苹果纯合基因型新种质的性状鉴定与培养
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摘要
【目的】苹果花药培养获得的纯合基因型株系来自不同的花粉粒,因此不同株系的植物学特征、生物学特性,以及诱导培养、生根的条件等都存在差异。本研究对不同个株系试管苗的倍性、表型特征、生根培养条件进行观察分析,选出苹果纯合基因型株系适宜的生根条件及综合性状较好的株系,加强苹果种质资源创新。【方法】采用花药培养获得的‘嘎啦’‘富士’‘红星’纯合基因型株系,利用流式细胞仪分析每个株系倍性,探索组培苗的生根培养条件,观察每个株系的根系、叶片形态特征。【结果】经苹果花药培养获得32个纯合基因型株系,其中单倍体1株、三倍体1株、四倍体3株、二倍体27株,‘红星’苹果纯合基因型株系的倍性分化率最大,为28.57%。IBA浓度影响再生株系的生根率、根长及根数,苹果纯合基因型株系的最适宜生根浓度为IBA 2—3 mg·L-1。各个株系的叶数、根长、根数、株高、叶形指数(叶长度/叶宽度)、叶柄有一定差异。‘红星’纯合基因型DH0-1、DH0-3、DH0-4、DH0-7株系,‘富士’纯合基因型DH1-3株系,‘嘎啦’纯合基因型DH2-2、DH2-4、DH2-12、DH2-20株系综合性状较好(植株高、根系长、叶数和根数多)。‘红星’纯合基因型株系的移栽成活率最高,为28.57%。【结论】苹果纯合基因型株系的主要倍性为二倍体。与二倍体株系相比,单倍体株系叶基窄、叶柄细、叶色和叶缘锯齿较浅,而多倍体株系表现为叶基更阔、叶柄较粗、叶色和叶缘锯齿变深。
【Objective】Homozygous genotype lines were obtained by anther culture of different apple pollen grains, so their botanical characteristics, biological characteristics, and their induced culture, root conditions were different. In this study, the ploidy, characteristics and root conditions of each line were observed and analyzed. Appropriate rooting conditions and good combinations of lines for selecting apple homozygous genotypes are of great significance to the innovation of apple germplasm resources.【Method】The homozygous genotype lines of ‘Gala', ‘Fuji' and ‘Red Star' were obtained by apple anther culture. The ploidy level of homozygous genotype lines was studied using flow cytometry, root and leaf morphology and root conditions of each line were observed and recorded. 【Result】 A total of 32 homozygous lines were obtained by apple anther culture. The results of flow cytometry showed that there were 1 haploid, 1 triploids, 3 tetraploids and 27 diploids. The ploidy rate of 'Red Star' homozygous genotype was the highest, about 28.57%. The root rate, root length and root number of homozygous lines were affected by IBA concentration of rooting medium. When the concentration of IBA was 2-3 mg·L-1, the root features of apple homozygous lines were the best. The leaf number, root length, root number, plant height, leaf length/width and petiole length of each line were different. ‘Red Star' homozygous genotype lines(DH2-1, DH0-3, DH0-4, DH0-7), ‘Fuji' homozygous genotype line(DH1-3) and ‘Gala' homozygous genotype lines(DH2-2, DH2-4, DH2-12, DH2-20) have a good traits(plants are taller, roots are longer, and the number of roots and leaves are more). The survival rate of ‘Red Star' homozygous genotype was the highest, about 28.57%. 【Conclusion】The main ploidy of apple homozygous genotype was diploid. Compared with the diploid lines, the haploid lines had a narrow leaf base, finer petiole, the leaf color and the leaf margin were shallow, while the polyploid lines showed wider leaf base, thicker petioles, leaf color and leaf edge serrated deep.
【Objective】Homozygous genotype lines were obtained by anther culture of different apple pollen grains, so their botanical characteristics, biological characteristics, and their induced culture, root conditions were different. In this study, the ploidy, characteristics and root conditions of each line were observed and analyzed. Appropriate rooting conditions and good combinations of lines for selecting apple homozygous genotypes are of great significance to the innovation of apple germplasm resources.【Method】The homozygous genotype lines of ‘Gala', ‘Fuji' and ‘Red Star' were obtained by apple anther culture. The ploidy level of homozygous genotype lines was studied using flow cytometry, root and leaf morphology and root conditions of each line were observed and recorded. 【Result】 A total of 32 homozygous lines were obtained by apple anther culture. The results of flow cytometry showed that there were 1 haploid, 1 triploids, 3 tetraploids and 27 diploids. The ploidy rate of 'Red Star' homozygous genotype was the highest, about 28.57%. The root rate, root length and root number of homozygous lines were affected by IBA concentration of rooting medium. When the concentration of IBA was 2-3 mg·L-1, the root features of apple homozygous lines were the best. The leaf number, root length, root number, plant height, leaf length/width and petiole length of each line were different. ‘Red Star' homozygous genotype lines(DH2-1, DH0-3, DH0-4, DH0-7), ‘Fuji' homozygous genotype line(DH1-3) and ‘Gala' homozygous genotype lines(DH2-2, DH2-4, DH2-12, DH2-20) have a good traits(plants are taller, roots are longer, and the number of roots and leaves are more). The survival rate of ‘Red Star' homozygous genotype was the highest, about 28.57%. 【Conclusion】The main ploidy of apple homozygous genotype was diploid. Compared with the diploid lines, the haploid lines had a narrow leaf base, finer petiole, the leaf color and the leaf margin were shallow, while the polyploid lines showed wider leaf base, thicker petioles, leaf color and leaf edge serrated deep.
