In Vitro Plant Regeneration And LFY Gene Transformation Of Paulownia

Paulownia (Paulownia spp.) is one of the important fast-growing trees in timber production and garden planting in China. Some scientists have been doing much works on selection of good cultivar and new variety breeding recent years. But little progress was made in application of gene engineering and cell engineering for variety improved. The emphases were put on the system development of in vitro organogenesis and somatic embryogenesis and Agrobacterium mediated antisense LFY transformation of Paulownia in this thesis, which laid a solid foundation for its new cultivars breeding in applying biotechnological methods.The results indicated that MS medium was optimum for inducing calli from 5 species of Paulownia plant leave. Callus induction media of leaves and their bud induction ones of Pauliwnia tomentosa, P. australis, P. fortunei, P. elongata and P. tomentosa xP. fortune were MS+0.5NAA+4BA, MS+0.3NAA+2BA, MS+0.5 NAA+4BA, MS+0.3NAA+6BA and MS+0.5NAA+8BA, MS+0.3NAA +12BA MS+0.3NAA+12BA, MS+0.5NAA+12BA, MS+0.5NAA+12BA and MS+0.7NAA + 12BA respectively; And that 1/2MS+0.1NAA, 1/2MS+0.1NAA, 1/2MS, l/2MS+0.5NAA and 1/2MS+0.5NAA were the shoot rooting media.The best media for embryogenic callus induction from young stem segments and leaves of P. tomentosa, P. elongata and P. fortunei were MS+0.3NAA+17BA, MS+0.3NAA+11BA, MS+0.3NAA+8BA and MS+0.3NAA+17BA, MS+0.3NAA+ 8BA, MS+0.3NAA+14BA respectively. Somatic embryogenesis undergoes 5 phases as following, pre-embryo, globular embryo, heart-shaped embryo, torpedo-shaped embryo and cotyledonary embryo. The course is just similar to zygotic embryogenesis. The induction of embryogenic callus, its embryogenesis and even the plant regeneration might be carried out on the same media.DNA extracted from P. elongata leaves with four different methods (CTAB-I, CTAB-II, SDS-I and SDS-II ) belonged to CTAB and SDS categories, which were tested by ultraviolet spectrophotometer analysis, agarose gel electrophoresis, restriction enzyme digestion and RAPD reaction in the thesis, were all suitable for digestion with two restriction enzymes and RAPD analysis. Besides, the DNA production rate are 3.5-5.2ug per 10 mg of fresh leaves, A260/A280=1.7-1.8, A260/A230>2.0, the molecular weights above 23 Kb. But SDS-I was the best method as comprehensive consideration.Plantlet regenerated from P. elongata leaves infected with agrobacterium mediated antisense LFY gene and confirmed by GUS and PCR amplification showed that the transformation was successful and the optimum parameters in transformation procedures were as follows: 30mg.L-1 Km selection pressure concentration, 250 mg. L-1 Cef concentration, 4 d preculture and 0.35 agrobacterium concentration (OD600).