‘嘎啦’苹果花药培养种质创新

doi:10.3864/j.issn.0578-1752.2017.14.015

摘要/Abstract

摘要： 【目的】单倍体花药离体培养是农作物包括果树育种中种质资源创新的最有效方法之一，苹果是染色体高度杂合且自交不亲和树种之一。在当前苹果主栽品种中，‘嘎啦’具有早熟、丰产、稳产、多抗的优良性状，是苹果育种的重要种质资源之一。花药单倍体育种也是苹果新品种培育的重要手段。本研究通过‘嘎啦’苹果花药培养诱导胚状体并获得纯合再生植株，为创制新的纯合体种质资源，加速苹果新品种培育进程提供材料。【方法】采集‘嘎啦’苹果单核靠边期到双核早期的花药（未开放的花蕾）, 低温处理后进行离体培养，经胚状体诱导，分化培养形成再生苗，再经生根培养获得花药再生植株。之后利用FACS流式细胞仪对再生植株进行倍性分析。取再生植株叶片分离DNA，选用80个来源于苹果HIDRAS数据库的SSR标记对所有植株进行PCR扩增，经过凝胶电泳和荧光毛细管电泳鉴定再生植株纯合基因型。移栽成活后，对每个再生株系进行形态学特征观察及统计分析。【结果】过去3年中，共接种的74 200个‘嘎啦’花药，从未被污染的5万多个花药中成功诱导形成386个胚状体（胚状体诱导率0.7%），经分化培养获得64株再生苗（植株再生率16.6%），最终经生根培养、移栽获得30个成活再生株系。其中包括28个二倍体株系，1个单倍体株系和1个四倍体株系。SSR标记用于纯合性鉴定，PAGE结果表明再生株系均为花粉（小孢子）单倍体细胞来源。为了鉴定这些再生植株基因型，从80个SSR中筛选出17个SSR标记（其余SSR标记不具有多态性或带型杂乱）对所获得的30个再生株系进行基因型鉴定。17个SSR标记所对应的PCR扩增物能有效区分鉴定不同再生植株基因型。继代培养60 d后的形态学观察显示不同再生株系的株高、叶长、叶宽等特征差异明显。不同二倍体纯合植株的植物学特征也存在差异：Gala 5植株相对较高，叶基变宽，叶尖渐尖；Gala 7叶片变小、变厚，叶柄变短且基部宽大，叶色深且有很强的光泽度；Gala 18叶片较小，叶数较多。纯合二倍体再生植株长势弱于‘嘎啦’杂合供体，但强于单倍体和纯合四倍体。【结论】采用优化花药培养技术，成功获得了一批苹果纯合体再生植株种质并建立了SSR标记鉴定体系。这些新的种质很大程度丰富了苹果育种亲本种质资源，为挖掘‘嘎啦’苹果优良性状基因提供了重要材料，为后期的田间性状筛选，杂交育种奠定了基础。

