Establishment And Optimization On The Transgenic Technique By Stem Cell System Of Larix Spp

Larch (Larix Mill) is an important timber and plantation coniferous species in North China. To facilitate the genetic improvement of larch is very important for modern plantation forestry. Somatic embryogenensis, the development of which is similar to zygotic embryo, is considered as an ideal experimental system for research and application of artificial embryo, and play important roles in forest improvement, propagation in vitro and other basal researches. By introducing useful foreign genes into the genome efficiently, the system of transformation on larch is essential for research on gene function identification, gene regulation, and larch improvement.In the paper, 638(Larix leptolepis), 109(L. olgensis), 113(L. leptolepis) were chosen from 39 cell lines of larch as material to research the effects of 3 antibiotics for an optimal schedule of antibiotic usage, and optimize factors involved with transformation mediated by Agrobacterium tumefaciens. The results were as followings:1.In this part of experiment, all 39 cell lines preserved in our lab were cultured on proliferation, mature somatic embryo induction and development medium step by step. From the 39 cell lines, cell lines 638, 109, 113 with good regeneration capability were chosen as transformation materials.2.To identify the optimal antibiotics and the concentration used in the transformation of larch based on somatic embryogenesis, we studied the effects of Kanamycin (Kan) and Hygromycin B (Hyg) at various concentrations on embryogenic tissues of larch cell lines 638, 109 and 113. The effects of Cefotaxime (Cef) and Carbenicillin (Cb) on cell line 109 were carried on as well. The embryogenic tissues were subcultured on proliferation medium supplemented with Kan (0-35mg/L), Hyg (0-9mg/L), Cef (300-500mg/L) or Cb (300-500mg/L). According to the appearance of embryogenic tissues after subcultured for 30d, we found that: The critical selection concentrations of Kan on cell lines 638, 109 and 113 were 25mg/L, 20mg/L, 15mg/L respectively and that of Hyg on the three cell lines were 5mg/L, 3mg/L, 3mg/L respectively. At the concentration of 300-500mg/L, Cef showed a positive impact on the proliferation of cell line 109 while Cb showed a negative impact. So it is suggested that Cef be used to eliminate bacteria during transformation of larch using emryogenic tissues of cell line 109.3.In this part, the Agrobacterium tumefaciens GV3101 harboring plasmid pCHF3-gp or pSuper1300+-HC were used to transform cell lines 638,109, 113, and the factors including genotype, physiological state of plant material, bacteria concentration, inoculation time and duration of co-culture were analyzed and optimized. The optimal transformation procedure was as following: Embryogenic tissues were affected by Agrobacterium (OD600=0.4) for 10-20min, followed by 2d co-cultivation and then washed by solid medium supplemented with Cefotaxime. After recovery for 7d and selection for several times by hygromycin, we would obtain 0.94 antibiotics-resistant tissues from 1g embryogenic tissues for transformation on average, and all of them will show positive by PCR confirmation.By this research above all, we have established the system of transformation mediated by Agrobacterium successfully, which would be a preferred method for genetical engineering of Larch.