农杆菌介导陆地棉棕彩选1号茎尖遗传转化

为建立高效的棕色棉茎尖遗传转化体系,以陆地棉棕彩选1号为材料,采用农杆菌介导茎尖的遗传转化方法,以培养7 d的棕彩选1号幼苗茎尖为受体,设计正交试验对共培养时间、共培养温度、菌液OD600、侵染时间等条件进行优化;此外,还研究了受体茎尖的大小及真空渗透等因素对转化效率的影响,初步建立了棕色棉茎尖农杆菌介导的遗传转化技术体系。结果表明,带有0.5 cm下胚轴的茎尖适合转化,适宜的农杆菌介导遗传转化条件为农杆菌菌液浓度OD600=1.0,侵染时间20 min,共培养时间3 d,共培养温度24℃。此外,真空渗透处理有助于抗性苗的发生。再生植株经在含100 mg·L~(-1)卡那霉素的培养基上筛选,获得102株抗性植株,再进行PCR鉴定和Southern杂交检测,共获得24株转基因植株,转化率为0.4%,且为单拷贝基因插入。本试验建立的优化条件为棕色棉的遗传转化奠定了理论基础。

Agrobacterium-mediated Genetic Transformation of Stem Apexes of Brown Fiber Cotton Zongcaixuan 1

In order to establish an efficient genetic transformation system for stem apexes of brown cotton,Zongcaixuan 1 that belongs to upland cotton was used as materials for genetic transformation using Agrobacterium-mediated method.In this paper,the shoot tips from 7-day-old seedlings of Zongcaixuan 1 were selected as receptors,and co-culture time,co-culture temperature,bacteria OD600 and infection time were optimized by orthogonal test.In addition,the effects of the size of the shoot tip and the vacuum infiltration on the transformation efficiency were also studied,and the genetic transformation system of stem apexes from brown cotton was initially established.The results showed that the stem apexes with 0.5 cm hypocotyl were suitable for transformation,and the suitable conditions for Agrobacterium-mediated transformation were Agro bacteria concentration of OD600 =1.0,infection time for 20 min,co-culture time for 72 h and co-culture temporature for 24℃.Moreover,vacuum osmotic treatment was helpful to the occurrence of resistant seedlings.At first,plantlets were screened on the medium containing 100 mg· L-1 kanamycin,and 102 resistant plants were obtained.Subsequently,these plants were further detected by PCR and Southern blot,and in total 24 transgenic plants were obtained with a single copy gene insertion.The transformation rate was 0.4 ％.Furthermore,the optimization conditions for Agrobacterium-mediated genetic transformation of stem apexes of Zongcaixuan 1 were established in this study,which laid a foundation for the genetic transformation of brown cotton.