The Genetic Transformation Of The Fir

Cunninghamia lanceolata Hook.,native to China, distributes in different provinces of southern drainage area of Yangtse River.It is an important commercial and fast growing conifer in southern.In the past decades,the dieases of Cunninghamia lanceolata Hook. became more and more severity.That is the intention and significance of the disease-resistant transgenic experiments of Cunninghamia lanceolata Hook. On the basic of tissue culture and reproduce of system, We carried on a series of concerned experiments by plantlet stems as explants:establishment of transgenic receptor system,studies including in instantaneous expression and stabilization expression of transgenic experiments. We observed the effect of several factors on induction buds ,such as pre-culture time,sucrose,6-BA,TDZ,Cef,Km.And finally we established a high-efficient tissue culture system.The results indicated that for stem induction buds the optimal medium was DCR+6-BA1.0mg/L+ TDZ0.002mg/L+ NAA 0.1mg/L+sucrose 20g/L.Pre-culture for 2 days,the bud induction frequency, average number of bud and bud forming capacity all reach the maximum,which was 100%,6.35,6.35,respectively.The substrate was used to buds inducement in transgenic experiments. Utilizing enzymology trait of GUS gene,we did instantaneous expression experiments so that we could optimize the transgenic conditions in short run. Comparing the influence of seven factors on the quantity GUS+ expression of stems, we preliminarily optimize the transgenic system:2 days for pre-culture time,3 days co-culture time,pH5.60,OD600=0.20.4-8 minutes for infectionand undecided low concentration AS. We made use of bacteriostasis effect of GAFP gene which was integrated into EHA105. After studying the influence of mode of culture and of material adoption on stem state and differentiation,we got the statistic of different AS concentration.It indicated that dark light was unfit for induction stem.As a whole,the differentiation was tiptop in evidence as AS 60μmol/L,and the drown stems was most as AS 40μmol/L.Under strict filtration we got two Kmr shoot.And one was proved negative by PCR analysis,the other grew too slowly to analysis at present.We thought that is the main reasons of so low transgenic ratio——the complex internal-environment of tree and unfound higher efficency reproduce system.