Mutagenic Effect Of Irradiation To Grape Seeds And Identification Of Early Selected Single Plants

Grape, one of the main fruits in the world, belongs to Vitis L. of Vitaceae family. Table grape is the major cultivated type in China. From its production point of view, Zhejiang province is one of the main producing areas in southern China. A lot of germplasms were introduced from abroad but few of them were acclimatized to the local environment. In this study, seeds of grape cultivar’Zuijinxiang’ were used as the experimental materials. They were subjected to137Cs gamma rays irradiation treatments. Biological mutation effects in terms of seed germination, seedling growth, chlorophyll fluorescence parameters etc were studied under different doses. These mutant plants screened at early stage were confirmed by the molecular and genetic diversity analysis using SSR markers. The purpose of this research was to provide scientific guidance for the grape irradiation breeding and industrial development. The main results were as follows:(1) Effects of137Cs gamma rays on grape seeds were obvious, it significantly decreased seed germination rate, germination vigor and germination index, prolonged the average germination days, decreased the emergence rate, seedling survival percentage, seedling height, stems thickness, delayed stem lignification time, increased etiolated seedlings, albino seedlings, and revealed different types of variant seedlings. There were also noted that irradiation effect was dose dependent. Results showed that137Cs gamma rays changed the chlorophyll fluorescence parameters, but it was not as significant as on seeds. There were observed that mutation effect was not linear with irradiation dose. The fluorescence parameters were significant different with untreated control at higher level of treatment (50Gy). It was found that effects of30Gy on all above mentioned parameters were satisfactory to generate the variation and selection of plant materials for mutation breeding. Thus we concluded that20-40Gy was an appropriate dose range of gamma rays for grape seed mutation breeding.(2) Five PCR ingredients e.g. Mg2+, dNTPs, Taq polymerase, primers and template DNA, were optimized using L16(45) orthogonal design experiments. The SSR-PCR reaction system for grapes was established, the optimal reaction system contained2μL10×PCR Buffer,1.5mmol/L Mg2+,0.2mmol/L dNTPs,1.0U Taq polymerase,0.4μmol/L primer, and50ng template DNA, ddH2O complemented to a total volume of20μL.(3) Forty-eight promising SSR primers were selected from122pairs, and the optimal annealing temperature for each primer was confirmed, ranging between50-60℃. Using these primers,64grape germplasms were amplified by SSR-PCR, a total of275bands were amplified including254bands showing polymorphic and the percentage of polymorphism was92.4%. Each primer could amplify3-10alleles, with an average of5.7. The amplified fragments were100-500bp, mostly between150-350bp. It was observed that polymorphic loci of the primers were abundant, the polymorphic rate of31primers reached100%.(4) The genetic similarity coefficient and UPGMA cluster analysis of64samples were analyzed from SSR results. The data showed that the genetic similarity coefficient among64samples ranged from0.644to0.891. The lowest was between CK-6and30-6, and the highest was between30-7and50-1. It was found that all samples were differentiated from each other by SSR markers. At similarity coefficient of0.77, all samples could be divided into8groups. Individuals of CK-6,30-6, CK-4,20-1, CK-9and20-3, each was considered as a single group. CK-10, CK-8, CK-5and CK-3were aggravated in one group. The remaining53individuals and parent ’Zuijinxiang’were clustered into one category. Each sample had a certain genetic difference with’Zuijinxiang’. Plants of CK-4, CK-6, CK-9etc without radiation had large genetic difference with’Zuijinxiang’, with the clear indication that grapes have high degree of natural variation, which was conducive to seedling selection breeding. Thus we concluded that radiation treated plants i.e.20-1,20-3,30-6etc, had showed the great genetic diversity with parent ’Zuijinxiang’ and the controls, were expected to become the new varieties afer further investigation.