrose – using the resorcinol and aniline-phthalate methods of paperchromatography of I.G. Zavadsky and others (1962); pectins (water-soluble pectin and protopectin)– using the carbazolic method (Ermakov, etc., 1987); the sums of anthocyanic pigments – using themethod of T. Swain, W. E. Hillis (1952) constructing a calibration curve on cyaniding crystal104

obtained from fruits of black chokeberry and cleared using the technique of J.G. Skorikova andE.A. Shaftan (1968) and employing S.S. Tanchev’s formula evaluation (1980); anthocyanins –using the method of L.O. Shnaidman and V.S. Afanasyeva (1965); the flavonol sums – accordingto the method of L.Sarapuu and H.Miydla (1971); the catechine sums – according to thephotometric method with the use of a vanillin reagent (Zaprometov, 1964); phenol-carboxylic acids(in terms of chlorogenic acid) – according to the method of descending chromatography on paper(Mzhavanadze, etc., 1971); tannins – according to the titrimetric method of Levental (StatePharmacopoeia of the USSR, 1987); lignins – using the modified method of Klason (Ermakov, etc.,1987); benzoic acid – using the method of M.I. Kalebin and A.A. Kolesnik (1949); fatty oils –using the method of V.A. Sapunov and I.I. Fedunyak (1958); triterpenic acids (in terms of ursolicacid) – using the method of A.V. Simonyan and others (1972). All analytic determinations havebeen carried out in triplicate. The data has been statistically processed using the computer programExcel.When estimating the genotypic variability of the parameters that influence the accumulation of theabove-mentioned compounds we were guided by the coefficients of variation (V) of thecharacteristics under study within the range of Vaccinium corymbosum L. taxons that wereexamined during a long-term cycle of observation. The values of variation coefficients indicate thatthey depend on a cultivar, i.e. the higher the variation coefficient is, the stronger this dependence isand, therefore, the level of genetic determinacy of the characteristic goes down, and vice versa.According to S.N. Sennov and V.F. Kovyazina (1990), series variability for biological objects isregarded low if it is equal to 11-30 % and is considered high if it exceeds 31 %. When examiningthe data presented in this paper, we should take into account the active reaction of an alien crop tothe breeding process. This reaction allows the crop to resist the breeding process to a certain extentas well as to regulate the biochemical composition of generative organs within geneticallydetermined variation ranges of each characteristic. It allows one to narrow down the abovementioned limits of small series variability for the indicators under study to 15 %. Accordingly, itsaverage range will be characterized by the level of variability within 16 – 30 %, while themaximum one is over 31 %.Results and DiscussionThe biochemical screening of the three groups of Vaccinium corymbosum L. cultivars characterizedby different terms of ripening made it possible to identify the taxons with the highest and thelowest content of wholesome substances in fruits. These substances refer to different classes ofchemical compounds. At the same time, there have been discovered some essential intervarietaldistinctions concerning the accumulation of certain compounds in the dry weight of fruits, which isconfirmed by the wide range of changes of their quantitative characteristics that during a three-yearcycle of observations made up (Table 1): for titratable acids – 1.5 – 11.1 %; for ascorbic acid –296-941.0 mg% (mg of ascorbic acid in 100 g of dry weight of sample); phenol-carboxylic acids –543-1189 mg%; benzoic acid – 0.82 – 1.59 %; soluble sugars – 10.9 – 29.2 %, including glucose –3.2 – 7.1 %, fructose – 5.2 – 19.3 %; sucrose – 0.4 – 4.5 %; pectins – 3.6 – 7.9 %, includinghydropectin – 1.3 – 3.2 %, protopectin – 1.8 – 5.2 %; for the total content of bioflavonols – 1935 –3110 mg %, including anthocyanic pigments – 9 – 58 mg %, catechines – 383 – 1404 mg %,flavonols – 1471 – 2251 mg %; tannins – 0.9 – 2.5 %; fat oils – 1.6 – 5.5 %; major mineralelements: N – 0.57 – 1.34 %, Р – 0.10 – 0.24 %, K – 0.44 – 0.99 %, Ca – 0.26 – 0.45 %, Mg – 0.07– 0.12 %. The content of solid in the fresh weight of fruits changed from 10.9 % to 17.6 %.The analysis of the information presented in the table has revealed among the given taxons ofVaccinium corymbosum L quite a wide range of coefficients showing variation in the quantitativeindicators of the biochemical composition of fruits both during individual years and during thewhole period of observation. That indicated different levels of their dependence on the genotype ofa plant. This fact enabled us to identify the characteristics possessing the highest and the lowestdegree of varietal distinctions and to order them according to the decrease in genetic determinancy.The majority of the parameters showing accumulation of reactants in blueberries within the periodof observations were characterized mainly by an average variability (V = 15 – 30 %). Aconsiderable part of the indicators were notable for a low (V

characteristics under study have shown a high level (V > 30 %) of variability indicating the highestdegree of varietal distinctions.Table 1. Averaged quantity indicators of the biochemical structure of fruits (dry weight) andvariation coefficients in a long-term cycle of supervision for a varietal row of Vacciniumcorymbosum L.2006 2007 2008PeriodIndex mean V, % mean V, % mean V, %averageV, %Soluble solids, % of freshfruit13.9 12.8 13.9 10.1 14.1 12.1 11.7Free organic acids, % 5.7 36.4 3.8 45.7 6.7 39.1 40.4Ascorbic acid, mg % 601.2 20.3 426.6 26.3 604.8 30.3 25.6Glucose, % 5.34 16.4 4.49 15.8 4.96 27.5 19.9Fructose, % 18.74 2.9 14.22 3.9 7.26 22.3 9.7Sucrose, % 3.19 19.8 2.08 20.2 0.56 22.1 20.7Sum of soluble sugars, % 27.25 4.1 20.79 5.1 12.79 6.8 5.3Fructose/ Glucose 3.6 19.2 3.2 16.5 1.7 46.0 27.2Monose/Disaccharide 7.9 19.4 9.4 19.8 22.7 21.5 20.2Sugar-acid index 5.4 31.2 6.5 39.5 2.5 75.9 48.9Hydropectin, % 1.98 24.3 2.18 23.8 2.37 18.0 22.0Protopectin, % 3.45 22.4 2.60 19.8 3.35 21.0 21.1Sum of pectins, % 5.43 16.5 4.77 19.2 5.71 17.8 17.8Protopectin/Hydropectin 1.8 33.8 1.2 22.9 1.4 17.1 24.6Anthocyanins, mg % * 7.6 45.1 2.0 91.5 17.1 35.5 57.4Leucoanthocyanins, mg% 17.4 23.2 12.1 20.6 24.1 29.1 24.3Sum of anthocyanicpigments, mg %25.0 19.7 14.1 21.5 41.2 27.1 22.8Catechines, mg % 984.3 20.2 923.4 27.6 570.1 15.4 21.1Flavonols, mg % 1766.6 5.5 1626.0 11.0 1890.6 12.2 9.6Flavonols/Catechines 1.9 27.1 1.9 36.3 3.4 20.9 28.1Sum of bioflavonols, mg % 2776.0 6.9 2563.5 11.7 2501.8 9.2 9.3Phenol-carboxylic acids,mg %781.4 18.0 800.3 24.4 787.3 24.3 22.2Benzoic acid, % 1.15 6.1 1.11 18.2 1.18 14.3 12.9Tannins, % 1.21 19.5 1.72 23.2 1.83 16.3 19.7Fat oils, % 3.61 29.9 3.17 13.0 3.25 21.9 21.6Nitrogen, % 0.76 15.1 0.91 16.2 1.10 10.3 13.9Phosphorus, % 0.17 24.0 0.14 11.5 0.14 13.0 16.2Potassium, % 0.53 10.0 0.57 8.7 0.76 11.3 10.0Calcium, % 0.31 10.0 0.42 4.9 0.40 4.9 6.6Magnesium, % 0.09 11.9 0.08 8.8 0.11 6.8 9.2* mg% - mg of substance in 100 g of dry weight of sample.The variability level of a particular characteristic only in some cases remained stable throughoutthe whole 3-year period of observation. For example, such stability of a low variability level wastypical for the parameters of the accumulation of solid, flavonols, potassium, calcium, magnesium,the total amount of sugars and the sum of bioflavonols in fruits whereas the parameters of theaccumulation of fructose, benzoic acid, fat oils, nitrogen and phosphorus in fruits were notable forlow variability only during one or two seasons. Similarly, the high level of genotypic variabilityduring the whole period of observation was typical for the parameters of the accumulation of freeorganic acids, anthocyanins and the values of a sugar-acid index whereas for the ascorbic acidcontent it has been notable only in 2008. Moreover, even within a particular area of variability ofthe characteristics analyzed there have been identified some essential interseasonal distinctions.This fact unequivocally indicates a dependence of the genotypic variability of quantity indicators ofbiochemical composition of Vaccinium corymbosum L. fruits on the hydrothermal mode of itsformation period.106

In our opinion, the best estimate about the degree of variability of the indicators in the varietal rowcan be given by the averaged values of variation coefficients calculated within a 3-year period ofobservation. In this case the characteristics under study can be provisionally divided into 3 groupsin an increasing order of genotypic variability, and, hence, intensification of varietal distinctions:1 – with small variability (V = 5.3 – 13.9 %) – the content of solid, fructose, total of solublesugars, flavonols, total bioflavonols, benzoic acid, nitrogen, potassium, calcium and magnesium infruits;2 – with average variability (V = 16.2 – 25.6 %) – the content of phosphorus, ascorbic acid,glucose, sucrose, hydropectin, protopectin, leukoanthocyanins, catechines, phenol-carboxylic acids,tannins and fat oils in fruits;3 – with high variability (V = 40.4 – 57.4 %) – the content of free organic acids,anthocyanins proper and values of a sugar-acid index in fruits.ConclusionThe biochemical screening of the fruits of 16 representatives within three groups of introducedblueberry cultivars with different terms of ripening (early-ripening, mid-ripening and late-ripening)according to 30 indicators specifying the content of some organic acids, carbohydrates,bioflavonols, terpenoids and chemical elements under vegetative seasons of 2006 – 2008 made itpossible to reveal the essential intervarietal distinctions in the accumulation of certain compoundsin the dry weight of fruits, confirmed by a wide range of changes of their quantitativecharacteristics. On the basis of the comparative analysis of the averaged variation coefficientscalculated for a varietal row of Vaccinium corymbosum L. during a long-term cycle of observationit has been established that the parameters of accumulation of solids, fructose, total soluble sugars,flavonols, total bioflavonols, benzoic acid, nitrogen, potassium, calcium and magnesium in fruitscan be characterized by the smallest degree of genotypic variability and, consequently, ofintervarietal distinctions, whereas the content of free organic acids, anthocyanins proper and thevalues of a sugar-acid index in fruits are characterized by the highest degree of genotypicvariability.References1. GOST 8756.2-82, (1982) Methods of definition of solids. М: Publishing house of standards, 5 p. (InRussian).2. The State pharmacopoeia of the USSR (1987) Vol.1. The general methods of the analysis, М: Medicine,pp.286-287. (In Russian).3. Ermakov A.I., et al. (1987) Methods of biochemical research of plants. М, VO Agropromizdat,. 430 p.(In Russian).4. Zavadskaja I.G., Gorbacheva G. I and Mamushina N.S. (1962) Quantitative definition of carbohydratesby resorcinol and aniline-phthalate methods by means of a paper chromatography. In: Kretovich V.L.(eds) The Technique of a quantitative paper chromatography of sugars, organic acids and amino acidsat plants, Moscow-Leningrad, Publishing house of AS of the USSR,. pp. 17-26. (In Russian).5. Zaprometov M. N. (1964) Biochemistry of catechines, Moscow, Science, 325 p. (In Russian).6. Kalebin M. I, and Kolesnik A.A. (1949) Research of fresh fruits, vegetables and products of theirprocessing. In: Tserevitinov F.V. (etc) Research of foodstuff, Мoscow, Gostorgizdat, pp. 218-245. (InRussian).7. Mzhavanadze V.V., Targamadze I.L., and Dranik L.I. (1971) Quantitative definition of chlorogenic acidin leaves of a bilberry Caucasian V. arctostaphylos L.) Proc. of АS of GSSR, Vol. 63, N 1, pp. 205-210.(In Russian).8. Sapunov V.А., and Fedunjak I.I. (1958) Methods of an estimation of forages and the zootechnicalanalysis. Minsk, 190 p. (In Russian).9. Sarapuu L., and Mijdla H. (1971) The phenolic constituents of an apple-tree. Uch. West Tart. SU, N 256,pp. 111-113.10. Sennov S.N., and Kovjazin V.F. (1990) Forestry: the Manual, Leningrad, LTA, 91 p. (In Russian).11. Simonjan A.V., Shinkarenko A.L., and Oganesyan E.T. (1972) Quantitative definition of triterpenes inplants of Thymus sort. Chemistry of natural compounds, N 3, pp.293-295. (In Russian).12. Skorikova J. G, and Shaftan E.A. (1968) Technique of anthocyanins definition in fruits and berries. In:Kefeli V.I. (etc) Proc. 3 Un. Seminar on biologically active (medical) substances of fruits and berries.Sverdlovsk, pp. 451-461. (In Russian).107

13. Tanchev S.S. (1980) Anthocyanins in fruits and vegetables. Мoscow, Food ind., 304 p. (In Russian).14. Fomenko K.P., and Nesterov N.N. (1971) Technique of definition of nitrogen, phosphorus andpotassium in plants from one assay. Chemistry in agriculture, N 10, pp. 72-74. (In Russian).15. Shnajdman L.O., and Afanasjeva V.S. (1965) A definition technique of anthocyanic substances. In:Kretovich V.L. (eds) Proc. of 9th Mendeleevsky Congress on Gen. and Applied Chemistry, Мoscow, pp.79-80. (In Russian).16. Swain T., and Hillis W., (1959) The phenolic constituents of Prunus Domenstica. 1. The quantitativeanalysis of phenolic constituents. J.Sci. Food Agric. Vol. 10, N 1, pp. 63-68.MICROPROPAGATION OF HIGHBUSH BLUEBERRY CULTIVARSAUGSTKRŪMU MELLEĥU ŠĖIRĥU MIKROPAVAIROŠANAJiri Sedlak and Frantisek PaprsteinResearch and Breeding Institute of Pomology Holovousy Ltd., 508 01 Horice, Czech Republic;e-mail: sedlak@vsuo.czAbstractThe aim of this study was to determine an efficient micropropagation system for the highbushblueberry cultivars ‘Spartan’, ‘Bluecrop’ and ‘Berkeley’. The shoot tips of the selected threegenotypes were successfully established in vitro using mercuric chloride in a concentration of 0.15% as a sterilization solution. Anderson’s rhododendron medium (AN), half-strength Murashige andSkoog medium (half-MS) and McCown woody plant medium (WPM) containing cytokinin zeatinin concentrations 0.5, 1 or 2 mg l -1 were tested. Multiplication rates varied depending on thecultivar, medium and concentration of the zeatin. The highest multiplication 4.8 ± 0.2 was noted for‘Berkeley’ on WPM medium with zeatin (2 mg l -1 ). Out of three media tested, the WPM mediumwas found to be more effective than the AN medium and half-MS medium for shoot multiplication.The in vitro rooting on the WPM medium is also reported.KopsavilkumsPētījuma mērėis bija noteikt augstkrūmu melleĦu šėirĦu ‘Spartan’, ‘Bluecrop’ un ‘Berkeley’efektīvāko mikropavairošanas sistēmu. Izvēlēto trīs genotipu dzinumu gali tika veiksmīgi ievietotiin vitro izmantojot 0.15% koncentrācijas dzīvsudraba hlorīdu kā sterilizācijas šėīdumu. Tikapārbaudīta Andersona rododendra barotne (AN), pus-stiprā Murashige un Skoog barotne (pus-MS)un McCown koksnaino augu barotne (WPM), kas satur citokinīna zeatīnu 0.5, 1 vai 2 mg l -1koncentrācijā. Pavairošanas ātrums bija atkarīgs no šėirnes, barotnes un zeatīna koncentrācijas.Augstākais pavairošanas koeficients 4.8 ± 0.2 bija vērojams šėirnei ‘Berkeley’ WPM barotnē arzeatīnu (2 mg l -1 ). No trim pārbaudītājām barotnēm dzinumu pavairošanai WPM barotne bijaefektīvāka par AN barotni un pus-MS barotni. WPM barotrnē tika novērota arī dzinumu in vitroapsakĦošanās.Key words: explant, in vitro, zeatin, multiplication, rootingIntroductionThe highbush blueberry (Vaccinium corymbosum L.), native to North America, is a commerciallyimportant fruit crop (Zmarlicki, 2006). Although plants of the Vaccinium genus have not beencultivated on a large scale in the Czech Republic, there is potential for commercial highbushblueberry production in some mountain regions. A number of research programs continue to findsuitable high yielding highbush blueberry cultivars with superior berry qualities for commercialgrowing in the rural countryside of the Czech Republic (Paprstein et al., 2006; Paprstein andLudvikova, 2006). In recent years, the blueberry has received considerable attention for itsnutritional quality and health benefits (Howell, 2009).If suitable cultivars are to receive wide distribution rapid propagation techniques will be essential.Highbush blueberry can be propagated vegetatively by multiple-node softwood or hardwoodcuttings. This method, although generally successful, is slow and labor intensive. Success with108

