【作者】 纪薇；

【导师】 王跃进；

【作者基本信息】 西北农林科技大学 ， 果树学， 2013， 博士

【摘要】 无核葡萄育种是世界葡萄研究的重要方向之一。无核葡萄胚挽救育种是提高无核葡萄育种效率的有效方法，胚挽救技术也可以培育多倍体葡萄新品种。本研究主要以培育优质、无核的葡萄新品种为育种目标，采用以下两种途径进行无核葡萄新种质的创建：(1)无核葡萄胚挽救育种及分子辅助育种：用无核品种×无核品种、无核品种×有核品种获得杂种群体，通过早期分子辅助筛选获得无核葡萄单株；(2)多倍体葡萄新种质创制及倍性鉴定：用二倍体无核×四倍体进行杂交，通过细胞流式仪对获得的杂种幼苗进行倍性鉴定，筛选出三倍体无核新种质。本实验初步探讨了杂交组合中不同亲本基因型及其组合，取样接种时期，低温预处理，培养基组成等对胚挽救效率的影响，并对胚挽救技术进行优化。同时，提出将葡萄胚挽救过程中出现的畸形苗进行分类研究，初步探讨了胚挽救过程中畸形苗的形成原因，并对其转化利用进行了研究，以期为通过降低畸形苗的出现比例来提高利用胚挽救技术进行无核葡萄育种效率，提供一定的试验依据。得到以下结果：1、不同的种子败育型无核葡萄品种做母本进行胚挽救时的适宜取样时期不同，本研究获得的各杂交组合的最佳取样时期分别是：“火焰无核×北醇”为花后39天；“红脸无核×双优”为花后54天；“粉红无核×北醇”为花后54天；“DA7×双优”为花后44天；“红脸无核×无核白”为花后54天；“粉红无核×火焰无核”为花后54天；“DA7×红脸无核”为花后44天；“红宝石无核×黑奥林”为花后63天；“DA7×京优”为花后44天；“火焰无核×藤稔”为花后39天；“黑大粒×巨峰”为花后72天。2、无核葡萄胚挽救过程中的各个培养阶段有其适宜的培养条件。本研究表明，在胚珠发育阶段：MM4作为基本培养基，添加香蕉泥500mg/L的胚形成培养基在胚形成的作用上效果较好；在胚萌发阶段：WPM作为基本培养基，添加BA0.2mg/L的胚萌发培养基在胚萌发的作用上效果较好。3、胚挽救育种过程中常常产生畸形苗，胚挽救杂种苗的畸形率主要受母本基因型影响。本研究提出根据外观形态的不同，将胚挽救过程中产生的畸形苗分为以下七类：(1)单子叶畸形苗；(2)无叶无根苗；(3)子叶扭曲褶皱状畸形苗；(4)分化过程中形成的白化苗；(5)下胚轴形成短根，但没有子叶；(6)上胚轴形成子叶，但没有根；(7)萌发后早期停止生长的小苗。4、将胚挽救过程中出现的多胚分开为单一的胚，并将其逐一接种到新鲜的胚萌发培养基上，可以消除畸形苗的产生，并且胚萌发率也可以达到100%。对采摘的幼果进行低温(4°C)预处理20d后再接种其胚珠，不仅可以有效的降低畸形苗的数量，同时，胚的萌发率也得到了显著性增加。从降低畸形苗形成的角度考虑，用MS+香蕉泥500mg/L作为胚形成培养基，WPM+6-BA0.2mg/L+香蕉泥500mg/L作为胚萌发培养基的效果最好。5、畸形苗转接至转化培养基后，从外观形态上表现出3种转化结果：(1)转化形成具有正常茎和叶的植株；(2)组织周围形成大量簇生芽；(3)退化形成愈伤组织。将转化产生的簇生芽或正常苗转接至生根培养基后，2周左右即可形成健康的小苗。以2MS+6-BA0.4mg/L+IBA0.1mg/L+香蕉泥500mg/L作为转化培养基，在对单子叶畸形苗和子叶扭曲褶皱状畸形苗进行正常植株的转化的成功率较高。胚萌发后4周是转化的适当时期。6、本研究通过无核特异探针(GSLP-1)对杂种幼苗的DNA分子鉴定，获得了11个可以扩增出569bp特异条带的株系，分别命名为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和JW-4-5；用流式细胞仪对杂种苗的染色体倍性水平鉴定，获得了3个三倍体株系JW3-1，JW3-2和JW3-3和2个单倍体株系JW1-1和JW1-2。并对成苗扩繁后进行了炼苗移栽，已成活436个单株。更多还原

【Abstract】 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.更多还原