通过基因枪和农杆菌介导用BADH基因转化小麦(英文)

Transformation of Wheat with BADH Through Gene Gun and Agrobacterium

土壤盐碱是一种严重障碍作物生产的环境因子,甜菜碱醛脱氢酶BADH基因是一种重要的可赋予植物渗透调节抗性的基因。本研究用基因枪法及农杆菌介导法向小麦幼胚和成熟胚愈伤组织导入了BADH基因。用PDS-1000/HE基因枪轰击2 933块幼胚愈伤组织,分化出了45株再生植株,分化率为1.53%。PCR分析表明,其中的5株为BADH基因转化植株。用PPT涂抹其叶片,进一步证实了PCR的结果。以小麦成熟胚愈伤组织为受体,用农杆菌介导转化1 968块愈伤,仅再生出了5株绿苗,PCR检测结果均为阴性。但对其转化愈伤组织的PCR检测表明,外源基因已在受体细胞中实现了整合。以幼胚愈伤组织为受体,用农杆菌介导转化2 933块愈伤,共再生出了21株绿苗。对其进行PCR检测,仅有5株为BADH基因转化植株。转化处理过的幼胚愈伤组织的绿苗再生率(0.72%)高于成熟胚愈伤(0.25%)。与对照相比,所有的转化植株均能够在0.5%NaCl(w/w)条件下正常生长,表明外源BADH基因已经整合并表达。

Salinity is very severe environmental problem hampering crop production. BADH is an important gene for conferring osmotic stress tolerance to plants. In this study gene gun and Agrobacterium were employed for the delivery of BADH into callus of wheat. Bombardment of 2 933 calli derived from immature embryos resulted in regeneration of 45 plant-lets after selection on phosphinothricin （PPT） with net regeneration rate of 1.53%. PCR analysis confirmed the presence of BADH gene in five plants. Results of PPT-leaf painting conformed to PCR test. In case of Agrobacterium mediated transformation five plant-lets regenerated from inoculation of 1968 calli derived from mature embryos but none was PCR positive. PCR test of calli from mature embryos exhibited the successful delivery and integration of transgene. Inoculation of 2 933 calli from immature embryos resulted in regeneration of 21 plant-lets. PCR evaluation of regenerated plantlets indicated the presence of BADH in 5 plants only. Net regeneration rate for immature embryos （0.72%） was higher than that of mature embryos （0.25 % ）. Transformed plants obtained through Agrobacterium and gene gun were able to thrive in 0.5% NaCl（w/w） without stunted growth and wilting as compared with check plants indicating the successful integration and expression of BADH.