cuttings also varies markedly with the individual genotype, age of the stock plant and thevegetation period (Kosina and Sedlak, 2006).The micropropagation can potentially multiply selected cultivars more rapidly than traditionalnursery methods utilizing softwood or hardwood cuttings. Over the last three decades, in vitroculture propagation methods on various basal media using axillary bud proliferation andadventitious shoot regeneration has been achieved with varying success (Reed and Abdelnour,1991; Noe et al., 1998; Gajdosova et al., 2006; Li et al., 2006). Unfortunately, these results are notbroadly applicable, because the effectiveness of the medium and morphogenesis of Vaccinium invitro plants seems to be highly dependent on plant growth regulators and the media used for theculture, and this dependence is genotype specific (Ostrolucka et al., 2004; Debnath, 2007).Moreover, the response of individual genotypes can vary during the whole cycle ofmicropropagation (Mehri-Kamoun et al., 2004). The suitability and genetic stability of blueberryplants micropropagated in tissue culture have also been discussed for a long time (El-Shiekh et al.,1996; Smolarz and Chlebowska, 1997).As a part of the project to introduce highbush blueberry culture to the Czech Republic, studies wereconducted to investigate new genetic resources and their possible multiplication by in vitro culture(Paprstein et al., 2005). The objective of this study was to compare various basal media withdifferent zeatin concentrations for shoot proliferation and to determine the best one formicropropagation of three highbush blueberry cultivars.Materials and MethodsFor the in vitro culture establishment, twenty actively growing shoot tips (5 to 15 mm in length)were cut from shoots of three blueberry cultivars (‘Spartan’, ‘Bluecrop’ and ‘Berkeley’) sproutingin laboratory conditions. The donor shoots were removed from mature shrubs growing in fieldgermplasm collection of RBIP Holovousy in March. After removal of most of the leaves, the initialexplants were dipped in a 0.15 % solution of HgCl 2 with a wetting agent added (0.05 % Tween-20)for 1 min. This was carried out under sterile conditions under a laminar flow hood. Followingsterilization, the tips were rinsed in sterile distilled water and cultured in 200 ml glass culture flasks(seven shoots per flask), each with 35 ml of WPM (woody plant medium) according to Lloyd andMcCown (1981). The initial WPM medium was with 1 mg l -1 zeatin. Culture vessels were glassbottles capped with clear permeable polypropylene caps. The contamination rate, the survival anddevelopment of shoots from excised shoot tips were analyzed after sterilization. Uncontaminatedshoots established on WPM medium were transferred after one month to a fresh proliferationmedium. All shoot cultures were serially subcultured for at least 4 months on a WPM mediumsupplemented with 2.0 mg l -1 zeatin. This provided a stock collection of shoots for proliferationstudies.All initiation and multiplication media contained 7.0 g l -1 Difco agar. The pH of the media wasadjusted to 5.2 before autoclaving at 120 °C at 100 kPa for 15 minutes. Cultures were grown inrooms under cool-white fluorescent tubular lamps at 60 µmol.m -2 .s -1 (16-hour photoperiod) at 22 ±1 °C.For the multiplication phase, three basal nutrient media WPM, AN (Anderson’s rhododendronmedium) according to Anderson (1980) and modified MS (Murashige and Skoog, 1962) mediumcontaining half macro and micronutrients (half-MS) were tested. The shoot tip cultures weremultiplied by removing several elongating shoots from the basal mass and subculturing the shootson a fresh medium. To induce new shoots, basal nutrient media included three concentrations 0.5, 1or 2 mg l -1 of the cytokinin zeatin. The zeatin was filter sterilized (25 mm, Acrodisc Syringe Filter0.2 µm, Pall Gelman, USA) and added to multiplication media after autoclaving. Uniform singleshoot tips (5 to 10 mm in length) excised from apical parts of established proliferating cultureswere used in all multiplication experiments. The morphological appearance of the shoots (primarilycallus formation, hyperhydricity etc.) was also noted.The multiplication rate was defined as the number of newly formed shoots (>10 mm) per initialshoot tip after four weeks of culture. The shoot formation was recorded between the fifth andfifteenth subculture. In all experiments 25 shoot tips were used. Each experiment was repeated fourtimes. Data from four independent experiments were pooled and expressed as the mean. Toevaluate the accuracy of the estimate of the mean of population, treatment means were compared109

with the standard error (SE) of the mean as a measure of variance. For three genotypes (‘Spartan’,‘Bluecrop’, ‘Berkeley’), shoots (10 to 20 mm in length) derived from the best proliferationmedium, were excised and rooted on WPM medium with 1 mg l -1 IBA. Although several mediawere evaluated for the induction of roots, only results from medium that showed maximal rootinduction are presented in this report. Culture conditions during root initiation and root growthwere the same as during shoot culture. A hundred microcuttings were used for this treatment. Thenumber of rooted in vitro plants was recorded five weeks after transfer to rooting medium. Thetreatment means were compared with the standard error (SE) of the mean. Shoots with roots wererinsed in water to remove remnants of the medium and then transferred to Jiffy 7 peat pellets (ASJiffy Products, Norway) soaked with water. The shoots were misted with water to prevent wiltingduring transplanting. The Jiffy 7 pellets with rooted plants were placed on a greenhouse benchequipped with transparent plastic covers (100 % air humidity) under the standard greenhousecondition. The plants were gradually acclimated by opening the covers over fourteen days.Results and DiscussionThe numbers of uncontaminated explants that survived and developed into shoots are shown inTable 1. Selected three genotypes were successfully established in vitro using mercuric chloride ina concentration of 0.15 % as a sterilization solution. Bacterial and fungal contamination wasinfrequent. Of the 60 shoot tips taken only one explant of ‘Spartan’, one explant of ‘Berkeley’ andtwo explants of ‘Bluecrop’ were visibly contaminated with micro-organisms. These explants werelater discarded. The use of mercuric chloride had a direct beneficial effect and overcame thecontamination from the microflora of the field germplasm collections of blueberry. On the otherhand, the toxicity to tissues caused by mercuric chloride was high. In the case of the cultivars‘Spartan’ and ‘Bluecrop’, about 50 % of initial uncontaminated explants did not develop shoots andturned brown. However, the remaining uncontaminated initial explants of these two cultivars had agreenish color and produced shoots. Debnath and McRae (2001) reported that althoughregeneration from primary explants is a first necessary step in any micropropagation of Vacciniumgenus, the regeneration frequency has no effect on the further success of the micropropagationprogram. Many shoots could be obtained from a few clean shoots regenerated from the primaryexplant.Table 1. Surface sterilization of highbush blueberry cultivars by 0.15 % mercuric chlorideExplants died without Established explants whichExplants contaminatedCultivarscontaminationdeveloped shootsNumber (%) Number (%) Number (%)Spartan 1 5 11 55 8 40Bluecrop 2 10 10 50 8 40Berkeley 1 5 5 25 14 70After 5 months in the culture, all surviving explants showed active and uniform shoot growth andmultiplication. Dividing and subculturing the basal shoot mass did not cause tissue breakdown orexudation. The results of the multiplication of highbush blueberry cultivars are shown in Tables 2 -4. The number of newly formed shoots varied with the cultivar, the medium tested and theconcentration of zeatin. Across all experiments, the highest multiplication rate (4.8) was obtainedfor ‘Berkeley’ on WPM medium with the highest concentration 2 mg l -1 of zeatin. On three testedmedia, ‘Berkeley’ was the cultivar with the highest ability to produce vigorous multiple shootcultures. On the contrary, for the cultivar ‘Spartan’, neither of the three tested media containingdifferent concentrations of zeatin promoted markedly in vitro shoot formation and the number ofnewly formed shoots was thus very low (from 1.3 to 1.8). Within the same range of zeatinconcentration, the three genotypes gave higher multiplication rates on the WPM medium. Thewoody plant medium (WPM) was found to be more effective than an AN and a half-MS mediumfor the initiation of new shoots in our study. The lowest multiplication rates were noted for‘Bluecrop’ and ‘Berkeley’ on a half-MS medium. The lowest multiplication rates for ‘Spartan’were noted on the AN medium. Short shoots (shorter than 10 mm) were frequently observed in thecase of the cultivar ‘Bluecrop’ on half-MS medium with all concentrations of zeatin tested. The110

shoots shorter than 10 mm were not counted for our multiplication studies. These small shoots didnot elongate and were difficult to use directly in further procedures. The half-MS medium provedto be less suitable for the multiplication of three highbush blueberry cultivars.Table 2. Multiplication rates for highbush blueberry cultivars on WPM medium with zeatinCultivarZeatin (mg l -1 )Spartan Bluecrop Berkeley0,5 1.4 ± 0.1 1.3 ± 0.1 3.0 ± 0.11 1.8 ± 0.1 1.5 ± 0.1 4.0 ± 0.22 1.7 ± 0.1 2.0 ± 0.1 4.8 ± 0.2Table 3. Multiplication rates for highbush blueberry cultivars on AN medium with zeatinCultivarZeatin (mg l -1 )Spartan Bluecrop Berkeley0.5 1.4 ± 0.1 1.2 ± 0.0 1.4 ± 0.01 1.3 ± 0.1 1.2 ± 0.1 2.2 ± 0.12 1.3 ± 0.1 1.9 ± 0.1 2.3 ± 0.1Table 4. Multiplication rates for highbush blueberry cultivars on half MS medium with zeatinCultivarZeatin (mg l -1 )Spartan Bluecrop Berkeley0.5 1.5 ± 0.1 1.2 ± 0.0 1.2 ± 0.11 1.3 ± 0.1 1.2 ± 0.1 1.2 ± 0.12 1.5 ± 0.1 1.5 ± 0.1 1.6 ± 0.1In the case of the cultivars ‘Bluecrop’ and ‘Berkeley’, the increasing zeatin concentration in thetested media also increased the shoot multiplication without excessive callus formation. Zeatinproved its ability to stimulate adventitious shoot development in Vaccinium in vitro culture. Thehighest multiplication rates were always noted on media with the highest concentration of zeatin (2mg l -1 ). The zeatin level 2 mg l -1 can be recommended for the multiplication of the cultivars‘Bluecrop’ and ‘Berkeley’. Earlier reports indicated that zeatin was an important plant hormone forefficient multiplication and growth in Vaccinium micropropagation (Reed and Abdelnour, 1991;Debnath and McRae, 2001; Ostrolucka et al., 2004; Jiang et al., 2009). According to Reed andAbdelnour (1991), the cultivation medium with relatively high levels of zeatin (4 mg l -1 ) promoteda significantly higher initiation of axillary shoots in eight of twelve Vaccinium corymbosumgenotypes than on the control medium. On the contrary, Gajdosova et al. (2006) pointed out theeffectiveness of zeatin in low concentration (0.5 mg l -1 ) for inducing multiple shoot development inmeristem cultures of Vaccinium sp. Zeatin concentrations of 2 mg l -1 and higher promoted callusformation and suppressed shoot regeneration in Gajdosova’s experiments, which is contradictory toour findings. In our experiments on all media, any physiological disorders or morphologicalabnormalities such as excessive callus formation or the production of abnormally narrow leaveswere not observed during the in vitro shoot proliferation stage. For the cultivar ‘Spartan’ thehighest multiplication rate 1.8 was noted on media with the zeatin concentration of 1 mg l -1 .However this multiplication rate (1.8) achieved on a WPM medium with 1 mg l -1 of zeatin can besufficient only for in vitro culture establishment and maintenance, but is not satisfactory for largerscale in vitro shoot production. Future research and testing of other media and plant growthregulators is needed in the case of ‘Spartan’.The results of rooting are summarized in Table 5. There was considerable variation in the rootingpercentage of used blueberry cultivars. WPM medium with a high concentration of IBA (1 mg l -1 )was effective for root induction in the case of cultivars ‘Berkeley’ and ‘Bluecrop’. Root initiationstarted within two weeks. The percent of rooting was 70 % for the cultivar ‘Berkeley’ and 61% for‘Bluecrop’. However, the same treatment yielded considerably fewer rooted plants (9 %) in thecase of ‘Spartan’. On an average, IBA promoted development of two to six good quality roots pershoot without callusing at the basal portion of shoots. Roots originated directly from the base of themain shoot. High survival (more than 80 %) was obtained after acclimatization of rooted plants inex vitro conditions. These plants showed normal growth and developmental characteristics,111

compared to conventionally grown plants. Ostrolucka et al. (2007) reported 80 % rooting and 80 –90 % survival after transfer to ex vitro conditions for V. corymbosum genotypes with both in vitroand ex vitro rooting using 0.8 mg l -1 IBA for root induction.Table 5. Rooting of highbush blueberry on WPM medium with 1 mg l -1 IBACultivar Rooting shoots (%) Root number per shoot ± SESpartan 9 6.0 ± 0.6Bluecrop 61 2.3 ± 0.1Berkeley 70 2.6 ± 0.1ConclusionsMicropropagation techniques described in this paper increased multiplication mainly in thehighbush blueberry cultivar ‘Berkeley’ on WPM medium. A rapid in vitro shoot multiplicationprocedure could have a crucial impact on our ability to rapidly proliferate and maintain desirablehighbush blueberry cultivars, while at the same time allowing for initial plant material availabilitythroughout the year. By using a zeatin supplemented WPM medium for shoot initiation andproliferation, thousands of plants a year could be produced from a single initial shoot. Incomparison, conventional nursery techniques using multiple-node softwood or hardwood cuttingsproduce only a few plants annually. However, some cultivars of highbush blueberry would stillrequire further research to optimize the proliferation media.Acknowledgements: This work was realized in the framework of project QH82232 from NationalAgency for Agricultural Research (MZe CR).References1. Debnath S.C. and McRae K.B. (2001) In Vitro Culture of Lingonberry (Vaccinium vitis-idaea L.): Theinfluence of Cytokinins and Media Types on Propagation. Small Fruits Review, 1(3), pp. 3-19.2. Debnath S.C. (2007) Strategies to propagate Vaccinium nuclear stocks for the Canadian berry industry.Canadian Journal of Plant Science, 87, pp. 911-922.3. El-Shiekh A., Wildung D.K., Luby J.J., Sargent K.L. and Read, P.E. (1996) Long-term effects ofpropagation by tissue culture or softwood single-node cuttings on growth habit, yield, and berry weightof ‘Northblue’ Blueberry. Journal of the American Society for Horticultural Science, 121 (2), pp. 339-342.4. Gajdosova A., Ostrolucka M.G., Libiakova G., Ondruskova E. and Simala D. (2006) Microclonalpropagation of Vaccinium sp. and Rubus sp. and detection of genetic variability in culture in vitro.Journal of Fruit and Ornamental Plant Research, 14, pp. 103-118.5. Howell A.B. (2009) Update on health benefits of cranberry and blueberry. Acta Horticulturae, 810, pp.779-784.6. Jiang Y., Yu H. Zhang D., He S. and Wang Ch. (2009) Influences of media and cytokinins on shootproliferation of 'Brightwell' and 'Choice' blueberries in vitro. Acta Horticulturae, 810, pp. 581-5867. Kosina J. and Sedlak J. (2006) Rooting of softwood cuttings and micropropagation of selected highbushblueberry cultivars. In: Blueberry and cranberry growing (with ecological aspects), Research Institute ofPomology and Floriculture, Skierniewice, Poland, pp. 131-137.8. Li Y., Tang X., Wu L. and Zhang Z. (2006) Effect of cytokinins on in vitro leaf regeneration ofblueberry. Acta Horticulturae, 715, pp. 417-419.9. Lloyd G. and McCown B. (1981) Commercially-feasible micropropagation of mountain laurel, Kalmialatifolia, by use of shoot tip culture. Combined Proceedings of International Plant Propagators Society,30, pp. 421- 427.10. Mehri-Kamoun R., Mehri H., Faidi A. and Polts V. (2004) Micropropagation of six OHxF (Old Home xFarmingdale) pear rootstocks. Advances in Horticultural Science, 18 (2), pp. 53-59.11. Murashige T. and Skoog F. (1962) A revised medium for rapid growth and bioassays with tobacco tissuecultures. Physiologia Plantarum, 15, pp. 473-497.12. Noe N., Eccher T., Del Signore E. and Montoldi A. (1998) Growth and proliferation in vitro ofVaccinium corymbosum under different irradiance and radiation spectral composition. BiologiaPlantarum, 41 (2), pp. 161-167.13. Ostrolucka M.G., Libiakova G., Ondruskova E. and Gajdosova A. (2004) In vitro propagation ofVaccinium species. Acta Universitias Latviensis, 676, pp. 207-212.112

14. Ostrolucka M.G., Gajdosova A, Libiakova G, Hrubikova K. and Bezo M., (2007) Protocol formicropropagation of Vaccinium corymbosum L., SAS. Nitra, Slovakia, 13 p.15. Paprstein F., Ludvikova J. and Sedlak J. (2005) Cultivars and propagation of highbush blueberries.Scientific Papers of Pomology, 19, pp. 147-152.16. Paprstein F., Holubec V., and Sedlak J. (2006) Introduction of Vaccinium culture in the Czech Republic.Acta Horticulturae, 715, pp. 455−459.17. Paprstein F. and Ludvikova J. (2006) Preliminary results of evaluation of highbush blueberry cultivars inHolovousy. In: Blueberry and cranberry growing (with ecological aspects), Research Institute ofPomology and Floriculture, Skierniewice, Poland, pp. 138−144.18. Reed B.M. and Abdelnour A.E. (1991) The Use of Zeatin to Initiate in Vitro Cultures of VacciniumSpecies and Cultivars. HortScience, 26, pp. 1320-1322.19. Smolarz K. and Chlebowska D. (1997) Growth vigour and yielding of highbush blueberry cv. Bluecroppropagated from semi-woody cuttings and in vitro. Journal of Fruit and Ornamental Plant Research. 2,pp. 53-60.20. Zmarlicki K. (2006) Production and marketing of blueberries in Europe, USA and in Canada. In:Blueberry and cranberry growing (with ecological aspects), Research Institute of Pomology andFloriculture, Skierniewice, Poland, pp. 181−186.INFLUENCE OF DRYING TECHNOLOGY ON THE QUALITY OF DRIED CANDIEDCHAENOMELES JAPONICA DURING STORAGEKALTĒŠANAS TEHNOLOĂIJU IETEKME UZ CHAENOMELES JAPONICA SUKĀŽUKVALITĀTI UZGLABĀŠANAS LAIKĀDalija Seglina, Inta Krasnova, Gunta Heidemane, Silvija RuisaLatvia State Institute of Fruit-Growing, Graudu 1, Dobele LV-3701, Latvia, e-mail: dalija.seglina@lvai.lvAbstractIn the Baltic region, the development of Japanese quince as a fruit crop started in Latvia in 1951.The fruits of Chaenomeles japonica are very firm, acidic, with too low sugar content to beconsumed fresh, but they are useful for processing and valuable because of the high content oforganic acids, vitamin C, phenolic compounds and fresh aroma. The interest for growing Japanesequince in Latvia reached a peak in 1993, but the processing was not solved. Only juice and pureewere produced. Therefore a new Japanese quince processing technology was worked out andpatented (RL patent Nr. LV 12779 B) at Dobele Horticultural Plant Breeding Experimental Station(presently Latvia State Institute of Fruit-Growing) and Latvia University of Agriculture in 2002.In order to enlarge the product spectrum made from Japanese quince, investigations wereconducted at the Latvia State Institute of Fruit-Growing during the 2007. The aim of the presentwork was to characterize the influence of the drying technology on the quality of dried candiedJapanese quince during storage for six months. Two different drying technologies were used: withforced air circulation and vacuum-microwave. The sweet dried Japanese quince products weretested for content of vitamin C, phenolic compounds and changes of colour by using the CIEL*a*b* colour system.The obtained data showed that the content of vitamin C decreased on average by 40 %, but thecontent of phenolic compounds by 17 % in the product. The over colour of the product changedafter two months of storage.KopsavilkumsBaltijas reăionā krūmcidoniju selekcija uzsākta Latvijā kopš 1951. gada. Chaenomeles japonicaaugĜi ir cieti, skābi, ar pārāk mazu cukura daudzumu, lai tos varētu patērēt svaigā veidā, bet,pateicoties augstajam organisko skābju, C vitamīna, fenolu savienojumu saturam un patīkamāaromāta dēĜ, tie ir noderīgi pārstrādei. Interese par Japānas krūmcidoniju audzēšanu Latvijā virsotnisasniedza 1993. gadā, bet netika atrisināta augĜu pārstrāde. Galvenokārt tika ražota sula unbiezenis. TādēĜ Dobeles Dārzkopības selekcijas un izmēăinājumu stacijā (šobrīd Latvijas ValstsaugĜkopības institūtā) un Latvijas Lauksaimniecības universitātē 2002. gadā tika izstrādāta un113

