葡萄特异种质的繁殖保存和利用
发布时间：2019-05-19 10:50
【摘要】：本论文围绕葡萄特异种质资源的保存与利用,对25种中国野生葡萄和6种圆叶葡萄抗病特异种质材料进行了植物组织培养和绿植扦插等繁殖和保存;同时研究了这6种圆叶葡萄植株对扇叶病毒的抗性,发现圆叶葡萄Trayshed(Muscadina rotundifolia Tray shed)对扇叶病毒抗性最强。研究了不同葡萄品种外植体材料在不同诱导培养基下的诱导率,筛选出了最佳诱导条件;建立了不同葡萄品种茎尖作为外植体诱导器官发生的再生途径,筛选出了最适合诱导葡萄分生愈伤组织的植物生长调节剂组合。另外,建立了农杆菌介导的葡萄遗传转化系统,以及利用番茄和葡萄转化系统,研究了VqDUF642基因的生物学功能,发现过量表达VqDUF642的转基因葡萄植株对白粉病和灰霉病的抗性有一定程度提高。最后,进行了葡萄染色体加倍诱导和鉴定的相关研究。具体研究结果如下:1.利用植物组织培养和绿植扦插的方法分别繁殖和保存了中国野生葡萄和圆叶葡萄特异种质材料,比较不同中国野生葡萄经过不同表面消毒处理对单芽茎段存活率的影响和不同类型培养基对单芽茎段生根率的影响,以及不同圆叶葡萄经过不同植物生长激素预处理对单芽茎段生根率的影响,得到每种葡萄材料最适合的表面消毒方法和最适合生根的激素组合。实验结果表明不同葡萄特异种质的繁殖和保存存在基因型差异性。同时,将六种圆叶葡萄分别与感染了葡萄扇叶病毒的葡萄植株嫁接,通过实时荧光定量P CR(qPCR)方法定量分析嫁接四个月后的圆叶葡萄叶片的病毒转录水平的表达,研究不同品种圆叶葡萄对扇叶病毒的抗性。从而获得了对扇叶病毒抗性最佳的圆叶葡萄Tra yshed(Muscadina rotundifolia Trayshed)。2.比较了4种鲜食葡萄 无核白(Vitis vinifera Thompson Seedless)、红地球(V.vinifera Redglobe)、玫瑰香(V.vinifera Muscat)和里扎马特(V.vinifera Rizamat)的2种不同外植体材料(花药和子房)在3种诱导培养基(PIV、MC和MSI)上胚性愈伤组织的诱导率。研究表明花药是最适合诱导胚性愈伤组织的外植体材料,PIV和M C是最适合诱导胚性愈伤组织的培养基。胚性愈伤组织的诱导存在葡萄品种差异,其中PIV培养基最适合红地球和里扎马特胚性愈伤组织的诱导,而MC培养基最适合无核白和玫瑰香胚性愈伤组织的诱导。3.建立了以霞多丽(V.vinifera Chardonnay)、无核白(V.vinifera Thompson Seedless)、红地球(V.vinifera Redglobe)、赤霞珠(V.vinifera Cabernet Sauvignon)、St.George(V.rupestris St.George)和101-14 Mgt(101-14 Millardet et de Grasset(V.r iparia x V.rupestris))的茎尖作为外植体材料诱导器官发生的再生途径。其中,无核白分生愈伤组织诱导率最高(98.4%),其次分别是霞多丽(97.6%)红地球(90.2%)赤霞珠(86.2%)St.George(85.4%)101-14 Mgt(79.6%)。BA(benzylaminopu rine)和NAA(naphthaleneacetic acid)是最适合于诱导葡萄分生愈伤组织的植物生长调节剂组合。4.获得了霞多丽、无核白、红地球、赤霞珠、St.George和101-14 Mgt的转基因植株。其中霞多丽转化效率最高,其次是无核白St.George红地球赤霞珠101-14 Mgt;本研究还通过对转化材料不同组织GFP表达的观察提高转化植株纯合率。5.克隆了中国野生毛葡萄丹凤-2(V.quinquangularis Danfeng-2)DUF642基因,命名为VqDUF642)。VqDUF642全长序列中含有1,107-bp的开放阅读框,编码368个氨基酸,与来源于欧洲葡萄的一个DUF642蛋白高度同源。亚细胞定位结果表明VqDU F642定位在细胞壁。番茄遗传转化实验表明过表达Vq DUF642加速了转基因番茄植株的发育,同时降低了转基因番茄对灰霉菌的感病性。此外,与野生型对照植株相比,过表达VqDUF642的转基因无核白植株对白粉菌和灰霉菌的抗性提高。6.分析了有丝分裂抑制剂(秋水仙素和安磺灵)的种类、浓度(250μM、625μM和1250μM;5μM、15μM和30μM)和处理时间(24小时、48小时和72小时)以及处理的组织器官对植株染色体加倍的效果,建立了有效的诱导葡萄染色体加倍的方法。分别获得了Trayshed和101-14 Mgt这两个葡萄品种的杂交后代(07107-075)的四倍体植株以及4种无核葡萄无核白(V.vinifera Thompson Seedless)、红脸无核(V.vinifera Blush Seedless)、红宝石无核(V.vinifera Ruby Seedless)、美丽无核(V.vinifera Be auty Seedless)的四倍体植株。
[Abstract]:The breeding and preservation of the disease-resistant and specific germplasm materials of 25 Chinese wild grape and 6 round-leaf grape were carried out by the preservation and utilization of the grape-specific germplasm resources, and the resistance of the six kinds of round-leaf grape plants to the leaf-leaf virus was also studied. Muscadina rotunda was found to be the most resistant to leaf-leaf virus. The induction rate of different grape variety explants in different induction culture media was studied, the best induction conditions were selected, and the regeneration route of the stem tip of different grape cultivars as explants was established. The combination of plant growth regulators that is most suitable for inducing the callus of the grape is selected. In addition, agrobacterium-mediated grape genetic transformation system was established, and the biological function of VqDUF642 gene was studied by using tomato and grape transformation system, and it was found that the resistance of transgenic grape plants expressing VqDUF642 to powdery mildew and gray mold was improved to some extent. Finally, the studies on the double induction and identification of the grape chromosomes were carried out. The specific results are as follows:1. The specific germplasm materials of Chinese wild grape and round leaf grape are respectively propagated and preserved by the method of plant tissue culture and green planting and insertion, the