摘要
以籼稻胚性愈伤组织作为基因枪法转化的靶材料，建立了可重复的、高效的籼稻转化系统。从籼稻成熟胚诱寻生长3～4周的愈伤组织，经过2～6周继代培养后，可以形成足够量的颗粒状胚性意伤组织。用含有bar基因B.t.δ-内毒素基因的质粒pFWZ16轰击转化胚性愈伤组织，在含2～4mg／LBasta的培养基上进行筛选、预再生、再生及长根培养，并通过干燥处理增加再生频率和出苗数。从接种到获得转化小植株只需要4～5个月。供试的两个品种为华南地区推广的优良籼稻品种，共轰击821块胚性愈伤组织，获得48个系的477株Basta抗性植株。经PCR扩增分析和Southernblot杂交分析证实外源的bar基因和B.t.基因己整合到转基因水稻植株基因组中，Basta抗性鉴定和PNA点杂交分析表明这两个基因在转基因植株中得到表达。87.5％的转基因植株可育。Basta喷施实验和Southernblot分析证明bar基因和B.t.基因已遗传至子代，并且主要按孟德尔规律进行分离。
A reproducible and efficient transformation system for indica rice was developed based on microprojectile bombardment of embryogenic callus. Sufficient globular conceived October 30, 1997; revision receired January 6, 1998 Supported by the Rockefeller Foundation's international Program on Rice Biotechnology embryogenic calli were produced within 2-6 weeks from 3-4 week old calli derived from mature embryos. The embryogenic calli were bombarded with the plasmid pFWZ16 containing bar gene and B. t. δ-endotoxin gene. Selection, pre-regeneration, regeneration and rooting were carried out on media with 2-4mg / L Basta. Partial desiccation treatment was used to increase the efficiency of regeneration. Transformed plantlets were recovered within 4-5 months after matur seeds were plated. From 821 bombarded embryogenic calli of two elite rice varieties popularized in South China, 477 Basta resistant plants of 48 lines were obtained. Results of PCR, Southern blotting, RNA dot blotting, and topical application of Basta demonstrated that the foreign bar gene and B. t. gene were integrated into the genome of transgenic rice plants, and also expressed. 87.5% of the transgenic plants were fertile. Mendelian segregation of the foreign genes was indicated by Basta application and Southern blot analysis of R_1 plants.
基金
美国Rockefeller基金
关键词
籼稻
胚性愈伤组织
基因枪法转化
育种
indica rice, Embryogenic callus, The biolistic method, bar gene, B. t. δ-endotoxin gene
