Breeding seedless grapevine via embryo rescue and marker-assisted selection in hybrid progenies

Abstract: 【Objective】Embryo rescue is one of the efficient breeding methods for seedless grapevine.However, there are many factors limiting a high rate of plantlet generation through embryo rescue like cultivars used as parents, time of sampling of hybrid fruit, medium for culturing, culture methods, addition of plant growth regulators, etc.The objective of our studies were to improve breeding efficiency of seedless grapevine through embryo rescue and to provide the theoretical basis for more accurate sampling of young embryos.【Methods】8 crosses were made among 7 stenospermic cultivars and 2 seeded cultivars.The hybrid ovules were removed from fruits and cultured in the medium for further embryo development.After 10 weeks in dark condition, embryos were picked from ovules and moved to the medium for development and germination.The number of plantlets were calculated after 1 month.Proliferation of plantlets was conducted in the subcultures to guarantee that every strain could be kept.The prime seedlings were trasplanted to the pots and the survived plantlets were then planted in the vineyard after seedling adaptation.GLSP1-569 and SCF27-2000 were utilized to detect seedless gene in filialgenerations.Cytological observation of embryo development and abortion of'Beauty Seedless' was made using paraffin section and Iron-vitrol hematoxylin hematoxylin staining.Sampling started after full-bloom stage which was regard as 0 d.Under the natural pollination condition, fruits in different growth periods of the same cultivar were picked at a sampling frequency of 2 days.At early stages, the whole fruit was collected.When the ovules could be separated from fruit, they were better choice.Materials were transfered into Carnoy's Fluid (glacial acetic acid:anhydrous ethanol =1:3) to fix for 24 h, and then washed with 70% alcohol.After dehydration in different levels of ethanol, clarification in xylol, saturation in paraffin and embedding, materials were kept in paraffin block and then cut at the thickness of 8 to 10 μ m with paraffin slicing machine.After staining the internal construction of the embryos were observed with light microscope.【Results】7 842 hybrid ovules and 468 hybrid strains wrere obtained from 8 crosses through embryo rescue and the average rate of plantlet generation was 5.97%.Crosses with different females had various rate of plantlet generation.The highest plantlet percentage (9.48%) was in the cross between'Kunxiangwuhe'and'Xinyu', while the lowest (0.0%) was in the cross between'Centennial Seedless' and'Crimson Seedless'.There was a significantly high seedling generation rate of'Kunxiangwuhe'×'Xinyu'than the rate of'Sultanina Rose'×'Xinyu'.And'Ruby Seedless' ×'Crimson Seedless' is significantly high than'Centennial Seedless' ×'Crimson Seedless' in seedling generation rate.When'Kunxiangwuhe'was used as female, seedling generation rates varied with crosseswith different male parents ('Beichun':7.35%, 'Ruby Seedless' :8.37%, 'Xinyu':9.48%) .The maximum of embryo germination rate and plantlet rate were found:41 DAP (days after pollution) in the cross'Flame Seedless' ×'Kunxiangwuhe' (66.67% and 7.35%) , 47 DAP in th ecross'Kunxiangwuhe'×'Ruby Seedless' (86.11% and 11.89%) and 55 DAP in th ecross'Ruby Seedless' ×'Crimson Seedless' (95.65% and 8.21%) .Embryos generated from the combination'Flame Seedless' ×'Kunxiangwuhe'and'Ruby Seedless' ×'Crimson Seedless' produced more seedlings on solid medium, while no significant difference was found between the seedling rates of embryos generated from the combination'Kunxiangwuhe'×'Beichun'regardless of whether they were cultured on solid medium or on solid-liquid medium.GLSP1-569 could be applied to detect seedless gene of hybrids generated from the crosses with'Flame Seedless' as female parent.SCF27-2000 should be used to test the hybrids in the crosses with'Kunxiangwuhe''Rudy Seedless' 'Crimson Seedless' and'Sultanina Rose'as females parents.The process of embryo development of'Beauty Seedless' began at the stages of dormant period, over dyad, tetrad and octant period, and stopped at globular embryo stage.So the embryo of'Beauty Seedless' was aborted on 38 days after flowering.【Conclusion】Female genotype had a more significant effect on the rate of embryos formation and rate of plantlet generation in embryo rescue although male genotype also affected the result to some extent.'Kunxiangwuhe'and'Ruby Seedless' were superior for being used as female parent in seedless grape breeding.The sampling times for highest embryo germination rate and plantlet rate varied with female parents.The media also varied with female parent.When the'Beauty Seedless' was collected as the female parent for the hybridization, the optimum sample time was 34-36 days after flowering.