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LITHUANIAN UNIVERSITY OF AGRICULTURE            RamunėKuprienė      DEVELOPMENT OF YELLOW-SEEDED RAPESEED (BRASSICA NAPUSL.) BY BIOTECHNOLOGICAL AND CLASSICAL BREEDING METHODS       Summary of doctoral dissertation Biomedical sciences, agronomy (06B)               Akademija, 2006 5
This doctoral dissertation was prepared at the Laboratory of Genetics  Biotechnology of the Lithuanian University of Agriculture in 2002  2006.   Scientific supervisor:  Ass. prof. dr. Liuda ilėnaitė University of Agriculture, biomedical (Lithuanian sciences, agronomy  06B).   Scientific consultant:  Ass. prof. dr. Natalija Burbulis (Lithuanian University of Agriculture, biomedical sciences, agronomy 06B).   The dissertation will be defended in the Council of Agronomy Science at the Lithuanian University of Agriculture:  Chairman:  Prof. dr. habil. Rimantas Velička (Lithuanian University of Agriculture, biomedical sciences, agronomy  06B).  Members:  Prof. dr. habil. Vidmantas Stanys (Lithuanian Institute of Horticulture, biomedical sciences, agronomy  06B).  Prof. dr. Ilona Miceikienė(Lithuanian Veterinary Academy, biomedical sciences, biology  01B).  Dr. Irena Brazauskienė Institute of Agriculture, biomedical sciences, (Lithuanian agronomy  06B).  Ass. prof. dr. Vytautas Ruzgas (Lithuanian Institute of Agriculture, biomedical sciences, agronomy  06B).   Opponents:  Prof. dr. habil. Gvidas idlauskas (Lithuanian University of Agriculture, biomedical sciences, agronomy  06B).  Dr. Bronislovas Gelvonauskis (Plant Gene Bank, biomedical sciences, agronomy  06B).    Defense of the doctoral dissertation will take place during the public meeting of the Council of Agronomy Science on the 15th December 2006, at 11 a.m. in room No. 322, of centralbuilding of the Lithuanian University of Agriculture. Address: Lithuanian University of Agriculture Sudentu 11, LT-53361 Akademija, Kaunas district, Lithuania. Phone: (370) 37 752254, Fax: (370) 37 3975500. The summary of the doctoral dissertation was distributed on the 15th November, 2006. of The doctoral dissertation is available in the libraries of the Lithuanian University of Agriculture and the Lithuanian Institute of Agriculture. 6
Daktaro disertacijos santrauka Biomedicinos mokslai, agronomija (06B)          Akademija, 2006  
Disertacija rengta 2003  2006 metais Lietuvos emėsūkio universiteto Genetikos ir biotechnologijos laboratorijoje.   Mokslinėvadovė: Doc. dr. Liuda ilėnaitė em (Lietuvosėsūkio universitetas, biomedicinos mokslai, agronomija  06B).  Mokslinėkonsultantė: Doc. dr. Natalija Burbulis (Lietuvos emėsūkio universitetas, biomedicinos mokslai, agronomija  06B).    Disertacija ginama Lietuvos emėsūkio universiteto Agronomijos mokslo krypties taryboje:  Pirmininkas: Prof. habil. dr. Rimantas Velička (Lietuvos emėsūkio universitetas, biomedicinos mokslai, agronomija  06B).  Nariai: Prof. habil. dr. Vidmantas Stanys (Lietuvos sodininkystės ir darininkystės institutas, biomedicinos mokslai, agronomija  06B). Prof. dr. Ilona Miceikienė (Lietuvos Veterinarijos akademija, biomedicinos mokslai, biologija  01B). Dr. Irena Brazauskienė (Lietuvos emdirbystės institutas, biomedicinos mokslai, agronomija  06B). Doc. dr. Vytautas Ruzgas (Lietuvos emdirbystės institutas, biomedicinos mokslai, agronomija  06B).   Oponentai: Prof. habil. dr. Gvidas idlauskas (Lietuvos emėsūkio universitetas, biomedicinos mokslai, agronomija  06B). Dr. Bronislovas Galvonauskis (Augalų genų biomedicinos mokslai, bankas, agronomija  06B).   Disertacija bus ginama vieame Agronomijos mokslo krypties tarybos posėdyje 2006 m. gruodio mėn. 15 d. 11 val. Lietuvos emėsūkio universiteto centrinių rūmų 322 auditorijoje. Adresas: Lietuvos emėsūkio universitetas Studentųg. 11, LT-53361 Akademija, Kauno raj., Lietuva. Tel. (8-37) 752254, faks. (8-37) 3985500. Disertacijos santrauka isiuntinėta 2006 m. lapkričio 15 d. Disertaciją galima periūrėti Lietuvos emėsūkio universiteto ir Lietuvos emdirbystės instituto bibliotekose.  8
