中国农业科学 ›› 2012, Vol. 45 ›› Issue (19): 4050-4056.doi: 10.3864/j.issn.0578-1752.2012.19.017

• 园艺 • 上一篇    下一篇

木薯脆性胚性愈伤组织原生质体培养与植株再生

 文峰, 肖诗鑫, 聂扬眉, 马秋香, 张鹏, 郭文武   

  1. 1.华中农业大学园艺林学学院/园艺植物生物学教育部重点实验室,武汉 430070
    2.广西壮族自治区亚热带作物研究所,南宁 530001
    3.中国科学院上海生命科学研究院植物生理生态研究所植物分子遗传国家重点实验室,上海 200032
  • 收稿日期:2012-04-25 出版日期:2012-10-01 发布日期:2012-08-02
  • 通讯作者: 通信作者郭文武,Tel:027-87281543;E-mail:guoww@mail.hzau.edu.cn
  • 作者简介:文 峰,E-mail:wenfengw83@163.com
  • 基金资助:

    国家“973”基础研究课题“木薯分子育种技术集成与种质创新”(2010CB126606)

Protoplasts Culture Isolated from Friable Embryogenic Callus of Cassava and Plant Regeneration

 WEN  Feng, XIAO  Shi-Xin, NIE  Yang-Mei, MA  Qiu-Xiang, ZHANG  Peng, GUO  Wen-Wu   

  1. 1.华中农业大学园艺林学学院/园艺植物生物学教育部重点实验室,武汉 430070
    2.广西壮族自治区亚热带作物研究所,南宁 530001
    3.中国科学院上海生命科学研究院植物生理生态研究所植物分子遗传国家重点实验室,上海 200032
  • Received:2012-04-25 Online:2012-10-01 Published:2012-08-02

摘要: 【目的】建立有效的木薯原生质体再生体系,为原生质体融合以及原生质体转化等研究奠定技术基础。【方法】酶解木薯品种TMS60444 的脆性胚性愈伤组织(FEC)的悬浮系,分离原生质体的产量最高达3.5×106个/g,FDA检测其活性约90%。用TM2G培养基以液体浅层法分别在5×105个/mL和2×105个/mL密度下培养,培养过程中前30 d用0.3 mol•L-1 TM2G新鲜培养基每10 d更换1次,此后每10 d用0.25 mol•L-1 TM2G新鲜培养基更换。原生质体培养45 d后即可挑出1—2 mm大小的愈伤组织分别在MSN培养基上分化胚、CMM上促胚成熟、CEM上茎伸长、MS上生根。【结果】在5×105个/mL的原生质体培养密度下,长出的都是致密型愈伤组织(能分化胚状体),而在2×105个/mL的密度下培养长出的有致密型愈伤组织,也有空泡型愈伤组织(不能分化胚状体)。本试验共挑出1 479个致密细胞团,分化获得757个子叶胚,已再生完整植株186棵。【结论】本研究分离的原生质体产量和活性有较大提高,对前人所指出的瓶颈问题有所改进,原生质体再生植株效率有较大提高。

关键词: 木薯, 脆性胚性愈伤组织(FEC), 原生质体, 植株再生

Abstract: 【Objective】The objective of this study is to establish an efficient system of protoplast regeneration for further developing protoplast fusion and transformation in cassava. 【Method】 Protoplasts were isolated from suspension cultures derived from friable embryogenic callus (FEC) of cassava genotype TMS60444. The highest protoplast yield obtained was 3.5×106 protoplasts/g fresh weight. Viabilities of the protoplasts assessed by the fluorescein diacetate (FDA) were approximately 90%. Protoplasts were cultured in TM2G medium with liquid thin layer culture at densities of 5×105 p/mL or 2×105 p/mL. During the first 30 d, the medium was refreshed by 0.3 mol•L-1 TM2G fresh medium every 10 d. After that, the medium was refreshed by 0.25 mol•L-1 TM2G fresh medium every 10 d. After cultured for 45 d, calli of 1-2 mm were picked out and separately developed into embryos on MSN medium, into mature embryos on CMM medium, into shoots on CEM medium and into roots on MS medium. 【Result】It was showed that all protoplasts cultured at density of 5×105 p/mL developed into compact calli (could develop into embryos), protoplasts cultured at density of 2×105 p/mL developed into compact calli and vacuolar calli (could not develop into embryos). A total of 1 479 compact calli were picked out and developed into 757 cotyledon embryos and regenerated 186 plants in the experiment. 【Conclusion】The yield and viability of isolated protoplasts had been greatly increased, the bottleneck of predecessors mentioned was improved, and the efficiency of plant regeneration from protoplasts was promoted. 

Key words: cassava, friable embryogenic callus (FEC), protoplast, plant regeneration