Establishment Of Agrobacterium Tumefaciens-mediated Transformation System Of Hevea Brasiliensis

Natural rubber performes better at elasticity, ductibility, and heat transfer properties than synthetic rubber. Rubber tree (Hevea brasiliensis Muell. Arg) is the most important source of natural rubber. Rubber tree is a perennial woody plant, developping a new Hevea variety by crossing method will take 20-30 years, but genetic transformation provides an effective alternative for shortening breeding cycle. The rubber tree planting area in China is located in the north edge of tropical area, cold damage brought great loss to the rubber production. Therefore,improving the cold resistant ability of elite clones by genetic engineering is very important. CBF1 transcriptional factor could induce the expression of several cold-resistant genes, which improved cold resistant ability of transgenic plant much more than only one functional gene transformation.Since the establishment of genetic transformation system of Hevea have been studied for 10 years in China, but failed. It limits genetic engineering breeding in Hevea. So, In order to establish Agrobacterium tumefaciens-mediated Hevea anther calli transformation system, we surveyed the factors influencing transformation frequency. Firstly, the optimal acceptors for Agrobacterium infection and clones with high somatic embryogenesis were screened, then the factors influencing transformation frequency, including strains of Agrobacterium, co-culture temperature, co-culture time and acetosyringone (AS), and the methods for transformed embryos regeneration, were surveyed, finally, the successful genetic transformation system of Hevea was established. And based on the system, Arabidopsis transcriptional factor CBF1 was transformed to Hevea elite clone CATAS7-33-97 and regenerated transgenic plantlets. The main results as follows:1.Selection of the acceptors for transformationAnther and inner integument calli were used as acceptors to study their reaction to Agrobacterium. The results showed that the inner integument calli browned easily after infection and was hard to resume their gowth; however, anther calli could endure infection, and was easy to eliminate bacteria, resume gowth fast. So, anther calli was more optimal for transformation.2. Selection of the clones with high embryogenesis efficiency Five elite clones with some important agronomic traits were selected for comparison of their regeneability. Three were good at yield and timber, one was elite in comprehensive tratits. The results showed that two genotypes, namely, CATAS6-62 and CATAS7-33-97 have predominant somatic embryogenesis ability. The identification of these two elite clons will afford good acceptor variety for genetic transformation in Hevea and accelerate the application of transgenic material in breeding.3. Selection of antibiotics for transformationThe sensitivity of 30-days old anther calli of Hevea brasiliensis to antibiotics was measured. Two groups of antibiotics were tested:antibiotics commonly used to eliminate Agrobacterium from tissue culture (carbenicillin, cefotaxime and timentin), and antibiotics for the selection of transformed tissue (kanamycin, paromomycin and hygromycin).Results show that, except 500 mg·L-1 timentin inhibited callus growth but promoted embryogenesis, the first group significantly increased callus growth but inhibited embryogenesis; while the second group inhibited callus growth and embryogenesis, their inhibited ability ranked as follows:hygromycin> kanamycin> paromomycin; but hygromycin completely inhibited embryogenesis, while paromomycin had no obvious effect on embryogenesis.So in Agrobacterium-mediated transformation of Hevea brasiliensis anther callus, timentin could be used to eliminate Agrobacterium, optimal concentration was 500 mg·L-1; kanamycin to select transformant, optimal concentration was 50 mg·L-1.4. Factors influencing transformation frequencyGUS transient expression frequency was significantly affected by Agrobacterium strains and co-culture temperature/time, which was 5.07 blue spots/callus for EHA105,higher than that for GV3101 and LBA4404. The highest GUS transient expression frequency was 8.43 and 19.10 blue spots/callus under 22℃co-cultured temperature and 6 days co-cultured time respectively. The AS was not helpful for improving the transformation efficiency.5. Establishment of Agrobacterium tumefaciens-mediated Hevea genetic transformation systemTwenty two thousand anther embryogenic calli were transformed by EHA105 with pCAMBIA2301.In the results,11 transgenic plantlets were obtained through GUS staining, PCR, sequencing of PCR products and Southern detection. Subsequently,253 plants derived from 8 trangenic plantlets survived in the field.6. Acclimation of transgenic plantsThree methods were used for acclimation of transgenic plants:directly acclimation without propagation, budding then acclimation, and acclimation after propagation by secondary embryogenesis of transgenic embryos. Three planted directly in the field died;three as scions were budded onto seedlings, producing five budded transgenic plants; five were multiplicated at embryo phase by secondary embryogenesis, producing 676 transgenic plantlets, among which 248 survived in the field.7. CBF1 gene transformation into HeveaAbove genetic transformation system was used to transfer the Arabidopsis transcriptional factor CBF1 to Hevea. In the result, two transgenic plantlets were regenerated through GUS staining, PCR, sequencing of PCR products. These two plantlets were multiplicated by mini-seedling budding and transgenic embryo secondary embryogenesis, and 44 plantlets were obtained and acclimated,34 suvived in soil.