Study On The Embryo Rescue Of Seedless Grape With Cold Resistance And The Functional Analysis Of VaERD

Seedless grapes have good quality but low chilling resistance, this weakness limit grapeindustry in cold regions. China has very abundant wild grape resource, in which the Chinesewild Vitis amurensis have the strongest cold resistance. Thus, combination the cold resistanceof Chinese wild Vitis amurensis cold resistance with the seedlessness and good quality ofseedless grape is a effective practice strategy for cultivating of the new seedless grape withthe resistance to the low temperature. In this study, we used seedless cultivars of Vitis viniferaas female parents, Vitis vinifera×Vitis amurensis with cold resisrance as male parents, for thecold-resistance embryos rescue breeding. And the basic characteristics analyses of coldregulated genes VaERD on Chinese wild Vitis amurensis which from our research grouppreliminary study have done.1Embryo rescue of seedless grape with cold resistance（1）The influence of different medium in embryo rescueOvules from eight cross combinations were cultivated on ER medium, ER+CH500mg/Lmedium, ER+CH500mg/L+GA30.5mg/L+IAA1.5mg/L Medium, respectively. The resultindicated that different media had different effect on germination rate of ovules and seedlingratio. But the added CH and GA3+IAA had no appreciable impact on proportion of embryosdeveloped and seeding ratio.（2）The effect of supplementation different amid acid in ER medium on embryo resuceThe ovules from Perlette×Beichun were cultivated on ER media added Ser, Asp, Nia,Cys, Gly, Pro, respectively, comparison with ER culture medium. The result indicated thatcompare with the proportion of embryo development on ER media which was8.89%,supplementation of Ser made the proportion of embryo development reach20.74%;supplementation of Asp and Cys made the proportion of embryo development have a litterincrease, reached10.67%and11.67%, respectively. But supplementation of Nia, Gly, Prodecreased the proportion of embryo development compared with CK, which were1.67%,5.19%and6.00%, respectively. Compared with the other five amino acid, the Ser had bettereffect for seedling ratio, which reached7.41%. The Chi-Square test results indicated that, at 0.05level, the supplementation of Ser and Nia had appreciable impact on proportion ofembryos developed; the supplementation of Ser had appreciable impact on. supplementionof Ser has remarkable promoting effect, while, supplemention of Niacin in the media performcontrarily by inhibiting the development of the embryo.（3）The effect of parents genotype on embryo rescueDurings the five cross combination used00-1-5as male, when used Jingdajing asfemale,got the biggest proportion of embryos developed and seeding ratio which were36.66%and4.16%, respectively. This result indicated Jingdajing was more suitable than otherfour seedless grape when was used as a female. The Chi-Square test results indicated that, at0.05level, used Jingdajing as a female had a appreciable impact on proportion of embryosdeveloped and seeding ratio. Among the two cross combinations of stenospermocarpicseedless cultivar ‘Royal Autumn’בBeichun’ and ‘Sultanina Rose’בBeichun’,‘RoyalAutumn’ was good to be a female as well as ‘Sultanina Rose’.When used ‘Sultanina Rose’and ‘Royal Autumn’ as female, Beichun had a bit improve on proportion of embryosdeveloped and seeding ratio than00-1-5, but the result is not significant. Thirty nine newseedless grape germplasms with cold resistant were obtained by embryo rescue technology inthis study.2Basic characterristica analysid of the cold responsive gene VaERD which from V.amurensis Heilongjiang seedling（1）Subcellular localization function of VaERDConstruction of the fusion expression vector, by gene gun-mediated transienttransformation technology, according to the characteristics of the GFP protein in theexcitation light, observed the green fluorescent location of VaERD-GFP fusion protein inonion epidermal cells by confocal microscopy. The results show that, the green fluorescent offusion expression vector35S: VaERD-GFP only in onion epidermal cell nucleus, but the greenfluorescent of negative control GFP vector was in all onion epidermal cells show greenfluorescence, this results indicated that VaERD has nuclear localization function.（2）VaERD transcription activation analysisThe ORF of VaERD was cloned into expression vector PGBKT7. Fusion expressionvector and the positive control and negative control were transformed into yeast AH109byfreezing and thawing method. The transformed yeast was coated on the appropriate SDselection medium inverted cultured at30°C conditions, until colonies appear. The selectionof transformants were crossed in a lack of SD (-Trp) medium and SD (-Trp-His-Ade) mediumcontaining X-α-gal three missing3d later, the results show VaERD transformants can growin SD (-Trp) medium and SD (-Trp-His-Ade) medium containing X-α–gal, and can activate the second reporter gene MEL1. Conversion of bacteria presented of blue in the substrate X-α-gal. At the same time, the negative control containing only screening marked Trp, onlygrowed on SD (-Trp) medium, because of absence of transcriptional activation, the negativecontrol can not grow in the SD (-Trp-His-Ade) medium. This result further proved VaERDand positive control GAL4have transcriptional activation in yeast.（3）The study of ectopic over-expression of VaERD inArabidopsisConstruction of the over-expression vector pCAMBIA3301-ERD， imported intoagrobacterium GV3101by electroporation. Ectopic over-expression of VaERD in Arabidopsis.20lines was got by antibiotics screening, furthermore,17positive lines was got by PCRdetection.