柑桔基因转化新方法的研究

尽管应用基因转化进行果树品种改良已日益引起重视,但是在受体的应用和转化方法上还存在着诸多困难。一方面,大多数果树尚不能从细胞或原生质体再生成完整植株,即使少数已可以再生的果树树种,也并非众多品种都能再生成功,而是存在着明显的基因型差异性。同时,还有再生植株童期过长的问题。另一方面,目前在植物基因转化中常用的两种方法即DNA直接吸入法和农杆菌介导的载体法,若以细胞或原生质体为受体,不仅存在再生困难的问题,而且再生过程费时长;若以叶盘、愈伤组织或珠心组织等为受体,既需要在转化后除去农杆菌,又需要排除转化与非转化组织的嵌合性。这些因素都大大地限制了基因转化在果树中的应用。因此,根据多年生果树的生长特点,建立一种适用的基因转化技术已成当务之急。本文采用农杆菌介导的附体腋芽转化-离体扩繁鉴定的方法,成功地将GUS基因转入沙田柚。结果证明这是一种简单、快速、高效的基因转化方法。

A NEW APPROACH FOR GENE TRANSFER INTO CITRUS

Agrobacterium- mediated in vivo transformation of lateral buds of citrus plants has been developed and been combined with in vitro cloning. This new approach has ingeniously detoured plant regeneration and the long juvenile stage of regenerated plants from protoplasts, which limits gene transformation of most fruit trees including citrus. It makes the elimination of remaining bacteria and the selection of transformants be easy and efficient by using the natural growth of buds. In addition, this approach has combined the cloning of transformants in order to increase population rapidly,In demonstrating experiment, 21 out of 112 treated buds of Shatian pummelo ( Citrus gran-dis Osb.) developed into primary branches which gave 76 secondary buds. 4 in 76 secondary buds (5.26%) were proved to be positive by GUS activity assay. Among 14 cluster buds differentiated from the in vitro culture of 4 positive buds, 4 showing highest GUS activity and being positive in Southern hybridization were regenerated into true plants. This approach could be used as a simple and effecient method for the transformation of a wide range of fruit tree species.