大豆愈伤原生质体的制备和培养方式探究  

Study on the Preparation and Cultivation of Soybean Callus Protoplasts

作　　者：苏彤 姚陆铭[1] 张鑫[2] 王彪[1] 武天龙[1] SU Tong;YAO Lu-ming;ZHANG Xin;WANG Biao;WU Tian-long(School of Agricultural and Biology, Shanghai Jiao Tong University, Shanghai 200240, China;College of Life and Environment Science, Shanghai Normal University, Shanghai 200234, China)

机构地区：[1]上海交通大学农业与生物学院,上海200240 [2]上海师范大学生命与环境科学学院,上海200234

出　　处：《大豆科学》2018年第5期741-747,共7页Soybean Science

基　　金：国家重点研发计划(2017YFD0101500);国家自然科学基金(31601321;31871645)

摘　　要：具有植物细胞全能性的原生质体是探究植物遗传转化和基因功能的理想材料,为了高效率制备大豆原生质体及其稳定培养,以交大05-133大豆未成熟子叶诱导的愈伤组织为材料,采用正交设计,对纤维素酶、果胶酶和离析酶等酶解液成分进行分析,研究原生质体高效制备方法的酶解液配比,同时探讨不同酶解时间对原生质体产量的影响,以确定最佳的原生质体酶解条件;通过对比在低熔点琼脂固体液滴培养与液体培养这两种不同培养方式下细胞的增值速率,以期建立高效的愈伤原生质体再生体系。结果显示,最佳酶解液配比为:2%纤维素酶+0. 1%果胶酶+1%离析酶,在该酶解液配比下酶解5 h,所得到原生质体产量和活力最高,达到(3. 976±0. 86)×10~6个·g^(-1)。用KP8培养基对原生质体进行培养,结果显示固体液滴的培养方式更适合原生质体的分裂,原生质体植板率高于液体培养,并且在25 d内分裂形成致密的细胞团。Protoplasts with cell totipotency are ideal materials for exploring genetic transformation and gene function of plants. In order to product and culture effectively protoplast of soybean, the callus induced by immature cotyledons of Jiaoda 05-133 was used as materials preparing for protoplasts in this study. Cellulase onozuka R-10, pectolyase Y-23 and macerozyme R-10 were designed according to the orthogonal array, at the same time, the influence of different enzymatic hydrolysis time on the protoplast yield were explored for optimizing protoplast preparation conditions. Two culture methods with agarose bead and liq-uid culture were compared to establish an efficient protoplasm regeneration system. The results showed that, the enzyme com-bination containing cellulase onozuka R-10 （2%）, Pectolyase Y-23 （0. 1% ）, macerozyme R-10 （1%） was the optimal for protoplasts isolation of Jiaoda 05-133 callus. Under this condition, the appropriate time for Jiaoda 05-133 callus protoplasts isolation was 5 h in enzyme solution and the amounts of protoplasts reaching （3. 976 ± 0. 86） × l0^6·g^-1 with high quality and vigour were attained. Comparing the two culture methods, the results showed that the agarose bead culture with higher plating rate is more suitable for the cell division, and there are many dense cell clusters only within 25 d.

关 键 词：大豆 愈伤组织 原生质体 再生 正交设计 

分 类 号：S565.1[农业科学—作物学]