I N T R O D U C T I O N One of the main objectives of spring rapeseed (Brassica napusL. ssp. oleifera annua Metzg.) breeding is to improve seeds quality, using both classical and modern breeding methods. To enrich genetic diversity,in vitro (tissues of anthers, microspores technologies and tissue cultures etc.) are successfully applied, providing not only new traits (resistance to diseases, herbicides) to the cultivars, but also speeding up the development of genetically stable lines. Recently intensive studies have been carried out to develop yellow seeded cultivars of rapes. The quality of such seeds is better, because they contain more oil and proteins (Rashid et al., 1995), more aminoacids (Han,1995; Symbaya et al., 1995) lower content of glucosinolates (Raney, Rakow, 1995) and fibre (Grisebach, 1981). The yellow seed coat contains less cellulose and tannins (Li et al., 2003), therefore yellow-seeded rapeseeds are more suitable for the production of alimentary oil. Such an oil is cheaper due to a shorter production process. Genotypes of rapeseeds producing yellow seeds were not found in nature. The first yellow-seededBrassica napusplant was developed in 1960, having crossedB. oleraceaand B. campestris 1960). Breeders yellow-seeded rapeseeds have been developed (Olsson, applying different combinations of interspecific crosses (Rashid et al., 1994; Rahman, 2001; Relf-Eckstein et al., 2003):B. campestrisandB. alboglabra(Chen et al., 1988); [B.napusx B.juncea] and [B.napus xB.carinata] (Rashid et al., 1994). It was tried to transfer genes controlling the colour of seed coat directly fromB.carinataC genome toB. napusC genome (Qi et al., 1993). In the Laboratory of Genetics-Biotechnology at the Lithuanian Agricultural University, yellow-seeded spring rapeseeds were developed for the first time without interspecific crosses (Burbulis, 2001). All cultivars of yellow-seeded rapeseed have one essential drawback  unblocking of pigmentation takes place in other generations and seeds of different colours (yellowish brown, brown or black) are formed. Breeders, working with the cultivars of yellow-seeded rapeseed, admit that environmental temperature is one of the factors limiting the manifestation of the trait. Yellow seeds of rapeseed are formed only under high environmental temperature (Henderson, Pauls, 1992; Van Deynze et al., 1993; Rakow et al., 1999; Baetzel et al., 2003). It is presumed that stabilization of the yellow colour may be complicated due to allotetraploidy, interaction of genes and climatic conditions (Van Deynze and Pauls, 1994). Hypothesis it is possible to develop the stable yellow-seeded spring rapeseed  cultivars, in which seed coat color not influenced by environmental temperature, by biotechnological methods without interspecific crosses. The aim of the studies was to develop and evaluate genetic diversity of spring  rapeseed for the selection of yellow-seeded plants by biotechnological and classical breeding methods. Seeking to achieve the aim, the following objectives were raised:  To study the influence of ambient temperature on seed coat pigmentation intensity of spring rapeseed DH lines.  To study the dependance between seed pigmentation of DH lines, resistance to white stem rot (Sclerotinia sclerotiorum) and biometric plant parameters.  spring rapeseed DH lines, seed qualityTo estimate biometric parameters of developed and germination.