effects of different surface disinfection treatments on the survival rate of single-bud stem segments and the effect of different types of culture medium on the rooting rate of single-bud stem segments are compared, and the effect of different round-leaf grapes on the rooting rate of single-bud stem segments is affected by different plant growth hormone pretreatment, The most suitable surface disinfection method for each grape material and the most suitable root-taking hormone combination are obtained. The results showed that there were genotypic differences in the reproduction and preservation of different grape-specific germplasms. At the same time, six kinds of round leaf grape were respectively grafted with the grape plants infected with the grape leaf virus, and the expression of the virus transcription level of the leaves of the round-leaf grape after four months was quantitatively analyzed by the real-time fluorescence quantitative P-CR (qPCR) method, and the resistance of the different varieties of round leaf grape to the leaf-leaf virus was studied. So as to obtain the best round-leaf grape (Muscadina rotundifolia Trayed) with the best resistance to the leaf-leaf virus. The induction rate of the embryogenic callus of four different explants (anther and ovary) of four fresh grape-free white (Vtids viginifera Thompson Seedless), red earth (V. viginifera Redglobal), rose-flavor (V. viginifera Muscat) and Rizzmatte (V. viginifera Rizamat) were compared. The results show that anther is the most suitable for inducing the embryogenic callus, and PIV and M C are the most suitable medium for inducing the embryogenic callus. There was a difference in the induction of the embryogenic callus, among which the PIV medium was the best for the induction of the embryogenic callus of the red earth and the rizimmata, and the MC culture medium was the best for the induction of the embryogenic callus of the non-nuclear and the rose. The stem tip of V. viginifera Chardonnay, V. viginifera Thompson Seedless, Red Earth (V. viginifera Redglobal), Cabernet Sauvignon, St. George (V. rrupsteris St. George) and 101-14 Mgt (101-14 Millard et de Grasset (V. r iparia x V. rrupis) was established as the regeneration pathway for inducing the formation of an organ. Among them, the callus induction rate was the highest (98.4%), followed by Chardonnay (97.6%), red earth (90.2%), Cabernet (86.2%), St. George (85.4%),101-14Mgt (79.6%). Transgenic plants of Chardonnay, non-nuclear, red earth, Cabernet Sauvignon, St. George and 101-14Mgt were obtained. In which, the transformation efficiency of the nepheline is the highest, and the second is the non-nuclear-free St. George red Earth Cabernet Sauvignon 101-14Mgt; the present study also improves the purity of the transformed plant by observing the expression of the GFP in different tissues of the transformation material. The DUF642 gene, named VqDUF642, is cloned. The full-length sequence of VqDUF642 contains an open reading frame of 1,107-bp, encoding 368 amino acids, which is highly homologous to a DUF642 protein derived from the European grape. The subcellular localization indicated that VqDU F642 was located in the cell wall. The results of the genetic transformation of tomato showed that the expression of Vq DUF642 accelerated the development of transgenic tomato plants and decreased the sensitivity of the transgenic tomato to the gray mold. In addition, compared with the wild-type control plants, the resistance of the transgenic non-nuclear-free plants overexpressing the VqDUF642 to the powdery mildew and the gray mold is improved. The effects of the