| Breeding for seedless grapes is one of the targets of grape breeders in worldwide. Theembryo rescue technology is an effective measure to improve the efficiency of seedless grapebreeding and could also be used to cultivate new varieties of polyploid grapes. This projectaimed at breeding seedless grape varieties by embryo rescue through two hybridizationmethods:(1) the early molecular assistant breeding technology of seedless grape: obtainingthe hybrids from cross-breeding between seedless V. vinifera cultivars and wild Chinese Vitisspp or crossing with two seedless cultivars, and then screening the seedless grape strains byusing the early molecular assistant breeding technology; and (2) the evaluation of polyploidnew seedless germplasm by flow cytometry: hybridization with diploid seedless×tetraploidgrape, and then identification and selection new triploid seedless seedlings by flow cytometry.At the same time, we optimized the embryo rescue technique and focused on the effects of thedifferent parental genotypes and their combination, media type and composition, andsampling time, low temperature pretreatment on the embryo germination and plantdevelopment rate. Moreover, we classified the abnormal seedlings into seven differentcategories based on morphology and investigated the formation and preliminarytransformation of the abnormal seedlings during embryo rescue in order to improve theefficiency of seedless grape breeding by the embryo rescue technology. The main results werepresented in the following:1. The efficiency of seedless grape combinations breeding was influenced by thematernal genotype by means of the embryo rescue technology. The differentcross-combinations gained their highest ovule germination rate at different sampling times.The best sampling times for different combinations were showed in following:‘FlameSeedless×Beichun’(DAF, days after flowering,39d);‘Blush Seedless×Shuangyou’(DAF54d);‘Pink Seedless×Beichun’(DAF54d);‘DA7×Shuangyou’(DAF44d);‘BlushSeedless×Thompson Seedless (DAF54d)’;‘Pink Seedless×Flame Seedless’(DAF54d);‘DA7×Blush Seedless’(DAF44d);‘Ruby Seedless×Black Olympia’(DAF63d);‘DA7×Jingyou’(DAF44d);‘Flame Seedless×Fujiminori’(DAF39d); and ‘Big Black×Kyoho’(DAF72d).2. Each cultivating stage had corresponding appropriate culture conditions in the process of seedless grapes embryo rescue. At the stage of ovule culture, the best results were foundwhen MM4was used as the basic medium and additional of mashed banana500mg/L; at thestage of embryo germination, the best results were detected when WPM was used as the basicmedium and additional of BA0.2mg/L.3. There were abnormal seedlings produced in the process of embryo rescue breeding.The deformity rate of the hybrid seedlings from embryo rescue was mainly influenced byfemale parent genotype. The abnormal seedlings were classified into seven differentcategories based on morphology:(1) epicotyl appears only one cotyledon;(2) neithercotyledon nor shoot;(3) the cotyledons were distorted fold-like;(4) the formation of thealbino seedlings in the process of differentiation;(5) hypocotyls formed short root, but nocotyledon;(6) epicotyl formed cotyledons, yet no root;(7) the plantlet earlydevelopment-stopped. The occurrence and proportion of each variety of malformationseedlings were random and uncertain.4. Separating polyembryony occur in the process of embryo rescue into single-embryoand inoculating each one into fresh embryo germination medium could completely preventthe generation of abnormal seedlings, and the embryo germination rate could reach100%.The treatment of pre-chilling (4°C,20d) on the immature fruit before the ovules werecultured could significantly reduce the number of abnormal seedlings and increase the embryogermination rate. In the aspect of formation of abnormal seedlings, MS+mashed banana500mg/L as the best effect of embryo development medium; WPM+6-BA0.2mg/L+mashedbanana500mg/L as the embryo germination medium effect best. As the aspect of reducingdeformity plantlet, the use of MS+mashed banana500mg/L as the embryo developmentmedium and WPM+6-BA0.2mg/L+mashed banana500mg/L as the embryo germinationmedium worked best.5. There were three kinds of variations after the deformity seedlings were transferred toconversion medium:(1) formation of normal seedlings with healthy stems and leaves;(2)formation of buds clustered around the tissues;(3) dedifferentiation and formation callus.Generally, those abnormal plantlets could develop into normal healthy plants after transferringthe clustered buds or the transformed normal seedlings to the rooting media after two weeks.The results showed that2MS+6-BA0.4mg/L+IBA0.1mg/L+mashed-banana500mg/Las the transformation medium showed the highest success rate in converting the deformityseedlings to normal plantlets. Four weeks after embryo germination was the proper time forthe transformation of abnormal seedlings.6. Through the detection of the seedlessness using seedless specific probe GSLP1,11grape lines amplified the569bp specific band and were named as JW-1-1, JW-1-2, JW-1-3, JW-2-1, JW-2-2, JW-3-1, JW-4-1, JW-4-2, JW-4-3, JW-4-4, and JW-4-5; through thedetection of chromosome ploidy identification by flow cytometry,3lines were confirmed astriploid, named as JW3-1, JW3-2, and JW3-3; and2lines were confirmed as haploid, namedas JW1-1and JW1-2, respectively. We carried out the subculture and multiplication andobtained436survival seedlings. |
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