 hybrids of spring rapeseed and to evaluateTo develop stable yellow-seeded DH lines, the possibility to use them for the development of yellow-seeded spring rapeseed cultivars.  influence of spring rapeseed parent forms on seed colour inheritance.To determine the  To study morphogenesis of yellow-seeded spring rapeseed DH lines in the cultures of zygotic embryos, tissues and isolated anthers, and to estimate the efficiency of applied in vitromethods for the development of yellow-seeded rapeseed regenerants.  spring rapeseed cultivars, hybrids and derived DH lines by RAPDTo evaluate DNR of method.Novelty. Applying the culture of isolated microspores, yellow-seeded DH lines were developed, in which the synthesis of pigments failed to take place under low environmental temperature (20/16oC). Yellow-seeded hybrids were derived having applied reciprocal crosses (initial material  yellow-seeded DH lines and black-seeded cultivars of spring rapeseed). Resistance test of DH lines to oxalic acid was applied, according to which sensitivity to the white stem rot Sclerotinia sclerotiorum is estimated. Factors determining the formation of primary and secondary embryos in the culture of immature zygotic embryosin vitro of spring rapeseed were evaluated. The conditions of hypocotyl and cotyledon callus induction and regeneration in the tissue culture of yellow-seeded rapeseed DH lines were optimized. Practical value. Seven DH lines of yellow-seeded spring rapeseed were derived, the pigmentation of which is blocked in the climatic conditions of Lithuania. By reciprocal crosses populations of hybrids were developed, among which individuals suitable for the development of yellow-seeded spring rapeseed cultivars were selected. Plants regenerated in the cultures of somatic tissues and isolated anthers may be used as the source of genetic variability for the production of initial breeder material, while optimized culture of immature zygotic embryos - to shorten the breeder process of yellow-seeded spring rapeseed. Approval of the research work. The main results of the doctoral thesis were presented in scientific conferences: International Conference Plant Tissue Culture: From Theory to Practice (Salaspils (Latvia), 2004); International Conference Growth and Development of Plants. Theoretical and Practical Problems (Babtai (Lietuva), 2004); Scientific Conference Ecological and Biotechnological Aspects of Increasing Plant Adaptivity (Babtai (Lietuva), 2006). Study results were announced in two prereviewed ISI publications and two reviewed TDB publications, included into the list adopted by the Lithuanian Council of Science, as well as in three proceedings of conferences. Structure and volume of the work. The dissertation is written in Lithuanian. It consists of the introduction, literature review, materials and methods, results, conclusions, publications on the theme of the dissertation and a list of other publications, references and appendices. The dissertation comprises 103 pages, including 20 tables, 31 figures, 231 literature sources. M A T E R I A L S A N D M E T H O D S L o c a t i o n o f s t u d i e s .In the Laboratory of Genetics-Biotechnology of the Lithuanian Agricultural University in 2003  2006 the studies of double haploid (DH) lines of yellow-seeded spring rapeseedin vitrowere carried out, while in 2003 - 2005 hybrids were derived. In 2004  2005, to evaluate colour stability under Lithuanian climatic conditions, DH lines, hybrids and regenerants were grown in the selection and hybrid plantations of spring rapeseed in the Experimental Station of the Lithuanian Agricultural University. D e v e l o p m e n t o f d o u b l e h a p l o i d s o f t h e s p r i n g r a p e s e e d 10
In 2003, three DH lines: NL-301, NL-302, NL-303 were developed from yellow-seeded DH 268-2, NL-310-1 and NL-360 double haploid lines (DH), by microspore culture. The colour of seeds of the derived DH lines was evaluated in 2003  2005. T h e m i c r o s p o r e c u l t u r e . Donor plants DH 268-2, NL 310-1 and NL 360 were grown in the growing chamber (temperature 20/16 °C, photoperiod 16/8 h (day/night)). Flower buds of a proper size were sterilized for 2 minutes in 70% ethanol, afterwards 3 times rinsed with distilled sterile water. Sterile floral buds were crushed in 13% sucrose solution and filtrated through a double Nytex filter. Microspores were deposited 3 times at 5 minutes, centrifugating at 1000 rev/min. rate. To prepare the suspension of isolated microspores, a modified NLN culture medium was used (Fletcher et