【作者】 唐冬梅；

【导师】 王跃进；

【作者基本信息】 西北农林科技大学 ， 果树学， 2010， 博士

【摘要】 无核葡萄是世界范围内主要的葡萄育种目标之一。现有的无核葡萄品种多属于欧亚种群(Vitis vinifera L.),抗逆性普遍较差,市场上急需培育出抗逆、大粒及符合各种消费和农艺生产要求的优质无核葡萄新品种。传统的无核葡萄育种方法,由于无核品种的合子胚在发育进程中发生败育,只能以有核品种做母本获得有发芽力的杂交种子,而以无核品种作父本来传递无核性状,但用这种有核×无核杂交方式获得的无核后代较少,育种效率很低。利用胚挽救技术,可以用无核×无核的杂交方法来有效的提高杂交后代的无核比率。同时在杂交培育三倍体无核葡萄品种时,胚挽救技术有助于克服二倍体和四倍体之间杂交胚珠的败育。为了增强无核葡萄的抗逆性,充分利用原产我国的野生葡萄抗病和抗寒资源,用无核葡萄和中国野生葡萄进行杂交;同时为培育三倍体大粒无核葡萄品种,用二倍体和四倍体品种之间进行杂交,借助胚挽救技术获得杂交后代新种质,是选育抗逆、大粒、优质的无核葡萄新品系的有效途径。本研究主要以欧亚种(Vitis vinifera L.)无核葡萄品种‘无核白’、火焰无核、底来特、黎明无核、红宝石无核、森田尼无核、无核紫、皇家秋天等9个品种和欧蘡杂种00-3-1([爱莫无核×蘡薁(V. thunbergii Sieb.et Zucc.)])为母本,与中国野生刺葡萄(V. davidii Foex.)塘尾和雪峰、山葡萄(V. amurensis Rupr.)双优及欧山杂种(V. vinifera×V. amurensis)北醇,无核品种火焰无核、森田尼无核、皇家秋天、奥兰多无核,欧亚种有核葡萄品种红地球、新葡一号、新郁、紫霞、里扎马特进行杂交,以及二倍体欧亚种底来特、黎明无核、森田尼无核、新郁与四倍体欧美杂种(V. vinifera×V. labrusca)品种巨峰、藤稔、紫珍香、高妻之间进行杂交,共计32个杂交组合,运用胚挽救技术创建无核葡萄新种质;同时以优无核、奇妙无核、奥迪亚无核及以上无核葡萄品种和组合为试材,在前期试验的基础上,对葡萄胚挽救育种技术加以改进完善,主要获得了以下结果:1.通过无核葡萄胚挽救技术获得了葡萄田间杂交32个组合的葡萄新种质1337份。其中无核葡萄×中国野生葡萄或欧山杂种杂交组合6个,获得杂交植株153个;无核葡萄×无核葡萄杂交组合6个,获得杂交植株401个;无核葡萄×有核葡萄杂交组合9个,获得杂交植株353个;二倍体×四倍体组合7个,四倍体×二倍体组合4个,共获得杂交植株430个。分批次对这些杂交苗进行炼苗移栽,已有509个株系在大田移栽成活。2.对以无核白和火焰无核作亲本的191个杂交株系用无核探针GSLP1进行检测,有77个株扩增出569bp特异带,占40.3%。对火焰无核×塘尾杂交组合后代84个株系用抗黑痘病RAPD标记S183-1354进行检测,有41个株系扩增出1354bp特异带,占48.8%。综合两种标记的检测结果,获得同时能扩增出与无核和抗黑痘病分子标记的24个株系。3.用倍性分析仪对二倍体和四倍体之间的12个杂交组合409个后代株系进行倍性鉴定,得到8个三倍体杂种株系。用胚挽救获得的成苗率明显高于成熟种子播种萌发成苗率;以2x×4x的杂交方式,比4x×2x,能得到更多的杂交后代;但4x×2x的杂交后代中三倍体的获得比率高于2x×4x。?4.对10个无核葡萄品种的胚挽救难易程度和适宜取样接种时期进行了试验评价。以相同果粒中获得的最多发育胚数和萌发胚数为评价标准,各品种胚挽救从易到难的顺序为:奥迪亚无核、奇妙无核、火焰无核、优无核、无核紫、皇家秋天、底来特、黎明无核、无核白、森田尼无核;其适宜的取样接种时期分别为:花后40d、60d、40d、50d、70d、70d、60d、55d、35d、40d。5.在前期研究的基础上,对无核葡萄胚挽救过程各个培养阶段的适宜培养条件进一步完善确定,认为:在胚珠发育阶段,以MM3和Nitsch+GA30.5 mg l-1+IAA1.5 mg l-1作胚珠发育培养基,其次是ER培养基;在常温下进行固液双相暗培养,有助于提高胚发育率;在胚萌发阶段,使用WPM + BA 0.2 mg l-1培养基,添加2~5 mM腐胺有利于提高无核葡萄的幼胚萌发率,胚的萌发率与胚的大小相关;在萌发成苗阶段,胚萌发形成正常苗的比率与其母本胚的发育程度有关,白化苗的发生主要与母本基因型有关,畸形苗在MS + BA 2.0 mg l-1 + IBA 0.5 mg l-1中易于诱导形成正常茎叶,之后接种在生根培养基1/2 MS + IBA 0.2 mg l-1上可形成正常完整小植株;在胚挽救苗的炼苗移栽阶段,宜选择30d苗龄的壮苗,对基质用高压灭菌及无菌水浇透,每周浇1/4 B5培养基大量元素溶液有利于胚挽救苗的成活生长。6.为提高胚珠败育较早的无核葡萄品种火焰无核、无核白、森田尼无核的胚挽救成苗率,试验确定了针对不同品种外施植物生长调节剂的适宜的药剂种类浓度和施用时期:森田尼无核为花前14d施用100 ~ 500 mg l-1的矮壮素;无核白为花前10d施用500 mg l-1矮壮素;火焰无核为花前20d施用30 mg l-1的BA。更多还原

【Abstract】 Development of seedless grapes is one of principle objectives of grape breeders worldwide. Since the major commercial seedless grape cultivars are derived from Vitis vinifera, a species susceptible to diseases, it is necessary to develop new resistant, big berries and high quality seedless cultivars for meeting the demand of consumers and grapevine growers. In conventional seedless grape breeding program, seeded grape varieties were used as female parents, while seedless cultivars were used only as male parents to transmit seedless trait due to the zygotic embryo abortion. But the proportions of seedless offsprings were quite low and the breeding programms were inefficient. The development of embryo rescue techniques had ensured more seedless progenies using seedless cultivars as female and male parents, and utilized to obtain triploid hybrid seedlings from