引文
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[22]TESTILLANO P,GEORGIEV S,MOGENSEN H L,CORONADO M J,DUMAS C,RISUENO M C,MATTHYS-ROCHON E.Spontaneous chromosome doubling results from nuclear fusion during in vitro maize induced microspore embryogenesis.Chromosoma,2004,112(7):342-349.
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[2]王炜,陈琛,欧巧明,叶春雷,罗俊杰.小麦花药培养的研究和应用.核农学报,2016,30(12):2343-2354.WANG W,CHEN C,OU Q M,YE C L,LUO J J.Research and application of wheat anther culture.Journal of Nuclear Agricultural Sciences,2016,30(12):2343-2354.(in Chinese)
[3]CARDOSO J C,MARTINELLI A P,GERMANàM A,LATADO R R.In vitro anther culture of sweet orange(Citrus sinensis L.Osbeck)genotypes and of a C.clementina×C.sinensis‘Hamlin’hybrid.Plant Cell,Tissue and Organ Culture,2014,117(3):455-464.
[4]ZHAO J,ZOU X X,ZHANG Z Q,YANG B Z,ZHOU S D.Influences of carbon sources and plant growth regulators on anther culture efficiency of pepper.Agricultural Science and Technology,2010,11(4):102-105.
[5]H?FER M.In vitro androgenesis in apple.Gartenbauwissenschaft,2003,60(1):287-292.
[6]李佳.百合单倍体培养及细胞学观察[D].北京:中国农业科学院,2011.LI J.Haploid culture and cytology observation in lilium[D].Beijing:Chinese Academy of Agricultural Sciences,2011.(in Chinese)
[7]ZHANG C F,SATO S,TSUKUNI T,SATO M,OKADA H,YAMAMOTO T,WADA M,MATSUMOTO S,YOSHIKAWA N,MIMIDA N,TAKAGISHI K,WATANABE M,CAO Q F,KOMORI S.Elucidating cultivar differences in plant regeneration ability in an apple anther culture.The Horticulture Journal preview,2016,doi:10.2503/hortj.MI-094.
[8]薛光荣,牛健哲,杨振英,史永忠,费开韦.苹果花药培养技术及8个主栽品种的花粉植株培育成功.中国农业科学,1990,23(3):86-87.XUE G R,NIU J Z,YANG Z Y,SHI Y Z,FEI K W.The technique of apple anther culture and the successful culture of pollen plantlets of 8main apple cultivar.Scientia Agricultura Sinica,1990,23(3):86-87.(in Chinese)
[9]ZHANG C,TSUKUNI T,IKEDA M,SATO M,OKADA H,OHASHI Y,MATSUNO H,YAMAMOTOT,WADA M,YOSHIKAWA N,MATSUMOTO S,LI J,MIMIDA N,WATANABE M,SUZUKI A,KOMORI S.Effects of the microspore development stage and cold pre-treatment of flower buds on embryo induction in apple(Malus×domestica Borkh.)anther culture.Journal-Japanese Society for Horticultural Science,2013,82(82):114-124.
[10]温鑫,邓舒,张春芬,侯丽媛,石江鹏,聂园军,肖蓉,秦永军,曹秋芬.‘嘎啦’苹果花药培养种质创新.中国农业科学,2017,50(14):2793-2806.WEN X,DENG S,ZHANG C F,HOU L Y,SHI J P,NIE Y J,XIAO R,QIN Y J,CAO Q F.Regeneration of new germplasms using anther culture of apple cultivar‘Gala’.Scientia Agricultura Sinica,2017,50(14):2793-2806.(in Chinese)
[11]H?FER M.Regeneration of androgenic embryos in apple(Malus×domestica Brokh.)via anther and microspore culture.Acta Physiologiae Plantarum,2005,27(4):709-716.