al., 1998). The suspension was poured into sterile Petri dishes and kept in dark under 30°C temperature. After 14 days since isolation, the culture was transfered on the shaker (60 mov./min.), where it was kept for 14 days in room temperature. After 28 days since the isolation of microspores, embryos of morphological maturity were transfered on agarized B5 medium and stored in the growing chamber (temperature 4°C, photoperiod - 8 hours). After 10 days, growing regime was changed into warm incubation (air temperature 27 ± 2°C, photoperiod 12 hours). After 30 days, regenerants, having a developed root system and 2-3 true leaves, were transfered into the soil. Regenerants were grown in the growing chamber (temperature 20/16 °C, photoperiod - 16/8 h (day/night)). For the poliploidization of haploid plants, 0.34% kolchicin solution was used. In the budding period each plant was covered with an isolator, seeking to sustain the purity of the line. After flowering, the isolators were removed and the seeds of each plant were collected separately. The colour of seed coat was scored visually. D e v e l o p m e n t o f t h e h y b r i d s o f y e l l o w - s e e d e d s p r i n g r a p e s e e d C r o s s e s o f y e l l o w - s e e d e d D H 2 6 8 - 2 0 l i n e w i t h s p r i n g r a p e s e e d c u l t i v a r s . Yellow-seeded DH 268-20 line of double haploids was crossed with the black-seeded cultivars Star, Bolero and Dynamite; in 2003 were derived F0hybrids, applying reciprocal crosses. According to the analysed trait, hybrids of F3generation were evaluated (2005). Having assessed F3 hybrids only with the seed coat of yellow or generation, yellowish brown colour were selected for sowing. C r o s s e s o f y e l l o w - s e e d e d D H l i n e s . Crosses (60 different combinations of crosses) were made in 2004 between yellow-seeded DH line populations NL-301- and NL-302-. Reciprocal crosses were applied to derive hybrids.  S o m a t i c e m b r y o g e n e s i s o f s p r i n g r a p e s e e d Induction of somatic embryogenesis of spring rapeseed in the culture of immature zygotic embryos. To study the induction of somatic embryogenesis, yellow-seeded double haploid lines: NL-302-01, NL-302-02, NL-302-25, developed by microspore culture, were used. Donor plants were grown in the growing chamber under controlled conditions: temperature 22±2oC, light intensity - 5000 lx, photoperiod  16/8 h (day/night). I s o l a t i o n o f e x p l a n t s a n d i n d u c t i o n o f s o m a t i c e m b r y o g e n e s i s . For the studies of somatic embryogenesis, mature and immature zygotic embryos were used. The age of immature zygotic embryos was counted by days after pollination (DPA) - from 14 to 29 days. Explants were sterilized in 70 % ethanol for 2 min., afterwards 3 times rinsed with sterile distilled water. The embryos under aseptic conditions were transfered to Petri dishes of 90 mm in diameter, containing 25 ml of basic MS (Murashige, Skoog, 1962) culture medium, supplemented with 2% medium sucrose and 8 g l-1Difco-Bacto agar. Medium pH  3.5; 4.0; 5.0. For the regeneration of embryos, a modified B5 (Fletcher et al, 1998) culture medium, supplemented with 0.1 mgl-1GA3, 30g l-1sucrose and 8 g l-1Difco-Bacto agar, was used. 11
Explants were grown under controlled conditions: light intensity - 5000 lx, photoperiod  16 hours, temperature 25±2oC. After three days of cultivation, immature zygotic embryos were transfered on the same fresh media and then cultivated for 28 days under the same conditions. The experiment was done with three replications. The percentage of formed primary (PE) somatic embryos and the number of somatic embryos per explant were estimated. Part of primary somatic embryos of the cotyledonary stage were carefully separated and transfered to a fresh culture media of the same composition and then cultivated for 28 days under the same conditions. Another part of primary embryos was transfered to B5 regeneration medium. After 28 cultivation days, the percentage of formed secondary somatic embryos and their number per primary embryo was estimated. The percentage of plants regenerated from primary and secondary embryos in B5 medium was estimated. Morphogenesis of spring rapeseed in the culture of somatic tissues. The studies of indirect somatic embryogenesisin vitro made with yellow-seeded lines of double were haploids: NL-302-01, NL-302-02, NL-302-25, derived by microspore culture. Donor plants were grown under the same conditions as in the studies of direct somatic embryogenesis, e.i. in