作 者 ：杜建中，孙 毅 *，郝曜山，王亦学，王长彪，王 铭
期 刊 ：西北植物学报 2012年 2期 页码：231~240
Keywords:Basta, non-tissue culture transformation method, tolerance, transformation frequency,
摘 要 ：以玉米‘昌7-2’、大豆‘晋遗19’和高粱‘晋粱5号’的种子为受体，质粒pCAMBAR.CHI.11为供体，采用非组培转化法导入bar基因。PCR扩增和Southern杂交分析结果证明，‘昌7-2’、‘晋遗19’和‘晋粱5号’的划胚种子都获得了转化植株，转化率均在5%以上，未划胚种子的转化率为0。共培养液中加入浓度为200 μmol/L的乙酰丁香酮（AS）能使玉米种子转化率比对照提高8倍，赋予300 W强度的超声波处理，转化率可再提高60%。划胚处理对种子的出苗率影响较大，为17%左右。划胚种子于实验室条件下的Basta耐受试验结果表明，不同作物对Basta的耐受性存在差异，玉米、大豆和高粱转bar基因植株的Basta耐受临界值分别为0.15%、0.1%和0.05%，Basta处理后的幼苗成活率在各作物间差异不大。田间喷雾Basta溶液结果证明，按Basta耐受性大小排列顺序为高粱＜大豆＜玉米，但按对Basta敏感性的排序为大豆＜高粱＜玉米。不同生育期喷雾除草剂的实验结果(T2)证明，在生育期后期作物植株对除草剂的敏感性比前期有所降低，生长前期喷雾后玉米、大豆和高粱的苗存活率分别为57%、49%和52%，生长后期喷雾后苗存活率分别为77%、70%和73%，但各个群体的PCR扩增阳性结果率均在80%以上，证明存活率差异是由植株对除草剂耐受性和敏感性的差异引起的。多代转基因植株的检测结果证明目的基因可稳定遗传。
Abstract:Used plasmid pCAMBAR.CHI.11 as donor,the seeds of maize ‘Chang 7-2’,soybean ‘Jinyi 19’ and sorghum ‘Jinliang 5’ as acceptor,bar gene was introduced into crop plants by non-tissue-culture transformation method.The results of PCR amplification and Southern hybridization analysis showed that the transgenic plants derived from wounded-embryo seeds of maize,soybean and sorghum were obtained.The transformation frequency was up to 5%,in contrast,while the transformation frequency of normal seeds was 0.Acetosyringone solution at final concentration of 200 μmol/L in medium could make transformation frequency of maize seeds 8 times higher than that of CK.In addition,the supersonic wave (300 W) treatment further enhanced 60% of transformation frequency.Compare with CK,the ratio of seedling of each crop wound-embryo seeds were reduced greatly,only 17%.The Basta-tolerance activity experiment of 3 crops with wounded-embryo seeds was conducted in the laboratory.The investigation results of the experiment suggested that a little difference of Basta-tolerance activity of 3 crops was existed among them.The critical values of maize,soybean and sorghum that can endured damage of herbicide were 0.15%,0.1% or 0.05%,respectively.The results of Basta-sprayting experiment in the field further also confirmed that the crop rank according to their Basta-tolerance activity was sorghum＜soybean＜maize.However,the crop rank according to their Basta-susceptibility became soybean＜sorghum＜maize.We sprayed Basta solution at different development stage in the field,the results (T2) showed that,compared with that of spraying Basta at early development stage,the Basta-susceptibility of each crop had a little reduced when spraying Basta at later development stage.Spraying Basta at early stage of growth,the ratio of living seedlings of maize,soybean and sorghum were 57%,49% and 52%,respectively,but spraying Basta at later stage of growth,it became 77%,70% and 73%,respectively.PCR amplification results of all samples come from these material displayed the ratio of PCR positive-result was up to 80%.The result indicated that the difference of the Basta-tolerance activity or Basta-susceptibility of each crop plant resulted in the difference of the ratio of living seedling.The results of PCR amplification and southern hybridization analysis to T1～T3 plants showed the target transgene could inheritance steadily.