Establishing A High-Efficient Tissue Culture System And Exploring Gene Transformation In Cunninghamia Lanceolata Hook.

Cunninghamia lanceolata Hook.is one of the evergreen coniferous species, native to the People's Republic of China. It is an important commercial and reforestation tree for the following reasons it was fast growing, produces high quality wood and with short rotation. In this thesis, five types of initial explants, including immature embryos, mature embryos, shoot sections, cotyledons and hypocotyls of seedlings, were taken to study its organogenesis and somatic embryogenesis. Factors have been tested for their effects on organogenesis and somatic embryogenesis. The somatic embryos were induced successfully from mature embryos, cotyledons and hypocotyls of seedlings with this species for the first time in the world. A high-efficient tissue culture system was established from shoot sections. This system can be used for transforming Cunninghamia lanceolata Hook by Agrobaterium tumefaciem mediated method. We have observed the origin and development of somatic embryos and adventitious buds through the paraffin method. Seven factors affecting transformation were investigated and effective genetic transformation system was established. This unique system provides a key platform for gene engineering and molecular breeding for Cunninghamia lanceolata Hook.Using mature embryos and immature embryos as the initial explants, we studied the effect of the following factors on induction callus, subculture callus and differentiation adventitious buds: basal medium, plant hormones, gelling agent, organic ingredient, development stages of zygotic embryo. Results indicated that the optimal medium for mature embryos induction callus was l/2MS+2,4-D 2mg/L+6-BA 1.0mg/L+KT 1.0mg/L+ sucrose 30g/L. With this medium, soft, transparent callus can be induced from immature embryos of embryo selection or tissue and organ differentiation stages. The induction frequency was 2-6%. Immature embryos of tissue and organ differentiation or embryo mature stages induction callus were similar in state with those of mature embryo. The callus induction frequency was significantly improved with the maturity of immature embryos. The optimal media for subculture callus and differentiation adventitious buds were 1/2MS+2, 4-D lmg/L+6-BA 0.5mg/L+KT 0.5mg/L+ sucrose 30g/L and DCR+6-BA 1.5mg/L+KT 1.5mg/L+ sucrose 20g/L, respectively.Using mature embryos, cotyledons and hypocotyls of seedlings as initial explants, we studied the effect of such factors as basal medium, plant hormones and seedling age on direct organogenesis, somatic embryogenesis and adventitious buds induction roots. We have observed the origin and development of somatic embryos and adventitious buds through the paraffin method. The results indicated that the induction frequency of organogenesis and somatic embryogenesis were greatly influenced by basal medium, 6-BA, TDZ and seedling age, whereas the effect of ZT and KT was not significant. The medium of DCR+6-BA 1.0mg/L + TDZ0.002mg/L+NAA 0.1 mg/L was optimal for mature embryos to induce adventitious buds,and for cotyledons and hypocotyls of seedlings to induce somatic embryos. The optimal medium for mature embryos to induce somatic embryos and cotyledons, and for hypocotyls of seedlings to induce adventitious buds was DCR+6-BA 1.0mg/L+TDZ0.003 mg/L+ NAA 0.1 mg/L. Adventitious buds can elongate effectively on the DCR mediurrfwithout plant hormone. The optimal medium for adventitious buds to root was 1/4MS+IBA0.2 mg/L +NAA0.1 mg/L. Histological observation indicated that there were two ways in which adventitious buds originate. The first way of origination was from meristematic tissue developed from epiderm cells and three or four liners under epiderm. The other was that the little vascular bundle dedifferentiates into meristematic tissue, which in turn develops into adventitious buds. Both are direct organogenesis. Somatic embryo was regenerated directly from the surface of the initial explants, but not from callus. So it was direct somatic embryogenesis.Using plantlet shoot sections near the shoot tip as initial explants, we observed the effect of s...