| Seedlessness is an important trait for research and breeding of table grapes all over the world.The application of embryo rescue technique in hybridization of stenospermacarpic grapes as female parents not only increases the seedless ratio in the hybrids,but also reduces the breeding period.For stenospermacarpic grapes,during seedless fruit development,it is necessary to take a note on the genes related to seed abortion,and their expressions and regulations.Clear understanding about the functions and interrelationships of these genes has significant value in science research and practical application.Taking the stenospermacarpic grape?Thompson Seedless?as a sample,this paper recovered observation of the ovule morphology and anatomical structure during the process of seed abortion and changes of endogenous hormone content.It also screened out genes relevant to seed abortion based no comparison and analysis of different gene expressions in the process of seed abortion/development of?Thompson Seedless?and?Pinot Noir?the seeded grape.Furthermore,it created new seedless and disease-resistant germplasms with embryo rescue technique and hybridization of the stenospermacarpic grapes as female parents and Chinese wild grapes and their progeny as male parents.The major conclusions are listed as below:1.The changes in morphology,anatomical structure,and endogenous hormone levels during seed abortion of?Thompson Seedless?.During 32-42 DAF（days after flowering）,ovules of the?Thompson Seedless?in Yangling Area grow into varied sizes,with the integument turning brown and shrinking at 37 DAF.Paraffin embedded slices of the ovule present embryo sacs shrink and endosperm degradation,together with testa and endotesta separation from 32 DAF.Importantly,the globular embryos in the?Thompson Seedless?are significantly smaller than those in?Ruby Seedless?at 42 DAF,indicating delayed growth.The content of endogenous GA3and ZR in the ovules of?Thompson Seedless?decreases 36DAF;endogenous IAA decreases 37 DAF;while endogenous ABA constantly increases from 34 DAF.（GA3+IAA）/ABA ratio decreases since 34 DAF,while ZR/ABA decreases since 36 DAF.Analysis about the expression of hormone synthesis and signal transmission genes,the expression of Vvi GA2ox-3 and Vvi GA3ox-1 decrease,and the expression of auxin inhibitors Vvi AUX/IAA4 and Vvi AUX/IAA8,and abscisic acid signaling genes Vvi Sn RK2.1and Vvi Sn RK2.2 increase from 30 to 45 DAF.2.Differentiated genes expressions are found in seed development or abortion,transcription factors and SA（salicylic acid）synthesis and signaling pathway between?Thompson Seedless?and?Pinot Noir?in the eight seed abotion/development stages.From20～55 DAF,during the eight periods,the relative expression levels of the seed coat development related gene Vvi AGL11 and the endosperm development related genes Vvi FIS2and Vvi IKU2 in?Thompson Seedless?keep decreasing,while that of the?Pinot Noir?keep increasing;the expression levels of endosperm development related gene Vvi PHERES1decreases after initial increase in?Thompson Seedless?but increase continuously in?Pinot Noir?;the expression levels of Vvi WRKY3,Vvi WRKY52,and Vvi HB7 increase in?Thompson Seedless?,but give no visible changes in?Pinot Noir?.The expression level of Vvi WRKY6increases before decreasing in?Thompson Seedless?,but stayed almost unchanged in?Pinot Noir?.The expression of the SA synthesis and signaling pathway genes Vvi EDS1.2,Vvi NDR1 and Vvi SID2 in?Thompson Seedless?increases,inspiring autoimmune defense response during abortion process to facilitate seed abortion.Tissue specific expression analysis demonstrates that Vvi AGL11,Vvi IKU2,Vvi WRKY3,Vvi WRKY6 and Vvi WRKY52had the highest expression levels in the ovules of seedless grape and seeded grape.Vvi PHERES1 had the highest expression level in the ovules of seeded grape.3.Through embryo rescue technique,stenospermacarpic seedless grapes were used as female parents to hybrid with Chinese wild grape or European seedless grape as male parents,producing 2259 new seedless and disease-resistant hybrid progenies from 24cross-combinations.The highest embryo rescue efficiency occur when?Ruby Seedless?,?Flame Seedless?and?Kunxiang Seedless?as the female parents,while?Tangwei?,?Shuangyou?,?Beichun?,?Shine-Muscat?,?Ruby Seedless?,?Thompson Seedless?and?Aishen rose?as the male parents.When?Thompson Seedless?,?Ruby Seedless?,?Flame Seedless?,?Kunxiang Seedless?and?Crimson Seedless?is taken as the female parent,the best time for embryo rescue is respectively 39 days after flowering,57-58 days after pollination（DAP）,40-41 DAP,50 DAP,and 42 DAP.By optimizing embryo rescue technique program,exogenous application of 5 mg/L brassinolide before flowering of?Flame seedless?could visibly increase embryo development rate and the amount of polyembryony;SA synthesis inhibitor added in basic medium ER（Emershed and Ramming medium）can improve embryo growth,while WPM（Wood Plant medium）added with 1.0μmol thidiazuron（TDZ）could significantly increase seedling rate of hybrid embryos.4.A bunch of new seedless and disease-resistant grapes are achieved through marker-assisted selection and natural field investigation on disease-resistance.Among the1662 survived strains after field transplantation,484 were found carrier of the objective bands via seedless trait molecular markers GSLP1 and SCF27,while 25 carriers of the objective bands via downy mildew resistance molecular markers S382 and S294.Field disease-resistance survey identifies 51 powdery mildew-resistant and 96 downy mildew-resistant strains.In summary,the transcription level of seed coat development related gene Vvi AGL11decrease to promote seed coat browning,shrink,together with testa and endotesta separation;the transcription levels of endosperm development related genes Vvi FIS2,Vvi PHERES1 and Vvi IKU2 decrease to promote endosperm degradation;the changes in endogenous GA3,IAA,ZR and ABA content,together with SA synthesis and signaling pathway genes inspiring defense response to promote seed abortion of?Thompson Seedless?.And the degradation of seed coat and endosperm occur simultaneously. |
