In Vitro Culture And Optimization Of Agrobacterium-Mediated Transformation System Of Chimonanthus Praecox Link.

Chimonanthus praecox Link. is the deciduous bush of Calycanthaceae Chimonanthus,whose old name; is HuangMei. C.praecox has ancient origin and few species. It is the plant of tertiary relict species and the rare and endangered species of secondary-grade state protection. As one of Chinese traditional famous flowers and the ornamental plant in common usage, C.praecox is called the-four-nival-friends with Prunus mume, Camellia japomica and Narcissus tazetta. It is not only has high appreciation,but aslo can be used as economic plant,environment protecting plant and offcinal by people. Although C.praecox has centuries-old cultivated history,the development research of it mainly began from the 1980's in china,and the area of concentration was from macroscopical germplasm resources and morphological classification to microcosmic cytology and molecular biology. In recent years, the development of both plant in vitro culture and the technology of genetic transformation provides a new way to make use of C.praecox. The usual reproductive methods of C.praecox are graft,division and sowing,but these methods are rigorous in operational demands while low in growth coefficient. By means of plant in vitro culture, it could not only be achieved to rapidlly propagate and conserve germplasm resources,but could aslo lay a foundation for C.praecox's gene engineering. Whereas the research on woody plant's tissue culture starts later than herbaceous plant,moreover,there are not further studies on it, and tree species that can be producted in large scale by tissue culture are still few. Being a kind of woody flower plants,there are certainly some difficulties in the tissue culture of C.praecox, such as having difficulty in cytodifferentiation and being browned easily etc. On the other hand, it could come ture to break the limit on conventional breeding,shorten the age of breeding and improve the characteristics such as resistance,flower colors and shape by means of the research and establishment of C.praecox's genetic transformation system,which will make C.praecox as an archaic species bloom once again. At present,the study on C.praecox's tissue culture in china is mainly concentrated on its rapid propagation. There are not any reports about the establishment of regeneration system by means of cytodifferentiation and the establishment of genetic transformation system of C.praecox,.This experiment is intended to induce the generation and cytodifferentiation of callus by use of cotyledon of C.praecox as explants,and then establish the genetic transformation system of C.praecox preliminarily on the basis of generation and cytodifferentiation of callus.(1) In vitro culture of C.praecoxThe optimum medium to induce the generation and cytodifferentiation of callus was screened out by use of the cotyledon of C.praecox as explants and the improved MS medium and MS medium as the basic medium in this experiment,which were added in different kinds of plant hormones. The half-developmental cotyledon was cut into pieces with 1cm×1cm and inoculated in the inducing medium to induce cytodifferentiation. Cuts of buds from cytodifferentiation were inoculated in the multiplication medium next. After the bud growing to as high as 3～4 cm,it should be inoculated in the rhizogenic medium when it's basal callus had been cut off. The results showed that the generation and cytodifferentiation of callus were induced in the miliorated MS+6-BA 1.0 mg/L+NAA 0.5 mg/L+KT 0.5 mg/L+IBA 0.2 mg/L+2,4-D 0.2 mg/L,and the ratio of cytodifferentiation was 11.7%.The regeneration plant had the best growth coefficient in the miliorated MS+6-BA1.0 mg/L+NAA0.1mg/L,which could be 2.7.The optimum medium for rhizogenesis was 1/2MS+NAA0.1 mg/L,where the ratio of rhizogenesis could be more than 80% .However,plants rooted very slowly in 1/2MS without any plant hormones,where the ratio of rhizogenesis was, only 10%.(2) The preliminary establishment and optimization of C.praecox's genetic transformation systemWhen selective concentration of Km and Cef was confirmed and GUS gene was shifted into callus by means of agrobacterium-mediated genetic transformation by use of callus as explants,the genetic transformation syetem of C.praecox was preliminarily established. By detecting the transient expression of GUS gene, this study probed into different factors effected on C.praecox's transformation,which were concentration of agrobacterium,time of infection,different types of infectious liquid,addition of AS in the infectious liquid and time of coculture. The reasults showed that these were important factors in C.praecox's transformation. In C.praecox's transformation system mediated by Agrobacterium tumefaciens EHA105-pIG121,the selective concentration of Cef was 200 mg/L,and Km was 35 mg /L. The optimal method of transformation was as follows:the callus was diced in 1cm×1 cm,and precultured in the miliorated MS+6-BA1.0 mg/L+NAA0.5 mg/L+KT0.5 mg/L+IBA0.2 mg/L+2,4-D0.2 mg/L in darkness for 3 days After the bacterial liquid whose OD₆₀₀ was 0.4 by the 2nd activation was centrifugated, thalli were collected. The OD₆₀₀ of bacterial liquid should be 0.4 after thalli was resuspended in liquid MS+AS100μmol/L.Then, the callus that had been precultured was put into the resuspended bacterial liquid to be infected for 10min in the shaking bed. The bacterial liquid on the surface of callus should be soaked up by aseptic paper in super clean bench before the callus with agrobacteria was cocultured in the miliorated MS+6-BA1.0 mg/L+NAA 0.5 mg/L+KT0.5 mg/L+IBA0.2 mg/L+2,4-D0.2 mg/L in darkness for 2 days.2 days later,when there was bacterial plaque around callus being coculrured,it was time to wash bacteria off on the surface of callus in the liquid MS+Cef1000mg/L in 2 hours. And next,after water on the surface of callus was soaked up by aseptic paper ,which had been washed by aseptic water 5 times, the callus was selectively cultured in the miliorated MS+6-BA1.0 mg/L+NAA 0.5 mg/L+KT0.5 mg/L+IBA0.2 mg/L+2,4-D0.2 mg/L+Km35 mg/L+Cef200 mg/L. Under these optimum. conditions,the rate of transient expression of GUS gene in C.praecox could be 37.5%.