the culture of isolated immature zigotic embryos. I s o l a t i o n o f e x p l a n t s . The seeds were washed under running water, sterilized in 70% ethanol for 5 min. and 3 times rinsed with sterile distilled water. Sterile seeds were germinated in Murashige and Skoog (MS) (Murashige, Skoog, 1962) culture media without growth regulators, supplemented with 8 g l-1Difco-Bacto agar. They were incubated under controlled conditions: temperature 22±2o- 5000 lx, photoperiod  16 hours.C, light intensity For the experiments, 4-5-days-old hypocotyls and cotyledons having 1-2 mm stalk were used. Hypocotyls were cut into 5-7 mm long segments. Explants of hypocotyls and cotyledons were cultivated in MS medium supplemented with different combinations of growth regulators: 4.0 mg l-1BAP (6-benzylaminopurine)+ 0.05 mg l-1NAA (α-naphtylactic acid)+ 2.5 mg l-1AgNO3; 3.0 mg l-1BAP + 0.1 mg l-1NAA + 2.5 mg l-1AgNO3; 2.0 mg l- 1BAP + 0.15 mg l-1NAA + 2.5 mg l-1 AgNO3 mg l; 6.0-1ZT (zeatin)+ 0.1 mg l-12,4-D (2.4-dichlorphenoxyacetic acid) + 2.5 mg l-1AgNO3; 4.0 mg l-1ZT + 0.15 mg l-12,4-D + 2.5 mg l-1AgNO3; 2.0 mg l-1 mg lZT 0.15-12,4-D + 2.5 mg l-1AgNO3 without; MS + growth regulators + 2.5 mg l-1AgNO3; control  basic MS (without growth regulators and AgNO3). Culture media was supplemented with 30 g l-1sucrose and 8 g l-1Difco-Bacto agar. Media pH  5.5±0.1. MS culture media was sterilized at 115oC for 30 min. Growth regulators and AgNO3were filtered through 0.22 µm sterile filter. Explants were incubated at 22±2oC, light intensity - 5000 lx, photoperiod  16/8 h (day/night). During the experiment, at 40 explants of each variant were grown, experiment was done in triplicate. Adventitious shoots formed after 4 weeks were transfered to different rooting media: B5 +  0.1 mgl-1GA3+ sucrose 20 mg l-1 MS + 0.1 mg l; ½-1NAA + sucrose 10 mg l-1. Culture media was supplemented with 8 g l-1Difco-Bacto agar. The media pH  5.7±0.1. Morphogenetic potential of somatic tissue was evaluated analysing morphological parameters of structures formed in the explant. The rate of callus formation (%), shoot formation (%) and shoot rooting (%) was estimated. Evaluation of selection material under field conditions O b j e c t o f s t u d i e s . 105 DH lines: NL301  53 unit, NL302  27 unit, NL303  25 unit, hybrids (derived from crosses between yellow-seeded DH lines with black-sedded cultivars, as well as crosses among yellow-seeded populations) and plants regenerants developed in 2003 by microspore culture. G r o w i n g c o n d i t i o n s . DH lines, hybrids and plants regenerants were grown in 2004 2005 in Experimental Station of the Lithuanian Agricultural University. -12
E s t i m a t i o n o f b i o m e t r i c p a r a m e t e r s . In the budding period, at 5 plants of each line were isolated, and the selected DH lines were isolated by squares. Studying selection material of spring rapeseed DH lines, in the period of seed maturation the height of plant (cm), number of branches per plant and the number of pods per plant were estimated (from 10 plants). Seeds were collected from each isolated plant separately, when the pods reached physiological maturity. The number of seeds per pod (unit); weight of 1000 seeds (g) and the productivity of one plant (g): (number of pods (unit) x number of seeds per pod (unit) ) x 1000 mass of seeds (g) / 1000 were estimated (Diepenbrock, 1999). For evalueted seed weight (mg) and seedcoat percent (% of seed weight), 200 seeds (yellow, yellowish-brown, black) were incubated on moist filter paper 12 h at room temperature. Seedcoats were manually separated from embryos. Seedcoats and embryos were dried at 70oh and weighed. Experiment was done in triplicate (Rashid, Rakow,C for 18 1995). E v a l u a t i o n o f r e s i s t a n c e t o the white stem rot(Sclerotinia sclerotiorum). In the flowering period of DH lines the resistance toSclerotinia sclerotiorumwas evaluated based on oxalic acid test. Assessment was done visually according to L. Kott protocol (2003). At five leaves were picked from one plant of each DH line. The leaves were immersed into dishes with four different concentrations of oxalic acid: 40 mM, 80 mM, 160 mM and 200 mM, water was used as the control. The immersed leaves were assessed after 18 hours. The experiment was done with three replications. Resistance to the white stem rot (Sclerotinia sclerotiorum), based on the test with different concentrations of oxalic acid, was evaluated in the system of three points: 1 point  resistant  the leaves are green, rigid; 2 points  averagely