patentēta (LR patents Nr. LV 12779 B) jauna krūmcidoniju pārstrādes tehnoloăija. Pētījums tikaveikts Latvijas Valsts augĜkopības institūtā 2007. gadā, lai paplašinātu krūmcidoniju pārstrādesproduktu piedāvājumu. Pētījuma mērėis bija novērtēt kaltēšanas tehnoloăiju ietekmi uzkrūmcidoniju sukāžu kvalitāti sešu mēnešu uzglabāšanas laikā. Pārbaudītas divas atšėirīgaskaltēšanas tehnoloăijas: piespiedu gaisa cirkulācija un kaltēšana vakuma mikroviĜĦu iekārtā.Krūmcidoniju sukādēm tika noteikts C vitamīna un polifenolu daudzums, kā arī krāsas izmaiĦasCIE L*a*b* krāsu sistēmā. Iegūtie rezultāti rāda, ka pēc 6 mēnešu uzglabāšanas C vitamīna satursproduktā samazinājās vidēji par 40 %, bet polifenolu saturs – par 17 %. Uzglabāšanas laikāprodukta krāsa sāk būtiski izmainīties pēc diviem uzglabāšanas mēnešiem.Key words: Chaenomeles japonica, products, technology.IntroductionThe fruits of Chaenomeles japonica are used in the food industry for processing because of theirhigh content of biologically active compounds: organic acids, vitamin C, phenolic compounds,pectin and aroma components (Lesinska et al., 2006; Ruisa, 1996). Vitamin C content in fruitsdiffers from 41.2 to 105.8 mg 100 g -1 , phenolic compounds range from 523.9 to 1271.7 mg 100 g -1(Krasnova et al., 2007). The juice of Japanese quince can be a useful ingredient for the foodindustry due to its flavour and high acidity. Another property of great interest is its presumed highantioxidant capacity due to the content of vitamin C and phenolic compounds. The content ofvitamin C is 45 to 109 mg 100 ml -1 and phenolic compounds in juice of the Chaenomeles japonicawas 210 – 592 mg 100 ml -1 (Hellin et al., 2003). However, juice and puree from the fruits are notdemanded in the market.To get healthy products from Chaenomeles japonica fruits with better market demand, a newprocessing technology was developed at Dobele in cooperation with the Faculty of FoodTechnology of Latvia Agricultural University. As a result two products were obtained – sweetdried candies and syrup. The technology was patented in 2002 (RL patent Nr. LV 12779 B). Theway of obtaining dried candies is based on cell destruction (mechanically or using lowtemperatures).Various methods can be used for drying: convective drying (COD), vacuum-microwave drying(VMD) and others. Drying in vacuum-microwave influences the quality of product slightly,however, it facilitates production on the industry scale.Small fruit and vegetable companies prefer convective drying, yet increased temperatures are usedvery often to get faster results, which in turn reduce the nutritive value of the product (Diaz-Morotoet al., 2002). Vacuum-microwave drying of food is becomming more and more popular due to itsadvantages and simple usage. By this method microwaves penetrate the interior of the materialcausing water to boil at a relatively low temperature. This creates a high vapour pressure in thecentre of the material, allowing rapid transport of moisture out of the product (Sham et al., 2001).For example, the quality of herbs: parsley and oregano (colour, content of volatile oil, aroma) washigher after drying in VM if compared to CO drying (Boehm et al., 2002). In addition the qualityof Mexican oregano (Lippia berlandieri Schauer) was similar to the results obtained by process ofsublimation, which can be characterized as one of the more cautious drying method (Jaloszinsky etal., 2008; Yousif et al., 2000). Similar results were obtained by testing samples of strawberries andcarrots – in the process of VM drying the content of vitamin C and phenolic compounds keepsbetter and antiradical activity is higher compared to drying in free air admission (Wojdylo et al.,2009; Böhm et al., 2006).The aim of the present work was to characterize the influence of the drying technology on thequality of dried candied Japanese quince during storage for six months.Material and methodsThe experiment was carried out at the Experimental Processing Laboratory of Latvia State Instituteof Fruit-Growing 2007. The object of the study – sweet dried quince candies were madecorresponding to the patented technology (RL patent Nr. LV 12779 B). Sugar was used as asweetener, preservatives and colours were not used in the production. The vegetable chopper114

„Metos RG-350” (http://www.metos.fi/); was used for cutting up Japanese quince fruits; size ofpieces – 13x13x4.5 mm. Two drying methods were used:• Convective drying (COD) – using equipment „ORAKAS” (www.lkv.com/marlemi) onsieves with forced air circulation at +60 ºC; duration of drying on average 8 hours, until themoisture of the product reaches 32±3%.• Vacuum-microwave drying (VMD) - using the equipment „Musson-1”(www.ingredient.spb.ru) by an individual program; moisture of the product: 32±3%;duration of drying depends on the initial moisture content. Duration of drying on averagefor 12 kg was 155 minutes.Ready sweet dried candies were packed in sealed polypropylene (PP) bags (thickness 25 µm),inserted in paper bags. The mass of the product in each bag was 100 ± 5.0 g. Packed candies werestored at a room temperature of 20±2 °C; relative air humidity ~60±2%.The duration of the study was 6 months. Three separate replications of product preparation weremade. The analyses of the product were carried out before packaging (0 days) and two times permonth. At each time of testing, three identical packages were analyzed.The following properties of dried sweetened quince candies were determined:• Vitamin C – the content of ascorbic acid (vitamin C) (mg 100 g -1 ) was determined by theHPLC method LVS EN 14130:2003.• Phenolic compounds - the total content of phenols (mg 100 g -1 ) was determined by themethod of spectrometry, by spectrometer UV-1650-PC at a wave length 765 nm (Singletonet al., 1999).• Colour changes - measured during the storage in CIE L*a*b* colour system using ColourTec PCM/PSM device. The product was placed in a glass container (diameter 30 mm); thechanges in colour were determined for three packages; the measurements were repeated for15 series.The data were statistically evaluated using SPSS (version 11.5) for Windows and MS Excelvariance analysis, significance level at P < 0.05.Results and DiscussionThe colour of the product is one of the important organoleptic parameters for consumers. Sharpchanges in the colour of dried candied quince were observed during storage. These changes occuras a result of the destruction of carotenoids, vitamins and other substances and the action ofenzymes. The changes influence the colour forming component L*, a* and b* values of CIE L* a*b colour system (MacDougall, 2002).Greater colour intensity L* (0=black, 100=white) at the beginning of the study was observed forthe product dried in the microwave vacuum equipment (value 72.4). However, the product dried inforced air circulation was darker (L* value 66.9). The colour of the product changed during storage– colour intensity value L* and b* (+ yellow, - blue) reduced during first 2 months, but a* (+ red, -green) value increased for both product types. Statistically significant differences depending on thedrying method were ascertained between the colour intensity values L* and b* of the product(p=0.004; p=0.009 respectively), but a* value did not differ significantly (p=0.86).The total changes of the colour component of the product during storage characterize the sum ofdifferences – total difference ∆ E ∗ , formed by L*, a* and b* values. The colour changes of theproduct were observed on average after 2 months of storage – products became darker independentof the drying method. Calculating the total colour difference ∆ E ∗ after 48 days of storage one canconclude that differences do exist but are hard to evaluate organoleptically (Figure 1). However,the total colour differences of the product were significant at the end of the study - the value forCOD product was 4.75, but for VMD 10.25.115

8.06.04.95.9∆ E4.02.00.0CODVMDFigure 1. The colour difference ∆E of dried candied quince product, after 48 days of storage(COD - convective drying, VMD - vacuum-microwave drying)Since the drying temperature for both drying methods did not exceed the 60 °C necessary for theMaillard reaction (between amino acids and reducing sugars), there is a reason to assume that theaction of enzymes could cause the browning of Japanese quince candies during their storage.Plant enzymes like oxireductases (polyphenoloxidases, lipoxidases and peroxidases) enables theoxidation and reduction processes of biologically important substances. Enzymatic browning ofplant material is caused mainly by polyphenoloxidases. They are released from membranes becauseof damage occurring to fresh fruits and come into contact with plant phenolic compounds.Polyphenoloxidases dehydrogenate phenolic compounds form into unstable chinons, thereforecausing the formation of melanin (black, brown or red polymer colour). Peroxidases found in plantproducts in turn transfer hydrogen peroxide, oxidizing hydrogen donors. Peroxidases becomeinactive after heating. However, it has been found frequently that inactivated peroxidase after sometime can become active (Baltess, 1998).Phenolic compounds are a large group of organic substances determining fruit taste and aroma.However, the aroma in the skin, flesh and juice of Japanese quince fruits constitutes an essencewhich is a solution of aromatic components in water and not an essential oil. These substances aresusceptible to changes of temperature, so after freezing fruits loose their aroma. Taste of theobtained product – sweet dried candies is good after adding sugar and drying at high temperature.The total content of the phenolic compounds of both dried products at the beginning of theinvestigation was similar: for the product dried in forced air circulation (COD) it was 467.2 mg 100g -1 , but 481.5 mg 100 g -1 after drying by vacuum-microwave (VMD) equipment (Figure 2). Thecontent of phenolic compounds was reduced on average by 9 % in products obtained in COD andby 4.7 % if obtained in VMD, after 2 months of storage. But at the end of the study the totalcontent of phenolic compounds reduced on average by 17 % apart from the drying method.Significant differences (p=0.037) between the quince products obtained by the two drying methodswere ascertained during storage, the higher total content of phenolic compounds remained in VMDproduct method.Several investigations on the biochemical composition of fruits, berries and herbs present evidencethat the VMD method is the better way to preserve the phenolic compounds compared to the CODmethod.Research by Mejia-Meza (2008) at Washington State University on dried bilberries (Vacciniumcorymbosum L.) indicates that the total content of phenolic compounds is maintained the best bydrying at low temperatures (sublimation) followed by the VMD method and after that the CODmethod. Similar results were obtained at Jena and Dresden Universities investigating different VMdrying regimes and vacuum-microwave drying combined with convective pre- and post-drying toimprove the quality of dried strawberries (Böhm, 2006). Convective drying and VM dryingdecreased the content of ascorbic acid to approximately 40 % of the initial value, phenoliccompounds to approximately 35 % in dried strawberries. Jaloszynski et al. (2008) found that thecontent of phenolic compounds were reduced sharply by using CO drying for herbs at 60 – 70 °C.116

500480Phenols,mg 100g -14604404204003800 12 24 36 48 60 72 84 96 108 120CODVMDStorage time, daysFigure 2. Changes of total content of phenolic compounds during storage(COD - convective drying, VMD - vacuum-microwave drying)The content of vitamin C in the products was on average 51 mg 100 g -1 at the beginning of theinvestigation – immediately after drying (Figure 3). A sharp reduction of vitamin C was observedafter 24 days of storage. After 2 months of storage the content of vitamin C was reduced by 29.5 %using the COD method and by 33 % using the VMD, but by 37.9 % and 40.2 % respectively at theend of the investigation. Drying methods used in this study did not influence the content of vitaminC (p=0.71).5550Vitamin C,mg 100g -145403530250 12 24 36 48 60 72 84 96 108 120COD VMD Storage time, daysFigure 3. Changes of the content of vitamin C during storage(COD - convective drying, VMD - vacuum-microwave drying)Due to the high content of total phenolic compounds in fruits vitamin C was retained during theprocessing. Several researchers emphasized that drying by the vacuum-microwave method thevitamin C in the product was preserved better than by the CO or spraying methods. Tein et al.(1998) compared the effect of different drying methods on the quality of dried carrot slices andestablished fact that a higher content of vitamin C and carotene was found in the product dried bythe VMD method compared to the COD method. The content of vitamin C was influenced not onlyby the drying method, but also by the microwave power intensity. Nurul Asyikin et al. (2007)studied the content of vitamin C in the dried fruits of papaya depending on the drying temperatureand concluded that lower microwave intensity helped to keep a higher content of vitamin C in theproduct.ConclusionsThe drying methods used in this study influenced the quality of the dried candied Japanese quince.A higher content of total phenolic compounds remained in the product dried by the vacuum-117

microwave method. The drying method did not influence the content of vitamin C in the product(p=0.71). The colour of the product started to change after two months of storage.For the industrial production of sweet dried candies it is recommended to use the vacuummicrowavedrying method, and the optimal storage time of the product without significant losses ofquality is two months.References1. Bohem M., Bade M., Kurz B. (2002) Quality stabilization f herbs using a combined vacuum-microwavedrying process. Advances in Food Science, 24 (2), pp. 55-61.2. Böhm V., Kühnert S., Rohm H., Scholze G. (2006) Improving the nutritional quality of microwavevacuumdried strawberries : A preliminary study. Food science and technology international, 12 (1), pp.67-75.3. Diaz-Moroto M.C, Perez-Coello M.S., Cabezudo M.D. (2002) Effect of different drying methods on thevolatile components of the parsley (Patroselinum crispum L.). European Food Research andTechnology, 215, pp. 227-230.4. Hellin P., Vila R., Jordan M.J., Laencina J., Rumpunen K., Ros J.M. (2003) Characteristics andComposition of Chaenomeles Fruit Juice. Japanese Quince Potential Fruit Crop for Northern Europe.Edited by K.Rumpunen, Department of Crop Science, Balsgard, pp. 93-98.5. Jaloszynski K., Figel A., Wojdylo A. (2008) Drying kinetics and antioxidant activity of oregano. ActaAgrophysica, 11 (1), pp. 81-90.6. Krasnova I., Ruisa S., Seglina D. (2007) Investigations of the Biochemical Composition of Chaenomelesjaponica fruits. Chemine Technologija, Kaunas, 4 (46), pp. 16-20.7. Lesinska E., Przybylski R., Eskin N.A.M. (2006) Some Volatile and Nonvolatile Flavor Components ofthe Dwarf Quince (Chaenomeles japonica), 53 (3), pp. 854-856.8. Mac Dougall D.B. (2002) Colour measurement of food. Colour in food: Improving quality. pp. 43.9. Mejia-Meza E.I., Yanez J.A., Davies N.M., Rasco B., Younce F., Remsberg C.M., Clary C. (2008)Improving Nutritional Value of Dried Blueberries (Vaccinium corymbosum L.) Combining Microwave-Vacuum, Hot-Air Drying and Freeze Drying Technologies. International Journal of Food Engineering,4 (5), Article 5. Available at: www.bepress.com/ijfe/vol4/iss5/art5, 08.04.2009.10. Nurul Asyikin M. Z., Muhamad I.I., Salleh M.L. (2007) Drying Characteristics of Papaya (CaricaPapaya L.) During Microwave-Vacuum Treatment. International Journal of Engineering andTechnology, 4 (10), pp. 15-20.11. Ruisa S. (1996) Investigations on Organic Acid Content in Fruits, Processing Products and Seed OilContent of Chaenomeles japonica. Problems of Fruit Plant Breeding, Collection of Scientific Articles,Jelgava, pp. 24-31.12. Sham P.W.Y., Scaman C.H., Durance T.D. (2001) Texture of vacuum microwave dehydrated applechips as affected by calcium pretreatment, vacuum level and apple variety. Journal of Food Science, 66(9), pp. 1341-1347.13. Singleton, V.L., Orthofer, R.M., Lamuela-Raventos, R.M. (1999) Analysis of total phenols and otheroxidation substates and antioxidants by means of Folin-Ciocalteu reagent. – Methods in Enzymology,299, pp. 152-178.14. Tein M.L., Timothy D.D., Christine H.S. (1998) Characterization of vacuum microwave, air and freezedried carrot slices. Food research international, 31 (2), pp. 111-117.15. Wojdylo A., Figiel A., Oszmianski J. (2009) Effect of Drying Methods with the Application of VacuumMicrowaves on the Bioactive Compounds, Color, and Antioxidant Activity of Strawberry Fruits.Agricultural Food Chemistry, 57 (4), pp. 1337–1343.16. Yousif A.N., Durance T.D., Scaman C.H., Girard B. (2002) Headspace volatiles and psychicalcharacteristic of vacuum-microwave, air, and freeze dried oregano (Lippia berlandieri schauer). Journalof Food Science, 65 (6), pp. 926-930.118

SHORT INFORMATION ABOUT THE HISTORY OF THE COMMERCIALCULTIVATION HIGHBUSH BLUEBERRY IN POLANDĪSA INFORMĀCIJA PAR AUGSTKRŪMU KRŪMMELLEĥU KOMERCIĀLASAUDZĒŠANAS VĒSTURI POLIJĀKazimierz SmolarzInstitute of Pomology & Floriculture, Pomologiczna 18, 96-100 Skierniewice, Poland,e-mail: ksmolarz@insad.plAbstractThe first notes concerning highbush blueberry cultivation in Poland appeared about the year 1935 –1939. The results obtained at that time were not positive. Also the first trials with the cultivation ofthis plants soon after the second world war were negative. The problem was in choosing impropersoil for the blueberry. The further experiments concerning the quality of soil and mineralfertilization brought interesting results and caused an increase of interest in blueberry cultivation onlarge scale. The experimental field in Skierniewice, belonging to the Warsaw AgricultureUniversity has plots with different long-term soil mineral fertilization and different pH status since1923. On those plots the first methodical experiment with the highbush blueberry was begun in1976 on drained soil with 1.3 % of humus. This experiment brought very interesting and importantresults. It explained some basic problems, first of all the meaning of pH value and fertilization ofsoil for blueberry cultivation. It appeared that blueberry did crop well when soil pH, determined inKCl is between 3.5 – 4.0. The soil should be rather light and level of ground water not higher than40 – 50 cm below the surface. It was also found that blueberry reacts badly for lack of nitrogen andto a lesser degree, for deficiency of P and K. The growth and yield of blueberry plants on singleplots was differentiated and depended mainly on soil pH and nitrogen fertilization. The plants ofcultivars Bluecrop and Jersey still grow well, though they are 32 years old. Now the rejuvenationpruning research is being done on them.KopsavilkumsPirmās ziĦas par augstkrūmu krūmmelleĦu audzēšanu Polijā ir atrodamas laikā no 1935. – 1939.gadam. Tomēr tā laika pētījumu rezultāti nebija pozitīvi. Arī pirmie izmēăinājumi par šo kultūruaudzēšanu, neilgi pēc otrā pasaules kara, bija negatīvi. Problēma bija saistīta ar nepiemērotuaugsnes izvēli krūmmellenēm. Turpmākie eksperimenti par augsnes kvalitāti un augu minerālobarošanos deva interesantus rezultātus un radīja interesi par krūmmelleĦu audzēšanu lielākosapjomos. Varšavas Lauksaimniecības universitātes Eksperimentālajā laukā Skiernevicē kopš 1923.gada ir ierīkoti ilgstoši izmēăinājumi ar dažādiem augu mēslošanas un pH līmeĦiem. Šajospētījumos pirmie eksperimenti ar krūmmellenēm tika sākti 1976. gadā drenētā augsnē ar 1.3 %humusa saturu. Šis eksperiments deva interesantus un nozīmīgus rezultātus. Tie izskaidroja dažaspamatproblēmas, pirmkārt par augsnes pH saturu un mēslošanu. Izrādījās, ka krūmmellenes labiražoja, kad augsnes pH KCL izvilkumā bija robežās no 3.5 – 4.0. Augsnei jābūt vieglai ungruntsūdenim jābūt ne augstākam par 40 – 50 cm. Tika arī konstatēts, ka krūmmellenes slikti reaăēuz slāpekĜa un, mazākā pakāpē, uz fosfora un kālija trūkumu. KrūmmelleĦu augšanas un ražībasatšėirības noteica, galvenokārt, augsnes pH un mēslošana ar slāpekli. Šėirnes ‘Bluecrop’ un‘Jersey’ joprojām aug normāli, lai arī to vecums jau ir 32 gadi. Pašreiz šajos lauciĦos turpināspētījumi par atjaunojošo apgriešanu.Key words: Vaccinium corymbosum, fertilization, soil pH, cultivar evaluation, pruning, yield.IntroductionThe highbush blueberry (Vaccinium corymbosum L.) grows in the wild in the North – East part ofUSA. The bushes grow up to 3 m high, are relatively frost resistant can stand the temperature up to-35 o C. The bushes can be found in forest, on high peat soil, as well as on light, acid mineral soil.The roots grow close under the soil surface, most of them are very thin, only some of them arethick enough to support the plant. The blueberries are very fruitful, the fruits are relatively large,tasty, mainly for fresh eating but also for processing.119

