Seedless Grape Embryo Rescue Breeding Techniques

Pollen was gathered firstly and cross-pollination was arranged in 2000,5~2001,2, Ovules of five cross combinations Delight × Olmo Seedless; interspecific hybridization of Olmo Seedless × Heilongjiang Seedling, Olmo Seedless × Bei Chun, Olmo Seedless × Jiangxi-2., Long-bunch Sultanina × Heilongjiang Seedling. These ovules were cultured by embryo rescue. Factors including sampling dates, basic medium, ratio of different external growth regulators, concentration of sucrose, LH ,low temperature, and so on were studied in this article.The results were as follows:1. 301 new plants were obtained by embryo rescue in this research. There were 100 plants from Delight × Olmo Seedless; 17 plants from Olmo Seedless × Jiangxi-2, 16 plants from Long-bunch Sultanina × Heilongjiang Seedling, 12 plants from Olmo Seedless × Bei Chun, 6 plants from Olmo Seedless × Heilongjiang Seedling; 68 plants from Sultanina, 47 plants from Flame Seedless, 35 plants from Delight.60 new plants in nutritive tube and transplanted in vineyard.2. The suitable dates of ovule cultured were attained among seven cross combinations. Ovules of seven cross combinations were cultured in different sampling dates. The suitable sampling dates of different cultivars and hybrids of cross combination were demonstrated the sharp different. The best sampling dates of nature cross-pollination of Delight and Delight × Olmo Seedless were 50 after pollination , nature cross-pollination of Sultanina was 37d after pollination ,Delight × Heilongjiang Seedling was 30d after pollination, Olmo Seedless × Bei Chun was 47d after pollination when its berry was soft, Olmo Seedless × Jiangxi-2 was 50d after pollination when its berry was soft, Long-bunch Sultanina × Heilongjiang Seedling. was 20d after pollination.3. The suitable basic medium was defined on embryo rescue. In-vitro ovule of Flame Seedless which obtained from 40d, 43d and 48d after nature-cross-pollination were cultured with N5, Do, B5. the best medium for embryo rescue was N5. The basic composition of N5 was Nitsch. It is essential to add 6% sucrose in the medium when ovules were cultured, then reduce to 2% sucrose in later two periods of culture.4. Different external growth regulators and their concentration ratio have different effect on ovules cultured. It was suitable to 2.0mg/L IAA, 0.5mg/LBA , 0.5mg/L GA3 for Delight × Olmo Seedless. It was suitable to 1.0mg/L IAA, 0.5mg/L BA, 0.5mg/L GA3 for nature cross-pollination of Sultanina.5. Simple procedure of embryo rescue. Firstly, in-vitro ovules were cultured on grade-1 medium which was ovule cultured medium to rescue immature embryo. After 80~90 days cultured, the development ovules were transferred into grade-2 medium which was germination of embryo medium. After 10~20 days germinated, these ovules were transferred into grade-3 medium which was established plant medium to develop plant directly.