Optimization Of Somatic Embryogenensis And Preliminary Study On Genetransfer Of Chinese Pine(Pinus Tabulaeformis C.)

Pinus tabulaeformis C.has the characteristics of thinness,wind resistance and strong adaptability to harsh environments.It is the main afforestation species in north China,northwest China and northeast China.Although P.tabulaeformis has a wide range of uses,due to its low reproductive efficiency,long growth cycle,and large variation in sexual reproduction,the rapid propagation techniques of this species are still stagnant,and in the study of conifer breeding techniques,Somatic embryogenesis is one of the effective methods for large-scale reproduction.In this study,P.tabulaeformis was used as experimental material,based on the previously establishment of the Chinese pine embryogenesis system.The factors affecting new cell line screening,embryonic cell proliferation,and somatic embryo maturation were analyzed in order to further improve the efficiency and optimize the somatic embryo system.And a preliminary study on genetic transformation was conducted based on somatic embryo system aimed at providing technical support for the large scale propagation of fine germplasm and the functional research of important genes of P.tabulaeformis.The main research results are as follows:(1)The immature zygotic embryo of P.tabulaeformis was used as the explant material to induce the new embryonic cell line and to increase the total number of embryonic cell lines.,A total of 17 embryogenic cell lines were obtained.Among them,15c4b,16c4g,and 17c4c were three cell lines that had the potential to undergo large-scale reproduction.(2)A embryonic callus proliferation system of P.tabulaeformis was established based on air-lift bioreactor.The highest embryogenic callus proliferation rate,216.18%was obtained using 10 g embryogenic callus,0.2 mg/L 2,4-D and 20 mL old culture medium inoculated in 100 mL liquid medium in the ALB,which was 3.15 times higher than that of suspension culture.(3)The maturation medium for somatic embryogenesis of P.tabulaeformis was optimized.The cell line 15c4b with good embryogenicity was used as the material to study the effects of factors such as ABA concentration,PEG concentration,sugar type and concentration,and concentration of phytagel on the maturation of embryogenic callus.The final result showed that adding 16 mg·L-1 ABA,75 g·L-1 PEG,5 g L'1 phytagel and 10+30 g·L-1 maltose+sucrose to the mature medium the maturation rate increased by 0.8 times(4)The genetic transformation of P.tabulaeformis was explored initially.Liquid suspension tissue of P.tabulaeformis was used for genetic transformation.The results showed that Kan=50-75 mg·L-1 was advantageous for transformation,Agrobacterium and embryogenic callus was in good condition,and the callus with positive GUS staining was obtained when OD value was between 0.4-0.6,infestation time was 5-15 min,and co-cultivation time was 48 h.