为实现杨树早期开花,缩短育种周期,利用农杆菌介导的转化方法,探讨影响热激启动子控制的FT 1 基因转化白杨派杂种无性系的因素,优化其转化条件,并实现FT 1 基因的转化,采用热激诱导方法诱导2~3个月大的转基因植株早期开花。结果表明: 叶盘转化前的预培养、菌液浓度、侵染时间及共培养时间对卡那霉素抗性植株再生率的影响均达到极显著水平; 叶盘在愈伤组织诱导培养基上暗培养6天,然后用活化至OD600=0.5的菌液侵染60 min,再在愈伤组织诱导培养基上共培养2天,该条件下FT 1 基因转化白杨派杂种的卡那霉素抗性植株再生率可达29.84%; 37 ℃每天1 h持续热激3周可使FT 1 基因顺利表达,促进转基因植株开花; 热激植株大小是影响转基因植株热激诱导开花的限制因素,低于20 cm的植株不能诱导开花; 热激诱导可产生相对较多的正常花器,但花器变异仍然十分广泛。
In order to achieve precociously flowering of poplar and shorten the genetic and breeding period, we studied factors affecting genetic transformation of FT 1 gene controlled by Heat Shock Promoter in hybrid aspen clone (Populus tremula×P. tremuloides) and optimized the Agrobacterium-mediated transformation conditions. FT 1 gene was transformed into hybrid aspen through optimized transformation protocol and the 2-3 months transgenic plants were successfully induced to precociously flower by heat shock. The results showed that regeneration frequency of kanamycin resistant plants of hybrid aspen were significantly affected by pre-culture of leaf disc, Agrobacterium concentration, incubation duration,and co-culture duration. Optimized transformation protocol was as follows: leaf discs were pre-cultured for 6 days on the callus-induction medium, incubated for 60 minutes in Agrobacterium solution to reach to the OD600 value of 0.5, and then co-cultured for 2 days. According to this protocol, regeneration frequency of kanamycin resistant plants can reach to 29.84%. Expression of FT 1 and precociously flowering of transgenic plants were induced by heat shock treatment of 37 ℃, 1 hour per day for 3 weeks. The size of transgenic plants was a limiting factor that affected the precociously flowering induced by heat shock, and plants with height less than 20 cm didn‘t show precociously flowering when induced by heat shock. Heat shock can induce more normal flowers, but extensive aberrance was still presented.
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