Transformation Of Bes1 Gene Into Rice By SAAT

Rice (Oryza sativa L.) is one kind of important worldwide cereal crops. It accounts for about fifty percent of cereal consumption in China. Plant hormones have been playing an important role in regulating the crops important traits, keeping the output formation and maintaining the quality. The attempt by researchers to improve and increase multiple traits of rice by regulating hormones inner levels has been a tremendous success. The important of brassinosteroid (BR) in regulating the seed dormancy and germination, the organs differentiation, the vascular tissue development, the flowering and the senescence can never be overestimated. The deeper analysis reveal such a fact that bes1 gene regulated BR. In this research, bes1 was introduced into two rice varieties via sonication-assisted Agrobacterium-mediated transformation (SAAT).The results were as follows:1. By comparison of the condition of the explants inducement with the state of the callus, a conclusion was drawn that the inducement rate of japonica rice was higher than that of indica rice. And the inducement rate of the immature embryos of japonica rice is the highest. The state of callus derived from their immature embryo was also the best. The inducement rate of Nipponbare was higher than that of Zhonghua 15, while callus derived from Nipponbare was more appropriate for suspension culture than that derived from Zhonghua 15 when it comes to the state of the callus.2. The selection and regeneration results of the two rice varieties suspension cells indicates that the best selection concentration of kanamycin for Zhonghua 15 and Nipponbare is 50mg/L and 30mg/L, respectively.3. The suspension cells were treated according to different ultrasound power and different time. When the ultrasound power is lower than 100 W, the growth of suspension cell is a bit better than control; when it was on 100 W, the growth tendency got its maximum; when it was upon 100 W, the drop of relative growth amounts is rapid. It can be concluded that the best and suitable ultrasound power is 100W.After being treated on the phase of 0,1,2,4,8 and 10 min respectively on 100w ultrasonic, the suspension cells were cultured in 27℃, 150 rpm and dark condition for the future research about fetching and measuring relative growth amounts. When the time of the ultrasound was treated is shorter than 4min, the station of the suspension cells growth is a bit better than that controlled. And when the treated time exceeds 10min, the drop of relative growth amounts is rapid. It can be discovered that the best ultrasound treated time is 2-4min.4. According to the intermediate stage, Nipponbare suspension cell was divided into two groups randomly. Group one was CK , another was handled with ultrasound (100 W , 2 min).Both of the groups were cultured in the period of 10d in the incubator at the condition of 27℃, 150 rpm .The relative growth amounts in these two groups were determined and growth curve was drew. The growth of the rice suspension cell is slow on the first 5 days, and the figure of relative growth amounts is low 150%. But when it is 380% that means it reached maximal figure in logarithmic phase, on the following 10-12d, it gets into stationary phase. After being treated by ultrasound, it was in the 3rd day when the growth rate was found different with CK firstly, and in the 5th day it is maximal. The gap between CK and treated group decreased and got the similar level in following two days. It served such a fact that the ultrasound stress is accommodated with suspension cell. The maximal cell amounts would be reached in the 12th day.5. The percentage of survival callus decreased with the increasing of ultrasonic power when Nipponbare callus were treated in the same ultrasond phase. In the power of 150 W, the percentage of survival callus decreased most. It decreased with the increasing of processing time when Nipponbare callus were treated under the same ultrasond power. The maximal percentage of survival calluses is reached under the condition of 100W ultrasond power and 3min processing time.6. After Nipponbare callus being transformed and selected with cephalosporins and kanamycin, 288 Nipponbare callus, 106 test-tube plantlets and 29 regenerated seedlings were obtained.7. The PCR technology initially proved that transgenic plants contain 1kb aimed fragment in the 29 positive transgenic plants, but the picture of electrophoretogram can not show this aimed subject clearly. Because the transgenic plants are small and weak, so it is approved cultivating in Hainan province as opposed to continuing this further research in PCR analysis. Unfortunately, all the transgenic plants died from the abnormal climate this year.