Study On Tissue Culture And Genetic Transformation Of Pinus Massoniana

In this study,the mature zygotic embryo of the mixed family of national Pinus massoniana from Xinyi city of Guangdong was used as explant,the adventitious bud induction,subculture,elongation and adventitious root induction were studied.On the basis of the above research,the genetic transformation of RCA was studied by Agrobacterium tumefaciens in order to obtain high photosynthetic productivity for P.massoniana.The main results were as following:1.The suitable sterilization method of mature seeds of P.massoniana was 70% alcohol disinfect 1min+0.1%mercury sterilization 10 min,the survival rate was 95%.2.The suitable adventitious bud culture medium was DCR+1.0mg/L 6-BA+0.05 mg/L NAA+30g/L sucrose+6.7g/L agar,pH=5.8.The highest induction rate was 78.54%,and the addition of 10mg/L Vc in the culture medium could inhibit browning.3.The suitable medium for the adventitious bud proliferation of P.massoniana was DCR+0.5mg/L 6-BA+0.2mg/L IBA+10mg/L Vc+30g/L sucrose+6.7g/L agar,pH=5.8.After the proliferation of green buds,robust growth,the proliferation coefficients were 12.74.4.The suitable culture medium for elongation of P.massoniana was DCR+0.15mg/L IBA+30g/L sucrose+10mg/L Vc+6.7g/L agar,pH=5.8,the highest adventitious bud height was 7.8cm,but stem elongation was not obvious.5.The suitable medium for induction of adventitious root was the 1/2DCR+0.5mg/L IBA+0.5mg/L NAA+30g/L sucrose+10mg/L Vc+6.7g/L agar,pH=5.8.The rooting rate was 10%,grew well and it is conducive to the survival of transplanting.6.In the determination experiment of the selection of fertilization and antibacterial antibiotic concentration,the screening marker gene was neomycin phosphotransferase gene(NPTII),and kanamycin and cephalosporin were used as genetic antibiotic and antibacterial antibiotics.After the gradient comparison test,the results showed that 50mg/L Kan,200mg/L Cef as the selection of the choice of pressure and antibacterial antibiotics concentration.7.In the study of genetic transformation system of mature zygotic embryo of P.massoniana,several factors affect the transformation of Agrobacterium tumefaciens(pre-culture time,bacterial concentration,infection time,co-culture time and acetosyringone concentration)were analyzed.The results showed that the pre-culture time was 3d,OD600=0.6,the infection time was 20 min,the co-culture time was 3d and AS concentration of 100?M is to obtain resistance to the transformation of buds.The results were relatively good.8.The real-time quantitative analysis of non-transgenic P.massoniana showed that the expression of RCA was the highest in the young leaves,followed by the stem and lowest in the root.