crosses between diploid and tetraploid cultivars. To introgress disease resistance of China wild Vitis species as pollen parent into seedless Vitis vinifera as female parent through controlled crosses, and to produce more efficiently triploid hybrids from crosses between diploid and tetraploid grapes, it should be reliable to breed new seedless and disease resistance, large-sized berries cultivars through using embryo rescue technique.Thirteen seedless cultivars/line (including‘Thompson Seedless’,‘Flame Seedless’,‘Deligh’t,‘Dawn Seedless’,‘Ruby Seedless’,‘Cenennial Seedless’,‘Black Monukka’,‘Autumn Royal’,‘Orlando Seedless’,‘Superior Seedless’,‘Fantasy Seedless’,‘Otilia Seedless’,‘00-3-1’) and twelve seeded cultivars (including‘Tangwei’,‘Xuefen’g,‘Shuangyou’,‘Beichun’,‘Kyoho’,‘Takatsuma’,‘Zizhenxiang’,‘Fujiminori’,‘Xinyu’,‘Red Globe’,‘Zixia’,‘Rizamat’,‘Xinpu-1’) were used in this study.‘Tangwei’and‘Xuefeng’are V. davidii cultivars.‘Shuangyou’is V. amurensis cultivar.‘Beichun’is European-Amur hybrid with V. vinifera and V. amurensis. Line‘00-3-1’is European-Wild hybrid with V. vinifera and V. thunbergii,‘Kyoho’,‘Takatsuma’,‘Zizhenxiang’,‘Fujiminori’are tetraploid cultivars and European-American hybrids with V. vinifera and North American species (V. spp.), while the other cultivars are diploid and V. vinifera cultivars. Thirty-two crosses, including seedless×China wild Vitis species (and hybrid‘Beichun’), seedless×seedless, seedless×seeded, interploid crosses between diploid and tetraploid cultivars, were carried out in order to obtain the novel seedless germplasm through embyo rescue. Meanwhile the cultivars and hybrid ovules above were used to improve the technique of embryo rescue on the basis of previous study, involved in demonstrating the effect of influence factors during different embryo rescue stages and the effect of exogenous application of plant growth regulators on development of ovule and subsequent embryo rescue of stenospermic grape. The main results were in the following:1. We had obtained 1337 hybrid seedlings from 32 crosses through embryo rescue technique, including 153 hybrid seedlings from 6 crosses of seedless×China wild Vitis species (or Beichun), 401 hybrid seedlings from 6 crosses of seedless×seedless, 353 hybrid seedlings from 9 crosses of seedless×seeded, 430 hybrid seedlings from 8 crosses of diploid×tetraploid and 4 crosses of tetraploid×diploid. The hybrid seedlings had acclimatized and transplanted into seedling nursery in batch es, 509 survival lines were obtained.2. The detection of the seedlessness in 191 hybrid lines was performed using seedless specific probe GSLP1, 77 lines (40.3%) amplified the 569 bp specific band. Assistant selection of RAPD marker S183-1354 of resistance to anthracnose was carried out for the cross of‘Flame Seedless’בTangwei’. 