At present the highbush blueberry is being cultivated in many countries of the world. Also inPoland, since many years the people have been interested in growing them, however the plants didnot grow well in the field. In the sixties of past century there was an idea to cultivate the plants inconcrete rings or in deep holes, covered with plastic and filled with acid peat or with the forest soilmixed with acid peat. In such conditions the plants grew very well and gave a good yield. Theobservation of those plants convinced us that the blueberries can be cultivated in our climatic andsoil condition. However the method of growing plants in concrete rings or deep hols could not berecommended for commercial cultivation from economical reasons. We started to look for thereasons, why in Poland the blueberries do not grow well in the open field, like it is in the USA andmany other countries.In seventies of past century some experiments were started to explain this phenomenon.Materials and MethodsIn 1976 one of the first experiments was set on the Experimental Field belonging to the Dept ofAgricultural Chemistry of Warsaw Agricultural College (SGGW) in Skierniewice. The plots of thisExperimental Field are being characterised by constant fertilization, since 1923, with the somemineral compounds. ( 0-control, CaNPK, NPK, PK, PN and KN). No organic fertilization is beingused. The experiment with blueberries was set on plots with mineral fertilizers and characterised bylow pH of soil. On those plots the nitrogen is being used only as ammonium sulphate.The soil on whole Experimental Field was drained, and belongs to the IV class according to Polishnomenclature. The same way of fertilization influences on the content of single compounds in thesoil, and differentiates its pH.Results and DiscusionBy planting the blueberry on such plots, it was possible to conclude after some years, what is theinfluence of single compounds on the growth and cropping of two blueberry cultivars – Bluecropand Jersey, used in this experiment (Table 1.)Table 1. Soil pH (KCl) and concentration of the nutrients (mg 100 g -1 soil)Treatments pH P K Ca Mg0 4.4 2.7 5.6 29.0 3.2Ca NPK 4.5 11.2 17.8 34.0 3.4NPK 3,6 10.8 13.5 14.1 2.1PK 4.5 11.9 20.2 25.0 2.6PN 3.5 10.6 4.7 15.2 2.0KN 3.7 3.3 13.7 13.8 2.1• After 60 years of treatmentThe main aim of this experiment was also to find out what are the possibilities of growingblueberries not only on light sandy soils and but also on more fertile soil of low pH 3,5 – 4,0 (Table2.) The method of determination of soil pH in USA and in Poland are different and this causedgreat misunderstanding in choosing the right soil for blueberry cultivation in our country. In Polishchemical stations pH is determined in KCl what means, that most suitable for blueberries soil withpH 3,5 – 4,0 ( this value according to American method of pH determination would be pH 4.0 –5.0). The Polish IV class soil ( the type which is dominated in Poland ) might be suitable forblueberry, in case that the other physical conditions required by this plant are proper. Those typesof soil are more fertile, do not require so much of irrigation in comparison to light sandy soil.Table 2. Average yield of two cultivars of highbush blueberry (kg/plant) (1988 – 1991 ).Treatments Bluecrop Jersey0 0.94 a 0.68 aCa NPK 0.90 a 1.34 bNPK 3.47 c 2.38 cPK 1.26 a 0.76 aPN 2.09b 1.53bKN 2.99 c 1. 67 b120

Evaluation of cultivars. After the resolving of the soil and its pH problem, the next experimentswere devoted to the evaluation of blueberry cultivars, their economical value and suitability forgrowing in Polish condition. In 1978 was performed the first experiment in Pomological Orchardof our Institute to compare the value of different blueberry cultivars. The plants were not irrigatedbut some of them gave relatively good crop. On the basis of this experiment the following cultivarswere selected as the most suitable for cultivation in our condition: Weymouth, Earliblue, Concord,Bluecrop, Jersey, Herbert, Darrow (Table 3).Table 3. Yield of highbush blueberry cultivars in kg/plant and mean weight of 100 berries in g fromyears 1984 – 1986 and 1988 – 1990Cultivars 1980 - 83 1984 - 86 1988 - 90 1993 - 94WheighTotal fromof fruits12 years(g)Earliblue 1.57 cd 2.40 abc 1.38 a 1.64 ab 6.99 a 109Weymouth 1.65 d 3.36 e 2.68 def 5.06 f 12.75 e 100Collins 1.01 a 2.56 bcd 2.08 bcd 1.18 a 6.83 a 131Bluecrop 1.42 bcd 2.68 cd 3.29 f 3.21 de 10.60 d 140Concord 1.38 bcd 2.43 bcd 2.86 ef 3.39 e 10.06 cd 83Ivanhoe 1.64 d 2.32 abc 2.51 cde 1.28 ab 7.75 ab 133Jersey 1.60 cd 2.00 ab 1.86 abc 1.99 bc bc 7.45 a 83Herbert 1.63 d 1.78 a 1.78 ab 3.42 e 8.61 abc 144Darrow 1.15 ab 2.11 abc 2.09 bcd 2.63 cd 7.98 ab 197Coville 1.34 bc 2.73 cd 2.29 b-e 3.19 de 9.55 bcd 131Lateblue 1.91 e 2.66 cd 2.45 b-e 3.72 e 10.74 d 12913-16-A 2.05 e. 3.05 de 2.87 ef 4.06 f 12.93 e 76* It is the first experiment with cultivar evaluation in PolandThe evaluation of newly bred cultivars are still being conducted in Skierniewice, some of them:Spartan, Duke, Bluegold, Nelson, Toro, Reka, Puru, Nui, Denise Blue, Brigitta, Bonifacy (obtainedby dr. Pliszka) have been introduced for cultivation.At present the following newest cultivars are tested: Aurora, Draper, Liberty, Chandler, Bonus andfew others. The suitability of cultivars: Rubel, Hard Blue are being studied for their suitability formechanical harvesting and processing of fruits.Some other experiments concerning pruning methods of different age plants, nitrogen fertilization,weeds control, irrigation and fertigation are being conducted at present (Table 4).Table 4. Influence of pruning on the yield of blueberry Bluecrop cvYield kg/plantPruning1993 r. 1994 r. 1995 r. 1996 r. 1997 r.Sum 1993 –1997Control 3,24 3,48 3,51 1,66 1,72 13,61Very strong 3,14 4,07 5,40 2,97 4,09 19,67Medium 3,26 4,68 5,17 2,44 3,27 18,82Light 2,98 3,53 3,46 1,28 1,76 13,01ConclusionThe blueberry can be cultivated on different types of soil, more or les fertile but characterized bygood physical condition and the proper pH levels.The pH of soil, the determined in KCl, should be 3.5 – 4.0.The blueberry reacts very strongly for lack of nitrogen in soil, but the doses per ha of thiscompound should not be higher than 100 kg NThe blueberry is less sensitive to the deficiency of potassium and phosphorous than for nitrogen.The cultivars, which can be recommended for cultivation are: Earliblue, Spartan, Duke, Bluecrop,Nelson.The recent results suggest that also Toro, Bluegold, Brigitta, Draper, Liberty can be recommend.121

The plants should be pruned since they start cropping. The rejuvenesce pruning should be donewhen the plantation is about 15 year old.It is recommended to use in rows organic substances like sawdust from coniferous trees, acid peat,bark and stow.It is also possible to spread in rows the black plastic.References1. Smolarz K. (1996) Wpływ wieloletniego nawoŜenia mineralnego na wzrost i plonowanie kilkugatunków roślin jagodowych. Monografie i Rozprawy, Inst. Sadow.Kwiac., Skierniewice.2. Smolarz K. (1997) Wzrost i plonowanie 12 odmian borówki wysokiej w środkowej części Polski. Zesz.Nauk. Inst.. Sadow. Kwiac., 4, pp. 97-110.3. Smolarz K., Chlebowska D. (2004) Wpływ rozstawy i nawoŜenia azotowego na siłę wzrostu iplonowanie borówki wysokiej. Zesz. Nauk. Inst. Sadow. Kwiac., 12, pp. 155-162.CLOUDBERRY BREEDING IN NORWAYLĀCEĥU SELEKCIJA NORVĒĂIJĀEivind Uleberg, Gunnlaug Røthe, Inger MartinussenNorwegian Institute for Agricultural and Environmental Research, Bioforsk Nord Holt, Box 2284, 9269Tromsø, Norway, e-mail: eivind.uleberg@bioforsk.noAbstractTwo female and two male cultivars have previously been released as a result of clone evaluation atBioforsk Nord Holt. The selection criteria were the number of pistils or stamens per flower, thenumber of flowers and the number of shoots per m 2 . Currently a new group of clones are beingevaluated with the aim of finding new cultivars for release. The preliminary results on floweringand berry production show a strong variance in these traits. Over three years of registration, thenumber of produced flowers varied from under 100 for the weakest clone to more than 1000 for thebest clone. The number of produced berries varied from 9 for the weakest clone to 242 for the bestclone. The three clones that produced the most flowers all had very low berry production anddeviated from the rest of the clones by having high numbers of flowers per harvested berry. Newselection criteria will be considered before selection of new cultivars for release. In addition to theproduction traits, new selection criteria will most likely include berry contents, such as the levels ofantioxidants.KopsavilkumsKlonu izvērtēšanas rezultātā Bioforsk Nord Holt tika izveidotas divas sievišėās un divas vīrišėāslāceĦu šėirnes. Izlases kritēriji bija drīksnu vai putekšĦnīcu skaits ziedā, ziedu un dzinumu skaitsuz kvadrātmetru. Pašlaik tiek izvērtēta jauna klonu grupa, lai iegūtu jaunas šėirnes. Iepriekšējierezultāti parāda lielas atšėirības starp klonu ziedēšanu un ogu ražošanu. Trīs gadu laikā ziedu skaitsuz m 2 svārstījās no mazāk nekā 100 sliktākajam klonam līdz vairāk nekā 1000 labākajam klonam,bet izveidoto ogu skaits – no 9 līdz 242. Trīs kloni, kuriem novēroja visbagātīgāko ziedēšanu,veidoja Ĝoti maz ogu un atšėīrās no pārējiem ar lielu ziedu skaitu, attiecībā pret izveidoto oguskaitu. Pirms jaunu šėirĦu izdalīšanas tiks izstrādāti jauni izlases kritēriji – neskaitot ražošanasīpatnības, tiks iekĜauts arī ogu sastāvs, piemēram, antioksidantu saturs.Key words: cloudberry, clone evaluation, selection criteriaIntroductionTwo female (‘Fjellgull’ and ‘Fjordgull’) and two male (‘Apollen’ and ‘Apolto’) cultivars ofcloudberry (Rubus chamaemorus) have previously been released as a result of clone evaluation atthe Norwegian Institute for Agricultural and Environmental Research. The evaluation consisted ofcomparisons of different clones under controlled environmental conditions. Selection criteria were122

the number of pistils (female clones) or stamens (male clones) per flower, the number of flowersand the number of shoots per m 2 (Rapp, 1991). Currently a number of clones collected fromdifferent wild populations are being evaluated at Bioforsk Nord Holt. The group consists of 11female and 1 male genotype collected around 1995. The aim of these evaluations is to find newgenotypes suited for cultivar release. Preliminary results from these evaluations are presented here,focusing on the female clones for the parameters of the number of flowers and numbers of berries.Materials and methodsThe evaluations were performed at Bioforsk Nord Holt in Tromsø (latitude 69º40’, altitude 30meters above sea level). The different clones were planted on peat in open benches outdoors. Thedifferent clones were planted in separated squares that contained 16 plants of the clone, and everyclone was represented in two different squares at random positions on the bench. The bench wasestablished in 2003, the square size within the bench was 0,8 m 2 . The squares were separated toavoid rhizomes propagation between the squares.Table 1. Description of the evaluated clones.Number Origin Sex Latitude Altitude002 England Female 54º30’ .102 Aust Agder Female 58º30’ 50104 Aust Agder Female 58º30’ 350105 Aust Agder Male 58º30’ 650106 Aust Agder Female 58º30’ 650202 Hedmark Female 62º30’ 50206 Hedmark Female 62º30’ 650208 Hedmark Female 62º30’ 950304 Nordland Female 66º30’ 350306 Nordland Female 66º30’ 650404 Finnmark Female 70º30’ 350602 Svalbard Female 78º38’ 80Table 1 gives a brief description of the evaluated clones. Ten clones were collected from fourdifferent Norwegian counties; two in Northern Norway (Finnmark and Nordland), and two inSouthern Norway (Aust Agder and Hedmark). In addition, one clone was collected in England andone at Svalbard. The clones were collected at altitudes ranging from 50 to 950 meters above sealevel. One of the clones was male. In addition, 5 male clones were located on nearby benches, thus6 different male clones were available for the pollination of the evaluated female clones.The first berries were produced in 2005, and from 2006 flowering and berry production have beenregistered. Number of flowers are registered once every season, thus both number of flowers,number of buds and number of withered flowers are registered and added up to a measure of totalnumber of flowers. Berries are harvested at maturity and are registered several times every season.Registrations are made for number of drupes and berry weight.Results and DiscussionFigure 1 shows the number of flowers produced by the different clones. There are large differencesin the total number of flowers over the three years, with clone 106 being the best with just over1000 flowers registered and clone 202 the poorest with fewer than 100 registered flowers.123

Figure 1. Number of flowers produced by the different clonesin the years 2006, 2007 and 2008.Figure 2 shows the berry production of the clones. The results are surprising based on flowerproduction as the clones that produced the most flowers would be expected to produce mostberries. In this case, the three clones that produced most flowers have a very low production ofberries. The three clones that produced the most berries, clones 002, 102 and 208, all hadintermediate flower production.Figure 2. Number of harvested berries produced by the different clonesin the years 2006, 2007 and 2008.Table 2 combines the results for the number of flowers and the number of berries and shows thenumber of registred flowers per harvested berry. In general, most clones show similar results,ranging from 1,88 to 2,90 flowers per berry over the three years. Three clones deviate from this.Clones 104, 106 and 404. Again, these are the clones that produced the most flowers and lessberries.Based on the registrations on flowering, the clones with high numbers of flowers per producedberry do not seem to be neither particularly late nor early flowerers and it also seems that therewere male flowers available at the time of flowering. Thus, deviating flowering times do notexplain the small amount of berries produced from the abundance of flowers, but as flowering isregistered only once per year it still may be that some tendencies that the registered data are notshowing up.In this case there were 6 different male clones available for pollination. It may be that the femaleclones with low berry production combine poorly with the available male clones, which mightexplain the low production.124