关键词: &lsquo, 嘎啦苹果&rsquo, ；花药离体培养；单倍体育种；SSR标记；纯合基因型

Abstract: 【Objective】Anther culture is one of the most effective techniques to create new germplasms in modern breeding. Apple is one of the most highly genetically heterozygous and self-incompatible fruit tree. Among the current major apple germplasms, ‘Gala’ cultivar shows traits of early maturity, high yield and enhanced anti-biotic stress, thus is a very important genetic resource for apple breeding programs. Haploid breeding has already been employed to regenerate new germplasm in apple breeding research. In this study, the plantlets were regenerated through embryogenesis during “gala” cultivar anther culture lines to enrich parental apple breeding germplasms. 【Method】The anther culture was used to regenerate gala plants. The ploidy level of regenerated plantlets was determined using flow-cytometry. Subsequently, using selected SSR (HIDRAS) markers, the genotypes of regenerated lines were characterized by gel electrophoresis as well as fluorescent capillary electrophoresis. The anther of ‘Gala’ cultivar collected at early stage were firstly treated at low temperature and then cultured in vitro through embryos induction and differentiation phases and the regenerated plantlets were obtained. The ploidy levels of regenerated plants were analyzed using FACS flow cytometry. To identify the genotype of the plantlets, the DNA was isolated from leaves and then subjected to PCR amplification based on 80 SSR primers selected from the apple HIDRAS database. The homozygous genotypes were determined using both gel electrophoresis and fluorescence capillary electrophoresis. After transplantation, the morphological characteristics of each regenerated plantlet were analyzed.【Result】In the past 3 years, by using the previously optimized anther culture technology, a total of 74 200 ‘Gala’ anthers were inoculated with embryo induction of 0.7% (contaminated anthers over 50 000), resulted in 386 embryos. With differentiation culture, 64 regenerated plantlets survived with regeneration rate of 16.6%. After root induction phase, finally 30 regenerated lines including 28 diploids, 1 haploid and 1 tetraploid were obtained. The PAGE analysis showed that all the regenerated lines were originated from haploid pollen. For genotyping these regenerated plantlets, 17 of 80 SSR markers were further selected (the remaining SSR markers were not polymorphic thus cannot be used for the genotyping) for PCR amplification. Results showed that the panel of 17 SSRs each located in 17 linkage groups, respectively, well distinguished genotypes of the 30 individual regenerated lines. Subsequent morphological observation at the time of 60 days subculture showed that the height of plantlets and morphology of the leaves varied largely. In addition, variations regarding variant ploidy levels were also observed: Gala 5 line was relatively high with wider leaf base, while Gala 7 line showed smaller and thicker leaves and Gala 18 line showed smaller leaves and less amount of leaves. Moreover, diploid plantlets showed a trend of weaker growth than that of the parental ‘Gala’ but stronger than that of haploid and homozygous tetraploid.【Conclusion】By using the technique of anther culture, a set of apple germplasm was successfully obtained and a SSR marker identification system was established. More importantly, the regenerated lines have greatly enriched the apple germplasms and will lay a foundation for apple haploid breeding.

Key words: Gala, anther culture, haploid breeding, SSR markers, homogeneous genotype

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[J]. 中国农业科学, 2017, 50(14): 2793-2806.

WEN Xin, DENG Shu, ZHANG ChunFen, HOU LiYuan, SHI JiangPeng, NIE YuanJun, XIAO Rong, QIN YongJun, CAO QiuFen. Regeneration of New Germplasms Using Anther Culture of Apple Cultivar ‘Gala’[J]. Scientia Agricultura Sinica, 2017, 50(14): 2793-2806.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2017.14.015

https://www.chinaagrisci.com/CN/Y2017/V50/I14/2793

参考文献

[1] Dwivedi S L, Britt A B, Tripathi L, Sharma S, Upadhyaya H D, Ortiz R. Haploids: Constraints and opportunities in plant breeding. Biotechnology Advances, 2015, 33(6): 812-829.

[2] Forster B P, Heberle-Bors E, Kasha K J, Touraev A. The resurgence of haploids in higher plants. Trends in Plant Science, 2007, 12(8): 368-375.

[3] Germana M A. Gametic embryogenesis and haploid technology as valuable support to plant breeding. Plant Cell Reports, 2011, 30(5): 839-857.

[4] Chiancone B, Germana M A. Microspore embryogenesis through anther culture in Citrus clementina Hort. ex Tan. Methods in Molecular Biology, 2016, 1359: 475-487.

[5] Dong Y Q, Zhao W X, Li X H, Liu X C, Gao N N, Huang J H, Wang W Y, Xu X L, Tang Z H. Androgenesis, gynogenesis, and parthenogenesis haploids in cucurbit species. Plant Cell Reports, 2016, 35(10): 1991-2019.

[6] Nguyen Q T, Bandupriya H D, Lopez-Villalobos A, Sisunandar S, Foale M, Adkins S W. Tissue culture and associated biotechnological interventions for the improvement of coconut (Cocos nucifera L.): A review. Planta, 2015, 242(5): 1059-1076.

[7] Ochoa-Alejo N. Anther culture of chili pepper (Capsicum spp.). Methods Molecular Biology, 2012, 877: 227-231.

