作　　者： ; ; ; ; ; ;

机构地区： 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

出　　处： 《中国农业科学》 2012年第19期4050-4056,共7页

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-1TM2G新鲜培养基每10 d更换1次,此后每10 d用0.25 mol.L-1TM2G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。 [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 × 10^5 p/mL or 2×10^5 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 0.25mol·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 5×10^5 p/mL developed into compact calli （could develop into embryos）, protoplasts cultured at density of 2×10^5 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.

关 键 词： 木薯 脆性胚性愈伤组织 原生质体 植株再生

领　　域： [农业科学]