| Potatoes will become a very important strategic reserve staple food in China in 2014, and their sequencing had been finished in July 2011. The potato materials used for sequencing are doubled haploid potato materials DM1-3-516-R44(DM) and hybrid diploid potato materials RH89-039-16(RH). Based on the basis of the successful sequencing, countries around the world will start the research of functional genomics related to potato. In this study, by using the Agrobacterium-mediated genetic transformation, we introduce the successfully constructed the dual carrier T-DNA enhancer trapping plasmid into potato plants to obtain transgenic plants and screen and analyze mutant plants. Besides, combined with sequencing data has been released potato genome, we start to conduct research on potato functional genomics. The information in this study may be useful to build enhancer trapping bank.And accelerate the research of the potato functional genes.This study mainly made two progress within the recent two years:1. Creating a regeneration system of double haploid potato DM. We can obtain healthy plants by differentiating the stem and blade of potato. The medium used for potato stem callus induction is(MS+IAA 0.5mg/L+6-BA 2.5mg/L); The medium used for potato blade callus induction is(MS+NAA 1.0mg/L+6-BA 1.0mg/L); The medium used for Potato callus differentiation adventitious bud is(MS+6-BA 2.0mg/L+GA3 0.1mg/L).2. Creating a regeneration system of heterozygous diploid potato RH. We can obtain healthy plants by differentiating the stem and blade of potato. The medium used for potato stem callus induction is(MS+2,4-D 0.5mg/L+ZT 1.0mg/L); The medium used for potato blade callus induction is(MS+NAA 1.0mg/L+ZT 1.0mg/L); The medium used for Potato callus differentiation adventitious bud is(MS+ZT 2.0mg/L+GA3 10mg/L).3. We are optimized the conditions of transformation from agrobacterium to double haploid potato DM. Put the potato stem section Leaf blade in the callus induction medium culture for 10 days. Using OD600 0.3 agrobacterium dip for 10 minutes and put them into callus induction medium to culture for 3days. Clean thetissue for culturing and put them into the Callus induction medium within 300mg/L Cef and 8mg/L Hyg. Culturing for 10 days, and then we can obtain good callus. And put the callus into differential medium with antibiotic for 20 days culturing, then the adventitious buds generated.4. Using the basic culture medium which cantaining hygomycin preliminary screening in the adventitious bud, totally have 117 plants can take root. Then using hygromycin primers to test the transformed potato materials, obtain 30 Transformed plants.5. Using real-time PCR to analysis of foreign gene copy numbers in tansformed plants. We get 1 plant obtain 2 copys, others obtain single copy. |
