Changes Of Endogenous Hormones During Embryo Development Of Seedless Grape And Optimization Of Embryo Rescue Technology System

Seedless grapes are popular among consumers because of their excellent quality and convenient consumption.Traditional breeding of seedless grapes can only be done by hybridizing seeded grapes as female parents and seedless grapes as male parents,resulting in a low rate of seedlessness,a long breeding cycle,and low efficiency in the hybrid offspring.The embryo rescue technique can expand the female parent selection by using seedless grapes as the female parent,significantly increasing the rate of seedlessness in the progeny of crosses.However,the development rate,germination rate,and seedling rate of embryo rescue in seedless grapes are still low.Therefore,it is essential to study the changes in endogenous hormones during the process of seedless grapes embryo development and to optimize the embryo rescue technology system to improve the efficiency of embryo rescue.This study was carried out:（1）Taking the seeded variety‘Jingxiu’and its F₁ generation of stenospermocarpy variety‘Qinxiu’as the test materials,the changes of endogenous hormone content in ovules of the two varieties during different periods were studied.（2）Optimize embryo rescue technology system of seedless grapes.（3）The hybrid embryo rescued seedlings obtained were selected with the help of seedless gene molecular markers and transplanted to the field.These studies will provide a theoretical basis for revealing the mechanism of embryo abortion of the stenospermocarpic grape and optimizing the embryo rescue technology system to improve the embryo rescue efficiency and lay a material foundation for further breeding of new varieties of cold-resistant seedless grapes.The main results obtained are as follows:1.During the same sampling period,the weight of a single ovule of the seeded variety‘Jingxiu’was higher than those of the seedless variety‘Qinxiu’.Compared with‘Jingxiu’,the content of growth-promoting substances（IAA,CTK,GA₃,ACC,JA,SA,Put,Spm,and Spd）in ovules of‘Qinxiu’was lower.In comparison,the content of growth-inhibiting substances（ABA）was higher,and the ratios of（IAA+GA₃）/ABA,（IAA+ZT+GA₃）/ABA,Spm/PAs,（Spd+Spm）/PAs,and（Spd+Spm）/Put were lower,which may be the main reason for embryo abortion of seedless grape.2.Pre-flowering inflorescence dipping at 30-50 mg?L^-1 IBA at 14 d before flowering significantly increased the embryo development rate,germination rate,and seedling rate of‘Ruby Seedless’×00-1-5（‘Meiguixiang’בHeilongjiang seedling’）,‘Qinhong No.2’×00-1-5,‘Qinhong No.10’×00-1-5,and‘Flame Seedless’×00-1-5.The most effective IBA concentration for‘Ruby Seedless’,‘Qinhong No.2’,and‘Qinhong No.10’was 50 mg?L^-1,while that for‘Flame Seedless’was 30 mg?L^-1.In addition,spraying 0.5 mg?L^-1 TDZ on the female parents’inflorescences at 10 d before flowering was shown to significantly increase the development of ovules and the development rate,germination rate,and seedling rate of embryos of the seedless grape cultivar‘Qinhong No.2’open-pollination and its self-pollination,and‘Qinhong No.10’open-pollination and its self-pollination under in vitro culture conditions.3.Studies on embryo rescue of nine combinations including‘Qinxiu’×00-1-5,‘Qinhong No.2’×00-1-5,‘Flame Seedless’×00-1-5,‘Qinhong No.10’×00-1-5,‘Ruby Seedless’×00-1-5,‘Crimson Seedless’בJupiter’,‘Flame Seedless’בJupiter’,‘Ruby Seedless’בJupiter’,and‘Qinhong No.1’בJupiter’showed that the effects of embryo rescue were different when the same male parent crossed with different female parents.When the cold-resistant seeded hybrid 00-1-5 was used as the male parent,‘Qinhong No.2’,‘Qinhong No.10’,and‘Ruby Seedless’were used as female parents,the embryo development rate,germination rate,and seedling rate were higher.Among them,the embryo development rate,germination rate,and seedling rate of‘Qinhong 10’×00-1-5 were the highest,which were 9.52%,5.36%,and 4.17%,respectively.In contrast,the embryo development,germination,and seedling rates of the female parent‘Flame Seedless’were the lowest,only 4.85%,2.42%,and 1.82%,respectively.When‘Jupiter’was used as the male parent,the embryo rescue effects of‘Ruby Seedless’and‘Qinhong No.1’were better.The embryo development rate of‘Qinhong No.1’בJupiter’was the highest,which was 14.39%.The germination rate of‘Ruby Seedless’בJupiter’and‘Qinhong No.1’בJupiter’was the highest,both of which were 10.67%;the seedling rate of‘Ruby Seedless’בJupiter’was the highest,which was 10.67%.While the embryo rescue effect of‘Crimson seedless’בJupiter’was the worst,embryo development,germination,and seedling rates were only 9.55%,6.78%,and 1.76%,respectively.Overall,‘Ruby Seedless’,‘Qinhong No.1’,‘Qinhong No.2’,and‘Qinhong No.10’were suitable for embryo rescue as maternal parents,‘Flame Seedless’,‘Crimson Seedless’,and‘Qinxiu’were not appropriate.For the open-and self-pollination combinations of‘Qinhong No.2’and‘Qinhong No.10’,the embryo rescue consequences of the open-pollination combinations were significantly better than that of the self-pollination combinations.4.The ovules of four combinations were cultured on three different media.It was found that MM3 solid-liquid bi-phase medium was found to be more suitable than ER and NN solid-liquid bi-phase mediums as the embryo rescue ovule medium.In addition,the hybrid ovules of‘Qinhong No.2’×00-1-5 and‘Qinhong No.10’×00-1-5 were cultured on MM3 solid and solid-liquid bi-phase medium.The embryo development rate,germination rate,and seedling rate of the two combinations on MM3 solid medium were significantly higher than those grown on the solid-liquid bi-phase medium.In the germination and seedling formation,the naked embryo cultured on WPM solid medium was superior to the direct germination of ovules on the NN medium.Using 2×MS medium as the basic culture medium,additional with 0.2 mg?L^-1 6-BA,0.1 mg?L^-1 IAA,and 0.15 mg?L^-1 Zn SO₄could significantly improve the transformation rate of abnormal seedlings into normal seedlings.5.A total of 866 developing embryos of nine hybrid combinations were obtained by embryo rescue technology,of which 493 embryos germinated into seedlings.A total of 2797developing embryos were obtained from 4 combinations of open-and self-pollination of‘Qinhong No.2’and‘Qinhong No.10’,of which 2363 embryos germinated into seedlings.The487 embryo rescue seedlings of nine hybrid combinations were detected by seedless gene marker SCF27-2000,and 432 seedless lines were preliminarily screened.