Studies On Transgenic Rapeseed With Rice Chitinase Gene By Agrobacterium Tumefacien

Rapeseed is one of the most important crops in our country and in recent years,the studies of its genetic improvement by gene transformation are concerned by many scientists.However,due to the great difference of the abilities of plant regeneration and transformation efficiency among different rapeseed genotypes,it is necessary to establish a high-efficiency plant regeneration and transforrhation system before transformation.In this study ,a high-efficiency plant regeneration system was established based on rapeseed (B. napus) "jia na da shuang di " rapeseed (B. juncea) "lu zhou si ling' rapeseed (B. campestris) "xi shui bai", during the work of Agrobacterium tumefaciens mediated chitinase gene and bar gene tansformation , the best conditions for transformation were found. The results are shown as follows:(1) cotyledon (with 0. 1-0. 2cm leafstalk) hypocotyl leaf discs stem cutting of cultivars B1 B2 B3 were cultured on different mediums, the callus inducing and differentiation of them were observed and compared, the results showed that there was an obvious difference of the ability of callus inducing and differentiation among different explants, which indicated that the most efficient explant for callus inducing and differentiation was hypocotyl,then was cotyledon(with 0. 1-0.2cm leafstalk). leaf discs stem cutting. The genotypic difference among different cujtivars was obvious, which performed that the the callus inducing and differentiationability of B2 was strongerthan which of Bl B3(2) the best explants were cultured on mediums with varieties of hormone compositions. After observation and comparison of the results of callus inducing and differentiation, the best medium system was established: For Bl: MS+6-BA lmg/L+NAA 0. lmg/L+2, 4-D 0. 5mg/L,B2: MS+6-BA 2mg/L+NAA 0. lmg/L+2, 4-D lmg/L, B3: MS+6-BA lmg/L+NAA 0. lmg/L+2, 4-D lmg/L,(3) Based on the high-efficiency plant regeneration syst$m, the binary intermediate expression vector pCAMBAR.CHI.il containing chitinase gene and bar gene was transferred into rapeseed mediated by Agrobacterium tumefaciens, several factors influencing transformation efficiency were examined and a high- efficiency transformation system was established:Preculturing explant for 2 days, immersing it in the Agrobacterium tumefaciens suspension with OD600= 0.5 for 30 seconds, co-culturing for 2 days, culturing on resume-medium forl4-17days(BK B3), and for 10-14 days( B2), then adding the selection expression, the results also showed that acetosyringone (AS) could obviously improve the transformation.(4) Resistant seedling were obtained after selection by Hygromycin and Bastar, and was proved to have chitinase gene via PCR test. Transform frequency was about 30%, non-masculine frequency was 60%o(5) there is a transgenic plant of B3 which was proved to have chitinase gene via Southern test was obtained.