通过共转化和花药培养快速获得直链淀粉含量降低且无抗性标记的转基因水稻

Rapidly Obtaining the Markerless Transgenic Rice with Reduced Amylose Content by Co-transformation and Anther Culture

用根癌农杆菌共转化的方法将p13W8质粒(含反义蜡质基因)和pCAMBIA1300质粒(含潮霉素抗性基因)导入水稻。经PCR检测,发现在29个T1群体中发生抗潮霉素抗性基因和反义蜡质基因的分离;从1264个T1单株中筛选到183个只带反义蜡质基因片段的转基因植株。选择了4个直链淀粉含量降低的T1单株进行花药培养,获得正常结实的花培苗34株;经PCR检测,有23株为只含反义蜡质基因而无潮霉素抗性基因的植株。从转化到获得直链淀粉含量降低、遗传稳定的转基因植株仅用了一年半时间。

The plasmid p13W8 carrying antisense fragment of waxy gene and plasmid pCAMBIA1300 containing hpt gene were introduced into rice by Agrobacterium tumefaciens-mediated co-transformation, and 86 transgenic plants were obtained, 32 of them showed positive bands for antisense waxy gene by PCR analysis, the wary-positive plant frequency is 37.2%. The segregation of antisense fragment of waxy gene and hpt gene was observed by PCR using hpt gene primers and waxy gene primers respectively in 29 T1 population. One hundred and eighty-three plants containing only the antisense fragment of waxy gene were identified in 1 264 T1 plants, the wary-positive plant frequency is 14.4% (Table 1). The amylose content of seeds derived from transgenic plants with only the antisense fragment of waxy gene weredetermined, varying degrees of reduction in amy-lose content were found in some plants (Table 2). Four T, plants with reduced amylose content were selected through anther culture. Thirty-four anther culture plants seed normally, 23 of them were shown to contain only the antisense fragment of waxy gene (Table 3) by PCR analysis, and the amylose content was reduced to 5%-12% (Table 4). It took only one and half years to obtain the stably inherited markerless transgenic rice with reduced amylose content by co-transformation and anther culture technique.