【作者】 王飞；

【导师】 李嘉瑞； 王跃进；

【作者基本信息】 西北农林科技大学 ， 果树学， 2002， 博士

【摘要】 本文通过葡萄无核品种×无核品种（种内杂交），葡萄无核品种×葡萄有核品种（种间杂交），共13个组合，一个自然授粉、一个自交。种间杂交的组合是红光无核×黑龙江实生。受莫无核×左山1号，长穗无核白×北醇，无核白×蘡奠，红宝石×红地球，无核白×左山1^#，红宝石×北醇，大粒红×河岸，郑州无核×山葡萄，京早晶×黑龙江实生，京早晶×蘡奠；种内杂交的是大粒红×无核白，红宝石×爱莫；自然授粉组合的为黎明无核；自交组合的为无核白。对影响无核葡萄品种胚珠培养的时间，基本培养基，胚发育培养基，萌发培养基，成苗培养基，胚挽救试管曲的茎段培养，移栽炼苗等进行了研究；并对黎明无核、北醇，杂交种葡萄无核品种×葡萄有核品种，红光无核×黑龙江实生的胚发育进行连续解剖学和形态学观察；测定了葡萄无核品种，葡萄有核品种、葡萄无核品种×有核品种以及杂交种胚发育过程中POD、CAT、SOD酶活性，可溶性淀粉、可溶性糖、可溶性蛋白质在胚发育和败育过程的变化。 一、主要取得以下研究结果 1．通过胚挽救技术，以葡萄无核品种为母本的13个组合，共获得苗子87株，其中种内杂交的大粒红无核×无核白17棵。种间杂交的无核白×蘡奠6棵，红光无核×黑龙江实生12棵，长穗无核白×北醇12棵，爱莫无核×左山1号10棵，另外还有18棵黎明自然授粉，13棵自交的无核白。 2．筛选出最适宜胚挽救的基本培养基和各杂交组合最适宜胚发育、萌发，成苗培养基。 以自然授粉40d的黎明无核，SP₁₃₇无核，离体胚珠分别接种在B₅、MS，Nitsch三种基本培养基中培养，结果表明，以Nitsch为基本培养基效果最好，进行胚珠培养时，在Nistch发育培养基中添加6％的蔗糖、1％的PVP和500mg·L^-1的酪蛋白，待胚发育后接到3％的蔗糖，300mg·L，0.1％PVP的胚萌发培养基上培养，幼胚萌发10—20天后转接到2％蔗糖的1/2MS成苗培养基中，使肝发育、萌发、成苗效果较佳。 以Nistch培养基为基础添加(IBA 2.5mg·L^-1+GA₃ 0.5mg·L^-1+6-BA 0.5mg·L^-1+2T0.1mg·L^-1)构成胚发育培养基（A₃培养基），在此培养基上表现较好的组合为：红光无核×黑龙江实生，无核白×左山1号，红宝石×北醇，大粒红无核×河岸葡萄，京早晶×黑龙江实生，京早晶×蘡奠（安林2号）。在发育培养基（A₂培养基）(IBA 2.Omg·L^-1+GA₃ 0.5mg·L^-1+6-BA 0.5mg·L^-1+2T0.1mg·L^-1)表现较好的为红宝石×红地球，长穗无核白×北醇，大粒红无核×无核白、红宝石×爱莫无核、无核白自交、黎明自然授粉、爱莫无核×山葡萄、郑州无核×黑龙江实生。 以Nistch基本培养基为基础添加(IBA 1.5mg·L^-1+6-BA1.0mg·L^-1+GA₃ 0.2mg·L¹)构成胚萌发培养基，在此培养基（B₂）表现较好的组合为长穗无核白×北醇，无核白×蘡奠（泰山1号）、大粒红×无核白，黎明自然授粉、受莫×左山1号，在萌发培养基（B₃）（IBA 2.0mg·L¹+6-BA1.0mg·L¹+GA₃ 0.2mg·L¹）表现较好的为红光无核×黑龙江实生，无核白×无核白。但红宝石×北醇，红宝石×爱莫无核，郑州无核×黑龙江实生，京甲晶×黑龙江实生，京早晶×蘡奠的胚萌发培养基需探讨。 以1/2MS基本培养基添加（IBA0.15rmg·L¹+6-BA 0.02mg·L¹）构成苗培养基（C₂），在此培养基上表现较好的有红光无核×黑龙江实生，长穗无核白×北醇，大粒红无核×无核白，无核白X无核白，黎明自然授粉、无核白X（塑奠），爱莫无核X左山1号。 以1／ZMS为基础培养基添加（0．08mg·L IBA）构成的试管苗茎段培养基（D），在D；培养基上表现较优的为无核白X泰山 1号，医穗无核白XJL醇，爱莫X左LIJI号。在（l／ZMS＋0．cling1IBA）的 D。培养基上表现较优的为红光无核 X黑龙江实生，黎明囱然授粉、无核白 X无核白。在（l／ZMS＋0．二Zing·L）的 D；；培养基上表现秋灯的为人粒红无核x无核白。 3．确定了各杂交组合的最佳取材培养时间 无核葡萄不同品种及其杂交组合胚挽救的最佳取材时期不同，大致介于授粉后30—45d之间，具体组合为，红光无核X黑龙江实生、黎明自然授粉、爱莫无核X左山1号，红宝石X北醇均是授粉后的大取胚珠培养效果好丁叨d：长穗无核白x北醇为授粉后30d，无核白X勇奠，人粒红无核X无核白，无核白X无核白，为授粉后35d。其余组合为郑州无核丫黑龙江实生，京早晶X黑龙江实生，京早晶X＠奠，人粒红X河岸，红宝石X红地球，无核白X左山二号，红宝石X受臭的取样时间有待进一步6)f究。 4．探索无核葡萄胚发育和胚败育的细胞学机理 以有核品种北醇，自然授粉黎明无核，杂种红光无核X黑龙江实生为材料，通过人蜡切片观察了授粉后0——55大胚珠的发育，发现葡萄无核品种、有核品种、杂种胚的发育都经历合于胚～二分体一多细胞时期一球形胚一心形胚一成熟胚。黎明无核自然授粉以及红光无核X黑龙江实生的杂种胚发育到心形胚后期的少。通过细胞学的观察，发现葡萄无核品种及其杂种胚败育的机理为①合子胚发育不良，引起胚退化。②珠心珠被发育异常，引起胚发育缓慢、异常，导致胚退化。③胚乳提前解体?更多还原

