葡萄特異種質的繁殖保存和利用
發(fā)布時間：2019-05-19 10:50
【摘要】：本論文圍繞葡萄特異種質資源的保存與利用,對25種中國野生葡萄和6種圓葉葡萄抗病特異種質材料進行了植物組織培養(yǎng)和綠植扦插等繁殖和保存;同時研究了這6種圓葉葡萄植株對扇葉病毒的抗性,發(fā)現(xiàn)圓葉葡萄Trayshed(Muscadina rotundifolia Tray shed)對扇葉病毒抗性最強。研究了不同葡萄品種外植體材料在不同誘導培養(yǎng)基下的誘導率,篩選出了最佳誘導條件;建立了不同葡萄品種莖尖作為外植體誘導器官發(fā)生的再生途徑,篩選出了最適合誘導葡萄分生愈傷組織的植物生長調節(jié)劑組合。另外,建立了農桿菌介導的葡萄遺傳轉化系統(tǒng),以及利用番茄和葡萄轉化系統(tǒng),研究了VqDUF642基因的生物學功能,發(fā)現(xiàn)過量表達VqDUF642的轉基因葡萄植株對白粉病和灰霉病的抗性有一定程度提高。最后,進行了葡萄染色體加倍誘導和鑒定的相關研究。具體研究結果如下:1.利用植物組織培養(yǎng)和綠植扦插的方法分別繁殖和保存了中國野生葡萄和圓葉葡萄特異種質材料,比較不同中國野生葡萄經過不同表面消毒處理對單芽莖段存活率的影響和不同類型培養(yǎng)基對單芽莖段生根率的影響,以及不同圓葉葡萄經過不同植物生長激素預處理對單芽莖段生根率的影響,得到每種葡萄材料最適合的表面消毒方法和最適合生根的激素組合。實驗結果表明不同葡萄特異種質的繁殖和保存存在基因型差異性。同時,將六種圓葉葡萄分別與感染了葡萄扇葉病毒的葡萄植株嫁接,通過實時熒光定量P CR(qPCR)方法定量分析嫁接四個月后的圓葉葡萄葉片的病毒轉錄水平的表達,研究不同品種圓葉葡萄對扇葉病毒的抗性。從而獲得了對扇葉病毒抗性最佳的圓葉葡萄Tra yshed(Muscadina rotundifolia Trayshed)。2.比較了4種鮮食葡萄 無核白(Vitis vinifera Thompson Seedless)、紅地球(V.vinifera Redglobe)、玫瑰香(V.vinifera Muscat)和里扎馬特(V.vinifera Rizamat)的2種不同外植體材料(花藥和子房)在3種誘導培養(yǎng)基(PIV、MC和MSI)上胚性愈傷組織的誘導率。研究表明花藥是最適合誘導胚性愈傷組織的外植體材料,PIV和M C是最適合誘導胚性愈傷組織的培養(yǎng)基。胚性愈傷組織的誘導存在葡萄品種差異,其中PIV培養(yǎng)基最適合紅地球和里扎馬特胚性愈傷組織的誘導,而MC培養(yǎng)基最適合無核白和玫瑰香胚性愈傷組織的誘導。3.建立了以霞多麗(V.vinifera Chardonnay)、無核白(V.vinifera Thompson Seedless)、紅地球(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))的莖尖作為外植體材料誘導器官發(fā)生的再生途徑。其中,無核白分生愈傷組織誘導率最高(98.4%),其次分別是霞多麗(97.6%)紅地球(90.2%)赤霞珠(86.2%)St.George(85.4%)101-14 Mgt(79.6%)。BA(benzylaminopu rine)和NAA(naphthaleneacetic acid)是最適合于誘導葡萄分生愈傷組織的植物生長調節(jié)劑組合。4.獲得了霞多麗、無核白、紅地球、赤霞珠、St.George和101-14 Mgt的轉基因植株。其中霞多麗轉化效率最高,其次是無核白St.George紅地球赤霞珠101-14 Mgt;本研究還通過對轉化材料不同組織GFP表達的觀察提高轉化植株純合率。5.克隆了中國野生毛葡萄丹鳳-2(V.quinquangularis Danfeng-2)DUF642基因,命名為VqDUF642)。VqDUF642全長序列中含有1,107-bp的開放閱讀框,編碼368個氨基酸,與來源于歐洲葡萄的一個DUF642蛋白高度同源。亞細胞定位結果表明VqDU F642定位在細胞壁。番茄遺傳轉化實驗表明過表達Vq DUF642加速了轉基因番茄植株的發(fā)育,同時降低了轉基因番茄對灰霉菌的感病性。此外,與野生型對照植株相比,過表達VqDUF642的轉基因無核白植株對白粉菌和灰霉菌的抗性提高。6.分析了有絲分裂抑制劑(秋水仙素和安磺靈)的種類、濃度(250μM、625μM和1250μM;5μM、15μM和30μM)和處理時間(24小時、48小時和72小時)以及處理的組織器官對植株染色體加倍的效果,建立了有效的誘導葡萄染色體加倍的方法。分別獲得了Trayshed和101-14 Mgt這兩個葡萄品種的雜交后代(07107-075)的四倍體植株以及4種無核葡萄無核白(V.vinifera Thompson Seedless)、紅臉無核(V.vinifera Blush Seedless)、紅寶石無核(V.vinifera Ruby Seedless)、美麗無核(V.vinifera Be auty Seedless)的四倍體植株。
[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.
【學位授予單位】：西北農林科技大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S663.1
本文編號：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.
【學位授予單位】：西北農林科技大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S663.1
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