Transformation Of RIP Gene Into Rice(Oryza Sativa) Mediated By Agrobacterium

In this paper, using the embryonal callus induced from rice mature embryos as the acceptor, RIPⅠand RIPⅡgene were transformed into rice (oryza sativa) mediated by Agrobacterium. The regeneration of rice callus system was optimized and the factors affecting transformation were also investigated. The resistant plantlets were examined by PCR and RT-PCR and the result indicated that the target genes have been inserted into rice genome.Resistance to the blast of leaves in vitro was tested.From preliminary results of the inoculation, we can see an increase of blast resistance level by foreign genes into the rice.In this study comparing the combined effect of Jilin five rice varieties mature embryos callus induced by three media,the results showed that NB and N6 is suitable for five Japonica rice varieties. The concentrations of 2.5 mg/L of 2,4-D is the best treated by different concentrations of 2,4-D.the highest rate of induction was up to 93.7%. Different medium make up significant differences in inducing callus and callus grow the fastest, and the callus quality, compact, light yellow tablets in NB medium. MS subculture is the worst。the callus,which were from different subculture time and different genetype,was carried the differentiation experiments Statistics showed differentiation rate, and variance analysis the callus tissue of Fengyou301 different subculture time, the result showed that the callus materials, 14-21 days ,were suitable for differentiation. After generations different genotypes of rice have greatly increased the rate of differentiation. Fengyou 301 rice varieties have the highest rate of 98.9% divide and Jiyou3 have the lowest, 86.5%. Therefore, we should understand rice regeneration in the process of transformation, choose easy differentiation genetype as soon as possible, and enhance our efficiency.According to the sensitivity to concentration varied differently among different cultivars the suitable concentration of Hyg is 40~50mg/L. The factors affecting the transformation consist of the concentration of Agrobacterium,the infecting time ,co-culture time and dried treats. The factors affecting the transformation have been studied and optimized. Too high or low of Agrobacterium concentration would reduce the rate of resistant callus markedly and most suitable OD600 is 0.4~0.6. The infection time was suitable for 20 min. 3d co-culture is optimal. Two days dry treatment could improve callus quality and the rate of resistant callus.A total of 32 strains resistant seedlings was gained by embryogenic callus as receptor, Agrobacterium infection and hygromycin resistance selection, Fengyou307 for 4 strains, Fengyou 301 for 10 strains, Fuyuan 4 for 15 strains, Jijing 88 for 3 strains. Tested by PCR and RT-PCR, Fengyou 301 gained 10 strains, and Fuyuan 4 obtainned 15 strains, 0 strains of the other two species.The blast-resistant identification was tested by vitro leaves which were inoculated for Magnaporthe grisea. The results showed that Fengyou 301 transfered RIPI belong to the resistant types, Fengyou 301 transfered RIPII reached a middle level. Fuyuan4 transfered RIPI belong to the resistant types, Fuyuan 4 transfered RIPII gene reached a middle level. Preliminary results from the inoculation. can be seen the rice transfered into foreign gene increased the level of blast resistance.