水稻胚性愈伤诱导及其遗传转化的几个技术参数研究

以日本晴成熟种子及幼嫩种子为材料诱导形成胚性愈伤组织,经农杆菌介导将水稻隐花色素基因的4个RNAi和2个反义RNA表达载体分别转化日本晴愈伤组织,再经抗性筛选和分化培养诱导形成植株,经分子检测筛选出阳性转基因苗。对胚性愈伤诱导率、抗性愈伤率、分化出苗率及转化率进行统计分析。结果表明,成熟种子、新鲜成熟种子及幼嫩种子愈伤诱导率均大于81%,平均为86.67%,其中以新鲜成熟种子愈伤诱导率最高,达96.89%;抗性愈伤率成熟胚为22.21%~40.71%,平均为28.49%,幼胚为36.79%~43.21%,平均为40%,幼胚的抗性愈伤率高于成熟胚;分化出苗率成熟胚为59.29%~80.43%,平均为71.59%,幼胚为63.16%~72.73%,平均67.95%,幼胚与成熟胚差异不明显;转化率(Gus检测阳性率)成熟胚为43.07%~81.08%,平均61.59%,幼胚为58.33%~76.67%,平均67.50%,幼胚转化率稍高于成熟胚。

STUDIES ON RICE EMBRYOGENIC CALLUS INDUCTION AND SEVERAL TECHNICAL PARAMETERS FOR THEIR GENETIC TRANSFORMATION

Mature and young seeds of japonic rice cultivar "Nipponbare" were used as materials to induce and produce embryogenic callus.Four RNAi and two anti-sense RNA expression vectors of rice cryptochrome gene were transformed into these callus tissue mediated by agrobacterium tumefaciens.Induced plants were obtained by resistance screening and differentiation culture.Positive transgenic plants were identified through molecular detection.Statistic analysis of induced callus rate,resistance callus rate,seedling differentiation rate and transformation rate were then carried out.The results indicated that the callus induction rate for all matuer seeds,fresh mature seeds and young seeds was higher than 81%,averaged 86.67%,with the induction rate for fresh mature seeds highest up to 96.87%.The resistance callus rate for mature embryo was 22.21%～40.71%,averaged 28.49%,for young embryo was 36.79%～43.21%,averaged 40%,which means the resistance callus rate for the young embryo was higher than that for the mature embryo.The seedling differentiation emergence rate for mature embryo was 59.29%～80.43%,averaged 71.59%,for young embryo was 63.16%～72.73%,averaged 67.95%,showing no significant difference between the two kinds of embryos.The transformation rate(Gus positive rate) for mature embryo was 43.07%～81.08%,averaged 61.51%,for young embryo was 58.33%～76.67%,averaged 67.50%,which means the transformation rate for the young embryo was slightly higher than that for the mature embryo.