Research On84K Populus Regeneration And Genetic Transformation System

Popular84K is the successful cultivated hybridal clone of Populus alba×Populus glandulosa. It has become an important receptor species because of its fast growth, quick reproduction features in the populus genetic engineering. In our research, we utilize the Popular84K as the experimental material, and establish two different high-efficiency regeneration and transformation systems.(1) Based on the previous research, we optimize the system for Popular84K directly differentiating to the bud using6-BA、NAA、TDZ-three different hormones. The high-efficiency regeneration culture medium sytem:MS culture medium+0.002mg·L-1TDZ+0.5mg·L-16-BA+0.1mg·L-1NAA, the differentiation rate can reach100%. Every leaf blade can differentiate20-30adventitious buds.(2) We use different culture situations to separate the dedifferentiation with redifferentiation processes. We get an effective regeration system through the callus pathway utilizing the different hormones to deal with the Popular84K. The highly effective culture medium to induce the callus with the Popular84K leaf blade under dark is MS+0.5mg·L-1KT+0.2mg·L-12,4-D; Whereas, for stem, it is WPM+0.5mg·L-1KT+0.1mg·L-12,4-D; The culture medium to induce the callus to produce the buds is WPM+0.05mg·L-1TDZ+0.5mg·L-16-BA. The inductivity for leaf blade and stem can reach100%. The bud ratio can reach98%through the callus differentiation, and, every callus can produce about30buds.(3) In the induction budding and callus culture medium containing the20mg·L-1Kanamycin, the ability of induction bud and callus is totally suppressed:The ability for callus to differentiate to bud under the induction budding culture medium can be retained when the Kanamycin concentration is20mg·L-1. The Cephalosporin can suppress the agrobacterium growth effectively when its concentration is250mg·L-1, and has no effect on the exophyte and callus induction budding.(4) We use the L9(34)orthogonal test to analyze effects of preincubate time, concentration of the bacteria liquid, infection time and co-culture time on Popular84K exophyte direct differentiation. The results indicate, the exophyte cannot be preincubated before infection. The exophyte differentiation ability after preincubation has huge difference with the ones without preincubation. The concentration of infection bacteria liquid is OD600=0.4～0.6, the infection time is10to15mins, the optimal co-culture time is two days. The transformation rate of this system can reach28%, whereas, there still exist the false positive species, the false species can account for35%.(5)We establish the genetic transformation system through the Popular84K callus. Referenced to the previous optimal method, we do the selectable culture after the exophyte infected under the callus induction culture medium. We use the callus to induce the bud after getting the resistance callus. This transformation rate can reach33%. We do not detect the false negative species.