水稻原生质体高频分裂及基因转化  

Improvement of division frequency and exogenous gene transformation in the rice protoplasts

作　　者：徐明良[1] 杨金水[1] 胡容霞[1] 葛扣麟[1] 

机构地区：[1]复旦大学遗传学研究所

出　　处：《复旦学报（自然科学版）》1996年第6期637-642,共6页Journal of Fudan University：Natural Science

基　　金：国家自然科学基金

摘　　要：从粳稻品系８９８和籼稻不育系珍籼Ａ的悬浮细胞系中制备的原生质体，分裂频率分别达１８．９％和３５％．低压脉冲电泳仪介导外源基因转移的水稻原生质体，分裂频率也在１０％以上，转化的小细胞团中检测到外原基因Ｇｕｓ的表达．对原生质体的制备、培养及转化作了下述改进：（１）添加２μｇ／Ｌ２，４－Ｄ的Ｎ６培养基培养悬浮细胞及原生质体可获高频分裂，添加０．３μｇ／Ｌ６－ＢＡ或０．１μｇ／ＬＮＡＡ不利于分裂；（２）细胞团酶解时间缩短至３ｈ左右，且镜检到少量原生质体游离下来时即终止酶解；（３）酶解后的细胞团先洗涤后过滤可获大量活力很强的原生质体；（４）低压脉冲电泳介导外源基因转移的最佳处理条件为：脉冲时间３０ｓ、电压４０Ｖ、电泳３０ｍｉｎ．rotoplasts isolated from suspension cultures of Japonica 898 an indica Zhengshan 97 A divided vigorously with frequencies of 18. 9% and 35%,respectively. Transgenic protoplasts by low-voltage pulsed field electrophoretic drive also divided rapidly with a high frequency of 10%. Gus gene activity was identified in the small cell mass thus produced. Methodological improvements are as follows: (1) Medium N6 supplemented with 2 ig/L 2,4-D stimulates,wheras,the addition of 0. 3μg/L 6--BA or 0. 1 μg/L NAA inhibits protoplast divislonl (2)Time of cell digestion is reduced to about 3 hours. Stop the digestion when small amounts of protoplasts are dissociated from the cell mass; (3)Washing digested cell mass followed by filtering results in a large amount of viable protoplasts i (4)The optimmum of division frequency of transgenic protoplasts by low-voltage pulsed field electrophoretic drive is a pulse time of 30 s,40 V and 30 min on electrophoresis.

关 键 词：水稻 原生质体 基因转化 高频分裂 生物技术 

分 类 号：S511.032[农业科学—作物学] S188