Table 2. Number of flowers per harvested berry for the different clones in the years 2006, 2007 and2008.Clone 2006 2007 2008 Totally002 2,26 1,16 2,72 2,04102 2,88 2,07 2,56 2,45104 15,63 32,11 41,29 24,31106 92,00 63,25 389,00 112,22202 2,52 1,57 2,73 2,41206 2,37 1,39 3,61 2,34208 4,05 2,68 3,02 2,98304 2,93 1,69 2,38 2,34306 3,48 1,30 1,98 1,88404 15,54 27,11 73,33 26,64602 4,29 2,03 3,12 2,90The northern clones might be expected to be best adapted to the environmental conditions inTromsø. However, the clones with the highest berry production all had a southern origin. At thesame time other southern clones produced few berries, so there were no obvious tendenciesconcerning origin when berry production was considered. For flower production, the three clonesfrom Aust Agder (102, 104 and 106) all produced above average, while the clones from Nordlandand Hedmark produced at different levels. In total, origin did not explain the variations inproduction.Based on these evaluations, selection based on the number of flowers and the number of pistils maytheoretically entail a risk of selecting genotypes with high potential, but low ability for productionunder certain environmental conditions. Thus, there may be genotype-environment interactionsinvolved, such that specific genotypes are adapted to specific environmental conditions. Moreknowledge about the genetic and environmental basis for berry production is needed to find theoptimal selection criteria. The genotypes evaluated here are also planted at different natural sites inNorway. Registrations from these sites may provide additional information about the differentgenotypes and environmental factors influencing berry production.Further evaluations will be done before the selection of new cultivars for release. In addition toproduction traits, the new selection criteria most likely will include berry contents, such as the levelof antioxidants.References1. Rapp K. (1991) Selection for high berry yield and development of varieties of cloudberry (Rubuschamaemorus L.). Norsk Landbruksforskning, 5, pp. 359-367.FUNGAL DISEASES OF VACCINIUM MACROCARPON IN LATVIAVACCINIUM MACROCARPON SLIMĪBAS LATVIJĀLiga Vilka, Regina Rancane, Maija EiheLatvian Plant Protection Research Centre, Lielvardes 36/36, Riga, LV – 1006, Latviae-mail: regina rancane@laapc.lvAbstractThe American cranberry (Vaccinium macrocarpon Ait.) has been known for fifteen years, butfungal diseases have been investigated only last years in Latvia. Upright dieback and berries rotwere observed several years ago, but growers did not know the causal agents of these symptoms.The aim of the study was to detect the causal agents of cranberry diseases in Latvia. Samples ofupright dieback (in summer) and berries (during the harvesting) from different regions of Latviawere collected for causal agent detection. Botrytis cinerea, Fusiccocum putrefaciens, Phomopsisvaccinii, Pestalotia vaccinii, Discosia artocrea, Physalospora vaccinii were detected from upright125

dieback. Botrytis cinerea, Allantophomopsis cytisporea, Fusiccocum putrefaciens, Phomopsisvaccinii, Coleophoma empetri, Phyllosticta elongata, Physalospora vaccinii, Pestalotia vaccinii,Gloeosporium minus and Discosia artocreas were detected from rotted berries. In the futureFusiccocum putrefaciens and Phomopsis vaccinii could become the most harmful fungi in thecranberry plantations, because it is difficult to control them.KopsavilkumsLielogu dzērvenes (Vaccinium macrocarpon Ait.) Latvijā jau ir zināmas vairāk kā piecpadsmitgadus, bet to slimības pētītas tikai pēdējos gados. Lai gan audzētāji dzinumu atmiršanu un ogupuves pazīmes bija novērojuši jau iepriekš, tomēr neviens īsti nezināja, kas ierosina šīs slimības.Lai noteiktu slimību ierosinātājus, no lielogu dzērveĦu stādījumiem dažādos audzēšanas rajonosLatvijā vasarā tika ievākti vertikālo dzinumu atmiršanas paraugi, bet ogas - ražas vākšanas laikā.No vertikāliem atmirušiem dzinumiem tika noteiktas sekojošas slimības: Botrytis cinerea,Fusiccocum putrefaciens, Phomopsis vaccinii, Pestalotia vaccinii, Discosia artocrea,Physalospora vaccinii. No puves bojātām ogām tika noteikti: Botrytis cinerea, Allantophomopsiscytisporea, Fusiccocum putrefaciens, Phomopsis vaccinii, Coleophoma empetri, Phyllostictaelongata, Physalospora vaccinii, Pestalotia vaccinii and Discosia artocreas. Turpmāk nopietnusbojājumus varētu izraisīt Fusiccocum putrefaciens un Phomopsis vaccinii izplatība dzērveĦustādījumos, jo šo ierosināto slimību ierobežošana ir sarežăīta.Key words: cranberry diseases, upright dieback, berries rot, causal agent.IntroductionThe American cranberry (Vaccinium macrocarpon) is a perspective and marketable culture in themarket of Latvia. The climate and peat bogs are similar to the cranberry growing areas in NorthAmerica (Ripa, 1996). Fungal diseases are one of the most important problems, because theyreduce and damage the quality of the harvest in America. The cranberry is a well known cultivatedfruit crop for fifteen years in Latvia as well, and some investigations of cranberry diseases startedin 2004, but significant studies - in 2006. Mainly uprights dieback, blossom blight and berry rotwere caused by fungi in North America and in Latvia as well. Detection of cranberry diseases isimportant to make control options in future.The study aim was to detect the causal agents of cranberry diseases in Latvia.Materials and methodsEight cranberry plantations (Jelgava, Talsi, Riga, Kuldiga, Liepaja, Aluksne, Cesis and Gulbenedistricts) in 2007 were inspected during the flowering and harvesting time. From cranberryplantations in different regions in Latvia were taken samples of upright dieback, blossoms, ovariesin summer, but berries were taken at harvest time.Samples of upright dieback, blossoms and ovaries blight were put in a moisture camera (wet filterpaper in Petri dishes) and kept at room temperature (20 – 25 o C) in sunlight.At harvest from each farm 200 sound berries were collected randomly along a diagonal through theplantation; in total 1200 berries from six plantations. Berries were kept in plastic bags inrefrigerated storage at +5 o C for up to 4 months. At the end of each month (December – March)the berries were counted and the rotted berries were placed with the cut surface down on potatodextroseagar for causal agent of storage rot detectionThe samples of cranberry diseases before being placed on PDA were surface-disinfested in 70 %alcohol and then rinsed in sterile water twice and pieces of samples put on PDA. The growingfungal colonies were transferred on PDA and pure cultures were incubated at room temperature 20– 25 o C for 3 to 4 weeks. Fungi were identified directly on the isolation plates by comparing themorphological characteristics of the spores and spore bearing structures with descriptions in theliterature (Caruso et al., 1995; Kačergius et al., 2004; Гopлeнкo et al., 1996). Morphologicalcharacteristics of discovered fungi were fixed using microscope OLYMPUS CX31, magnifierMEIJI EMZ and camera SONY DSC – H2. Liga Vilka took the photos for fungi identification andcollected them for the archive database of cranberry diseases in Latvia.126

ResultsFirst time upright dieback in Latvia was observed in 2004. In the last three years the incidence levelof upright dieback was only 1-3 % (from 100 uprights). In the beginning of the summer uprights ofthe previous year usually were dark brown or red-brown, but young uprights - bronzing brown,with top slope and died. These symptoms could be caused by non parasitic diseases (sun, droughtor rain, fertilization problems, etc.) or by fungi.In Latvia from upright dieback 6 causal agents were detected (Table 1), and mainly they causedblossom and ovaries blight and berries rot. From berries damaged by rot 9 causal agents weredetected (Table 1).Table 1. Detected causal agents of cranberry diseases in Latvia, 2007 – 2008Causal agents from upright diebackCausal agents from berries rotBotrytis cinerea Pers.:Fr.Botrytis cinerea Pers.:Fr.Fusicoccum putrefaciens Shear vaccinii Groves) Fusicoccum putrefaciens Shear vaccinii Groves)Phomopsis vaccinii Shear in Shear, N. Stevens, & Phomopsis vaccinii Shear in Shear, N. Stevens, &H. BainH. BainDiscosia artocreas (Tode) Fr.Discosia artocreas (Tode) Fr.Pestalotia vaccinii (Shear) GubaPestalotia vaccinii (Shear) GubaPhysalospora vaccinii (Shear) Arx & E. Müller Physalospora vaccinii (Shear) Arx & E. MüllerPhyllosticta elongata G. J. Weideman in G. J.Weideman, D. M. Boone, & BurdsallColeophoma empetri (Rostr.) Petr.Allantophomopsis cytisporea (Fr.: Fr.) PetrakCausal agents survive and reproduce during the vegetation season on berries or other plants parts,increasing the incidence level of the diseases in the next year.Botrytis cinerea caused upright dieback, blossom and ovaries blight and yellow rot in Latvia.Flowers and ovaries were yellowish brown and later became dark brown. Upright dieback wasbronzing brown, the end of the top sloped. Berry rot was yellow or yellowish brown. Yellow rotmostly appeared in the field, few of berries were affected during storage. Yellow rot can be easilyconfused with end rot caused by Fusicoccum putrefaciens. The fungus grew rapidly at 20 – 24 o Con the PDA. At first the colonies were white, with loose aerial mycelium. Later the myceliumbecame pale gray-brown. On the surface after 10 days white sclerotia appeared, after maturationthey turned black. From black sclerotia developed conidiophores and on the top ovate or elliptical,green-grey conidia appeared. In the moisture camera on upright diebacks, blossoms and ovariesconidia appeared as well. According to the symptoms of cranberry disease and fungus peculiaritiesin the moisture camera and pure culture, upright dieback, blossom and ovaries blight and yellow rotwas caused by Botrytis cinerea Pers.:Fr. The causal agent of the disease was identified based onsymptoms and the morphological characteristics as described by Гopлeнкo et al., 1996 and CarusoF. L., 1995.Fusicoccum putrefaciens caused upright dieback, blossom and ovary blight and end rot in Latvia.Uprights, blossoms and ovaries turned brown and died. Some damaged berries of end rot wereobserved in the field, but mostly berry rot appeared in storage. Berries, damaged in field, were soft,wet, pale yellow, but those damaged during storage turned pale rosy or yellowish brown. Damagefrom rot on berries mostly appeared at the calyx; probably berries were infected by fungus duringblossoming. Later in storage life the rotted berries shrunk. Upright dieback and end rot caused byFusicoccum putrefaciens were the widely distributed cranberry diseases in Latvia. The fungus grewrapidly on PDA at 20 – 24 o C. Aerial mycelium was fluffy, compact, grey-yellow or olive-yellow.Pycnidia under mycelium matured, and on the surface appeared a pale orange cream spore mass.Separately conidia were hyaline, elliptic to fusiform, with aseptate or pseudosaptate, measurementon average 2.0 x 8.8 µm (1.5 – 3 x 6-11µm) (Figure 1).127

Figure 1. Conidia of Fusicoccum putrefaciens on PDA (400x).According to symptoms upright dieback, blossom and ovary blight and end rot were caused byFusicoccum putrefaciens Shear. The teleomorph stage caused by Godronia cassandrae Peck f.vaccinii Groves was not found in Latvia. The causal agent of the disease was identified based onsymptoms and the morphological characteristics described by Гopлeнкo et al., 1996 and Caruso F.L., 1995.Phomopsis vaccinii from upright dieback, blossom and ovary blight and viscid rot samples wasdetected. Uprights, blossoms and ovaries turned brown and died, but viscid rot was off-color,slightly mottled or yellowish brown and firm but wet inside with a viscous, sticky substance.Viscid rot was common in the field and during the first months of storage. Colonies on the PDAgrew up to 15 mm per day; white, circular, and near to centre dark rings were produced. The aerialmycelium was not compact, was grayish white, and toward the centre a wall was produced. Thepycnidia mostly set on the wall. They were 1 – 4 mm in diameter, partly embedded, leathery, palegrey and then turned black (Figure 2). From maturity pycnidia emitted a yellow creamy spore massin the moisture camera on uprights dieback and the pure culture. P. vaccinii had two types ofspores (Figure 3). Alfa conidia were hyaline, one-celled, ellipsoid, with two oil globules at bothends and measured 7.8 x 3.1µm (4.3 – 9.8 x 2.0 – 4.4 µm). Beta conidia were unicellular, hyaline,filiform – hook-shaped at the end, and measured 18.6 x 0.8 µm (13.4 – 22.1 x 0.3 – 1.2 µm).Fig. 2. Pycnidia of P.vaccinii on PDA(10 x).Fig. 3. α and β conidia of P. vaccinii onPDA (400 x).According to the symptoms of cranberry disease and fungus anamorph morphological peculiaritiesin the moisture camera and pure culture, upright dieback, blossom and ovary blight and viscid rotwere caused by Phomopsis vaccinii Shear in Shear, N. Stevens, & H. Bain. The teleomorph stage,which is caused by Diaporthe vaccinii Shear in Shear, N. Stevens, & H. Bain., was not detected inLatvia. The causal agent of the disease was identified based on symptoms and morphologicalcharacteristics as described by EPPO, 1997 and Kačergius et al., 2004.Discosia artocreas from upright dieback, blossom blight and berry rot was detected only in fewsamples. Young uprights were bronze, brown with end of the top sloped and the uprights of lastyear were dark brown. Blossoms damaged by disease were brown, but discosia fruit rot was withyellowish brown spots. Discosia artocreas mostly was detected from upright dieback. The fungus128

grew rapidly on PDA at 20 – 24 o C. Aerial mycelium was low, compact, leathery and pale gray.Mycelium produced paler irregular rings. Colonies were dark pale. In the moisture camera onuprights dieback and pure culture appeared pyriform, dark grey pycnidia. From maturity pycnidiaemitted a yellowish white, creamy spore mass. Separately conidia were hyaline or pale grey,oblong, measured 3.2 x 14.2 µm (2-4 x 12-17 µm); 2 – 3 septates, end of tops had two longappendages (Figure 4). In the pure culture septates and appendages of conidia hardly wereobserved.Figure 4. Conidia of Discosia artocreas in moisture camera and PDA (400 x).According to the symptoms of cranberry disease and fungus morphological peculiarities inmoisture camera and pure culture was caused by Discosia artocreas (Tode) Fr. The teleomorphstage of the fungus caused by Gnomonia setae was not detected. The causal agent of the diseasewas identified based on symptoms and morphological characteristics as described by Гopлeнкo etal., 1996.Pestalotia vaccinii caused upright dieback, blossom blight and pestalotia fruit rot in Latvia.Uprights were bronze, with end of the top sloped. They were spread only in several cranberryplantations and the incidence level was not high. In storage on some berries appeared yellowbrown,circular, slightly sunken rot spots with darker, concentric rings. Fungus grew rapidly on thePDA, at 20 – 24 o C. The aerial mycelium was fluffy, at the centre lemon-white, up to marginswhite appeared. Colonies were lemon-yellow. In the culture through mycelium appeared black,watery, and spore mass was scroll-shaped (Figure 5). In the moisture camera on uprights diebackacervuli matured also. The conidia were elongated fusoid, straight or slightly incurved andmeasured 5.8 x 27.5 (4.7 - 6.8 x 22 - 32 µm). The conidia had five-cells, the apical and basal cellswere hyaline, but inside three cells were green-brown (Figure 6). The conidia had appendages atboth ends. The end of the basal cell had on average a 13.7 µm (9.5 – 18 µm) long appendage, butthe end of the apical cell had 3 – 4 on average 23.9 µm (16 – 33 µm) long moustached appendages.In the culture appeared hyaline, ellipsoid, curved microconidia, in diameter 2.0 x 6.3 µm (1.3 - 2.7x 4.5 – 7.8 µm).Figure 5. Spore mass of P. vaccinii on PDA.Figure 6. Conidia of P. vaccinii on PDA(400x).According to the symptoms of cranberry disease and fungus morphological peculiarities in themoisture camera and the pure culture was caused by Pestalotia vaccinii (Shear) Guba. The causal129

agent of the disease was identified based on symptoms and morphological characteristics describedby Гopлeнкo et al., 1996.Physalospora vaccinii from upright dieback and blotch rot was detected. Uprights from last yearwere dark brown or red-brown and they were collected only from some cranberry plantations. Onberries pale rosy, circular, flattened or sunken spots were observed. Gradually the berries becamedried and shriveled. Only after three or more months in storage blotch rot was observed. Rotdamage on berries mostly appeared at the calyx, probably berries were infected by fungus duringblossoming. The fungus had two different strains. On the PDA the white colony type producedpoor, low, yellowish white mycelium, which was most common in Latvia. The dark colonyproduced poor, low, brownish grey or green-grey mycelium. In the pure culture both strains aftertwo weeks at 20 – 24 o C abundantly produced perithecia, but ascospores matured only after 5weeks. The perithecia of the dark strain were slightly smaller than the perithecia of white strain(Figure 7). They were globose to pyriform, dark brown, at the end of ostiole and had black spines.White stain on average was 199.2 x 42.1 µm (133–251 x 19.6 – 64.1 µm) large, hyaline, fusoidwith eight spores asci produced. Ascospores of white strain were acuminated obovoid, paleyellowish brown, with a punctate surface (Figure 7) and measured 43.5 x 17.4 µm (33.8 – 53.8 x12.3 – 24.9 µm). When ascospores were not mature they were hyaline. Ascospores of the darkstrain were slightly smaller (26.5 – 33.2 x 12.3 – 18.6 µm), broadly obovoid with blunt ends. Bothof strains had a lot of large paraphyses (Figure 8).Figure 7. Asci and perithecia of P. vacciniiparaphyses of P. vaccinii on white strain onPDA(100 x).Figure 8. Asci, ascospores and PDA(400 x).According to symptoms of cranberry disease and fungus teleomorph morphological peculiarities inpure culture, blotch rot and upright dieback were caused by Physalospora vaccinii (Shear) Arx &E. Müller. P. vaccinii has no anamorph stage known in the world. The causal agent of the diseasewas identified based on symptoms and morphological characteristics as described by Caruso F. L.,1995 and Oudeman 1998.Phyllosticta elongata caused fruit rot only in storage. At first on the berries appeared small, lightcoloredspots then they developed soft, watery rot. In the centre of the rot spots dark red ringsappeared. Fungus on the PDA grew rapidly, produced dark, ranges in color from blue-gray togreen-grey and thick colonies. The aerial mycelium was floury, pale blue-grey. In the culture afterfew days at 20 – 24 o C there appeared globose and black and a lot of pycnidia. From maturitypycnidia emitted a pale grey spore mass. The conidia were hyaline, single-celled, obovate tooblong and measured 13.5 x 5.6 µm (10.1 – 16.4 x 3.9 – 7.3 µm) (Figure 9). At the end the conidiahad a mucilaginous, long appendage. When conidia were flown off, the pycnidia turned black.130

Figure 9. Conidia of Physalospora vaccinii on PDA (400 x).According to symptoms of cranberry rot and fungus morphological peculiarities in pure culture,fruit rot was caused by Phyllosticta elongata G. J. Weideman in G. J. Weideman, D. M. Boone, &Burdsall. The teleomorph stage Botryosphaeria vaccinii (Shear) Barr) in the laboratory was notdetected. The causal agent of the disease was identified based on symptoms and morphologicalcharacteristics described by Caruso F. L., 1995 and Weidemann 1983.Coleophoma empetri from ripe rot in storage was detected. Berries were off-colored, soft, wateryinside, and squirted fluid when squeezed. Symptoms of rot were similarly to end rot caused byFusicoccum putrefaciens. Ripe rot is common in Latvia. Fungus on the PDA produced a dark,thick, colony, but started it with a whitish color. Aerial mycelium was low, fluffy and dark grey. Inthe culture appeared dark grey – brown or black, globose at first, but then turned into a disc shapewith fluffy walls. Pycnidia formed in a ring near the outer edge of the colony by group or scatter.The conidia were hyaline, straight, uniformly cylindrical, and slightly punctuated and measured 3.0x 14.8 µm (2.6 – 3.4 x 12.2 – 17.08 µm).Figure 10. Pycnidia of Coleophoma empetri onPDA (10x).Figure 11. Conidia of C. empetri on PDA (400x).According to the symptoms of cranberry rot and fungus morphological peculiarities in pure culture,ripe rot were caused by Coleophoma empetri (Rostr.) Petr. Teleomorph stage was not detected yet.The causal agent of the disease was identified based on symptoms and morphologicalcharacteristics described by Caruso F. L., 1995.Allantophomopsis cytisporea caused black rot in the field, but mostly during the first months instorage. At first damage appeared like pale brown spots, afterwards berries became uniform blackor dark grey. Their mass was firm and dry, but gradually the berries became dried and shriveled.The fungus grew rapidly on potato-dextrose agar at 20 – 24 o C. The colonies were dark green-grayand produced poor, low aerial mycelium. In the moisture camera on berries and the pure cultureappeared globose to pyriform, dark grey pycnidia (Figure 12). From maturity pycnidia emitted ablack, little creamy spore mass. The conidia were hyaline, unicellular, allantoid to lunate,binucleate and measured 7.6 x 2.7 µm (6.6 – 8.6 x 2.2 – 3.8 µm), end of top mucoid appendageswere hardly observed (Figure 13).131

