【作者】 李桂荣；

【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】 葡萄是世界主要果树之一。无核葡萄育种是世界葡萄研究的重要方向，无核葡萄在目前鲜食品种种植中的比例逐渐增大。栽培的无核葡萄品种多属欧洲葡萄（Vitis viniferaL.）的品种，其优点是品质优良，但突出缺点是不抗病。因此，选育优质、大粒、抗逆的无核品种已成为葡萄育种研究的重要目标之一。近年来无核葡萄胚挽救育种是提高无核葡萄育种效率的重要技术和途径。本研究以4个欧亚种无核葡萄品种汤姆逊无核（Thompson Seedless），以下简称无核白，火焰无核（Flame Seedless），赫什无核（HeshiSeedless），红宝石无核（Ruby Seedless）等为母本，与中国野生葡萄塘尾刺葡萄（V. davidiiFoex）、雪峰刺葡萄和山葡萄（V. amurensis Rupr.）双优及欧山杂种（V.vinifera L.×V.amurensis Rupr.）北醇葡萄，欧亚种无核葡萄品种无核白、火焰无核、赫什无核、红宝石无核及有核葡萄品种玫瑰香（Muscat Hamburg）、红地球（Red Globe）等作父本进行杂交，共计36个杂交组合。主要研究内容是：通过测定作为母本的无核葡萄浆果和离体胚珠中的矿质元素，活性氧和酶，内源多胺以及内源激素含量的变化，揭示无核葡萄胚胎发育的生理变化对幼胚败育的影响；开展杂交座果率及胚挽救获得杂交后代成苗率提高的技术研究，获得一批新的无核葡萄杂交后代，同时对胚挽救过程中胚萌发畸形葡萄苗产生的影响因素及转变成正常苗的条件开展研究，获得了一些葡萄新种质；对胚挽救苗移栽驯化规范化操作技术开展了研究，目的是提高移栽驯化成苗率。获得的主要结果如下：1、测定母本的无核葡萄浆果和离体胚珠中的矿质元素，活性氧和酶，内源多胺以及内源激素等的含量，发现同一品种胚珠数量不同的浆果中内源多胺和激素含量有一定的差异；同一品种胚数量不同的离体培养胚珠的内源多胺和激素含量存在一定的差异，说明内源激素及多胺对于无核葡萄胚胎发育败育有一定的影响。在坐果过程中施加外源多胺(Put50mmol·l^-1+Spd50mmol·l^-1)和外源激素(IAA30mg·l^-1+ABA30mg·l^-1)有利于提高无核葡萄浆果内胚珠的发育。在胚挽救过程中添加外源多胺(Put2mmol·l^-1+Spm2mmol·l^-1+Spd2mmol·l^-1)和外源激素(IAA1.0mg·l^-1+6-BA2.0mg·l^-1+GA32.0mg·l^-1)有利于提高无核葡萄胚挽救成苗率。2、不同亲本杂交组合座果率不同，说明亲本杂交组合是决定座果率的主要因素。其中无核葡萄×中国野生葡萄杂交组合中红宝石无核×北醇座果率较高，达到15.8%；无核葡萄×无核葡萄杂交组合中红宝石无核×赫什无核座果率较高，达到9.7%；无核葡萄×有核葡萄杂交组合中红宝石无核×玫瑰香座果率较高，达到16.8%。3、无核葡萄胚挽救过程各个培养阶段的适宜培养基是不同的。在胚珠发育阶段，使用固液双相培养基，以改良MM3为固体培养基，ER为液体培养基，作胚珠发育培养基，在常温下进行暗培养，有助于提高胚发育率；在胚萌发阶段，使用WPM+IAA1.0mg·l^-1+6-BA2.0mg·l^-1+GA32.0mg·l^-1培养基，添加Put2mmol·l^-1+Spm2mmol·l^-1+Spd2mmol·l^-1有利于提高无核葡萄的幼胚萌发率和成苗率。研究结果发现不同杂交组合胚挽救率不同，无核葡萄×中国野生葡萄杂交组合中红宝石无核×北醇胚萌发率较高，达到28.1%，成苗率较高，达到61.0%。无核葡萄×无核葡萄杂交组合中无核白×红宝无核胚发育率较高，达到37.5%，成苗率较高，达到33.3%，其次是红宝石无核×火焰无核胚发育率较高，达到17.1%，成苗率较高，达到28.6%。无核葡萄×有核葡萄杂交组合中红宝石无核×红地球胚发育率较高，达到34.5%，成苗率较高，达到33.3%。4、葡萄胚挽救过程中父母本基因型和离体接种培养条件的影响，产生畸形苗的比率不同。畸形苗转变成正常葡萄苗的培养基是WPM+6-BA2.0mg·l^-1+IAA2.0mg·l^-1，转变百分比为24.6%，对于徒长苗来说14d转接一次，转变率较高，达到33.3%，在产生的畸形苗中观察发现所有的白化苗和玻璃化苗最后都没有转变成正常苗，说明这两种畸形苗不易再转变成正常苗。5、建立了一套比较适宜操作的胚挽救苗移栽驯化程序：胚挽救苗生根壮苗的培养基是1/2×MS+0.3mg·l^-1IAA；选择10cm高的试管苗移栽到装有M1基质（蛭石：泥炭土：椰糠=1：4：1）的18cm×16cm营养钵中，外施营养液成分为1/10×MS大量元素+1×MS其他成分的培养基溶液，有利于提高胚挽救苗移栽驯化成苗率。通过炼苗移栽无核葡萄胚挽救苗，已有371个株系在大田移栽成活。综上所述，无核葡萄果实发育过程中的生理变化对幼胚发育和败育有着决定性的影响，无核葡萄胚挽救育种过程中父母本基因型的选择是胚挽救成功的首要因素；其次，离体接种培养条件是幼胚离体条件下发育成功的重要影响因素；无核葡萄胚挽救育种成苗后移栽驯化过程中的试管苗生根、植株生长势状况、移栽基质和喷施营养液等是提高无核葡萄育种成苗和效率的主要因素。更多还原

【Abstract】 Grape is one of the major fruit trees. Seedless grape breeding has become an importantaspect of grape research, with increasing proportion of seedless grapes in fresh grapes. Atpresent, cultivated seedless grapes are most from Vitis vinifera L., which own fine quality, butsusceptible to pathogens. Therefore, for seedless grapes, the selection of high quality, bigberry, and high resistance has become one of important goals of grape breeding. Moreover,the embryo rescue is a