resistant  beside the veins the leaves have become yellowish and have started to soften; 3 points  sensitive  the leaves are soft, flabby and between veins the colour is yellowish brown. C h e m i c a l c o m p o s i t i o n o f t h e s e e d s o f D H l i n e s . Oleic acids, glucosinolates and fibre in the seeds of DH lines were determined by a computer system PSCO/ISI IBM-PC 4250 of infrared rays at the Experimental Station of the Lithuanian Agricultural University. To compare the results of biometric parameters and chemical composition of DH lines with the standard species of spring rapeseed, the data by R. Velička (2002) were used. S e e d c o l o u r a s s e s s m e n t . The colour of seed coat was visually scored. The seeds of each new line according to the colour of seed coat were grouped into four classes (Burbulis, Kott, 2005): yellow; yellowish brown  50 % of the seed coat is yellow, the rest part is brown; brownish yellow  about 75% of the seed coat is of brown colour, the rest part is yellow; brown. Evaluating DH lines, all indices are analysed separately in each group according to the colour of seeds: group I  amount of yellow seeds 100%; group II  amount of yellow seeds from 50 to 99%; group III  amount of yellow seeds from 20 to 49%; group IV  the amount of yellow seeds is lower than 20%. C y t o l o g i c a l s t u d i e s o f s e e d s . Mature seeds were kept for 12 hours in moist filtre paper, then cut with a microtome. Incisions in the seed coats were studied by an optical microscope with 20xand 40xenlargement eyepieces. The thickness of seed coat in µm was measured. A n a l y s i s o f s e e d g e r m i n a t i o n p o t e n t i a l a n d v i a b i l i t y . To determine germination potential and viability of napus B. seeds of different colour, a laboratory L. viability test was conducted according to the International Rules of Seed Investigation (1996). During the experiment, at 100 seeds of each colour of the same maturity stage, age and storage conditions were germinated. The study was done with three replications. The 13
seeds were germinated in light under 20oC temperature on moist filtre paper. Germination potential (%) of the seeds was evaluated after 3 days, viability after 5 days and germination completeness (percentage ratio from laboratory germination) - after 7 days. E v a l u a t i o n o f t e s t e d g e n o t y p e s b y t h e R A P D m e t h o d Total genomic DNA was isolated from leaf tissue using the cetyltrimethylammonium bromide (CTAB) method (Saghai-Maroof et al., 1984). Purification of DNA was done by an additional precipitation with 95 % ethanol and Na-acetate, followed by two washes with 70 % ethanol. DNA concentration was ascertained by a spectrophotometer. PCR reaction contained: 3 µl DNA; 2 µl of the primer ( GmbH, Germany); 2.5 µl - 10x PCR buffer; 2.5 µl - 10 mM dNTP mixture; 4 µl - 50 mM MgCl2; 0.2 µl - Taq polymerase; 10.8 µl - distilled and sterilized H2O; DNA amplification was done with Biometra thermocycler. PCR regime was applied for the amplification: 95oC for 2 min (1cycle); 95oC for 20 s; 36oC for 1 min; ramp 1oC/s to 72oC; 72oC for 1 min (35 cycles); 72oC for 7 min (1 cycle). Amplified products were separated by electrophoresis on 1 % agarose gels and visualized under UV light by staining with ethidium bromide. S t a t i s t i c a l a n a l y s i s o f t h e r e s u l t s The data of the investigations were calculated using the computer programe STAT for EXEL, ver. 1.55 and ANOVA for EXEL, ver. 2.1. from SELEKCIJA (Tarakanovas, 1999). Significant differences were calculated using the disperse analysis and grouped by Duncans criteria P0.05. Mean value and SE for each genotype were calculated based on the number of independent replication. Analysis of RAPD fragments was done using a set of Herolab GmbH Laborgeräte computer programs E.A.S.Y. Win 32.   R E S U L T S A N D D I S C U S S I O N  D e v e l o p m e n t o f d o u b l e h a p l o i d s o f s p r i n g r a p e s e e d It has been determined by earlier studies that the colour of seed coat of donor plants, used to develop new double haploids, depends on the temperature during growth period (Burbulis, Kott, 2005). Under 20/16oC temperature, plants of these genotypes produce consistenly darker seeds than that planted seeds. Planning this study, a hypothesis was raised that under this temperature less temperature-sensitive recombinations may be developed in the microspores of donor plants. By microspore culture, from three yellow-seeded lines 105 DH lines were developed: DH 268-2 (dark yellow colour of the seed coat)  53 unit; NL 310-1 (yellow colour of seed coat) 27 unit; NL 360 (brownish yellow colour of seed coat)  25 unit (Table 1). Under 20/16oC temperature double haploids produced yellow,  yellowish brown, brownish yellow and brown seeds (Table 1).  