Genetic Transformation Of Blackcurrant With Key Genes During Osmotic Stress Signal Transduction

Drought, high salinity and low temperature are common stress condition that adverselyaffect plant growth and crop production. Using new cultivars which had high resistance tothese stress is a good way to defense the stress. But blackcurrants (Ribes nigrum L. ) areperennate woody fruit crops. Because of long life cycles , genetic heterogeneity and most ofeconomic traits controlled by quantitative trait loci or multiple genes , genetic improvement inblackcurrant by traditional breeding is very difficult., improvement of source strength bymolecular technology. Transferring extraneous gene to crops by genetic engineering to breednew varieties has become the research trend in crop breeding. But as a perennate woody fruitcrops, technics in tissue culture of blackcurrant still remain many limitations, It is verydifficult to regeneration and lack of efficient method of transformation. According to above-mentioned problems, this study make use of blackcurrant as material tocarry on tissue culture and genetic transformation. In this research construct plant expressionvectors with osCDPK7 gene and osMAPK4 gene. The regeneration system of blackcurranthas been established. Both of the two genes were transformed into Blackcurrant mediated byAgrobacterium and particle bombardment, in order to get some transgenic plants to enhancethe stress-tolerant. The main results were summarized as follows.1. Vector construction Plant expression vector PBC7E12 was constructed, on which osCDPK7 gene was regulatedby the constitutive promoter E12, and NptⅡ gene as selectable marker. Plant expressionvector PBME12 was constructed, on which osMACDPK7 gene was regulated by theconstitutive promoter E12, and NptⅡ gene as selectable marker.2. Establishment of Blackcurrant regeneration system(1). Callus induction of Blackcurrant Callus induction with leaf, petiole and stem segment. Study the effect of different PGR on callus induction, it was the basis of the regeneration of callus for the next step.(2). Make certain the optimal differentiation medium of blackcurrant shoot tip culture is MS+ 1mg/L BA, 30g/L sucrose, 0.8%agar, pH5.8. The optimal medium of root regeneration from shoots was 1/2MS,30g/L sucrose, 0.8%agar, pH5.8.3. Transformation of blackcurrant shoot tip by particle bombardment The concentration of Km selection in phase of shoot differentiation was 25 mg/L , in phaseof root regeneration , this concentration was 20 mg/L. Transformation of blackcurrant shoot VII摘 要tip by particle bombardment,we get some resistant shoots. The percentage of resistant shootwas 8.4％.4. Transformation of blackcurrant shoot tip by Agrobacterium mediate We get some resistant shoots. The percentage of resistant shoot was 10.5％.5. Identification analysis of transgenic plant Resistant shoots from particle bombardment were detected. one PCR positive transgenicplants with gene osCDPK7 and one PCR positive transgenic plants with gene osMAPK4 wereacquired.