程远;万东艳;张宝林;王跃进;文颖强;

• 1:西北农林科技大学园艺学院·农业部西北地区园艺作物生物学与种质创新重点实验室·旱区作物逆境生物学国家重点实验室

摘要(Abstract)：

【目的】通过诱导、保存和扩繁葡萄原胚团,为葡萄外源基因转化及基因编辑研究提供受体材料。【方法】以4个欧亚种葡萄品种和1个野生种葡萄的花器官为材料,比较不同外植体在3种培养基上胚性愈伤组织的诱导率,通过二次再生途径诱导、保存和扩繁次生原胚团并利用改良苯酚品红染色法对其进行细胞学观察。【结果】‘无核白’葡萄的雄蕊接种在PIV培养基上胚性愈伤组织诱导率最高(10.8%);初生原胚团细胞核大、染色深且具有旺盛分裂能力,二次再生途径诱导的次生原胚团具有同样的细胞学特征且可以正常成苗。【结论】葡萄体细胞胚诱导受基因型、外植体类型影响显著;二次胚再生途径可以长期诱导、保存和扩繁原胚团,能为葡萄遗传转化持续提供受体材料。

关键词(KeyWords)： 葡萄;体细胞胚;二次胚再生;次生原胚团;细胞学观察

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金(31372022);; 陕西省科技研究发展(攻关)计划(2014K02-02-03)

作者(Authors): 程远;万东艳;张宝林;王跃进;文颖强;

