基因枪介导的海岛棉(Gossypium barbadense L.)茎尖遗传转化体系的建立

The Establishment of Biolistics Transformation System of the Apical Meristem of Gossypium barbadense L.

以新疆海岛棉品种新海13号的茎尖为转化受体,采用基因枪介导法,研究DNA包裹浓度、微载体种类、前渗处理时间、轰击条件、筛选方法等对茎尖转化的影响,建立了基因枪法介导的海岛棉茎尖转化体系。研究结果表明,采用1.5μg·μL-1的质粒DNA包裹1.0μm金粉微载体,前渗处理12 h,轰击压力为7.584 MPa(1100 psi),轰击距离为6 cm,后渗处理16 h,恢复处理24 h,在120 mg·L-1卡那霉素浓度下直接筛选35 d。在添加4g·L-1活性炭粒的生根培养基中诱导抗性幼苗生根,获得抗性再生植株。通过PCR和RT-PCR检测,共得到5株转抗除草剂基因EPSPS的阳性植株。该研究体系的建立为海岛棉的遗传转化奠定了基础,为海岛棉的育种工作提供了材料。

In this study,biolistics transformation system was established with apical meristems of Xinhai No.13,a variety of Gossypium barbadense L.cultured in Xinjiang.Several factors of the system are studied,which contained the wrapped concentration of DNA,the type of microcarrier,the early penetration processing time,bombardment condition and screening technique,et al.The optimal transformation system is as follows under other constant conditions.Microcarriers of a diameter of 1.0 μm were covered up with the DNA concentration of 1.5 μg· μL-1.Apical meristems were bombarded with the helium pressure of 7.584MPa（1100 psi）,and with the bombardment distance of 6 cm.Target materials were cultured for 12 h before being bombarded and 16 h after being bombarded in the osmotic medium.After recovering for 24 h in the recovery medium,target materials were moved to the kanamycin screening medium containing the concentration of 120 mg·L-1 kanamycin to screen directly for 35 days.Resistant seedlings were induced to take roots in the induction rooting medium adding 4 g· L-1 activated charcoal granule and the regenerating plants were obtained.After PCR and RT-PCR detection,five transgenic regenerated plants were obtained in the end in which the gene EPSPS were expressed.