[8] Seifert F, Bossow S, Kumlehn J, Gnad H, Scholten S. Analysis of wheat microspore embryogenesis induction by transcriptome and small RNA sequencing using the highly responsive cultivar "Svilena". BMC Plant Biology, 2016, 16: 97.

[9] 翟衡, 赵政阳, 王志强, 束怀瑞. 世界苹果产业发展趋势分析. 果树学报, 2005, 22(1): 44-50.

ZHAI H, ZHAO Z Y, WANG Z Q, SHU H R. Analysis of the development trend of the world apple industry. Journal of Fruit Science , 2005, 22(1): 44-50. (in Chinese)

[10] 翟衡, 史大川, 束怀瑞. 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)

[11] Harris S A, Robinson J P, Juniper B E. Genetic clues to the origin of the apple. Trends in Genetics, 2002, 18(8): 426-430.

[12] Igarashi M, Hatsuyama Y, Harada T, Fukasawa- Akada T. Biotechnology and apple breeding in Japan. Breed Science, 2016, 66(1): 18-33.

[13] Urrestarazu J, Denance C, Ravon E, Guyader A, Guisnel R, Feugey L, Poncet C, Lateur M, Houben P, Ordidge M, Fernandez-Fernandez F, Evans K M, Paprstein F, Sedlak J, Nybom H, Garkava-Gustavsson L, Miranda C, Gassmann J, Kellerhals M, Suprun I, Pikunova A V, Krasova N G, Torutaeva E, Dondini L, Tartarini S, Laurens F, Durel C E. Analysis of the genetic diversity and structure across a wide range of germplasm reveals prominent gene flow in apple at the European level. BMC Plant Biology, 2016, 16(1): 130.

[14] Guha S, Maheshwari S C. In vitro production of embryos from anthers of Datura. Nature, 1964, 204(4957): 497.

[15] Maluszynski M, Kasha K J, Forster B P, Szarejko I. Doubled Haploid Production in Crop Plants. Springer Netherlands, 2003: 287-292.

[16] Nakayama R, Saito K, Sato T, Tamura Y. Studies on the hybridization in apple breeding. II. Anther culture of the apple. Faculty of Agriculture, Hirosaki University, 1971, 17, 12-19.

[17] 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 8 main apple cultivar. Scientia Agricultura Sinica,1990, 23(3): 86-87. (in Chinese)

[18] Vanwynsberghe L, Kde W, Coart E, Keulemans J. Limited application of homozygous genotypes in apple breeding. Plant Breeding, 2005, 124(4): 399-403.

[19] 杨振英, 薛光荣, 苏佳明, 迟福梅, 牛健哲, 丛佩华. ‘富士’花药培养选育出苹果新品种‘华富’. 园艺学报,2005, 32(1):14.

Yang Z Y, Xue G R, Su J M, Chi F M, Niu J Z, Cong P H. A new apple cultivar ‘huafu’ bred by anther culture of ‘Fuji’. Acta Horticulturae Sinica, 2005, 32(1): 14.(in Chinese)

[20] Okada H, Ohashi Y, Sato M, Matsuno H, Yamamoto T, Hoytaek K, Tukuni T, Komori S. Characterization of fertile homozygous genotypes from anther culture in apple. Journal of the American Society for Horticultural Science, 2009, 134(6): 641-648.

[21] Höfer M. Regeneration of androgenic embryos in apple (Malus x domestica Brokh.) via anther and microspore culture. Acta Physiologiae Plantarum, 2005, 27(4): 709-716.

[22] 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)

[23] Zhang C, Tsukuni T, Ikeda M, Sato M, Okada H, Ohashi Y, Matsuno H, Yamamoto T, 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.

[24] 罗慧珍, 邓舒, 张春芬, 肖蓉, 王卉, 孟玉平, 曹秋芬. 低温诱导下苹果花药差异表达基因分析. 植物生理学报, 2016(3): 259-268.

