In Vitro Conservation Of Germplasm And Analysis Of Proteomics In Grape

In this experiment the grape(Vitis vinifera)was used as the experimental material. somegrape germplasm resources were collected in Fujian Province to conduct the following researches①establishment of the in vitro regeneration system in grape；②the in vitro conservation ofgrape germplasm；③proteomics researchduring grape in vitro conservation.1. Establishment of in vitro regeneration system in grape.The stem segments with a single node were used as explants in Vitis vinifera cv. ManicureFinger. The best season for sanpling was April. The primary culture showed that the best mediumwas GS Medium supplemented with0.2mg/L IAA. The genotype affected the induction rates inthe primary culture. The best medium for proliferation was MS(1/2N) Mediun with1.0mg/L6-BAand0.1mg/L IAA, with the proliferation rate as high as6.8on the30thday.2. In vitro conservation in grape.The effects of different concentrations of paclobutrazol and mannitol on the in vitroconservation were compared in in Vitis vinifera cv. Manicure Finger. The result showed that thebest conservation effect occurred on the medium GS supplemented with0.2mg/L PP333or15g/LMannitol. The comparisons of in vitro conservation of10grape cultivars cultered on the GSMedium supplemented with2mg/L PP333indicated that there were similar effects among them.The orthogonal experiment of the different concentrations and combinations of mannitol and PP333and sugar showed that GS Medium supplemented with30g/L suger,2mg/L PP333and14g/Lmannitol was optimum for grape in vitro conservation.3. Proteomics research during in vitro conservation in grape.①2-D electrophoresis of the plantlet proteins during in vitro conservation in grape.Two-dimensional gel electrophoresis coupled to mass spectrometry analysis was used toexamine for the first time during the in vitro conservation in grape. The same line of in vitroManicure Finger planlets was used in the test. Tube plantlets were cultured on GS Medium with 2mg/L PP333or not. Control and PP333-treated plantlets were sampled after10,20,30and40dafter transfer to the new medium. Examination of2-D maps derived from PP333-treatmentreavealed the presence of many spots displaying a differential expression pattern. The PP333-treatment group detected spots number was356,368,286and174. However, the control group was261,295,183,154, respectively. The MWs of most proteins were30-45kDa.②Identification of proteins by mass spectrometry and analysis of their functionsduring in vitro conservation in grape.49protein spots were selected for mass specteometry analysis.46protein spots weresuccessfully identified. Among the responsive proteins, some photosynthsis-related proteinsincluding several fragments of the enzyme Rubisco were identified. Several enzymatic antioxidantand pathogenesis-related proteins were also observed. The proteins which were successfullyidentified were involved in many biology processes, which mainly concentrated on oxidation/reduction, carbohydrate metabolic process, defense response. Meanwhile, from the point view ofthe molecular function, the proteins were concentrated on catalytic activity, oxidoredactase activity,magnesium ion binding, nucleotide binding. Among the known proteins, most were related to anti-oxidization and photosynthsis, which suggested that adding paclobutrazol improve that resistanceof grape plantlets.