[12]H?FER M,GRAFE C,BOUDICHEVSKAJA A,LOPEZ A,BUENO M A,ROEN D.Characterization of plant material obtained by in vitro androgenesis and in situ parthenogenesis in apple.Scientia Horticulturae,2008,117(3):203-211.
[13]H?FER M,FLACHOWSKY H.Comprehensive characterization of plant material obtained by in vitro androgenesis in apple.Plant Cell,Tissue and Organ Culture,2015,122(3):617-628.
[14]翟衡,史大川,束环瑞.我国苹果产业发展现状与趋势.果树学报,2007,24(3):355-360.ZHAI H,SHI D C,SHU H R.Current state and developing trend of apple industry in China.Journal of Fruit Science,2007,24(3):355-360.(in Chinese)
[15]张春芬,邓舒,肖蓉,孟玉平,曹秋芬.苹果花药培养再生植株的倍性鉴定及SSR分析.园艺学报,2015,42(S1):2580.ZHANG C F,DENG S,XIAO R,MENG Y P,CAO Q F.Ploidy identification and SSR analysis of regenerated plants in apple anther culture.Acta Horticulturae Sinica,2015,42(S1):2580.(in Chinese)
[16]任莹.苹果单倍体育种技术探究[D].太原:山西大学,2015.REN Y.Research on Haploid breeding technique in anther culture[D].Taiyuan:Shanxi University,2015.(in Chinese)
[17]蒲富慎.果树种质资源描述符—记载项目及评价标准.北京:中国农业出版社,1990:23-37.PU F S.Fruits Germplasm Descripto—Record Items and Evaluation Standards.Beijing:China Agricultural Press,1990:23-37.(in Chinese)
[18]崔佩佩,刘鹏,刘佳琪,王劲松,武爱莲,董二伟,丁玉川,焦晓燕.不同养分配比对高粱根系生长及养分吸收的影响.中国生态农业学报,2017,25(11):1643-1652.CUI P P,LIU P,LIU J Q,WANG J S,WU A L,DONG E W,DING Y C,JIAO X Y.Effect of different nutrient co mbinations on root growth and nutrient accumulation in sorghum.Chinese Journal of EcoAgriculture,2017,25(11):1643-1652.
[19]ZENG S H,CHEN C W,HONG L,LIU J H,DENG X X.In vitro induction,regeneration and analysis of autotetraploids derived from protoplasts and callus treated with colchicine in Citrus.Plant Cell,Tissue and Organ Culture,2006,87(1):85-93.
[20]KAWASE K,YAHATA M,NAKAGANA S,HARAGUCHI K,KUNITAKE H.Selection of autotetraploid and its morphological characteristics in Meiwa Kumquat(Fortunella crassifolia Swingle)(Breeding&Germplasm Resources).Horticultural Research,1975,4(11):141-146.
[21]H?FER M.In vitro androgenesis in apple-improvement of the induction phase.Plant Cell Reports,2004,22(6):365-370.
[22]TESTILLANO P,GEORGIEV S,MOGENSEN H L,CORONADO M J,DUMAS C,RISUENO M C,MATTHYS-ROCHON E.Spontaneous chromosome doubling results from nuclear fusion during in vitro maize induced microspore embryogenesis.Chromosoma,2004,112(7):342-349.
[23]GRIGGS R,ZHENG M Y.Nuclear fusion during early stage of microspore embryogenesis indicates chromosome doubling in wheat(Triticum aestivum).American Journal of Plant Sciences,2016,07(3):489-499.
[24]钟晓红,戴思慧,马定渭.核果类果树茎尖培养研究进展.果树学报,2003,20(5):388-392.ZHONG X H,DAI S H,MA D W.Advances of research in shoot-tip culture on stone fruit crops.Journal of Fruit Science,2003,20(5):388-392.(in Chinese)
[25]肖祖飞.童性对苹果砧木绿枝扦插生根的影响[D].北京:中国农业大学,2014.XIAO Z F.Impact of the juvenility on the adventitious rooting of leafy cuttings in apple rootstocks[D].Beijing:China Agriculture University,2014.(in Chinese)
[26]KAUSHAL N,MODGIL M,THAKUR M,SHARMA D R.In vitro clonal multiplication of an apple rootstock by culture of shoot apices and axillary buds.Indian Journal of Experimental Biology,2005,43(6):561-565.
[27]DOBRáNSZKI J,SILVA J A T D.Micropropagation of apple-a review.Biotechnology Advances,2010,28(4):462-488.
[28]谢璇,许轲,谢闽新,朱元娣.苹果茎尖培养快繁体系的优化.植物生理学报,2015,51(12):2152-2156.XIE X,XU K,XIE M X,ZHU Y D.Optimization of rapid micropropagation system of apple meristem-tip culture.Plant Physiology Journal,2015,51(12):2152-2156.(in Chinese)
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