棕色棉茎尖基因枪遗传转化

为了建立高效的棉花茎尖遗传转化体系,以棕色棉品种新彩棉5号为材料,采用茎尖培养和基因枪遗传转化方法,通过对凝固剂种类、活性炭含量、卡那霉素浓度、渗透处理时间、轰击压力、轰击距离等因素的优化,建立了棕色棉新彩棉5号茎尖基因枪遗传转化技术体系。结果表明,不同凝固剂种类、活性炭含量、卡那霉素浓度、渗透处理时间、轰击压力和轰击距离对棕色棉茎尖转化率有明显影响。最佳的遗传转化体系为:前渗处理4 h,轰击压力7.586 MPa(1100 psi),轰击距离9 cm,轰击后恢复培养4 d,然后在MSB+8 g·L~(-1)琼脂+125mg·L~(-1)卡那霉素筛选培养基中筛选25 d。获得抗性芽后,在添加1 g·L~(-1)活性炭的生根培养基中诱导抗性芽生根,并获得抗性再生植株。通过PCR和q PCR检测,5200个茎尖共获得16株阳性转基因植株,转化率为0.31%。该技术体系的建立为棕色棉的遗传转化奠定了基础,为彩色棉育种提供了新的材料。

Biolistic Genetic Transformation of Brown Cotton Shoot Tips

To genetically transform cotton shoot tips, we established a biolistic genetic transformation system though the plant shoot tip culture technique and the gene gun-mediated transformation method, using the shoot tips of brown cotton Xincaimian No.5, a Gossypium hirsutum L. variety cultured in Xinjiang as materials. Several critical factors of the system were assessed, including the type of coagulant, the activated charcoal concentration, the kanamycin concentration, the penetration processing time, and the bombardment pressure and distance. Results showed that each factor clearly influenced the transformation rate of brown cotton shoot tips. An optimal biolistic genetic transformation technology system was determined: (1) shoot tips were cultured for 4 h before being bombarded in the osmotic medium; (2) the shoot tips were bombarded with helium pressure of 7.586 MPa (1100 psi), and a bombardment distance of 9 cm; (3) after culturing for 4 d in the recovery medium, shoot tips were moved to MSB screening medium containing 8 g·L^－1 agar powder and 125 mg·L^－1 kanamycin and then screened for 25 d to obtain the resistant germs; (4) then, 1 g·L^－1 activated charcoal was added to the induction medium to induce rooting of the resistant germs to obtain resistant regenerating plants. After PCR and qPCR detections, 16 plants positive for the transgene were obtained from 5200 shoot tips, with a transformation rate of 0.31%. The establishment of this technology system lays a foundation for the genetic transformation of brown cotton and provides new materials for naturally-colored cotton breeding.