Studies On HbHMGR1Gene Clone,Genetic Transformation Into Fiable Embryogenic Callus And Plant Regeneration In Hevea Brasiliensis

Agrobacterium-mediated genetic transformation is the main method for rubber genetic transformation. In the resent reports about rubber genetic transformation, rubber tissue culture and somatic embryogenesis was often used for rubber genetic transformation. Because they are embryonic, long term subculture and friable, the friable embryogenic callus are used as the perfect infection explants. In this study, the HbHMGR1gene was cloned successfully and used to constructed pant expression vector pCAMBIA2301-35S-HbHMGR1. And the plant expression vector was transformed into Agrobacterium EHA105. The friable embryogenic callus of rubber tree clone Reyan8-79were used as recipients by the method of Agrobacterium-mediated transformation to study the influcing transformation elements, such as bacterial concentration of Agrobacterium, infection time, co-culture pH, co-culture temperature and co-culture time. The conditions of transformation were optimized as well.By using Ticarcillin to inhibit bacteria and kanamycin to screen the transformed callus, the resistant friable embryogenic callus, resistant embryos and transgenic plants. By the methods GUS staining, molecular detection including PCR, RT-PCR and Southern blotting, the HbHMGR1gene had been transformed into the genome of rubber tree. And the results are as follow:1. The influencing factors of Agrobacterium-mediated transformation were studied by single factor experiment. And the frequency of transient expression of GUS gene and the survival rete of the callus that were used as the indexes. The best bacterial OD600of Agrobacterium was0.4, the best infection time was4to8minute, the co-culture temperature was20to26centigrade, the best co-culture time was4days, and the best co-culture pH was5.7.2. During the progress of screening the resistant callus, the best inhibition concentration of Ticarcillin was200mg/L; and the best kanamycin concentration that was used to screening the resistant callus was75mg/L. Finally,15resistant friable embryogenic callus lines were got after the progress of screening.2944resistant embryos were obtained after embryo induction. After plant regeneration,8resistant plantlets were obtained.3. Molecular identification: The methods of GUS staining, PCR identification, sequence analysis, RT-PCR identification and Southern blotting analysis were used to identify the resistant friable embryogenic callus. And the final results showed that the gene HbHMGRl had been integrated into the genomic DNA of the rubber tree.