Ghosh Biswas G C, Iglesias V A, Datta S K, Potrykus I
Institute of Plant Sciences, Swiss Federal Institute of Technology, Zürich.
J Biotechnol. 1994 Jan 15;32(1):1-10. doi: 10.1016/0168-1656(94)90114-7.
We have established a reproducible procedure for transformation of protoplasts and regeneration of transgenic plants for an improved Indica rice cultivar IR43. Mature embryo-derived calli were placed in liquid culture medium containing maltose to establish meristematically active, embryogenic cell suspension lines. In order to obtain transgenic plants, a chimeric hygromycin phosphotransferase hph gene under the control of the cauliflower mosaic virus CaMV 35S promoter was introduced into protoplasts from these cell suspension lines using polyethylene glycol. Protoplasts were cultured in maltose-containing medium. Hygromycin B selection was applied to 14-day-old dividing cell colonies. Resistant calli were readily obtained after 3 weeks of selection. Seventy-three plantlets were regenerated from resistant calli from several independent experiments, and a few of the 29 plants grown in the greenhouse reached maturity. Stable integration of the transgene in the genome of these plants was confirmed by Southern blot analysis and the expression of the transgene in plants by hygromycin phosphotransferase assay. The procedure described yielded 5 to 18 resistant colonies and approximately four transgenic plantlets per million treated protoplasts.
我们已经建立了一种可重复的方法，用于改良籼稻品种IR43的原生质体转化和转基因植株再生。将成熟胚来源的愈伤组织置于含有麦芽糖的液体培养基中，以建立具有分生活性的胚性细胞悬浮系。为了获得转基因植株，使用聚乙二醇将受花椰菜花叶病毒CaMV 35S启动子控制的嵌合潮霉素磷酸转移酶hph基因导入这些细胞悬浮系的原生质体中。原生质体在含麦芽糖的培养基中培养。对14日龄的分裂细胞集落施加潮霉素B选择。选择3周后很容易获得抗性愈伤组织。从几个独立实验的抗性愈伤组织中再生出73株幼苗，温室中生长的29株植物中有少数达到成熟。通过Southern印迹分析证实转基因稳定整合到这些植物的基因组中，并通过潮霉素磷酸转移酶测定证实转基因在植物中的表达。所述方法每百万个处理过的原生质体产生5至18个抗性集落和约4个转基因幼苗。
