农杆菌介导的花生遗传转化体系的初步研究

A Primary Study on Transfer System of Peanut By Agrobacterium Tumefaciens

应用花生胚小叶再生体系,以农杆菌介导法对质粒上的基因转化进行初步探讨.通过对影响基因转化的各因素,如抗生素的筛选压、共培养时间、AS的影响、及外植体取材方式等进行研究,结果发现:外植体与农杆菌在加有AS(100μmol/L)的改良MS液体培养基中共培养4 d后,先进行10 d的恢复培养,继而转入加有Hy(25 mg/L)和Cef(200 mg/L)的抗性筛选培养基中进行筛选培养40～50 d,后转入含Hy(20 mg/L)的分化培养基中分化,3个月后在首批材料中得到6个再生植株,经PCR检测,有1株显示了800 bp的GUS基因核酸片段,初步证实了此转化体系合理有效.

Applying to leaflet regeneration system of peanut, genetic transformation of peanut using Agrobacterium-mediated approach were carrying on the research. The factors affecting the transformation, such as the selective pressure of antibiotic, co-cultivation, AS effect and the cultured effects of two kinds of leaflet were examined. The optimized procedure was as following： after co-cultivation for 4 days on modified MS liquid medium containing 100 μmol/L AS, 10 day＇ s recovering culture, the explants were transferred to selective media with 20mg/L Hy and 200g/L Cef, 40 - 50 days later, the explants were transferred to redifferentiation divided media with 20mg/L Hy. First batch of material gained 6 regeneration seedling in 3 months later. Out of these, one proven to contain a 800bp DNA segment of the GUS by PCR analysis. The integration of foreign DNA into the peanut genome was confirmed.