Study On Pomegranate Breeding And Establishment Of Its Regeneration System And Optimization Of Analysis Condition In RAPD

In this article, pomegranate breeding, synthetic valuation on pomegranate cultivars in anhui privince by economic characters, regeneration system of pomegranate by in vitro culture, isolation of genomic DNA of pomegranate and optimization of its RAPD analysis condition were all studied. The following results were obtained.Three new pomegranate cultivars were breeded through field survey and planting experimentation. The wanliuyihao was the variant of sanbaishiliu. And its seed was bigger than sanbaishiliu. The \vanliuerhao was the biger seed and better colour variant of manaozi. The wanliusanhao was the biger seed and better colour variant of yushizi. The stock plants and their clone progeny were stabile on heredity and their economic characters were excellent.According to the theory of fuzzy math synthetic valuation on 23 pomegranate varieties cultivated in Anhui province was done by yield, quality, mature period and resistance to preservation and diseases .The results shows that the economic characters of Wanliusanhao , Wanliuerhao and Manaozi are the best; those of Huaibeiruanziwuhao and other seven varieties are the better; those of Bopicao and other eight varieties are the general; those of Huohulu and other two varieties are the worst.The regeneration system of pomegranate had been established by using tissue and organ in vitro culture. The explants from twig cultured in MS medium supplemented with 2.0 mg . L-1 BA + 1.0 mg . L-1 GA generated callus 20 days after inoculation, and adventitious bud sprouted on the callus in succession. Being inoculated to MS medium supplemented with 5.0 mg . L-1 BA + 0.1mg . L-1 NAA the leaf tissues produced callus and sprouted 25 days after transplanting. The MS medium supplemented with 2.0 mg . L-1 BA + 0.3 mg . L-1 NAA was the most suitable one for axillary node segments taken from growing shoot, and the axillary bud could proliferate 4.9 times per 35 days. For inducement of adventitious root 1/2MSmedium supplemented with 0.5 mg . L-1 NAA + 0.1mg . L-1 AC + 20 g . L-1 sucrose was the best one tested, and the rooting rate of shoots was 95.8% and the mean number of roots was 5.6 calculated 10 days after transplantation. In addition primary medium supplemented with 0.1 %AC was efficient for adsorption to phenolic substances, prevention of explants browning and acceleration of rooting.The isolation effect of genomic DNA from mature leaf and tender leaf of pomegranate was studied respectively by SDS method. After comparing the quantity and quality of genomic DNA isolated from different materials the high-quality DNA was used as template to optimize the reaction condition of RAPD analysis system. The result showed that the genomic DNA isolated from tender leaf could be used for RAPD analysis and that isolated from mature leaf couldn't be used directly. The optimum reaction condition of RAPD analysis system for pomegranate was as follows: total reaction volume 50 μl, including about 80ng template DNA, 5 μ1 10×PCR Buffer purchased, 2.0mmol/L MgCl2, 120 μ mol/L dNTPs,, 3 unite Taq polymerase, 0.5 μ mol/L random primer; the program of heating: pre-denaturation 5min at 94 ℃ to make template DNA denarurating sufficiently, then undergo a proceeding of reaction cycles as denaturation 30sec at 94℃, anneal 45 sec at 36℃, polymerization 1 min and 30 sec at 72℃, 40 cycles, finally polymerization 7 min at 72℃.