main technique to improve breeding efficient in seedless grapes.In present research,36crossings with European seedless grape cultivars （including,Thompson Seedless, Flame Seedless, Heshi Seedless, Ruby Seedless） as the female parentsand3types cultivars （wild Chinese Vitis species: Tangwei, Xuefeng, Shuangyou, Beichun.Seedless grapes: Thompson Seedless, Flame Seedless, Heshi Seedless, Ruby Seedless, andseeded grapes: Muscat Hamburg, Red Globe） as the male parents are conducted. To reveal thephysiological mechanism of embryo abortion of seedless grape, the content of mineral, activeoxygen, enzyme, endogenous polyamine and hormone was measured in pulps and in-vitroovules. To obtain a new batch of hybrid progenies of seedless grape, the improvement of therate of hybridization fruit and embryo rescue was conducted. Meanwhile, we gained somenew grape germplasm resources by studying the affecting factors of abnormally germinatedembryos in the process of embryo rescue and the conditions of transforming into normalseedlings. Moreover, to improve the rate of transplantation, the standardization operation ofembryo rescue seedlings was studied in the process of transplantation and acclimation. Theresults obtained were as follows:1. Among the same variety, the contents of endogenous polyamine and hormone weredifferent in different pulps and in different kinds of in vitro ovules, suggesting that theendogenous hormones and polyamines had a certain influence on embryo abortion of seedlessgrape cultivars. We also found that spraying Put50mmol·l^-1+Spd50mmol·l^-1and IAA30mg·l^-1+ABA30mg·l^-1was helpful to improve the development of the ovules in fruit berryduring the fruit setting. Adding Put2mmol·l^-1+Spm2mmol·l^-1+Spd2mmol·l^-1andspraying IAA1.0mg·l^-1+6-BA2.0mg·l^-1+GA32.0mg·l^-1could increase the rate of embryorescue seedlings in seedless grapes.2. The fruit setting rate was different in hybridized combinations of different parents, so hybridized combination was a key factor to control fruit setting rate. Our results showed thatfor the fruit setting rate, Ruby Seedless×Beichun was higher （15.8%） among16combinations of seedless grapes×wild Chinese Vitis species; Ruby Seedless×HeshiSeedless was higher （9.7%） among12combinations of seedless grapes×seedless grapes;Ruby Seedless×Muscat Hamburg reached higher （16.8%） among8combinations of seedlessgrapes×seeded grapes.3. The different appropriate culture mediums were conducted during the process ofembryo rescue. For ovule development, improved MM3as a solid medium and ER as a liquidmedium were helpful to improve embryo development under the dark culture at roomtemperature; for embryo germination, the medium with WPM+IAA1.0mg·l^-1+6-BA2.0mg·l^-1+GA32.0mg·l^-1adding