Yan B, Srinivasa Reddy M S, Collins G B, Dinkins R D
Department of Agronomy, N122 Agricultural Science Center-North, University of Kentucky, Lexington KY 40546-0091, USA e-mail:
Plant Cell Rep. 2000 Nov;19(11):1090-1097. doi: 10.1007/s002990000236.
-mediated transformation of soybean [Glycine max (L.) Merrill. cv. Jack] using immature zygotic cotyledons was investigated to identify important factors that affected transformation efficiency and resulted in the production of transgenic soybean somatic embryos. The factors evaluated were initial immature zygotic cotyledon size, Agrobacterium concentration during inoculation and co-culture and the selection regime. Our results showed that 8- to 10-mm zygotic cotyledons exhibited a higher transformation rate, as indicated by transient GUS gene expression, whereas the smaller zygotic cotyledons, at less than 5 mm, died shortly after co-cultivation. However, the smaller zygotic cotyledon explants were found to have a higher embryogenic potential. Analysis of Agrobacterium and immature cotyledon explant interactions involved two Agrobacterium concentrations for the inoculation phase and three co-culture regimes. No differences in explant survival or somatic embyogenic potential were observed between the two Agrobacterium concentrations tested. Analysis of co-culture regimes revealed that the shorter co-culture times resulted in higher explant survival and higher somatic embryo production on the explants, whereas the co-culture time of 4 days severely reduced survival of the cotyledon explants and lowered their embryogenic potential. Analysis of selection regimes revealed that direct placement of cotyledon explants on hygromycin 25 mg/l was detrimental to explant survival, whereas 10 mg/l gave continued growth and subsequent somatic embryo development and plant regeneration. The overall transformation frequency in these experiments, from initial explant to whole plant, was 0.03 %. Three fertile soybean plants were obtained during the course of these experiments. Enzymatic GUS assays and Southern blot hybridizations confirmed the integration of T-DNA and expression of the GUS-intron gene in the three primary transformants. Analysis of 48 progeny revealed that three copies of the transgene were inherited as a single Mendelian locus.
研究了根癌农杆菌介导的利用未成熟合子子叶对大豆[Glycine max (L.) Merrill. cv. Jack]进行转化，以确定影响转化效率并导致产生转基因大豆体细胞胚的重要因素。评估的因素包括初始未成熟合子子叶大小、接种和共培养期间的农杆菌浓度以及选择方案。我们的结果表明，8至10毫米的合子子叶表现出较高的转化率，瞬时GUS基因表达表明了这一点，而小于5毫米的较小合子子叶在共培养后不久死亡。然而，发现较小的合子子叶外植体具有更高的胚性潜力。对接种阶段涉及两种农杆菌浓度和三种共培养方案的农杆菌与未成熟子叶外植体相互作用进行了分析。在所测试的两种农杆菌浓度之间，未观察到外植体存活率或体细胞胚性潜力的差异。对共培养方案的分析表明，较短的共培养时间导致外植体存活率更高，外植体上的体细胞胚产量更高，而4天的共培养时间严重降低了子叶外植体的存活率并降低了它们的胚性潜力。对选择方案的分析表明，将子叶外植体直接置于25毫克/升潮霉素上对外植体存活有害，而10毫克/升则能使外植体持续生长并随后进行体细胞胚发育和植株再生。在这些实验中，从初始外植体到整株植物的总体转化频率为0.03%。在这些实验过程中获得了三株可育的大豆植株。酶促GUS分析和Southern杂交证实了T-DNA在三个初级转化体中的整合以及GUS内含子基因的表达。对48个后代的分析表明，转基因的三个拷贝作为一个单孟德尔位点遗传。
