Study On High Efficient Regeneration System And Two Genetic Transformation Methods Of Rice

Rice(Oryza saliva L) is one of important grain crops.To date,it becomes the model plant in molecular area.Regeneration and genetic transformation are essential methods in rice molecular research and gene engineering.Because regeneration and transformation are depend on genetic different and genotype of rice cultivars.So it enlarge the random and complication.There are many problem should be studied in building a system with efficient and stable regeneration and transformation system.This paper studied on rice cultivars in Jilin province and optimized the tissue culture system.By biolistic and Agrobacterium-mediated transfer Bt into calli of mature embryos.Results are as follows: 1 .The tissue culture system of mature embryos on cultivated rice varieties in Jilin province was optimized.In order to improve the embryogenic calli induction rate from mature embryos,different media with different hormones are used in the embryogenic calli induction of five japonica cultivars.The experiment results indicated that selection suitable medium is basis to improve the induction rate and the quantity of embryogenic calli from mature embryos.The hormone treatments have certain effects on the calli induction ,with the effects having significant difference for different genotypes and selected media.The mature calli induction of different media is quite different. NMB, NB, CC gain very well induction efficiency and excess 50percent.During the different of hormones concentration 2,4-Dl-2mg/L can obviously enhance efficiency of induction and embryos induction. In experiment ,NMB and NB media can improve the growth of mature calli and canobtain white ,grain-shape ,embryogenic calli.In the same medium (NMB) and concentration of hormones ,adding D-Mannital 4g/Lcan improve the state and height of calli.During treament of calli for pre-regeneration before differentiation could increaseplant regeneration by 8 times of Fengyou 501.It also can improve the state of calli.The five differentiation efficiency is very different .It is between 35.0 to 95.0 percent.2.According the state and browning rate of calli,8-10mg/L PPT is the better conditionof calli selection.3.Parameters of Genegun(PDS-1000) transformation of mature embryo calli werestudied by Gus transient expression.When the biolistic target distance is 6cm,9cm,12cm,and the Gus transient expressionis 97.14,85.29,and 27.03 percent.While the target distance is fixed of 9cm,bomard for l,2,3times ,the Gus transcicentexpression is 85.29,91.89,96.43 percent respectively.4.Agrobacterium tumefaciens has been used extensively for gene engineering in rice.In experiment ,different co-cultured time of Agrobacterm the state and browning rateof calli is very different.I commend infection time for 20min is better in ricetransformation.Rice cultivars"Nongda 3","Fengyou 501" are more sensitive to Agrobaterium stainLBA4404 than other cultivars.Two ways of selection transformation rate is different.By adding Cef and Carb inculture media not including PPT, can bring down browning rate 50 percent.5.Bt gene has been delivered into rice by Agrobacterium tumefaciens.Thirty-fourtransgenic rice plants were obtained.Bt gene had been inserted into rice genomedemonstrated by PCR.