| Rice (Oryza sativa L.)is one of the most important grain crops which supply the energy and food for more than one third of people in the world. However, rice is sensitive to cold stress in several periods while it is growing up, such as early seedling, seedling and booting stage. Cold stress is one of the most important factors to decrease the productivity of rice severely, which is thought to be a matter of great urgency to be solved. About 3～5 billion kilograms of rice are lost due to the cold stress every year. Therefore, it is of great importance to enhance the ability of cold tolerance and increase the yield of rice.Recently, more and more scientists noticed the CBF/DREB gene family when they studied on the abiotic stress to plant such as high salty, drought, and cold. AtCS is also a kind of cold-resist gene of Arabidopsis in Genebank. The gene BrDREB was belong to the CBF/DREB gene family, and was cloned from celery Cabbage by RACE(Rapid Aplication of cDNA Ends). The other one BnCS was cloned from Brssica napus by the same way. All primers were designed according to the conserved sequences of the cold-resistant genes of Arabidopsis in Genebank.In this research, some varieties of rice cultivated in Heilongjiang Province suitable for tissue culture were selected among dozen of varieties at first. Then, a series of genetic transformation system of mature embryos for rice varieties was optimized. The two genes were transferred into the rice calli separately by the way of Agrobacterium -mediated. The last, the transgenic plants gained were detected by PCR and dot plot hybridization.The main results are as follows:1. Heijiang 19, Longjing 14, Longyu 390 and Puyou 17 are suitable for tissue culture.2. The critical technologies of transformation optimized are: a) NMB is the component of induction media, MS is that of regeneration media. b) Take off the bud after one week. c) Add 5mg/L ABA in the media before regeneration. d) The concentrate of selectable marker is G418 200mg/L, PPT 2.5mg/L. e) Dilute the Agrobacterium 4 or 5 times when the OD600=0.6 to infect the calli. f) Use AS 200μmol/L both in the infect media and co-cultivate media. g) Immerse the calli in 500～1000mg/L cef dilution for 30min after usual method.3. 22 BnCS transgenic plants were gained in which 15 are positive in PCR detection, and 12 are positive in dot plot hybridization. 6 BrDREB transgenic rice were obtained and all of which are positive in PCR detection. |
