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.
出处
《复旦学报（自然科学版）》
CAS
CSCD
北大核心
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
关键词
水稻
原生质体
基因转化
高频分裂
生物技术
rice protoplast
low-voltage pulse field electrophoretic drive gene transformation
