Study On Regeneration And Genetic Transformation System Of Populus Trichocarpa

Popolus trichocarpa has the characteristics of relatively small genome,small number of chromosomes, growth fastly,asexual reproduction easily and so on.It is the first woody plant of whole genome sequencing.The genetic transformation of Popolus trichocarpa has a very important significance for the study of functional genomics of woody plants. Stems and leaves of Popolus trichocarpa were used as explants to explore the effect of different combination of hormone,different sampling sites and inoculation way on induction of adventitious bud and an efficient regeneration system was established. Based on the regeneration system,the effect of Agrobacterium concentration,duration of infection,duration of co-cultivation,concentration of acetosyringone and addition method of acetosyringone on genetic transformation were explored and the efficient genetic transformation system was established. The main results were as follows:1. Twigs of Popolus trichocarpa were used as the basic material.The optimal aseptic processing condition was sterilized with 70% ethanol and 3% sodium hypochlorite for 30 s and 8 min, respectively. Then used sterile water washing three times and dried the surface of the twigs with sterile filter paper. Finally, the twigs were inoculated on rooting medium and the pollution rate was 0 after 15 days.2. The regeneration system of the stems and leaves of Populus trichocarpa were established.The optimum medium for adventitious buds regeneration of stem explants was WPM+0.03 mg·L-16-BA+0.02 mg·L-1 IBA+0.004mg·L-1 TDZ.The optimal sampling site was the second and third stem nodes which was from the top to the end of morphology.The adventitious bud frequency was up to 100% for twenty-five days culture,the number of shoots and the length of buds on average was 18.77 and 1.25cm, respectively.The optimum medium for adventitious buds regeneration of leaves explants was WPM+0.04 mg·L-1 6-BA+0.02 mg·L-1 IBA+0.005 mg·L-1 TDZ.The optimal sampling site was the third,fourth and fifth leaves which was from the top to the end of morphology.The adventitious bud frequency was up to 100% by the inoculation way of upper for twenty-five days culture,the number of shoots and the length of buds on average was 12.11 and 1.20cm, respectively; The best rooting medium was WPM containing with 0.1 mg · L-1 IB A and the rooting rate was up to 100%. Adventitious buds began to root for 5-7 days of inoculation and the rooting rate was 100%.3. An efficient genetic transformation system was established. The optimal selection pressure of Kan was 30 mg·L-1 during the shoots and rooting screening process. The optimal antimicrobial concentration of Cef was 200 mg·L-1. The transformation efficiency was relatively high when the explants were infected for 20 min in the condition of Agrobacterium concentration OD600 0.4 and co-cultured for 2 days. Besides, the co-culture medium and cell suspension were added 80μmol·L-1 acetosyringone and the transformation efficiency was 26.7%;5. GUS staining was performed on the resistant buds. Then the PCR and RT-PCR molecular detection of 28 transformed lines were obtained. This shows that the GUS gene was successfully transferred to the genome of Popolus trichocarpa.