Breeding New, Cold-resistant, Seedless Grape Germplasm Using Embryo Rescue Technology

Seedless grape take very important position in grape industry for the table and for drying. breeding of new, high-quality, seedless, grape cultivars are becoming a focus for researchers. Most seedless grape cultivars belong to Vitis vinifera, which have very good characters but has poor resistance to stresses, such as to cold. This limits the production of seedless grapes in cooler regions, and especially in northern China. China has rich germplasm resources of wild Vitis spp. This species has already been evaluated for its resistance to cold. Using its cold resistance by means of hybrid and seedless grapes seedless characteristic to breed new, cold-resistant seedless grape varieties. Conventional crossing breeding can only use seeded cultivars as female parent and seedless cultivar as parental,the seedless is very low, but useing seedless cultivars as the female parent by means of embryo rescue can increas the seedless offspring ratio. In this study we used the Chinese wild Vitis amurensis with resistance to cold-stress and it’s F1(Vitis vinifera×Vitis amurensis) as male parents; seedless cultivars of Vitis vinifera as female parents, for the cold-resistance embryos rescue breeding. In addition we reseach the main factors in embryo rescue to provide certain technical basis for improving the efficiency of embryo rescue breeding. Main results as following:1. The basal culture medium are important for in vitro plant culture。The result of the influence of ER medium and MM + 500 mg/L CH medium has showed that MM + 500 mg L-1 CH ovule cultured medium was more suitable for using embryo rescue for V. vinifera as the female parent with wild Chinese Vitis amurensis or its hybrids as the male parent.2. Early studies have shown that 1 mmol/L serine could promote the ovule development. We choose MM3 + 500 mg/L CH + 1 mmol/L serine as basal medium, adding IAA to reseach the influence of IAA on embryo rescue. the result is thar the effect of the addition of IAA to this embryo-rescue culture medium depends on the female genotype. The percent increase in embryo development can rise to as high as 57.7% for Ruby Seedless × 00-1-5, but IAA additions inhibit embryo development in Perlette × 00-1-5 and Neptune × 00-1-5, affecting the embryo rescue efficiency.3. The cross combination is sensitive of different concentration of 6-BA added on the MM3 + 500 mg /L CH + 1 mmol /L serine medium. When 6-BA concentration is 1.0 mg/L,the efficiency of embryo rescue is increased or not significant,but the concentration of 1.5 mg/L 6-BA inhibit the plantlet rate of ‘Flame Seedless’ בBeichun’, ‘Jupiter’בBeichun’, ‘Neptune’בBeichun’ hybrids.4. The genotype of the hybrid parent has significant effects on embryo rescue. Crimson Seedless, Ruby seedless and Flame Seedless are the most suitable genotypes as female parents whereas ‘Neptune’, ‘Perlette’, ‘Beauty Seedless’ and ‘Su-67’ do not seem to be very suitable. As the male parent contributing cold-resistance genes to a potential new seedless cross, ‘00-1-5’(V. vinifera × V. amurensis) would seem to be the most suitable candidate.5. Using the paraffin section technique, cytological observation was conducted on abortion and development of ovules,endosperms in 2 cultivars seedless grapes ‘Jupiter’ and ‘Neptune’,we determine the abortion stages and determine the suitable sampling time of ‘Jupiter’ and ‘Neptune’, which were as female parent of seedless grape embryo rescue.The best sampling time of ‘Jupiter’ and ‘Neptune’ were 38 days and 36 days after full bloom respectively.6. Using molecular markers identified original parents of hybrids and crossing offspring. 5 plants has seedless trait of 22 hybrid strains of ‘Beauty Seedless’ ×’Beichun’, ‘Flame Seedless’ ×’00-1-5’ by molucular probe GLSP1-569 linked to seedlessness gene. 48 plants has seedless trait of 87 hybrid strains of ‘Su-67’×’00-1-5’, ‘Neptune’× ‘00-1-5’, ‘Ruby Seedless’ × ‘00-1-5’ by molucular marker SCF27-2000 linked to seedlessness gene. 15 plants has the hardiness trait of 36 hybrid strains of ‘Beauty Seedless’ × ‘Beichun’, ‘Heshen Seedless’ × ‘Heilongjiang Seedling’, ‘Jupiter’× ‘Heilongjiang Seedling’, ‘Jupiter’× ‘Shuangyou’ by molucular marker S241-670 linked to the hardiness gene.