LUO H Z, DENG S, ZHANG C F, XIAO R, WANG H, MENG Y P, CAO Q F. Differentially expressed gene analysis of apple (Malus domestica) anther under low temperature induction. Plant Physiology Journal,2016(3): 259-268. (in Chinese)

[25] 冯丽云, 张春芬, 聂园军, 邓舒, 温鑫, 肖蓉, 李倩, 任莹, 罗慧珍, 曹秋芬. 苹果花药石蜡切片制片技术改良及其解剖学观察. 中国农学通报, 2016, 32(31): 62-67.

FENG L Y, ZHANG C F, NIE Y J, DENG S, WEN X, XIAO R, LI Q, REN Y, LUO H Z, CAO Q F. Improvement of paraffin section methods and structural observation of apple anther. Chinese Agricultural Science Bulletin, 2016, 32(31): 62-67. (in Chinese)

[26] Aubakirova K, Omasheva M, Ryabushkina N, Tazhibaev T, Kampitova G, Galiakparov N. Evaluation of five protocols for DNA extraction from leaves of Malus sieversii, Vitis vinifera, and Armeniaca vulgaris. Genetics and Molecular Research , 2014, 13(1): 1278-1287.

[27] Burbulis N, Blinstrubiene A, Sliesaravicius A, Venskutoniene E. Influence of genotype, growth regulators, sucrose level and preconditioning of donor plants on flax (Linum usitatissimum L.) anther culture. Acta Biologica Hungarica, 2005, 56(3/4): 323-331.

[28] Kadota M, Niimi Y. In vitro induction of tetraploid plants from a diploid Japanese pear cultivar (Pyrus pyrifolia N. cv. Hosui). Plant Cell Reports, 2002, 21(3): 282-286.

[29] Höfer M. In vitro androgenesis in apple-improvement of the induction phase. Plant Cell Reports, 2004, 22(6): 365-370.

[30] Höfer M, Touraev A, Heberle-Bors E. Induction of embryogenesis from isolated apple microspores. Plant Cell Reports, 1999, 18(12): 1012-1017.

[31] Germana M A, Chiancone B. Improvement of Citrus clementina Hort. ex Tan. microspore-derived embryoid induction and regeneration. Plant Cell Reports, 2003, 22(3): 181-187.

[32] 张圣仓, 魏安智, 杨途熙. 果树单倍体和加倍单倍体(DH)技术研究与应用进展. 果树学报, 2011, 28(5): 869-874.

ZHANG S C, WEI A Z, YANG T X. Advances in the research and application of haploid and doubled haploid technologies in fruit trees. Journal of Fruit Science, 2011, 28(5): 869-874. (in Chinese)

[33] Peixe A, Barroso J, Potes A, Pais M S. Induction of haploid morphogenic calluses from in vitro cultured anthers of prunus armeniaca cv. ‘Harcot’. Plant Cell, Tissue and Organ Culture, 2004, 77(1): 35-41.

[34] 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.

[35] Höfer 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.

[36] 张利义, 杨振英, 丛佩华, 张开春, 刘建利, 薛光荣. 苹果花药培养植株倍性及其纯合基因型的鉴定. 园艺学报, 2007, 34(2): 481-484.

ZHANG L Y, YANG Z Y, CONG P H, ZHANG K C, LIU J L, XUE G R. Analysis of homozygous genotype of apple plant obtained by anther culture. Acta Horiculturac Sinica, 2007, 34(2): 481-484. (in Chinese)

[37] Torkamaneh D, Laroche J, Belzile F. Genome-wide SNP calling from genotyping by sequencing (GBS) data: A comparison of seven pipelines and two sequencing technologies. PLoS One, 2016, 11(8): e0161333.

[38] 李林光, 王颖, 王玉霞, 张志宏, 伊凯, 刘志. 苹果四倍体品种天星的花粉形态及胚囊发育特征研究. 植物遗传资源学报, 2011, 12(4): 662-666.

LI L G, WANG Y,WANG Y X, ZHANG Z H, YI K, LIU Z. Pollen characteristic and embryonic development of tense apple. Journal of Plant Genetic Resources, 2011, 12(4): 662-666. (in Chinese)