type, concentration (250. mu.M,625. mu.M and 1250. mu.M,5. mu.M,15. mu.M and 30. mu.M) and the treatment time (24 hours,48 hours and 72 hours) of the mitotic inhibitor (colchicine and ansulfuron) and the treatment time (24 hours,48 hours and 72 hours) and the treatment of the plant chromosomes were analyzed, And a method for effectively inducing the chromosome doubling of the grape is established. The tetraploid plants of the hybrid progenies (07107-075) of Trayshed and 101-14Mgt, as well as the four seedless grape-free white (V. vinifera Thompson Seedless), the red-face seedless (V. viginifera Blush Seedless), the ruby-free (V. viginifera Ruby Seedless) and the beautiful seedless (V. viginifera Be auy Seedless) are respectively obtained.
【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S663.1
本文编号：2480665
[Abstract]:The breeding and preservation of the disease-resistant and specific germplasm materials of 25 Chinese wild grape and 6 round-leaf grape were carried out by the preservation and utilization of the grape-specific germplasm resources, and the resistance of the six kinds of round-leaf grape plants to the leaf-leaf virus was also studied. Muscadina rotunda was found to be the most resistant to leaf-leaf virus. The induction rate of different grape variety explants in different induction culture media was studied, the best induction conditions were selected, and the regeneration route of the stem tip of different grape cultivars as explants was established. The combination of plant growth regulators that is most suitable for inducing the callus of the grape is selected. In addition, agrobacterium-mediated grape genetic transformation system was established, and the biological function of VqDUF642 gene was studied by using tomato and grape transformation system, and it was found that the resistance of transgenic grape plants expressing VqDUF642 to powdery mildew and gray mold was improved to some extent. Finally, the studies on the double induction and identification of the grape chromosomes were carried out. The specific results are as follows:1. The specific germplasm materials of Chinese wild grape and round leaf grape are respectively propagated and preserved by the method of plant tissue culture and green planting and insertion, the effects of different surface disinfection treatments on the survival rate of single-bud stem segments and the effect of different types of culture medium on the rooting rate of single-bud stem segments are compared, and the effect of different round-leaf grapes on the rooting rate of single-bud stem segments is affected by different plant growth hormone pretreatment, The most suitable surface disinfection method for each grape material and the most suitable root-taking hormone combination are obtained. The results showed that there were genotypic differences in the reproduction and preservation of different grape-specific germplasms. At the same time, six kinds of round leaf grape were respectively grafted with the grape plants infected with the grape leaf virus, and the expression of the virus transcription level of the leaves of the round-leaf grape after four months was quantitatively analyzed by the real-time fluorescence quantitative P-CR (qPCR) method, and the resistance of the different varieties of round leaf grape to the leaf-leaf virus was studied. So as to obtain the best round-leaf grape (Muscadina rotundifolia Trayed) with the best resistance to the leaf-leaf virus. The induction rate of the embryogenic callus of four different explants (anther and ovary) of four fresh grape-free white (Vtids viginifera Thompson Seedless), red