Put2mmol·l^-1+Spm2mmol·l^-1+Spd2mmol·l^-1couldincrease embryo germination rate and plantlet development rate. In addition, our resultshowed that the rate of embryo rescue was different in hybridized combinations. Such as, theembryo germination rate was higher （28.1%） and plantlet development rate was higher（61.0%） in Ruby Seedless×Beichun among combinations of seedless grapes×wild ChineseVitis species; among combinations of seedless grapes×seedless grapes, the embryogermination rate of Thompson Seedless×Ruby seedless was higher （37.5%）, plantletdevelopment rate was33.3%, followed by embryo germination rate of Ruby Seedless×FlameSeedless （17.1%） and plantlet development rate （28.6%）; among combinations of seedlessgrapes×seeded grapes, embryo germination rate of Ruby Seedless×Red Globe was higher,34.5%and plantlet development rate was33.3%.4. The genotype of parents and culture condition of in-vitro affected the rate of abnormalseedlings induced. The medium WPM+6-BA2.0mg·l^-1+IAA2.0mg·l^-1was conducted totransform from abnormal seedlings to normal seedlings, and24.6%abnormal seedlings wastransformed into normal. For excessive growth-seedlings, the better transform rate was33.3%by inoculating every14days. We also investigated all induced albino seedlings and vitrifiedshoots, and they failed to transform into normal finally, indicating that the two kinds ofmalformed seedlings was difficult to change into normal ones.5. The factors influencing transplantation and acclimation, including the rooting oftest-tube plantlet, plant growth potential, matrix, and nutrient solution were investigated in2011and2012. We summarized a set of normalized procedures of the transplantation andacclimation of embryo-rescue seedlings. Such as, the medium1/2×MS+0.3mg·l^-1IAA asthe most suitable culture medium for the rooting and seedling; transplanting10cm highseedling into pots （18cm×16cm） with M1（peat: vermiculite: coconut shell=1:4:1） andspraying nutrient solution （1/10×MS macroelement+1×MS other culture medium）, helpful for improving the seedling formation rate of transplantation and acclimation of embryo-rescueseedlings. After seedlings adaption,371lines were transplanted and survived in the field.In summary, the physiological changes during the berry development of seedless grapehad a decisive effect on young embryo development and abortion; meanwhile, genotypes ofparents played a primary role in deciding the success of embryo rescue. Secondly, thecomponents in medium were an important factor influencing embryo development by in vitroculture. Moreover, rooting of test-tube plantlet, plant growth potential, matrix and nutrientsolution became main factors improving seedling rate during transplantation and acclimation.更多还原