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从水稻品种台北309成熟胚诱导生长3～4周的愈伤组织,经过1～3次继代培养后,可以形成大量颗粒状胚性愈伤组织,适用于基因枪转化实验.将分别含有雪花莲凝集素(GNA)基因、hpt基因的质粒pIP860和p35H混合包裹在金粉上轰击转化上述胚性愈伤组织,在含30～50mg/L潮霉素的培养基上进行筛选、预再生、再生及长根培养.使用干燥处理来增加再生频率和出苗数,并摸索出移栽再生小植株的有效方法.从接种到获得转化小植株只需要13～21周.三次转化实验共轰击536块胚性愈伤组织,获得199个系的2783株潮霉素抗性植株.DNA点杂交检测表明73.4%的抗性植株同时含有GNA基因和hpt基因,Southern杂交分析进一步证实了GNA基因在转基因水稻基因组中的整合.89.3%的转基因植株可育.分子证据表明GNA基因和hpt基因已遗传至子代,并且主要按孟德尔规律进行分离.
The callus from rice cultivar Taipei 309 mature embryo was induced to grow for 3 to 4 weeks, and after 1 to 3 subculturing, a large number of granular embryogenic callus could be formed, which was suitable for gene gun transformation experiments. Lectin (GNA) gene, hpt gene plasmid pIP860 and p35H mixed wrapped in gold powder bombardment transformation of the above embryogenic callus in 30 ~ 50mg / L hygromycin on the medium for screening, pre-regeneration, regeneration And long-root culture, using drying treatment to increase the regeneration frequency and seedling emergence, and to find out the effective method for transplanting and regenerating plantlets.It takes only 13 ~ 21 weeks from inoculation to obtain the transformed plantlets.Three transformation experiments bombarded 536 embryos Sexual calli, 2783 strains of hygromycin resistant plants were obtained from 199 lines.The results of DNA dot blotting showed that 73.4% of the resistant plants contained both GNA and hpt genes, and Southern hybridization further confirmed that the GNA gene in the transgenic rice genome , 89.3% of the transgenic plants are fertile, and molecular evidence suggests that the GNA and hpt genes have been inherited to progeny and are predominantly Mendelian.