DOI: 10.13925/j.cnki.gsxb.20170011

参考文献(References)：

• [1]贺普超,罗国光.葡萄学[M].北京:中国农业出版社,1994:10.HE Puchao,LUO Guoguang.Grape science[M].Beijing:China Agriculture Press,1994:10.
• [2]BISSON L,WATERHOUSE A,EBELER S,WALKER M,LAPSLEY.The present and future of the international wine industry[J].Nature,2002,418(6898):696-699.
• [3]BOUQUET A,TORREGROSA L,IOCCO P,THOMSA M R.Grapevine(Vitis vinifera L.)[M]//In:Meilan R&Ma C(ed)Agrobacterium Protocols Volume 2.Springer,2007:273-285.
• [4]REN C,LIU X,ZHANG Z,WANG Y,DUAN W,LI S,LIANG Z.CRISPR/Cas9-mediated efficient targeted mutagenesis in Chardonnay(Vitis vinifera L.)[J].Scientific Reports,2016,6:32289.
• [5]MARTINELLI L,GRIBAUDO I.Strategies for effective somatic embryogenesis in grapevine:an appraisal[M]//In:Roubelakis-Angelakis KA(ed).Grapevine molecular physiology&biotechnology.Springer,2009:93.
• [6]周起.无核白葡萄体细胞胚再生体系建立与转基因研究[D].杨凌:西北农林科技大学,2014.ZHOU Qi.Somatic embryogenesis and transformation of grapevine Thompson Seedless[D].Yangling:Northwest A&F University,2014.
• [7]DHEKNEY S A,LI Z T,DUTT M,GRAY D J.Agrobacterium-mediated transformation of embryogenic cultures and plant regeneration in Vitis rotundifolia Michx.(muscadine grape)[J].Plant Cell Report,2008,27(5):865-872.
• [8]DHEKNEY S A,LI Z T,DUTT M,GRAY D J.Optimizing initiation and maintenance of Vitis embryogenic cultures[J].HortScience,2009,44(5):1400-1406.
• [9]PERRIN M,GERTZ C,MASSON J E.High efficiency initiation of regenerable embryogenic callus from anther filaments of 19-grapevine genotypes grown worldwide[J].Plant Science,2004,167(6):1343-1349.
• [10]KIKKERT J R,STRIEM M J,VIDAL J R,WALLACE P G,BARNARD J,REISCH B I.Long-term study of somatic embryogenesis from anthers and ovaries of 12 grapevine(Vitis sp.)genotypes[J].In Vitro Cellular&Developmental Biology-Plant,2005,41(3):232-239.
• [11]GAMBINO G,RUFFA P,VALLANIA R,GRIBAUDO I.Somatic embryogenesis from whole flowers,anthers and ovaries of grapevine(Vitis spp.)[J].Plant Cell Tissue and Organ Culture,2007,90(1):79-83.
• [12]OLáH R,ZOK A,PEDRYC A,HOWARD S,KOVáCS L G.Somatic embryogenesis in a broad spectrum of grape genotypes[J].Scientia Horticulturae,2009,120(1):134-137.
• [13]王关林,方宏筠.植物基因工程原理与技术[M].北京:科学出版社,2004:250.WANG Guanlin,FANG Hongjun.Plant genetic engineering[M].Beijing:Science Press,2004:250.
• [14]AMAR A B,COBANOV P,BOONROD K,KRCZAL G,BOUZID S,GHORBEL A,REUSTLE G.Efficient procedure for grapevine embryogenic suspension establishment and plant regeneration:role of conditioned medium for cell proliferation[J].Plant Cell Reports,2007,26(9):1439-1447.
• [15]JAYASANKAR S,VAN AMAN M,CORDTS J,DHEKNEY S,LI Z,GRAY D.Low temperature storage of suspension culture-derived grapevine somatic embryos and regeneration of plants[J].In Vitro Cellular&Developmental Biology-Plant,2005,41(6):752-756.
• [16]支玉玺,张剑侠,王跃进.华东葡萄广西-2花药胚状体诱导与再生植株的研究[J].果树学报,2010,27(1):18-23.ZHI Yuxi,ZHANG Jianxia,WANG Yuejin.Studies on somatic embryogenesis and plant regeneration from anthers of Guangxi-2,a line of Chinese wild grape(Vitis pseudoreticulata)[J].Journal of Fruit Science,2010,27(1):18-23.
• [17]肖宇,张朝红,支玉玺,徐炎,李志谦,王跃进.葡萄花器官体细胞胚的诱导和植株再生[J].果树学报,2011,28(5):888-892.XIAO Yu,ZHANG Chaohong,ZHI Yuxi,XU Yan,LI Zhiqian,WANG Yuejin.Somatic embryogenesis from floral organ and plant regeneration of grapevine[J].Journal of Fruit Science,2011,28(5):888-892.
• [18]卢炳芝,于向荣,郭飞跃,李佩芬,鲍雪珍.葡萄花丝培养中的胚胎发生[J].果树科学,1995,12(增刊):37-40.LU Bingzhi,YU Xiangrong,GUO Feiyue,LI Peifen,BAO Xuezhen.The embryogenesis from culture in vitro of filaments of grapevine[J].Journal of Fruit Science,1995,12(Suppl.):37-40.
• [19]刘华英,萧浪涛,鲁旭东,吴顺.柑橘体细胞胚发生的组织细胞学研究[J].果树学报,2004,21(4):311-314.LIU Huaying,XIAO Langtao,LU Xudong,WU Shun.Studies on histocytology of somatic embryogenesis in Citrus sinensis cv.Valencia[J].Journal of Fruit Science,2004,21(4):311-314.
• [20]邓朝军,易干军,曾继吾,张秋明.荔枝体胚发生过程中的细胞学观察[J].果树学报,2006,23(5):760-762.DENG Chaojun,YI Ganjun,ZENG Jiwu,ZHANG Qiuming.Cytological observation of somatic embryogenesis in litchi[J].Journal of Fruit Science,2006,23(5):760-762.
• [21]曾黎辉,吕柳新,王平,卢秉国.荔枝、龙眼胚性愈伤组织的细胞组织学观察[J].福建农林大学学报(自然科学版),2002,31(3):331-333.ZENG Lihui,LU Liuxin,WANG Ping,LU Binguo.Cytohistological observation of embryogenic callus of litchi and longan[J].Journal of Fujian Agriculture and Forestry University(Natural Science Edition)2002,31(3):331-333.
• [22]冯新富,谢丽君,陈厚彬,李华平,徐春香.香蕉愈伤组织及体细胞胚诱导过程中的组织学观察[J].果树学报,2007,24(6):788-791.FENG Xinfu,XIE Lijun,CHEN Houbin,LI Huaping,XU Chunxiang.Histological observation on the callus and somatic embryo induction in banana(Musa AAA)[J].Journal of Fruit Science,2007,24(6):788-791.
• [23]何业华,罗吉,胡中沂,王瑞霞,高爱平,方少秋,赵春香,杨向晖,叶自行,王泽槐.菠萝非胚性愈伤组织的类型及其器官发生[J].果树学报,2008,25(1):65-68.HE Yehua,LUO Ji,HU Zhongyi,WANG Ruixia,GAO Aiping,FANG Shaoqiu,ZHAO Chunxiang,YANG Xianghui,YE Zixing,WANG Zehuai.Classification and organogenesis of non-embryogenic callus from Ananas comosus[J].Journal of Fruit Science,2008,25(1):65-68.
• [24]LI Z T,KIM K,JASINSKI J R,CREECH M R,GRAY D J.Largescale characterization of promoters from grapevine(Vitis Spp.)using quantitative anthocyanin and GUS assay systems[J].Plant Science,2012,196:132-142.
• [25]KANDEL R,BERGEY D R,DUTT M,SITTHER V,LI Z T,GRAY D J,DHEKNEY S A.Evaluation of a grapevine-derived reporter gene system for precision breeding of Vitis[J].Plant Cell Tissue and Organ Culture,2016,124(3):599-609.