随着生物技术的发展,转基因技术已经越来越多的应用到植物育种领域并发挥着重要的作用,将传统育种技术与转基因技术相结合,既可以缩短育种时间,并具有良好的经济效益.为最终获得能稳定遗传且性状优良的转基因植株,对其后代进行鉴定及筛选是非常重要的.本研究以抗苜蓿花叶病毒(AMV)转基因红三叶和抗白三叶花叶病毒(WCMV)的转基因红三叶人工杂交后代T1,T2代植株为研究对象,通过PCR及RT-PCR等分子生物学手段,对T1代34株植株进行PCR检测,筛选出12株阳性植株,其中兼有两种抗病基因的植株5株,各有一个抗病基因的植株7株,在这些植株中进行杂交组合,大部分杂交组合都结了种子,最终得到30株性状优良的T2代植株,对其进行PCR及RT-PCR检测,结果表明AMV与WCMV外壳蛋白基因能够在大部分T2代转基因植株中整合并表达,并且通过人工杂交,得到9株兼有两个抗病基因的杂交后代,外源基因稳定地从T0传至T2.同时也表明人工杂交是转基因植株基因累积的一种有效方法.%Thirty-four T1 generation plants of transgenic red clover (Trifolium pretense L.) were analyzed by PCR and 12 single or double resistant transgenic plants were obtained. Artificial hybridization was carried out between these positive plants of T1. Among 30 T2 progenies obtained, nine offspring expressed both AMV and WCMV CP genes and nine expressed single CP gene by PCR and RT-PCR analysis. The results of PCR and RT-PCR indicated that the exogenous genes had been integrated into the genome of transgenic red clover in most of the hybrid offspring and could transcribe correctly. Moreover, the exogenous genes could steadily inherit from T0 to T2. The results also demonstrated that artificial hybridization is an efficient way of gene pyramiding of breeding program.
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