Figure 12. Pycnidia of Allantophomopsiscytisporea on PDA(10x).Figure 13. Conidia of A. cytisporeaon PDA (400x).According to symptoms of cranberry rot and fungus morphological peculiarities moisture cameraand pure culture, black rot were caused by Allantophomopsis cytisporea (Fr.: Fr.) Petrak. Thecausal agent of the disease was identified based on symptoms and morphological characteristicsdescribed by Caruso F. L., 1995 and Гopлeнкo et al., 1996.DiscussionConclusions after two years (2007, 2008) investigations: in Latvia are distributed the same causalagents of cranberry diseases as in North America. According to the literature in the USA andCanada Colletotrichum acutatum, Pestalotia vaccinii, Phyllosticta vaccinii, Physalospora vaccinii,Phomopsis vaccinii and Coleophoma empetri are the most common fungi isolated from sound fruitin storage (Olatinwo et al. 2003; Oudemans et al., 1998). The fungus Fusicoccum putrefaciens wasrecovered in less than 1 % of the total isolations, it is a minor fungal pathogen in the USAaccording to investigations carried out in Michigan (1999 – 2001) and New Jersey (1994 – 1996)(Olatinwo et al. 2003; Stiles et al., 1999). Shear C. L. and Bain H. F. investigated the life cycle offungus in cranberry plantations and established that Fusicoccum putrefaciens grows well at lowtemperatures and in some seasons causes significant losses (Stiles et al. 1999). End rot was mostlyspread in Latvia (2007). The weather conditions are suitable for cranberry growing in Latvia.Upright dieback caused by Phomopsis vaccinii are widely distributed in the USA. In Latvia theincidence level of Phomopsis vaccinii is still low, but in the future it could be an economicallyimportant disease in Latvia as well.Probably causal agents with seedling material have been imported to Latvia. American cranberriesmostly propagate using uprights. It is the most common method in all the world. Next year afterover wintering fungi can develop and infect blossoms, ovaries, uprights and berries again. In thefuture a system should be developed how to control spread of cranberry diseases in plantations.Cranberry growers still do not know all specific agro technical methods of cranberry growing.Damage to cranberries can be caused also by non-parasitic diseases like drought and wrongfertilization.ConclusionUpright dieback of cranberry was caused by several agents. In the Latvian Plant ProtectionResearch Centre laboratory 6 fungi from upright dieback and 9 causal agents from fruit rot weredetected. Mainly the fruit rot symptoms were similar; therefore precise detection of the causalagent is so important.In the future causal agents Fusicoccum putrefaciens and Phomopsis vaccinii could be dangerous,because they cause upright dieback, blossom blight and fruit rot and their control could be difficultin the future.In the future research into to cranberry diseases should be continued.AcknowledgementsThis project was supported by the Ministry of Agriculture of Latvia (Contract No. 43/2007).132

References1. CABI, EPPO, (1997) Diaporthe vaccinei. Quarantine Pests for Europe, pp. 737 – 741.2. Caruso F. L., Ramsdell D.C., eds., (1995) Compendim of Blueberry and Cranberry Diseases, TheAmerican Phytopathological Society, pp. 27 – 87.3. Kačergius A., Jovaišiene Z., Valiuškaite A. (2004) Phomopsis vaccinii on Vaccinium corymbosum inLithuania. Botanica Lithuanica, 10 (1), pp.75 – 80.4. Oudemans, P. V., Caruso F. L., Stretch. A.W. (1998) Cranberry Fruit Rot in the Northeast: A Complexdisease. Plant Disease, 82 (11), pp. 1176 -1184.5. Ripa A. (1996) Amerikas lielogu dzērvene. (The American cranberry), Latvijas Zinību biedrība, 75 p, (inLatvian).6. Stiles C. M., Oudemans P.V. (1999) Distribution of cranberry fruit-rotting fungi in New Jersey andevidence for non-specific host resistance. Phytophatology, 89, pp. 218-225.7. Weidemann G. J., Boone D. M. (1983) Incidence and pathogenicity of Phyllosticta vaccinii andBotryosphaeria vaccinii on cranberry. Plant Disease, 67, pp. 1090 – 1093.8. Гopлeнкo C. B., Бyгa C. B. (1996) Бoлeзни и вpeдитeли клюквы кpyпнoплoднoй. Haвyкa i тэxнiкa,247 c.STORAGE ROTS OF VACCINIUM MACROCARPON SPREADAND DEVELOPMENT IN LATVIAVACCINIUM MACROCARPON OGU PUVES IZPLATĪBA LATVIJĀLiga Vilka, Regina Rancane, Maija EiheLatvian Plant Protection Research Centre, Lielvardes 36/36, Riga, LV – 1006, Latviae-mail: regina.rancane@laapc.lvAbstractAmerican cranberries (Vaccinium macrocarpon Ait.) have been cultivated for ten years in Latviaand their plantations have a tendency to enlarge every year. Latvian cranberry growers realizemainly fresh berries, because it is difficult to keep quality during prolonged storage. One of themain why quality is reduced is berry rot caused by different fungi. Berries from six cranberryplantations in different regions in Latvia for rot detection were taken at harvest time.Different berry rot appeared on 61 % of all berries in storage. In the berries Botrytis cinerea(yellow rot), Allantophomopsis cytisporea (black rot), Fusiccocum putrefaciens (end rot),Phyllosticta elongata (Botryosphaeria fruit rot), Physalopora vaccinii (blotch rot), Phomopsisvaccinii (viscid rot), Pestalotia vaccinii (Pestalotia fruit rot), Coleophoma empetri (ripe rot),Discosia artocreas (Discosia fruit rot) were detected. Causal agents of berry rot had differentdynamics of development. These fungi had different incubation periods. This is importantinformation for the growers that helps to determine the real time for realization, before rotting isstarted. After four months of storage, the amount of causal agents was different from eachplantation area.In further investigations it is necessary to establish conditions influencing the development ofcausal agents.KopsavilkumsLielogu dzērvenes (Vaccinium macrocarpon Ait.) Latvijā ir zināmas jau piecpadsmit gadus un toplatības turpina palielināties. Latvijas lielogu dzērveĦu audzētāji saražoto produkciju realizē svaigāveidā, pārstrādā un, ja iespējams, sasaldē, jo galvenokārt puve uz ogām parādās jau līdzdecembrim. Ražas laikā 2007. gadā, lai noteiktu ogu puves ierosinātājus, tika ievāktas ogas nosešām dažādām lielogu dzērveĦu audzēšanas vietām Latvjā.Uzglabāšanas laikā 61 % ogu bija puves bojātas. No puves bojātām ogām, galvenokārt, tikakonstatētas: Allantophomopsis cytisporea (ogu melnā puve), Fusiccocum putrefaciens (ogugalotnes puve), Phyllosticta elongata (Botryosphaeria ierosinātā ogu puve), Physalopora vaccinii(ogu gaišā puve), Phomopsis vaccinii (viskozā ogu puve), Coleophoma empetri (gatavo ogu puve).Ogu puves ierosinātājiem glabāšanas laikā ir atšėirīga attīstības dinamika, tas nozīmē, ka sēnēm ir133

dažādi inkubācijas periodi. Šī informācija būtu nozīmīga lielogu dzērveĦu audzētājiem, jo tā varētupalīdzēt realizēt produkciju pirms puves parādīšanās. Nosakot puves ierosinātājus glabāšanas laikā,varēja secināt, ka dažādos Latvijas audzēšanas rajonos to izplatība ir atšėirīga.Turpmāk būtu vēl jāveic pētījumi par ogu puves ierosinātāju izplatības veicinošiem iemesliemLatvijā.Key words: cranberry, storage rot, dynamicsIntroductionCranberries are a well known crop in world over 200 years, but for only 10 years in Latvia.Cranberry plantations in Latvia are up to 100 ha. Growers are interested to know how to keep freshberries in good condition during the storage time and what are the reasons for the yield losses.Storage rot fungi are those species that reduce fruit quality during storage.According to literature in the USA and Canada Colletotrichum acutatum, Pestalotia vaccinii,Phyllosticta vaccinii, Physalospora vaccinii, Phomopsis vaccinii and Coleophoma empetri are themost common fungi of sound fruit in storage (Olatinwo et al. 2003; Oudemans et al., 1999; Stileset al., 1999). The fungus Fusicoccum putrefaciens was recovered in less than 1% of the totalisolations. It is the minor fungal pathogen in the USA according to investigations carried out inMichigan (1999 – 2001) and New Jersey (1994 – 1996) (Olatinwo et al. 2003; Stiles et al., 1999).Investigations on the fungal diseases of American cranberries have been carried out since 2004 inLatvia, but storage rotting has not been investigated until 2007.Materials and methodsSix cranberry plantations (Talsi, Riga, Kuldiga, Liepaja, Aluksne, and Cesis districts) wereinspected during harvesting time in 2007. From each cranberry plantation in different regions inLatvia (Figure 1) 200 sound berries (in total 1200 berries) were taken at harvest for causal agentdetection. The cultivar ‘Stevens’ was used for the observations.Figure 1. Inspected cranberry plantations in Latvia.Berries were held in cold camera (+5 º C, RH – 50 %) in plastic boxes until March for causal agentsof storage rot detection. Berries damaged by rotting at the end of each month for four months werecounted. The causal agents of berry rot were isolated only in a pure culture, using potato dextroseagar (PDA). Plates were incubated at room temperature (20 – 25 ° C) for 3 to 4 weeks. Fungi wereidentified directly on the isolation plates by comparing the morphological characteristics of thespores and spore bearing structures with descriptions in the literature (Caruso et al., 1995;Kačergius et al., 2004; Гopлeнкo et al., 1996). The discovered fungi were identified in thelaboratory of Latvian Plant Protection Research Centre. All calculations were performed usingMicrosoft Excel 2003.134

Results und DiscussionBerries were taken from the field at the end of October or in the beginning of November. For thefirst time berries were evaluated at the end of December and on average 12% of all berries wererotted. The assessment of rotted berries was continued for the next three months until the end ofMarch. Spread of berry rot made equal progress during all the storage time (Figure 2).10080%604020122840610end of December end of January end of February end of MarchFigure 2. Dynamics of fruit rot during storage, %.At the end of March berry rot was on average 61 % of all collected berries from the inspectedcranberry plantations in Latvia. This means, berries should be very quickly realized, before they aredamaged by storage rot. It is very difficult to store fresh cranberry in cold cameras withoutfreezing.All rotted berries at the end of each month were sorted and put on the pure culture (used PDA) forcausal agent detection.The spread of berry rot from different growing regions in Latvia where berries were collecteddiffered. Most of all of the rotted berries – 74 % were in the Riga district, but only 52 % of thedamaged berries were in Talsi district (Figure 3). Probably weather conditions and plantation agehad a significant influence on the spread of the berry rot. The cranberry plantation in the Talsidistrict was younger and the climate was optimal in comparison with the plantation in Riga district.%1009080706050403020100746658 566052Aluksne dist. Riga dist. Cesis dist. Kuldiga dist. Talsi dist. Liepaja dist.Figure 3. Berry rot spread from different cranberry growing districts during storage, %.Climate has a strong influence on berry rot caused by different fungi. The winter 2006 was thewarmest of the last 84 years in Latvia. In November, December and January the air temperatureexceeded +10 – 12 ° C and was 7 – 13 ° C above normal. Usually such temperatures are found inSeptember or May. February was the coldest month. In the Aluksne district the air temperature fell135

to – 30 ° C and the snow layer was very thin, in some places it did not exist at all. March was verywarm in Latvia (the warmest of the last 100 years). The air temperature was +17 – 19 ° C, but at theend of month snow in the Aluksne district was observed. After such changeable weather conditionsduring the winter uprights were damaged very hard in the Aluksne district in comparison with otherinspected cranberry plantations in Latvia. In April there was very little rainfall in the Liepaja andRiga districts. May was very hot (25 – 30 ° C) and dry in the Riga district, but in other cranberryplantations was rich rainfall. In June the temperature throughout Latvia was very high up to 5 – 6° C over the average standard and significant rain was observed in the Liepaja, Kuldiga and Talsidistricts, but in the Riga and Aluksne districts first rainfall was only June 14 th and 17 th . Although allinspected cranberry plantations had irrigation systems, probably drought affected growth of theupright and development of the blossoms. During the cranberry flowering time in the Liepajadistrict it was very hot 27 – 30 ° C and wet. The rainfall exceeded three times norm of the month,rainfall was in the Kuldiga and Cesis districts as well. These conditions were favorable for parasiticdiseases, including flower blight and berry rot in the field and in storage. Berry formation wasaffected by heat in August in the Cesis and Aluksne districts, but beneficial conditions for thecranberry growth were in the Talsi and Kuldiga districts. There was rainfall in September in theTalsi, Kuldiga and Riga districts, but in the Aluksne was very dry and hot up to 6 – 7 ° C over thestandard norm. High rainfall amount could increase infection by fungi for berry rot development inthe Riga, Kuldiga and Talsi districts. (www.meteo.lv)From rotted berries in storage fungi Fusiccocum putrefaciens (end rot), Physalopora vaccinii(blotch rot), Phyllosticta elongata (botryosphaeria fruit rot), Allantophomopsis cytisporea (blackrot), Phomopsis vaccinii (viscid rot), Coleophoma empetri (ripe rot), Botrytis cinerea (yellow rot),Discosia artocreas (discosia fruit rot), Pestalotia vaccinii (pestalotia fruit rot) were detected.From inspected cranberry plantations in storage end rot caused by Fusicoccum putrefaciens was themost widespread (Figure 4). End rot developed very quickly during first months (December –January) in storage, and further incidence of berry rot decreased. The ripe rot (causal agentColeophoma empetri) mostly developed during the first months as well, but botryosphaeria fruit rotcaused by Phyllosticta elongata and blotch rot caused by Physalopora vaccinii mostly appeared inMarch. Causal agents of berry rot had different incubation periods. It is important for cranberrygrowers, if the end rot, which is economically the most important disease, was not spread duringthe previously year, fresh berries could be stored until January, other berry rots did not produceimportant material loses.2015%1050end of December end of January end of February end of MarchFusicoccum putrefaciens Phyllosticta elongata Coleophoma empetriPhysalospora vaccinii Allantophomopsis cytisporea Phomopsis vacciniiFigure 4. Causal agents of berry rot spread during storage, %.136

In literature Caruso eds. and Гopлeнкo wrote, the black rot (causal agent Allantophomopsiscytisporea) is common in the field or during the first months in storage (Caruso et al., 1995;Гopлeнкo et al., 1996) but in Latvia, black rot was common till the end of March.The incidence level of berry rot was different in each inspected cranberry plantation. End rot(Fusicoccum putrefaciens) was common in the Aluksne, Kuldiga and Cesis districts, but in theRiga and Liepaja districts pathogen was spread very infrequent (Fig. 5). In the cranberry plantationof the Riga district ripe rot (causal agent Coleophoma empetri) was widely spread and in Liepajadistrict botryosphaeria fruit rot (Phyllosticta elongata) and viscid rot (Phomopsis vaccinii) which isa quarantine organism were found. Phomopsis vaccinii was identified in Lithuania in 2002 as well(Kačergius et al., 2004). Blotch rot caused by Physalospora vaccinii in the cranberry plantationlocated in the Talsi district was widely spread, but this rot in the Liepaja district was not observed.In the cranberry plantations were common 5 - 6 causal agents of berry rot, but from samples takenin the cranberry plantation in the Cesis district, developed only three rots - end rot, botryosphaeriafruit rot and blotch rot.%4035302520151050Aluksne dist. Riga dist. Cesis dist. Kuldiga dist. Talsi dist. Liepaja dist.Fusicoccum putrefaciens Phyllosticta elongata Coleophoma empetriPhysalospora vaccinii Allantophomopsis cytisporea Phomopsis vacciniiFigure 5. Causal agents of berry rot spread in different regions in Latvia during storage, %.ConclusionFrom 6 inspected cranberry plantations in Latvia six causal agents of berry rot were detected in thestorage berries. The end rot caused by Fusicoccum putrefaciens was the most widespread.The incidence level of berry rot in all cranberry plantations was not similar; therefore in futureinvestigations should focus on what promotes the fungi in each cranberry plantation.References1. Caruso F. L., Ramsdell D.C., eds., (1995) Compendim of Blueberry and Cranberry Diseases. TheAmerican Phytopathological Society, pp.27-87.2. Kačergius A., Jovaišiene Z., Valiuškaite A. (2004) Phomopsis vaccinii on Vaccinium corymbosum inLithuania. – Botanica Lithuanica, 10 (1), pp.75-80.3. Olatinwo R.O., Hansos E. J., Schilder A. M. C. (2003) A first assessment of the cranberry fruit rotcomplex in Michigan. Plant Disease, 87 (5), pp. 550-556.4. Oudemans P. V., Caruso F. L., Stretch A. W. (1998) Cranberry fruit rot in the northeast: A complexdisease. Plant Disease, 82, pp. 1176-1184.5. Stiles C. M., Oudemans P.V. (1999) Distribution of cranberry feruit-rotting fungi in New Jersey andevidence for non-specific host resistance. Phytophatology, 89, pp. 218-225.6. Гopлeнкo C. B., Бyгa C. B. (1996) Бoлeзни и вpeдитeли клюквы кpyпнoплoднoй. Минск, Нayкa итexникa, 247 c.7. www.meteo.lv137

