Studies On AtLEC1 Gene Transformation Of Friable Callus In Hevea Brasiliensis

Agrobacterium-mediated genetic transformation is the main method of the present transgenic research and application. Recently, there are some reports about genetic transformation of rubber tree in China, based on rubber tree tissue culture and somatic embryogenesis. Somatic embryogenesis is a complicated process that was regulated and controlled by many factors. Generally speaking, somatic embryogenesis frequency and plant regeneration frequency of most clones of rubber tree are low. So they limit rubber tree clone genetic amelioration severely. Friable embryogenic callus of rubber tree is of soft structure. It has the ability of somatic embryogenesis, and grows fast and can be subcultured for a long time. So it’s a suitable recipient for genetic transformation. The Arabidopsis somatic embryogenesis key gene AtLEC1 was transformed into the friable embryogenic callus of rubber tree elite clone Reyan 7-33-97 in order to promote somatic embryo genesis in the present study. The influential factors such as Agrobacterium solution concentration, infection time of embryogenic callus, co-culture temperature, co-culture time, co-culture pH were stuied using single factor method. Thus the genetic transformation system was optimized. Resistant callus lines, resistant embryoids and putative transgenic plantlets were obtained through ticarcillin and kanamycin screening. It was preliminarily indicated that AtLECl gene had been integrated into the genome of rubber tree by GUS staining and moleculer detection. The main research results are the following:1. The influential factors such as Agrobacterium solution concentration, infection time of embryogenic callus, co-culture temperature, co-culture time, co-culture pH were stuied using single factor method. The optimal Agrobacterium solution concentration was 0.4-0.5 at OD600-The suitable infection time of embryogenic callus was 5-8 minutes, co-culture time was 4 days, co-culture temperature was 24-26℃, co-culture pH was 5.7, respectively.2. The optimal antibiotic concentrations for screening resistant embryogenic callus lines of rubber tree clone Reyan 7-33-97 were 200mg/L ticarcilllin and 75 mg/L kanamycin.3. In the present study,25 resistant callus lines,1140 resistant embryoids,6 regeneration plantlets were obtained through GUS tinction, PCR and RT-PCR detection.4 of the 6 plantlets were positive putative transgenic plantlets after GUS tinction, PCR and RT-PCR detection. And the embryoid inducing frequencies of 3 AtLEC1 transgenic callus lines were increased remarkably compared with the control.