冬小麦遗传再生体系及根癌农杆菌介导的转化研究

摘要:以冬小麦品种临优145的幼胚为外植体,消毒后用解剖刀挑取幼胚，盾片朝上接种于MS+2,4-D 2 mg/L+肌醇100 mg/L＋MES 400 mg/L＋CH 100 mg/L7]＋40g/L麦芽糖＋8g/L琼脂的培养基中诱导愈伤组织, 每两周继代一次，将诱导出的淡黄色、颗粒状Ⅱ型胚性愈伤组织接种于分化培养基（MS+ZT 1 mg/L+IAA 1mg/L+ MES 400 mg/L＋CH 100 mg/L+麦芽糖40g/L+gelrite 2.6g/L，PH5.8）上培养2－3周,然后转接到再生培养基(1/10MS+NAA 0.5mg/L+KT 0.5mg/L+蔗糖30g/L+ gelrite 2.6g/L，PH5.8)中进行再生成苗。接种1229块幼胚,得到987块愈伤组织, Hyg抗性愈伤组织123块，抗性再生植株34株。在建立了再生体系的基础上,用根癌农杆菌介导法将GUS基因导入幼胚愈伤组织,抗性植株的PCR检测呈阳性。X－gluc染色表明, 少部分愈伤组织出现肉眼可见的蓝色晕斑，说明GUS基因已经在小麦幼胚愈伤组织中表达。

A preliminary study of regeneration system of winter wheat immature embryo and its Agrobacterium transformation

Abstract: Immature embryos of a common wheat variety Linyou145 was used as explant to induce regeneration and transformation,respectively. The immature embryo were inoculated on the MS medium with 2 mg·L-1 2,4-dichlorophenoxyacetic acid to initiate calli. Then,they were inoculated on the MS medium with 1mg·L-1 ZT and 1mg·L-1 IAA, cultured for 2-3weeks for plant differentiation.Finally,the calli of differentiation were transferred into the MS medium with 0.5mg·L-1 NAA for plant regeneration.Following the above protocol,987calli,123resistant callus and 34 plantlets were obtained from 1229 embryosintotal, Furthermore, plasmid DNA of pCAMBI1301 containing GUS gene as the donor of alien gene was transformed into the immature embryo calli by Agrobacterium tumefaciens.By means of PCR amplification, hygomycin resistant plantlets were obtained. The result from X-gluc staining showed that GUS gene gave transient expression successfully in the transformed calli.Blue spots were observed in resistant calli.