Artificial hybridization was carried out between AMV (alfalfa mosaic virus) and WCMV (white clovermosaic virus) transgenic red clover plants in order to combine both genes in one plant. Almost all the pair crossesset seeds, the germination rate of T1 plants varied among different crosses, with those from 1 copy transgeneparents gave the highest values. Southern hybridization analysis demonstrates that some T1 plants got more thanone copies of transgene, some of them expressed both transgenes. Their resistance to viruses were assessed byvirus challenge and bioassay, which showed that the T1 plants with both transgenes were resistant against bothAMV and WCMV, from which immune plants were generated. Some of plants were only resistant to either AMVor WCMV, some were susceptible in spite of their positive results in Northern hybridization. Compared with genetransformation in transgenic plants, artificial hybridization between different transgenic plants is a simpler, time-saving way for gene pyramiding.%随着转基因技术的发展及其在植物育种中的应用,出现了一大批表现优良、经济效益高的转基因植物,但生产实践要求一种植物同时具有多个优良的转基因性状.目前解决这一问题的方法主要是基因累集(Gene pyramiding),就是将多个基因集中到同一植株,可以将已经转入1个基因的植株做原材料,用农杆菌介导或基因枪等方法将另一基因转进去.也可以采用人工杂交,在不同转基因植株之间进行人工授粉,从后代中筛选出所需要的基因型.本研究对抗苜蓿花叶病毒(AMV)的转基因红三叶和抗白三叶花叶病毒(WCMV)的转基因红三叶进行了人工杂交,以期获得兼有2个基因、同时抵抗2种病毒的新植株,为下一步的育种打好基础.几乎所有的杂交组合都结了种子,杂交一代的发芽率随杂交亲本转基因拷贝数的不同而有所变化,只有1个转基因拷贝的亲本其杂种发芽率最高.对所有幼苗先用PCR进行检测,从中挑出较理想的基因型作进一步的分子生物学分析.Southern和Northern印迹分析结果表明,有些杂交一代的转入基因多于1个拷贝,有一部分同时兼有2种抗病基因,并且得到了表达.为进一步检测其抗病性,对它们进行了温室接种和田间评估,结果显示,尽管有的个体Northern分析呈阳性,但在田间仍然对病毒敏感,不过其感病程度都比对照有显著降低.大多数兼有2种转入基因的植株都能同时抵抗2种病毒,可以作为免疫个体供下一步育种之用.与遗传转化相比,人工杂交以其操作简单、省时省力、预见性强和准确率高而具有明显优势,是常规育种方法在分子育种中的具体体现.
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