EXPERIMENTAL AND RESEARCH WORKS ON SOME OFVACCINIUM TAXA IN BELARUSDAŽU VACCINIUM ĂINTS SUGU EKSPERIMENTĀLIE UN IZPĒTES DARBIBALTKRIEVIJĀYakovlev A. 1 , Rupasova Zh. 1 , Volotovich A. 21 Central Botanical Garden of the NAS of Belarus, e-mail: yakovlev@it.org.by2 Polessky State University, e-mail: volant777@tut.byAbstractCarrying out of the experimental and research works on the study of plants of genus Vaccinium inBelarus can be divided into several stages. The first stage is the study of biological and resourcepotential of wild-growing berry plants: cranberry (V. oxycoccus L), bog blueberries (V. uliginosumL.), lingonberry (V. vitis-idaea L.), bilberries (V. myrtillus L.).The second stage is the introduction of the cultivars of large cranberry (V. macrocarpon Ait.), ofhighbush blueberry (V. corymbosum L.), of lingonberry under the conditions of Belarus to built thefirst experimental and industrial plantations.The third stage is the preparation of scientific manpower, the formation of scientific school, thedevelopment of production schedules of the estate berry culture.The fourth stage is the dilating of the assortment of Vaccinium taxa used in the culture and theaugmentation of the areas of plantings under berrylike cultures.KopsavilkumsEksperimentālos un izpētes darbus par Vaccinium ăints augiem Baltkrievijā var iedalīt vairākosposmos. Pirmais posms ir bioloăiskie pētījumi, kur tiek apsekoti potenciālie savvaĜā sastopamieogaugĜaugi: dzērvenes (V. oxycoccus L), zilenes (V. uliginosum L.), brūklenes (V. vitis-idaea L.) unmellenes (V. myrtillus L.).Otrais posms ir Baltkrievijas audzēšanas apstākĜiem piemērotu lielogu dzērveĦu šėirĦu (V.macrocarpon Ait.), krūmmelleĦu (V. corymbosum L.) un brūkleĦu ieviešana. Tiek ierīkotieksperimentālie un rūpnieciskie stādījumi.Trešais posms – tiek sagatavots zinātniskais personāls, veidojot zinātniskās skolas, izstrādājotražošanas tehnoloăijas katrai ogaugĜu sugai.Ceturtais posms ir Vaccinium ăints augu sortimenta un izmantoto augu stādījumu paplašināšana.Key words: berry plants, natural resources, Belarus, VacciniumIntroductionThere are 5 kinds from berry plants of Vaccinium L. genus, such as V. oxycoccus L., V.microcarpon Ait., V. uliginosum L., V. myrtillus L., V. vitis idaea L. in natural flora of Belarus. Allof them are rich with biologically active substances, possessing by capillary tonic action, by antiinflammatory,bactericidal, hematopoietic and antitumoral kinds of action. Berries of a cranberry, abilberry, a blueberry, a cowberry differ also by high level of anti-oxidative activity. Berriespromote the deducing of radioactive nuclides from an organism and in certain degree make positiveimpact on strengthening of immune system of the person.Requirements of a national economy for wood berry plants are only partially satisfied at theexpense of operation of natural resources which feel now the powerful man-caused andanthropogenic influence. There is a necessity for working out of actions for protection, rational useand intensive reproduction of wood berry plants.The first stage of rational use of plants of Vaccinium L. genus in Belarus is the inventory of wildgrowingbush and it is basically solved. So, according to researches of the Belarus research instituteof forestry with the use of forest regulation materials, it is established that the areas occupied undera cranberry in republic are made by 84514 hectares, and average long-term productivity is 260 kgper hectare (Sautin, etc., 1975). On the data of Ministries of statistics and the analysis of theRepublic of Belarus, the possible purchases of a cranberry at average productivity make 8.7thousand tons taking into account losses. V.I. Parfyonov etc. (1996) cite a little different data that138

the total area under a cranberry makes 552.2 thousand hectares, bacciferous is 364.5 thousandhectares; the general biological stock of berry plants is estimated in 198.9 thousand tons, at averageproductivity of 364.5 kg per hectare.Researches on studying of distribution of a bog bilberry bush and the account of their resourceshave been conducted also. So, according to V.E. Volchkov etc. (1982), in Belarus blueberryplantations occupy the space of 11.8 thousand hectares with a biological stock of berries of 9.7thousand tons. V.I. Sautin (1980) notices that blueberry resources on all territory of Republic ofBelarus make approximately about 10 thousand tons, productivity is about 20 – 235 kg per hectare,depending on ecotope. Possible purchases of a blueberry in woods of Belarus at averageproductivity of 120 kg per hectare can make about 1.7 thousand tons taking into account losses(Karas, 1983).Despite on considerable resources of wild-growing berry plants of Vacciniaceae family, volumesof industrial purchases of berries tend to decrease. They do not satisfy of growing requirements ofthe population for them, as well as they do not satisfy of valuable raw materials in pharmaceuticaland the food-processing industry. So, mid-annual preparations of a cranberry in 1961-1965 madeabout 9 thousand tons, in 1966 – 1970 – 5.8 thousand tons, in 1971 – 1975 – 2.3 thousand tons, andin 1980 – 1985 – did not exceed 1.5 thousand tons (Marsh cranberry, 1987). Last years, owing tothe radionuclide pollution of the large territories caused by failure on the Chernobyl atomic powerstation, volumes of berries purchases have considerably decreased, especially in southern andcentral areas of the Republic, and now make no more than 1 thousand tons (Environment, 2000).Industrial purchases of a blueberry last 25 years are not spent at all. It is connected with manyreasons, mainly, with reasons of anthropogenic character. The general tendency to decrease inresources of natural berry-field is revealed as because of drying land improvement and of workingout of peatbogs, and as a result of forest fires and deforestation (including clearings of care bywood) in the period of vegetation of berry-fields. Besides, the biological efficiency of wildgrowingberry plants rather low and considerably fluctuates on years that also causes decrease involumes of their purchases (Evtuhova, 1991).For today the maximum resources of fruit-berry plants are located on territory of Minsk (28 %) andGomel (26 %) regions. The greatest stocks as a whole on the Republic are marked a bilberry and acranberry (about 33.0 thousand tons or 66 % and 11.2 thousand tons or 22.5 %, accordingly). Bythe least stocks are marked a mountain ash (1.1 thousand tons, or 2.2 %) and a blueberry (1.3thousand tons, or 2.6 % from a biological stock of all principal kinds).On purchases volumes on the first place there are a bilberry (up to 5 thousand tons annually) and acranberry (about 1 thousand tons). The cowberry last years is prepared by the population basicallyfor own needs; blueberry preparations are rather insignificant.A principal cause of low level of use of natural stocks of berries is imperfection of technologiesand systems of the organization of their purchases. Purchases of wild-growing berries are carriedout till now by their purchase at local population that always bears in itself the elements of chance.To provide the greatest efficiency of the organized gathering of berries, the service of the accountof resources and the forecast of terms as well as a crop of berry production is necessary.Thus, all-round studying of biology of wild-growing berry-fields, their distribution, efficiency andstocks has allowed to develop scientific bases of rational use, protection, reproduction,introduction, breeding, technologies of cultivation, storage and production processing. Becameobvious that stocks of berry-fields should increase not only at the expense of protection, rationaland careful use of natural thickets, but also by the organization of industrial culture of berry plants,including a cranberry and a blueberry.Rational use of berry plants can be conducted in three directions: 1 – gathering of berries in themost productive grounds; 2 – the same, but with application of a complex agrotechnical andforestry actions; 3 – introduction of useful berry plants in culture by crops and landing, both in anatural area, and in new areas for the species.The elementary way of increase of productivity of wild-growing berry-fields is regular care ofnatural bush. In the Republic were created the semi-cultures of a cranberry and works on itsrestoration in a natural cover of bogs were conducted.Actions for creation of semi-cultures are developed for increase of biological efficiency of naturalbush of a blueberry with annual affordability of 22 rubbles per hectare (in the prices of the end of139

1980th) (Grimashevich, 1986). The average increase of productivity thus makes 180 kg per hectare.In necessity of carrying out of actions for increase of efficiency of natural bush specified by E.I.Proskurjakov (1937) in due time. However the creation of semi-cultures does not answer in the fullmeasure to problems of a forestry intensification. It is because difficult to mechanize processes ofcultivation of a cranberry and a blueberry (application of fertilizers, harvesting, rejuvenescence ofbushes etc.) under natural conditions therefore the expected effect is not always reached.The beginning of introductional works in the Republic is connected with occurrence in theseventies of XX century of the generalizing data about achievements of foreign countries. Since thesame time in the scientific institutions of Belarus it was studied the possibility of marsh cranberrycultivation (Vaccinium macrocarpon Ait). Long-term ecological and biological researches hadbeen proved advantage of cultivation in culture of the North American alien cultivars incomparison with a European cranberry.The first research-and-production plantation on the area of 10 hectares has been put in 1980-1983of "Glavpolesyevodstroy" of MWE of the USSR in Gantsevichi district of the Brest region(nowadays it is the experimental base of CBG of NAS of Belarus). Small skilled plantations of amarsh cranberry (of 2-3 hectares) are created at the same years in skilled timber enterprises ofInstitute of wood of NAS of Belarus. Successful cultivation of a marsh cranberry on skilledplantations was the concrete proof of high prospects of industrial culture of this species.On the basis of theoretical and practical elaborations in 1986 by the Council of Ministers of Belarusit was accepted Decision "About the organization in the Belarus Soviet Socialist Republic of marshcranberry manufacture" therefore the first industrial plantation of a North American cranberry hasbeen created in Pinsk district of the Brest region on the area about 60 hectares by concern"Polesyevodstroy" in 1985 – 1990. By this time the first domestic technologies of industrialcultivation of marsh cranberry is accepted for reception of a planting stocks and on berryproduction. In effect, each of technologies represents the complex of actions directed on receptionof the maximum productivity of plants. The basic operations on cultivation of a cranberry withmaintenance of conditions of probably full use of fertility of soils, their optimization of water-airand nutritious modes, with application of reliable measures of protection from extreme biotic andabiotic factors, all of these operations are working out.The successful decision of many put questions has created a necessary basis for adjustment of wideindustrial production of a marsh cranberry. By this time in the Republic functions about 70 hectaresof skilled and skilled-industrial plantations where 11 perspective varieties of the Americanbreeding are tested. Average productivity of berries has made 12-15 tons per hectare over the last 5years depending on a variety and a growth place.The considerable attention in the works devoted to a marsh cranberry has been given for researchof biochemical structure of its berries in connection with the optimization of a mode of a mineralfood, of definition of optimum terms of berries harvesting for a putting on storage and for anestablishment of optimum modes of the storage.In 1980 there were begun tests of 11 varieties of high-bush blueberry (Vaccinium corymbosum L.)of American breeding, received of the Main Botanical Garden of Academy of sciences of theUSSR (nowadays MBG of the Russian Academy of Sciences). The culture of this alien crop hasproved to be as perspective under the conditions of Belarus, and collection replenishment by newvarieties of early term of maturing has allowed to advance it in more northern areas of Belarus.Economic advisability of cultivation Vaccinium corymbosum L. as crop made active the works oncreation of plantations in economy with a various pattern of ownership. Today more than 50hectares of farmlands in the Republic of Belarus have been taken away under skilled landings ofthis North American alien crop.The bog bilberry, unlike of high-bush blueberry, neither in industrial crop abroad, nor in allterritory of the CIS countries is not cultivated. At the same time for areas with the short vegetativeperiod, with insufficient quantity of heat in the summer and with the severe winter it is enoughperspective species for introduction. Besides, this species is characterized by wide ecologicalamplitude (it grows on sphagnous bogs, in boggy woods, on sandy and stony soils), by high winterhardiness, by stability to fungus pathogens and by the expressed polymorphism (habitus of plants,the form of berries and other features).140

Skilled works on development of the bog bilberry culture are realized from the end of 70th years atInstitute of wood of NAS of Belarus. By this time the working out of technology of plantationcultivation of a bog bilberry on peat-bog soils has been already almost complete. The resultsreceived under skilled conditions testify to high profitability of the cultivation of the speciesmentioned above. Without considerable expenses on care of plants it is possible to receive annuallyabout 5 and more tons of berries from 1 hectare of plantations (Evtuhova, 1991). However nowthere are only skilled landings of a bog bilberry, created by Institute of wood of NAS of Belarus inthe Gomel and Vitebsk regions (it is near 1.5 hectares).The multi-plan researches on a cowberry are conducted in the Republic. In various agro-climaticzones of Belarus on the basis of studying of a shaped variety it is created the gene pool representingthe necessary base for practical breeding. Approbation as the crop (cultivation) is spent basicallyfor the purpose of revealing of the most valuable forms and development of the general strategy ofagro-technical actions. On a plot of sorts trial there are tested such Swedish cultivars as 'Sussi','Sanna'; such German cultivars as 'Ammerland', 'Erntedank', 'Erntecrone', 'Erntesegen'; such Polishcultivar as 'Masovia'; such Dutch cultivars as 'Koralle', 'Red Pearl'; and Russian cultivar 'Rubin'.Positive results of introduction and prospects of intensive development in our republic of culture ofwood berry plants put forward the variety of problems on research of ways and of control facilitiesby its efficiency. A special urgency thus find researches of physiological features of developmentof plants, and knowledge of character of their changes at a various combination of natural andanthropogenic influences. It has allowed during the period of 1985 throw 2000 to prepare highlyskilled experts in area of natural resource, of introduction and rational use of berry plants ofVaccinium L. species. The 9 dissertations for a candidate of science's degree of biological andagricultural sciences were prepared and protected this time; 1 thesis for a Doctor's degree. The next7 years there were the 2 more dissertations for a Doctor's degree of biological sciences and 1dissertation for a candidate of science's degree. It allows to tell us about the formation of theBelarus school in area of the unconventional berry plants cultivation which work results are widelyused so in the country and as far behind abroad.Last years the collection fund of Central botanical garden of NAS of Belarus has replenished withnew cultivars and forms of marsh cranberry, of European cranberry, of high-bush blueberry, oflow-bush blueberry and of their inter-specific hybrids. It also has replenished with new varietiesand forms of a cowberries of American, Canadian and Russian breeding. It gives additionalpossibilities for expansion of assortment of the cultivars and forms offered for division into districtsand for breeding, on the basis of revealing of the most perspective of them by results of a complexestimation not only of crop production and of bio-productive parameters, but also of nutritious andvitamin value of berries.At the same time, it is necessary to use the more rationally of earlier received own results, of theadvanced foreign experience and of qualification of the prepared experts for more active works oncreation of industrial plantations of non-traditional berry plants of Vaccinium L. species under soilenvironmentalconditions of Belarus. At the introduction of skilled plants as crops it is alsoimportant that they can be grown up on invaluable in the agricultural relation areas such as thedeveloped peat deposits, the boggy earths, sandy uncultivated plots and other kinds ofuncomfortable areas. Thus, wide cultivation of berry plants on the developed peat-bogs can notonly help with the decision of a problem of providing of the population by berries, but also promoterational use of the "waste" areas in such plough-land deficiency region as Belarus.References1. Environment and natural resources of Byelorussia: Proc. of art. of Statistics scientific research institute.(2000) In: Borovikov A.N. (etc), Minsk, Ministry of statistics and analysis of Belarus, 191 p. (InRussian).2. Evtuchova L.A. (1991) Plantation blueberry (Vaccinium uliginosum L.) cultivation under the conditionsof the southeast of Belarus. Dis. for cand. of agricultural sciences degree, 06.03.03, Gomel, 212 p. (InRussian).3. Grimashevich V.V. (1986) Bog bilberry (Vaccinium uliginosum L.) in Polesye and actions for increaseof its efficiency. Dis. for cand. of agricultural sciences degree, 06.03.03, Minsk, 222 p. (In Russian).4. Karas A.M. (1983) Collateral using in woods of the Belarus Soviet Socialist Republic. In: Volchkov V.E.(etc.) Resources of wild-growing fruit-berry plants, their rational use and the organization of plantation141

cultivation of economic-valuable kinds in the light of decisions of the Food program of the USSR. Rep.thes. of res.-manuf. conf., on September, 27-28 th , Gomel, pp. 64-67. (In Russian).5. Marsh cranberry in Belarus (1987) In: E.A. Sidorovich, M.A. Kudinov, N.N. Ruban, etc. The Science andtechnics, Minsk, 238 p. (In Russian).6. Parfyonov V.I., Golod D.S. (1996) Wood's resources of Belarus and questions of their rational use.Natural resources. N 1, pp. 41-54. (In Russian).7. Praskurjakov Е.I. (1937) Wild fruit-berry plants of BSSR. Minsk, St. issue of BSSR, 48 p. (In Russian).8. Sautin V.I. (1980) Distribution, efficiency and the account of resources of a blueberry in BSSR In:Yudina V.M. (etc.). Wild-growing berry plants of the USSR. Rep. thes. of All-Un. Meet., Petrozavodsk,on October, 1-3, 1980, Biology institute, pp. 158-161. (In Russian).9. Sautin V.I., Palamarchuk A.S., and Rajko P.N. (1975) Productivity and stocks of berries in woods ofBelarus Vegetative resources, 11 (3), pp. 320-328. (In Russian).10. Volchkov V.E., Sautin V.I., Valova Z.G., and Bobrovnikova T.I. (1982) Result of studying of wildgrowingberry plants of cowberry family in BSSR Housekeeping in pine woods of BSSR: Proc. of res.mater. of BelRIF. – Gomel, pp. 79-83. (In Russian).PLANT MORPHOLOGY AND RAPD MARKER CHARACTERIZATIONS OFVACCINIUM OXYCOCCUS LITHUANIAN POPULATIONSVACCINIUM OXYCOCCUS LIETUVAS AUGU POPULĀCIJU MORFOLOĂIJA UNRAKSTUROJUMS AR RAPD MARĖIERIEMJudita Žukauskien÷ 1 , Algimantas Paulauskas 1 , Remigijus Daubaras 2 , Laima Česonien÷ 21 Vytautas Magnus University, Vileikos 8, Kaunas, e-mail: a.paulauskas@gmf.vdu.lt ,j.zukauskiene@fc.vdu.lt2 Kaunas Botanical Garden of Vytautas Magnus University, Z.E. Zilibero 6, 46324, KaunasAbstractPlant morphogenesis is a useful tool for physiological, biochemical, and molecular studies. Thediversity in plant form is produced mainly because different parts of the plant grow at differentrates. Furthermore, the growth of an individual structure is different in various dimensions. Suchdifferential growth rates are very well determined by genetic factors.Understanding the relationships among wild cranberry morphologic and genetic characteristics mayprovide insights for the better utilization of germplasm. The objectives of this research were todetermine the relationships between the genotype and the development of morphologic features.Eleven morphologic characteristics (vegetative stage, flowering time, ripening start and ending,mass, volume and size of berry, berry form, leaf shape, steepness of upright) of 56 morphologicallydiverse genotypes from the 4 Lithuanian bogs were analyzed using random amplified polymorphicDNA (RAPD) classifications. To compare wild cranberry (Vaccinium oxycoccus) morphologiccharacteristic 213 RAPD bands were used. Comparing wild cranberry genetic and morphologicfeatures there were found some significant variations.KopsavilkumsAugu morfoăenēzes analīze ir noderīga metode fizioloăijas, bioėīmijas un molekulārajās studijās.Augu formu dažādība rodas galvenokārt tāpēc, ka dažādas auga daĜas aug dažādos ātrumos.Turklāt, atsevišėas struktūras augšana var notikt vairākās dimensijās. Šīs augšanas atšėiribas tiekgalvenokārt noteiktas ăenētiski.Sakarības izzināšana starp savvaĜas dzērveĦu morfoloăisko un ăenētisko raksturojumu var Ĝautlabāk izmantot dzērveĦu genofondu. Sī pētījuma mērėis bija noteikt sakarības starp genotipu unmorfoloăisko pazīmju attīstību. Kopumā tika analizētas 56 morfoloăiski atšėirīgi genotipi nočetriem Lietuvas purviem. Izmantojot klasifikāciju pēc RAPD marėieriem, tika pētītas vienpadsmitmorfoloăiskās pazīmes (veăetatīvās fazes ilgums, ziedēšanas laiks, nogatavošanās sākums unbeigas, ogas masa, tilpums un lielums, ogu un lapu forma, dzinumu stāvums). SavvaĜas dzērveĦu(Vaccinium oxycoccus) morfoloăisko atšėirību raksturošanai tika izmanotas 213 RAPD līnijas.142