earth (V. viginifera Redglobal), rose-flavor (V. viginifera Muscat) and Rizzmatte (V. viginifera Rizamat) were compared. The results show that anther is the most suitable for inducing the embryogenic callus, and PIV and M C are the most suitable medium for inducing the embryogenic callus. There was a difference in the induction of the embryogenic callus, among which the PIV medium was the best for the induction of the embryogenic callus of the red earth and the rizimmata, and the MC culture medium was the best for the induction of the embryogenic callus of the non-nuclear and the rose. The stem tip of V. viginifera Chardonnay, V. viginifera Thompson Seedless, Red Earth (V. viginifera Redglobal), Cabernet Sauvignon, St. George (V. rrupsteris St. George) and 101-14 Mgt (101-14 Millard et de Grasset (V. r iparia x V. rrupis) was established as the regeneration pathway for inducing the formation of an organ. Among them, the callus induction rate was the highest (98.4%), followed by Chardonnay (97.6%), red earth (90.2%), Cabernet (86.2%), St. George (85.4%),101-14Mgt (79.6%). Transgenic plants of Chardonnay, non-nuclear, red earth, Cabernet Sauvignon, St. George and 101-14Mgt were obtained. In which, the transformation efficiency of the nepheline is the highest, and the second is the non-nuclear-free St. George red Earth Cabernet Sauvignon 101-14Mgt; the present study also improves the purity of the transformed plant by observing the expression of the GFP in different tissues of the transformation material. The DUF642 gene, named VqDUF642, is cloned. The full-length sequence of VqDUF642 contains an open reading frame of 1,107-bp, encoding 368 amino acids, which is highly homologous to a DUF642 protein derived from the European grape. The subcellular localization indicated that VqDU F642 was located in the cell wall. The results of the genetic transformation of tomato showed that the expression of Vq DUF642 accelerated the development of transgenic tomato plants and decreased the sensitivity of the transgenic tomato to the gray mold. In addition, compared with the wild-type control plants, the resistance of the transgenic non-nuclear-free plants overexpressing the VqDUF642 to the powdery mildew and the gray mold is improved. The effects of the type, concentration (250. mu.M,625. mu.M and 1250. mu.M,5. mu.M,15. mu.M and 30. mu.M) and the treatment time (24 hours,48 hours and 72 hours) of the mitotic inhibitor (colchicine and ansulfuron) and the treatment time (24 hours,48 hours and 72 hours) and the treatment of the plant chromosomes were analyzed, And a method for effectively inducing the chromosome doubling of the grape is established. The tetraploid plants of the hybrid progenies (07107-075) of Trayshed and 101-14Mgt, as well as the four seedless grape-free white (V. vinifera Thompson Seedless), the red-face seedless (V. viginifera Blush Seedless), the ruby-free (V. viginifera Ruby Seedless) and the beautiful seedless (V. viginifera Be auy Seedless) are respectively obtained.
【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S663.1
【相似文献】
相关期刊论文 前7条
1 黄羽;彭宏祥;林玲;张瑛;卢江;;四个鲜食加工兼用型圆叶葡萄品种在广西引种栽培初报[J];广西农学报;2010年03期
2 黄羽;彭宏祥;林玲;张瑛;卢江;;美国圆叶葡萄引种表现及栽培技术要点[J];福建果树;2010年02期
3 程建徽;魏灵珠;梅军霞;李琳;吴江;;美国圆叶葡萄在浙江省的引种表现研究[J];现代农业科技;2012年23期
4 Radvanyi,T.;孔庆山;;八个圆叶葡萄品种的葡萄汁和葡萄酒的成分与质量[J];国外农学(果树);1982年02期
5 房玉林;刘东;宋士任;;圆叶葡萄‘Alachua’叶柄再生体系的建立[J];西北植物学报;2007年09期
6 李红玉;刘同才;;影响扦插与压条成活的因素[J];河北果树;2008年06期
7 ;[J];;年期
相关博士学位论文 前1条
1 谢晓青;葡萄特异种质的繁殖保存和利用[D];西北农林科技大学;2016年
相关硕士学位论文 前1条
1 宋士任;圆叶葡萄（Vitis rotundifolia）引种初步研究[D];西北农林科技大学;2005年
，本文编号：2480665
本文链接：https://www.wllwen.com/shoufeilunwen/nykjbs/2480665.html
