高效的水稻基因枪转化和可育转基因植株再生

Efficient Production of Fertile Transgenic Rice Following Particle Bombardment

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.

Many globular embryogenic calli suitable for particle bombardment were produced, after 3-4 week old calli derived from mature embryos of rice variety Taipei 309 were subcultured for 1-3 times. The embryogenic calli were bombarded with gold particles coated with the plasmid pIP860 containing snowdrop lectin (GNA) gene and the plasmid p35H containing hpt gene. Selection, pre-regeneration, regeneration and rooting were carried out on media with 30-50 mg/L hygromycin. Partial desiccation treatment was used to increase the efficiency of regeneration. The effective method of transplanting regenerated plantlets was established. Transformed plantlets were recovered within 13-21 weeks after mature seeds were plated. From 536 bombarded embryogenic calli, 2783 hygromycin resistant plants of 199 lines were obtained. DNA dot blot analysis showed that 73. 4% of resistant plants contained GNA and hpt genes. Southern blot analysis confirmed the integration of GNA gene in the genome of transgenic rice. 89. 3% of the transgenic plants were fertile. Mendelian segregation of the foreign genes was demonstrated by molecular analysis of R1 plants.