Induction Of Embryogenic Callus Of Chinese Pine (Pinus Tabulaeformis Carr.)

Chinese pine (Pinus tabulaeformis Carr.) is one of the important forest trees for timber and afforestation in north China. In recent years, Its propogation is still limited in the way of seeding and well-bred graft, which can't meet the production demand due to low propogation rate, long cycle period and so on. Tissue culture can overcome many obstacles existing in traditional propagation. Compared to organogenesis, somatic embryogenesis is more promising in future application. However, to our knowledge, no study about somatic embryogenesis of Chinese pine has been repoted. In present study, immature and mature zygotic embryos from Chinese pine were used as explants to induce embryogenic callus. This study was aimed to provide preferences and technologies for the somatic embryogenesis of Chinese pine and furtherly establish a foundation of in vitro rapid propogation and genetic transformation. The results are as follows:(1) The develpoment stage of explants inflenced the induction of callus and embryogenic callus. Zygotic embryos collected from early-July to mid-August, when the developmental was in the embryo selection stage, could initiate the callus. The highest callus induction was 60%from explants collected in late-June, however it is about all nonembryogenic. The highest embrogenic callus was 4.5%from explants collected in early-June. The callus induced from the explants of early-July had higher proliferation and differentiation potential, but the initiation was low, only 1.7%.(2) Plant growth regulators (PGRs) influenced the induction of callus and embryogenic callus. The highest callus initiation was 60%, when the explant was cultured on the DCR medium with 5.0 mg/L 2,4-D and 1.0 mg/L BA. No obvious regular pattern was observed about PGR affecting embryogenic callus induction. However, it seemed that DCR medium containing 1.0 mg/L 2,4-D was suitable for embryogenic callus induction. (3) Good proliferation was otbtained with 5×5mm2 callus cultured on DCR medium without PGR.(4) ABA was important for the differentiation and development of somatic embryos. And inositol was advantageous for maintenance of the embryogenic potential.(5) Of the 5 media (DCR,SH,LOB,B5,MSG) combinated with 3 different PGRs (2,4-D, BA, KT), DCR medium containing 1.0 mg/L 2,4-D gave the best embryogenic callus induction rate,7.5%. LOB medium was suboptimal.(6) Embryogenic callus of Chinese pine was fresh, white, moist and filamentous or lumpish. It was the structure of embryogenic suspensor mass (ESM) containing small embryo head with thick cytoplasm and highly vocuolized long embryo suspensor. Embryogenic callus was induced from zygotic embryo, and somatic embryos origined from single cells of the callus. Somatic embryos went through a developmental pattern similar with zygotic embryos.