优化农杆菌介导的月季遗传转化系统的研究

Optimization of Agrobacterium-mediated transformation of Rosa hybrida

为了建立月季品种‘萨蔓莎’根癌农杆菌介导的遗传转化系统,我们通过衡量GUS基因瞬间表达水平,探讨了各因子对基因转化的影响.结果表明:外植体、光照条件、共培养培养基中无机盐含量及乙酰丁香酮(AS)含量是重要的影响因子;共培养时间、共培养温度及农杆菌菌液浓度对根癌农杆菌的生长以至基因转化有重要影响.优化后的转化条件为:将月季胚性愈伤组织与OD600值为0.5～0.8的农杆菌菌液侵染20min,然后在含300μmolLAS并且无机盐减半的MS培养基上黑暗、23℃条件下共培养3d.

To develop a transformation protocol of Rosa hybrida ‘Samantha＇ via Agrobacterium tumefaciens, the authors examined the effect of different factors on T-DNA transfer by measuring transient expression levels of an intron-containing β-glucuronidase gene. The results indicate that explant, light condition, salt concentration and acetosyringone （AS） concentration in co-culture medium are the most important factors, and factors like co-culture temperature, co-culture period and bacteria density also have an important effect on the growth of bacteria and even T-DNA transfer. Optimized co-cultivation was performed by inoculation of embryogenic callus with bacteria at a density of OD600 = 0.5-0.8 for 20 min and co-culture in darkness at 23℃ on medium with 1/2 MS salt and 300μmol/L AS for 3 d.