Somatic Embryogenesis And Genetic Transformation Of MtlD Gene Of Larix Kaempferi

In order to obtain larch plants with strong environmental adaptability,the different stages of somatic embryogenesis,somatic embryogenesis and genetic transformation of mtl D gene of Larch were studied in this study using embryogenic callus of Larix kaempferi.?1?The best combination of ABA,PEG 4000 and Ag NO₃ was determined by orthogonal design,and the somatic embryos were inoculated to the germination medium with AC concentration of 0,0.2,0.4,0.6,0.8 and 1.0 g/L,then the number of germination was counted.The results showed that the optimal combination comprised ABA 20 mg/L+PEG 4000 120 g/L+Ag NO₃ 10 mg/L,and AC 0.4?0.6 g/L was conducive to somatic embryo germination.?2?Orthogonal design was used to explore the proliferation of proembryogenic mass at 2,4-D 0.15,0.3,0.5 mg/L,6-BA 0,0.15,0.3 mg/L and ABA 0,0.5,1.0 mg/L concentrations,factorial design was used to study the suspension cell growth at 2,4-D 0.15,0.3,0.5 mg/L and 6-BA 0,0.15,0.3 mg/L concentrations,and the optimum hormone concentrations of the two cultures were determined.Then,somatic embryogenesis of logarithmic proembryogenic mass and suspension cell were tested.Finally,the effects of GA₃ or IAA on somatic embryogenesis were investigated using a somatic embryogenic induction medium.The results showed that the optimal hormone combination comprised 2,4-D 0.15 mg/L and ABA0.5 mg/L,as well the optimal hormone combination comprised 2,4-D 0.15 mg/L and 6-BA 0.3 mg/L.Finally,we found that the mature somatic embryo number increased significantly when 10–20 mg/L GA₃or IAA was added.?3?The regulation effect of 6-BA on suspension cells was investigated by measuring 6-BA 0,0.15,0.3,0.6 mg/L fresh weight,activity,cell morphology,antioxidant index and lignin content in suspension cells.The results showed that 6-BA had significant influence on activity,cell morphology,antioxidant index and lignin content in suspension cells of Larix kaempferi.?4?Through proteomic sequencing analysis and GO functional annotation of globular and cotyledon embryos,it was found through GO functional annotation that the differentially expressed proteins involved in the oxidation-reduction process in biological processes were the extremely significant,and six of them belonged to the thioredoxin-like family proteins?5?The embryogenic callus of larch was transformed by agrobacterium method and gene gunshot,and the proembryogenic mass callus of mtl D gene was successfully obtained by gene gunshot.In this study,somatic embryos were successfully obtained.the optimal hormone combination for proembryogenic mass proliferation comprised 2,4-D 0.15 mg/L and ABA 0.5 mg/L,the optimal hormone combination for suspension cell growth comprised 2,4-D 0.15 mg/L and 6-BA 0.3 mg/L.In addition,we also found that 10-20 mg/L GA₃ and IAA could significantly increase the number of mature somatic embryos of Larix kaempferi.6-BA can maintain the activity of larch suspension cells through antioxidant regulation.Thioredoxin-like family proteins plays an vital role in the development of somatic embryos from globular embryo to cotyledon embryo.The proembryogenic mass of larch with mtl D gene can be successfully obtained by gene gunshot.