偃松体细胞胚胎发生与遗传转化技术体系的建立OA

Immature zygotic embryos from immature seeds of Pinus pumila were used as explants,with DCR as the basal medium.Somatic embryogenesis was induced by adjusting the concentrations of plant growth regulators(6-BA,NAA,and ABA),leading to the acquisition of 3 embryogenic cell lines.Based on this somatic embryogenesis system,an Agro-bacterium-mediated genetic transformation system suitable for P.pumila was finally established through the determina-tion of kanamycin selection concentration,infection time,and co-cultivation duration,and resistant calli of P.pumila were successfully obtained using this system.The results indicated that the somatic embryogenesis system of P.pumila included 6 stages:disinfection of immature seeds,induction of embryogenic callus,proliferation of embryogenic callus,induction of somatic embryos,post-germination culture,and rooting culture.The optimal medium for embryogenic cal-lus induction was DCR+1 mg/L 6-BA+2 mg/L NAA,with an induction rate of 1.11%;the optimal medium for embryo-genic callus proliferation was DCR+0.35 mg/L 6-BA+0.5 mg/L NAA,achieving a proliferation multiple of 3.73;the op-timal medium for somatic embryo induction was DCR+20 mg/L ABA,with an average of 86.67 somatic embryos per gram of embryogenic callus;after somatic enbryos post-germination,DCR+activated carbon was used for root induction,resulting in a rooting rate of 58%;and the optimal medium for rooting culture was WPM+0.1 mg/L IBA.For genetic transformation,the optimal conditions were a kanamycin concentration of 60 mg/L,an infection time of 15 minutes,and an Agrobacterium suspension concentration of OD₆₀₀ is 0.4;after 7 days of recovery culture,the tissues were transferred to a medium containing 60 mg/L kanamycin for selection and callus proliferation induction.Through verification by DNA-level identification and GUS histochemical staining,transgenic calli were successfully obtained,and a genetic transfor-mation system for P.pumila was initially established.