Research On In Vitro Regeneration Systerm And Micrografting Of Tea Plant

Tea（Camellia sinensis） is one of the most important woody plants which product non-alcoholic beverage drinks worldwide. It’s long growth cycle, low natural seed set and hybrid seed set make the need for conventional breeding and propagation20-25years. Plant tissue culture technology is an important means of innovating high quality varieties, is the basis of modern biotechnology applying to genetic and breeding research. To establish a rapid and effective tea regeneration system, we take axillary buds [C. sinensis cv.pingyangtezao] in late July and cotyledons [C. sinensis cv. shuchazao] in mid-October as explants. In the condition of in vitro culture, first, established a system of in vitro propagation of tea cotyledons, and compared the organogenesis and somatic embryogenesis regeneration systems, then established a system of in vitro propagation of tea axillary buds，third sloved the problem of rooting hard and time-consuming by micrografting. We hope to provide technical reference on the connection of tea plant tissue culture and industrial production. The main conclusions are shown as follows:1. Organogenesis pathway in the system of in vitro propagation from tea cotyledonsInduction rate of cotyledons without embryos increases with the increase of the concentration of2,4-D. In the medium MS+2.0mg L^-12,4-D+0.5mg L^-1 KT, induction rate is highest to83.6%. Ture to buds induction medium MS+2.0mg L^-16-BA+0.2mg L^-1N AA, seedling rate, proliferation multiple and the incidence of somatic embryo in cotyledons with embryos are higher than which in cotyledons without embryos, reach36.5%、12.3and38.2%respectively.2. Somatic embryogenesis pathway in the system of in vitro propagation from tea cotyledonsIn medium MS+2.0mg L^-16-BA+0.2mg L^-1 NAA+1.0mg L-1GA3, cotyledons with embryos and cotyledons without embryos can dedifferentiation to embryogenic callus,and then to somatic embryos and secondary somatic embryos, and germinate. The incidence of somatic embryo in cotyledons with embryos is85.7%, germination rate of somatic embryo is79.4%, proliferation multiple is17.0, are all higher than that of cotyledons without embryos.3. Establishment of in vitro propagation system of tea axillary budsTake axillary buds as explants, axillary buds germinated rapidly in the medium MS+2.0mg L^-16-BA+0.2mg L^-1N AA, after in vitro culture60days, seedling rate can reach82.3%. The plantiets will be divided into three treatments: branches, branches without terminal buds and some leaves, axillary buds with nodal segments and some petioles, then inoculated into1/2MS+1.0mg L^-16-BA+0.1mg L^-1N AA medium, the seedling rate and proliferation multiple of axillary buds with nodal segments and some petioles is highest, reach71.4%and5.5respectively. The treatment of branches without terminal buds and some leaves grow strongest there are somatic embryos in the bottom of branches.4. Subculture of embryogenic callusIn subculture of embryogenic callus, the incidence of somatic embryo, germination and the incidence of secondary somatic embryo are happen in the same time. The incidence of somatic embryo in embryogenic callus can reach the highest58.3%in M（MS without any hermone） medium, and lowest in P(MS+2.0mg L^-16-BA+0.2mg L^-1N AA+1.0mg L-1GA3) medium, but the incidence rate of secondary somatic embryo and the germination rate of somatic embryo is highest in the latter.5. Incidence of somatic embryoSomatic embryo occurred in cotyledon petiole directly, or occurred in cotyledon indirectly. Cotyledons with embryos have both directly and indirectly pathways, cotyledons without embryos have only indirectly pathway. In the indirectly pathway of somatic embryo, strumae occurred on cotyledon surface is a sign of dedifferentiation.6. Micrografting of regenerated microshootsSelect the well-growth regenerated microshoots, take1.5-3cm long as scions.when the seedings grow to two leaves and a bud, cut the shoots at3-5cm away from bottom, take the rest as root stocks. Cut ends of scions were pretreated with200mg L-12,4-D for3-5min, after20days survival rate can reach64%.