Table 1. The number of developed double haploids according to seed coat colour (under 20/16oC temperature) Donor plant Seed coat colour of double haploids brown brownish yellow yellowish brown yellow DH 268-2 9 25 16 3 NL 310-1 7 9 11 0 NL 360 8 12 5 0  Double haploids may be genetically pure  homozygotic according to all genes, however, regenerants may undergo mutations of genes and then, according to individual genes, the plant will be heterozygotic. Most developed plants of DH lines under 20/16oC temperature produced significantly lighter seeds than donor plants under the same temperature. Seed coat pigmentation, even of lines ascribed to brownish yellow class, is 14
lower than that of donor plants. Study results show that applying the method of isolated microspores culture, it is possible to derive recombinants, pigment synthesis of which would inhibite under lower temperature. The obtained results show that, even though donor plants are double haploids, regenerants developed in microspore culture produced seeds of different seed coat colour. Such a diversity of seed colour allows to presume that, according to the analysed trait, microspores had combinations of four types. From the selection viewpoint, the most valuable are genotypes which produced yellow and yellowish brown seeds under 20/16oC temperature.  E v a l u a t i o n o f d o u b l e h a p l o i d s i n t h e y e a r 2 0 0 4 Evaluation of the seed colour of DH lines. The seeds of each line were evaluated separately. Analysing study results, it was found that in most cases plants of the same line produced seeds of different colour. Besides, on one plant seeds of different colours were detected. Having estimated average amount of yellow seeds, lines in which at least one of isolated plants produced not less than 20 % of yellow seeds were selected. Along with new lines, donor plants, the seed colour of which was dark yellow (DH 268-2), yellow (NL 310-1) and yellowish brown (NL 360), were grown. In the study year these plants were extremely sensitive to ambient temperature, because they produced seeds of dark colour, as those grown under 20/16oC temperature. Maximal amount of yellow seeds of DH 268-2 plants comprised 2 %; NL 310-1  1 %, while NL 360  4 %. By microspore culture, three double haploids having yellow seeds were derived (Table 1). In next generation there was a dissidence according to the analysed trait. In the study years, the amount of yellow seeds of DH lines NL-301-01, NL-301-04 varied respectively from 5.0 to 84.2 % and from 58.0 to 100%, while isolated plants of DH line NL-301-46 produced brown seeds (data is not presented). By microspore culture, 32 DH lines, which produced yellowish brown seeds, were developed (Table 1). In respect of the analysed trait, five DH lines NL-301-29, NL-301-29, NL-301, NL-301-35, NL-301-36, NL-301-41 and NL-30209 were distinguished, at least one plant of which produced yellow seeds. The amount of yellow seeds of these lines varied from 0 to 100 %, except NL-301-35 and NL-30209 plants of DH lines, the minimal amount of yellow seeds of which was respectively 17.2 and 19.4 %. 46 lines of double haploids according to the colour of seed coat were ascribed to the brownish yellow class (Table 1). In the study years, analysing the changes of seed coat colour, distinguished were those lines in which at least one of isolated plants produced yellow seeds. Among DH lines, the seeds of the initial material of which were brownish yellow, should be mentioned the following ones: NL-301  03, NL-301-14, NL-301-44, NL-301-45, NL-301-47, NL-30220, NL-303-11, the percentage of yellow colour of which in the study year, depending on the line, varied from 4 to 100 %. 24 DH lines, derived in the culture of isolated microspores, had seeds of brown colour (Table 1). Analysing the change of seed colour, distinguished were NL-301-05, NL-301-11, NL-302-01, NL-302-02, of DH line, at least one plant of which produced yellow seeds. The amount of yellow seeds fluctuated from 9.1 to 100 %. Other DH lines, which according to the seed colour of initial material belong to yellowish brown, brownish yellow and brown class, in the study years produced seeds of different colours. The amount of yellow seeds in the lines variem from 2.3 to 90.0%.