41 lines (48.8%) amplified the 1354 bp specific band from 84 lines. After comprehensive detection using the two molecular markers, 24 lines of seedlessness and resistance to anthracnose as well were selected.3. Eight triploid hybrid progenies were obtained from 409 lines of the 12 crosses between diploid and tetraploid grapes by flow cytometry identification. The results showed that regeneration rate of hybrid seedings obtained by embryo rescue were obviously higher than that of obtained by sowing mature seeds. It could be confirmed that larger amount of hybrid seedlings were obtained from the cross form of 2x×4x than the cross form 4x×2x, while more triploid hybrid progenies were obtained from the cross form of 4x×2x than the cross form of 2x×4x.4. The investigation was conducted to evaluate the feasibilities and optimal sampling periods of 10 seedless grape cultivars. By measuring in terms of most number of developed embryos or germinated embryos per berries, the result showed that the order from ease to difficulty of embryo rescue was‘Otilia Seedless’,‘Fantasy Seedless’,‘Flame Seedless’,‘Superior Seedless’,‘Black Monukka’,‘Autumn Royal’,‘Delight’,‘Dawn Seedless’,‘Thompson Seedless’, and‘Centennial Seedless’. The optimal sampling dates of above cultivars for embryo rescue were 40 DAB, 60 DAB, 70 DAB, 40 DAB, 50 DAB, 70 DAB, 70 DAB, 60 DAB, 55 DAB, 35 DAB, 40 DAB, respectively.5. The effects of various correlative factors on embryo rescue in vitro were investigated at every culture stage on the basis of previous experiment. At the stage of ovule culture, the results indicated that MM3 and Nitsch +GA3 0.5 mg l-1+IAA 1.5 mg l-1 medium were most suitable for embryo development, and the next was ER medium.?Solid-liquid double phase medium and room temperature had positive effect on embryos development in vitro. At the stage of embryo germination, WPM + BA 0.2 mg l-1 medium, and supplemented with 2~5 mM Put had positive effect on embryos germination. It showed that embryo germination correlated with size of embryo. At the stage of plantlet regeneration, the results indicated that rates of normal plantlets was relative to zogyic embryo development degree of grape cultivars, and the emergence of albino seedlings connected with maternal genotypes. The optimal medium of converting?abnormal plantlets was MS + BA 2.0 mg l-1 + IBA 0.5 mg l-1, the plantlets with normal shape stems and leaves produced roots on MS medium supplemented with 0.2 mg l-1 IBA, therefore normal plantlets were regenerated from abnormal plantlets. At the stage of plantlet acclimatization and transplantation, the suitable seedling age was 30 days. It was beneficial to survival of embryo rescue seedlings that mixture substrate was autoclave sterilized and the solution of 1/4 B5 macroelement irrigated seedlings every week.4. The effects of exogenous application of plant growth regulators on embryo rescue of high abortion cultivars‘Centennial Seedless’,‘Thompson Seedless’and‘Flame Seedless’were discussed. The optimum concentration and period of application of plant growth regulators was determined: 100~500 mg l-1 chlormequat application in‘Centennial Seedless’14 days before bloom, 500 mg l-1 chlormequat application in‘Thompson Seedless’10 days before bloom, 30 mg l-1 benzyladenine application in‘Flame Seedless’20 days before bloom.更多还原