Study On Development Of Novel Triploid Grapevine Germplasm

The purpose of this research is to cultivate tirploid grapevine germplasm ultilizing interploid hybridization and subsequent embryo rescue. Twenty-one diferent cross combinations between diploid and tetraploid grapes were conducted during the2009and2010season.These diploid parent grapes used here were2004-6-12、Seto Giants, Balado, Manicure figure and Muscat of Alexandria, and tetraploid parent grapes were Gorby, Aki Queen and Suiho, which was noted separately as6, D, B, M, A, G, Q and A for short, and2004-6-12、Seto Giants, Balado were male-sterile (female flower) fine individuals but others are bisexual varieties.Immature fruits were collected on37,42,47,52and57days after pollination (DAP) respectively, after surface-sterilized, the immature zygotic embryos were excised and cultured until embryo germination. Then the plantlets were propagated and transplanted to the greenhouse after acclimation. In this study embryo rescue medium, ovule treatment and hybridization affinity between different parent and other factors were discussed, and the ploid level of these hybrid plantlets were detected using flow cytometry (FCM), chromosome observation, morphological comparation and molecular markers technology. Besides, seedings from mature seeds sowed at field and offsprings from tetraploid grapevine self-breed were also detected using the same techniques to select triploid plants. The main results obtained are as follows:1. Through hybridizations between diploid and tetraploid grapevine cultivars,41hybrids were obtained from embryo rescue and12hybrids from seed sowing at field. Their ploid testing and genetic identification were then actualized utilizing FCM, chromosome observation, morphological comparison and SSR at seedling stage, among which6Q47-1,6Q52-1,6Q52-3,6S52-3,6S52-5, DG42-1, DG42-2, BQ52-5, AS52-1, MS52-2, MS47-1, MS47-2, MS47-3, MS57-2, BG-003, BG-005, MS-002, MS-003were confirmed to be triploid, while others diploid.2. Somatic embryogenesis and plant regeneration were successfully established on Nitsch and Nitsch (NN) medium from immature zygotic embryos of interploid crossing6-12×Aki Queen.The optimum hormone combinations were1.75mg·L-1IAA and0.35mg·L-1GA3or1.80mg·L-1NAA for somatic embryogenesis directly from IZEs. Somatic embryogenesis and normal germinating occurred synchronically, and the frequency of somatic embryogenesis varied from11.11to16.67%among IZEs of different sampling period after pollination. After subcultured ongermination medium, somatic embryos converted into normal plantlets. The analysis of DNA content and genetic fidelity determined by flow cytometry and SSR of the regenerated plantlets clearly indicated that the four plantlets were triploid and the normal germinated plant and the SE-derived plants came from the same IZE.3. A fine individual2004-5-11were proved to be one offspring of tetraploid grape Suiho, and was triploid. Its growth and fruit-set chracteristic and possible origin was studied.