Tika atrastas dažas būtiskas atsėirības starp savvaĜas dzērveĦu ăenētisko un morfoloăiskoraksturojumu.Key words: genetic and morphological correlation; morphological characteristics; RAPDcharacterizationIntroduction.The berries of wild cranberry (Vaccinium oxycoccos L.) are very valuable because they possess alarge content of biologically active substances (flavonoids, acids, vitamins), that may help protectagainst heart disease, the ability of blood vessels to relax - in subjects with high blood cholesteroland atherosclerosis (Vorsa et al., 2002; Wang et al., 1996). Antioxidants found in this berries helpto neutralize harmful free radicals that are thought to be linked to most chronic diseases includingcancer, diabetes and heart disease (Budriuniene, 1998; Vedenskaja and Vorsa, 2004).Multiple characteristics provide good support for the recognition of species and the determinationof relationships among taxa. The strength of support for a lineage is based on the assumption thatmultiple characteristics have been acquired independently of each other. However, many suites ofcharacteristics, such as the complex flowers of Asclepiadaceae and Orchidaceae, may befunctionally integrated with each other and are probably not acquired independently. Others, suchas the overall size of different organs, may have a common genetic and developmental basis inaddition to functional integration (McLellan, 2005).Genetic basis play important role in evolution of plant morphology (Davis, 2001; Conner, 2002;Ungerer et al., 2002). Two possible mechanisms for these correlations are the association of morethan one phenotypic characteristic with a single genotype, and linkage, when independent genesthat determine different traits are inherited together because they are located near each other in thegenome (McLellan, 2005).The purpose of this paper is to examine correlations between eighteen different morphologicaltraits among four populations of the V. oxycoccus. Also to compare the morphological and RAPDmarker classifications of 56 morphologically diverse genotypes, and to determine the relationshipof their RAPD and morphologic features.Material and methods.Morphologic features analysis. Fifty six cranberry cuttings with clearly differing vegetativeindications (color, size, shape of berry and productivity) (Daubaras et all., 2004) were collectedfrom 4 populations in Lithuania bogs (Čepkeliai (54 o 00’-54 o 03’N, 24o25’-24 o 35’E), Žuvintas(54 o 23’- 54 o 30’N, 23 o 25’-23 o 40’E), Kamanos (56 o 15’-56 o 20’N, 21 o 35’-22 o 45’E) and Aukštatija(55o 40’ - 55 o 00’N, 25 o 80’ – 26 o 25’)) during 1995–1999. The cuttings were arranged in the KaunasBotanical Garden collection into special peat (pH 4.0-5.0) beds. These clones were selected forfurther evaluation ex situ. Eleven morphological properties of shoots, leaves and berries for themorphological characterization of the clones were used along the descriptor list for cranberries(Budriūnien÷, 1998). Depending on the size berries were classified in to 5 groups: very small (1.5 g). Flowering shootswere measured at the end of growth. The main phenological phases of the investigated clones wererecorded also. During four years (2001 - 2004) clones were observed and evaluated according tothe phenological parameters (stages) as: vegetative stage, beginning of blossoming, beginning ofberry ripening, and end of berry ripening. The length of generative shoots, volume of the berries,leaf length, leaf width, and berry weight was also measured. These morphological data were usedfor statistical analysis.DNA analysis. DNA extraction and RAPD analysis was done as it is described previously(Areškevičiūt÷ et all, 2006). Nine (OPA-1, OPA-4, OPA-5, OPA-9, OPA-10, OPB-11, ROTH-6,ROTH-8, ROTH-9) 10 base pair length primers of random sequence (Fermentas, Lietuva; Roth,Germany) were used. DNA amplification was performed in a thermocycler (Mastercycler,Eppendorf, Germany) under the following conditions: initial denaturation for 4 min at 94 °C, 44cycles of denaturation for 1 min at 94 °C, primers annealing for 1 min at 35 °C, extension for 2 minat 72 °C followed by a final extension for 5 min at 72 °C.143

Calculation of the observed number of alleles, Nei’s (Peer et all., 1994; Nei, 1973) gene diversity(H), Shannon’s Information Index (I), total gene diversity (Ht), gene diversity within populations(Hs), gene diversity among populations (Gst = (Ht–Hs) / Ht), gene flow (Nm = 0.5 (1–Gst) / Gst)and generation of a Nei’s genetic distance based dendrogram were carried out with POPGENE V1.31 software (Yeh and Yang 1999). Principal coordinates analysis were performed with theGenAlEx 6 program (Peakala and Smouse 2006).Statistical Analyses. The test for the significance of each 213 RAPD product band was done usingthe Pearson Chi-Square (p≥0.05) (Kish, 1987).To find out relatedness between RAPD fragmentsand phenological stages we searched for correlations. Stepwise multiple correlations (two-tailedsignificance level 0.05) were used to determine which combinations of genetic variables wereassociated with morphologic traits were carried out with SPSS 13.0 for Widows software.Results and DiscussionIn our study, RAPD markers proved to be a powerful method for the detection of spatial geneticvariation. Based on the literature (Nei and Li, 1979).we have chosen nine 6 OPA and 3 ROTHprimers. With nine primers, we obtained 213 fragments and could differentiate the 56 V. oxycoccusclones, reflecting a rich allelic diversity among the populations.The size of the amplified fragments ranged from 80 to 2750 bp, and all loci were polymorphic. Thenumber of bands per primer ranged from 13 (RAPD OPB-11) to 30 (RAPD ROTH-180-09). Thepolymorphism level for populations was as follows: Čepkeliai 52.11%, Žuvintas 37.09 %,Aukštaitija 43.19% and in Kamanos population 36.62 %.To estimate genetic variation between populations, the values of Shannon’s Information Index (I),Nei’sgene diversity (H) and the observed number of allelesper locus (Na), the number andpercentage of polymorphic loci were calculated (Table 1). For the total sample, Shannon’sInformation Index was 0.2 and Nei’s gene diversity 0.10. The observed number of alleles per locusranged from 0.1 in Čepkeliai population to 1.4 in Kamanos and Žuvintas population. The estimatedtotal proportion of diversity among populations (G ST ) and gene flow (Nm) were 0.15 and 2.9respectively.Shannon’s index estimates of intraspecific genetic diversity within V. oxycoccus were higher thanin other plant species (Jogait÷ et all, 2006). Shannon’s information index has general applicationsin ecology and is relatively insensitive to the skewing effects caused by the inability to detectheterozygous loci (Dawson, 1995).Table 1. Values of genetic diversity indicated in Lithuanian populations of V. oxycoccus.PopulationShanon’s InformationIndex (I)Nei’s genediversity (H)Observed mean numberof alleles (Na)The number ofpolymorphic lociThe percentageof polymorphicloci %(P)Čepkeliai 0.225 0.142 1.5211 111 52.11Žuvintas 0.120 0.071 1.3709 79 37.09Kamanos 0.086 0.045 1.3662 78 36.62Aukštaitija 0.189 0.120 1.4319 92 43.19Total 0.195 0.102 1.9953 213 99.53Owing to this life history trait on genetic diversity, a low genetic diversity within but a highdiversity among populations is expected (Kreher, 2000). Plants with highest genetic diversitywithin and among populations can better adapt to different environmental conditions.Analyzing molecular variance of Vaccinium and other plant species were observed that biggest partof molecular variance were within populations (Stewart and Excoffier, 1996; Jordano and Godoy2000; Jürgens et all., 2007). Average molecular variance within Vaccinium species populationswere 87.7% and within populations were 27.7 %. Highest molecular variance was detected withinamerican cranberry (V. macrocarpon) populations (more than 91 %) (Stewart and Excoffier, 1996).In V. ulingosum – 90.3 %(Kreher et all., 2000) in V. myrtillus – 86.19 %(Albert et all., 2004,Garkava-Gustavson et all., 2005).Thus, our study revealed a comparably low DNA polymorphismlevel in Vaccinium oxycoccus populations (Table 1). RAPDs indicate that Lithuanian V. oxycoccus144

appears to maintain a quite low level of the genotypic variance among populations (25 %). andwithin (75 %) Lithuanian V. oxycoccus populations was found compared to American V.macrocarpon and the other Vaccinium species.P rinc ipal C oordinatesČ epkeliaiK amanosC oord. 2Ž uvintasAukštaitijaC oord. 1Figure 1. Distribution of V. oxycoccus populations in principal coordinates analysis.To estimate the relationship between V. oxycoccus populations, Nei’s genetic distance betweenpairs of populations was calculated. To generate graphs principal coordinate analysis (PCA) wasused. Graphs show genetic distances among samples and populations. The PCA analyses haverevealed that Čepkeliai populations of V.oxycoccus had homogenous genotype and all samples arein one lineage. Kamanos, Žuvintas and Aukštaitija populations are found in one site and havemixed genotypes. (Figure 2). The resulting different and mixed lineages can confirm the predictionthat these three Lithuanian V. oxycoccus populations were derived from one population beforeglaciation and the Čepkeliai population differed from them. This prediction also confirms and PCAanalysis (Figure 1). Postglacial decolonization one more factor that could have influenced thesedifferences is because the populations Čepkeliai, Žuvintas, Kamanos and Aukštatija (Webb andBartelein, 1992). According to Lithuanian deglaciation periods these four populations geneticallyseparated into different lineages this can confirm glaciation stages in Lithuania (Figure 1). Thesecond factor explaining our results is wide river Nemunas separating Čepkeliai from otherevaluated bogs.Principal CoordinatesCoord. 2KamanosAukštaitijaČepkeliaiŽuvintasCoord. 1Figure 2. Distribution of V. oxycoccus populations by clones in principal coordinates analysis.Different population clones are marked by different marker.Using statistic analysis 119 out of 213 RAPD band products were identified as significantlycontributing to the genetic and morphologic classification (OPA-1,125, 275, 400, 450, 500, 550,625, 650, 700, 750, 850, 1131, 1200, 12800, 1300, 1750 bp. OPA-4 225, 550, 600, 810, 1040,1150, 1550 bp. OPA-5 280, 350, 375, 400, 440, 500, 550, 590, 600, 700, 750, 800, 900, 1031,1040, 1230, 2000 bp. OPA-9 320, 350, 390, 400, 465, 550, 630, 650, 680, 710, 750, 850, 1234,1500, 2900 bp. OPA-10 290, 325, 375, 400, 450, 500, 550, 690, 700, 750, 800, 850, 900, 690,1031, 1150, 1200, 1234, 1550, 2000 bp. OPB-11 300, 400, 650, 700, 750, 850, 1031, 1131, 1200,1700, 1900 bp. ROTH-6 175, 420, 460, 500, 520, 600, 700, 820, 1031, 1150 bp. ROTH-8 440, 600,950 bp. ROTH-9 110, 190, 200, 250, 300, 310, 320, 350, 420, 440, 450, 500, 580, 650, 680, 700,850, 900, 1031, 1350, 1750, 2400 bp.). Some significant molecular fragments and morphologicdata correlations were also found (Table 2).145

Table 2. Calculated significant fragments and phenological parameters correlations per primer inLithuanian populations of V. oxycoccus.Morphologiccharacteristicsroth 9 opa 1 opa 4 opa 5 opa 9 opa 10 opb 11 roth 6 roth 8 totalVegetative stage 4* 5 1 4 3 3 6 3 1 31beginning ofblossom3 4 -** 1 2 3 5 2 1 21beginning ofberries ripening1 1 - - 1 2 - - - 6end of berriesripening1 - 1 1 1 - 1 - 1 6length ofgenerative 2 2 - - 1 - 1 - - 6uprightsvolume of berry 3 - - 1 - - 1 - - 5leaf length 2 1 1 2 1 1 1 1 1 11leaf width 4 3 1 3 - 3 3 - - 17berry mass 5 - 1 2 1 4 - 1 - 14* Numbers inside table shows how many correlations were find with significant fragments and morphologicfeature per primer.** Trace shows that there were no correlation.According to correlations between phenological parameters and significant fragments per primer,we can see that biggest correlations were with vegetative stage and beginning of flowering. Lowcorrelations were with the beginning of berry ripening, the end of berry ripening, length ofgenerative shoots, and the capacity of the berries.ConclusionsAccording to correlations between morphologic features and primers fragments we can see that thebiggest correlations were with vegetative stage and the beginning of flowering. Low correlationswere between the beginning of berry ripening, the end of berry ripening, length of generativeshoots, and the volume of the berries.The resulting different and mixed lineages can confirm the prediction that these three Lithuanian V.oxycoccus (Žuvintas, Kamanos and Aukštaitija) populations belonged to one population beforeglaciations and the Čepkeliai population differed from them. This could be because of the influenceof the river Nemunas and of postglacial decolonizationAcknoweledgmentsThis work was partially supported by the Lithuanian Ministry of Education and Scienceprogramme and the Lithuanian state science and studies foundation.References1. Albert T., Raspe O., Jacquemart A.L. (2004) Clonal Diversity And Genetic Structure in VacciniumMyrtillus Populations from Different Habitats. Belgian Journal of Boany, 137 (2), pp. 155-162.2. Areškevičiūt÷ J., Paulauskas A., Česonien÷ L., Daubaras R. (2006) Genetic characterisation of wildcranberry (Vaccinium oxycoccos) from Čepkeliai reserve by the RAPD method. Biologija, 1, pp. 5–7.3. Budriuniene D. (1998). Investigation of Vaccinium macrocarpon Ait., Vaccinium oxycoccos L. varietiesin Kaunas Botanical Garden. Forestry Studies, 30, pp. 23-29.4. Conner J. K. (2002). Genetic mechanisms of floral trait correlations in a natural population. Nature, 420,pp. 407–410.5. Davis S. L. (2001) Phenotypic and genetic correlations among floral traits in two species of Thalictrum.Journal of Heredity, 94, pp. 361–366.6. Daubaras R., Česoniene L. and Labokas J. (2004). Phenotypic diversity of wild cranberry (Vacciniumoxycoccos) in Lithuania. Acta Horticulturae, 663, pp. 317-619.7. Dawson I. K., Simsons A. J., Waugh R. and Powell W. (1995) Diversity and genetic differentiationamong subpopulations of Gliricidia sepium revealed by PCR-based assays. Heredity, 74, pp. 10-18 .146

8. Garkava – Gustavson L.,Persson H.A., Nybom H., Rumpunen K., Gustavsson B.A., Bartish I.V. (2005)RAPD based analysis of genetic diversity and selection of lingolberry (Vaccinium vitis-idaea L.)material for ex situ conservation. Genetic Resources and Crop Evolution, 52, pp. 723 – 735.9. Jogait÷ V., Lazutka J.R., Stapulionyt÷ A., Naujalis J.R., Odland A. (2006) Analysis of DNApolymorphizm in wild populations of herb – Paris (Paris quadrifolia L., Trilliaceae) from Lithuania andNorway. Biologija, 1, pp. 18 – 23.10. Jordano P. and Godoy J.A. (2000) RAPD variation and population genetic structure in Prunus mahaleb(Rosaceae), an animal-dispersed tree. Molecular Ecology, 9, pp. 1293 – 1305.11. Jürgens A.H., Seitz B., Kowarik I. (2007) Genetic differentiation of Rosa canina (L.) at regional andcontinental scales. Plant Systematics and Evolution, 269, pp. 39-53.12. Kish L. (1987). Statistical Design for Research. New York: John Wiley and Sons pp. 276.13. Kreher S. A., Foré S. A. and Collins B. S. (2000) Genetic variation within and among patches of theclonal species, Vaccinium stamineum L. Molecular Ecology, 9 (9), pp. 1247-1252.14. McLellan T. (2005) Correlated evolution of leaf shape and trichomes in Begonia dregei (Begoniaceae).American Journal of Botany, 92 (10), pp. 1616–1623.15. Nei M. and. Li W-H. (1979) Proc. Natl. Acad. Sci. 76, pp. 5269–5273.16. Nei, M.P. (1973) Analysis of Gene Diversity in Subdivided Populations. Proc. Natl. Acad. Sci., USA, 70,pp. 3321–3323.17. Peakall R. and Smouse P.E. (2006) GENALEX 6: genetic analysis in Excel. Population genetic softwarefor teaching and research. Molecular Ecology Notes, 6, pp. 288-295.18. Stewart C.N., Jr., Excoffier L. (1996) Assessing population structure and variability with RAPD data:Application to Vaccinium macrocarpon (American cranberry). Evol. Biol., 9, 153-171.19. Ungerer M. C., Halldorsdottir S. S., Modliszewski J. L., Mackay T. F. C. and Purugganan M. D.. (2002)Quantitative trait loci for inflorescence development in Arabidopsis thaliana. Genetics, 160, pp. 1133–1151.20. Van de Peer,Y. and De Wachter,R. (1994) TREECON for Windows: a software package for theconstruction and drawing of evolutionary trees for the Microsoft Windows environment. Comput. Appl.Biosci., 10, pp. 569–570.21. Vedenskaja I. and Vorsa N. (2004) Flavonoid composition over fruit development and maturation inAmerican cranberry, Vaccinium macrocarpon Ait. Plant Science, 167, pp. 1043-1054.22. Vorsa N., Polashock J., Cunningham D., Roderick R., Howell A. (2002) Evaluation of fruit chemistry incranberry germplasm: potential for breeding varieties with enhanced health constituents. ActaHorticulturae, 574, pp. 215-219.23. Wang H., Cao G., and Prior R. L.. (1996) Total antioxdant capacity of fruits. J. Agric. Food. Chem., 44,pp. 701-705.24. Webb III T. and Bartelein P. J. (1992) Annual review of ecology and systematic, 23, pp. 141-173.25. Yeh F.C. and Yang R.C. (1999) POPGENE (Version 1.31): Population Genetic Analysis Software.Alberta, University of Albertaand Tim Boyle Center for International Forestry Research.147

Agronomijas Vēstis. – Nr. 12.Latvian Journal of Agronomy No. 12.Jelgava, 2009Latvijas Lauksaimniecības universitāteLatvijas Lauksaimniecības un meža zinātĦu akadēmijaParakstīts iespiešanai: 2009. gada 27. novembrisTirāža 150 eks.Sagatavots iespiešanai Latvijas Lauksaimniecības universitāte,Lauksaimniecības fakultāte,Agrobiotehnoloăijas institūtsLielā iela 2,JelgavaTālrunis: 630 05 682Fakss: 630 05 682e-pasts: Aleksandrs.Adamovics@llu.lvIespiests tipogrāfijā SIA “Drukātava”Liliju iela 95/1, Mārupe, LV-2167Tālr.: 673 68188e-mail: valdis@drukatava.lv148

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