【Abstract】 2 pollinations between seedless grapes( Dalihong Seedless X Sultanina, Ruby Seedless XOlmo Seedless), 11 pollinations between seedless grapes and Chinese wild grapes (Flame SeedlessXHeilongjiang Seedling,Olmo SeedlessXZhuoshan-l,Changsui Seedless X Beichun, Sultanina X Yingyu, Ruby Seedless X Red Globe, Sultanina X Zhuoshan-1, Ruby SeedlessX Beichun, Dalihong Seedless XHe’an, Zhenzou Seedless X Heilongjiang Seedling, Jingzaojin Seedless X Heilongjiang Seedling, Jingzaojin Seedless X Yingyu), open-pollinated Dawn Seedless and self-pollinated Sultanina were made to investigate effect of harvest time, basic medium for embryo rescue, medium for embryo culture, medium for embryo development, medium for embryo germination, medium for embryo rescue plant development, conditions of young plant tissue culture for propagation and transplanting. Anatomy and morphology of embryos were investigated in succession with materials of open-pollinated Dawn Seedless, open-pollinated Beichun(seeded hybrid) and grapes of Flame Seedless pollinated with Heilongjiang Seedling. Enzyme activities of POD, CAT and SOD, soluble starch(SS), soluble protein(SP) and soluble sugar(S) in embryos were determined during the embryo development of seedless grapes, seeded grapes.The main results were as follows.1.By embryo rescue, 87 plants were obtained from 13 crosses, including 17 plants from hybrid Dalihong Seedless X Sultanina, 6 plants from hybrid Sultanina X Yingyu, 12 plants from hybrid Flame Seedless X Heilongjiang Seedling, 11 plants from hybrid Changsui Seedless X Beichun, 10 plants from hybrid Olmo Seedless X Zhuoshan-1, 18 plants from open-pollinated Dawn Seedless and 13 plants from self-pollinated Sultanina.2.The suitable basic media were selected for embryo development, embryo germination and embryo rescue plant development. 40d after pollination, ovules of open-pollinated Dawn Seedless and open-pollinated SP137 Seedless were cultured in vitro on basic mediums of 65, MS and Nitsch and it showed that the best medium for embryo rescue was Nitsch Medium.It was found that basic medium with the addition of 6% sucrose, 0.1% PVP and 500mg·L-1 casein hydrody was best for embryo development; 3% sucrose, 0.1% PVP and 300mg·L-1 casein hydrody was best for embryo germination. 10-20 days after embryo germination, young plants were transferred to grow on 1/2MS with 2% sucrose.inFor embryo development, basic medium with the addition of 2.5mg -I/-1BA+O-Smg -I/-GAs+O-Smg -1/-0.1mg -L-1 6-BA +ZT was suitable for 6 crosses including Flame Seedless X Heilongjiang Seedling, SultaninaX Zhuoshan-1, Ruby Seedless X Beichun, Dalihong Seedless X He-an, Jingzaojin Seedless X Heilongjiang Seedling, Jingzaojin Seedless X Yingyu; 2.0mg·L--lBA+0.5mg·L~-GA3 +0.5mg·L-!6-BA + 0.1 mg·L-1 ZTwas suitable for 7 crosses including Ruby Seedless X Red Globe, Changsui Seedless X Beichun, Dalihong Seedless XSultanina, Olmo Seedless X Zhuoshan-1, Ruby Seedless X Olmo Seedless, Zhenzou Seedless X Heilongjiang Seedling, open-pollinated Dawn Seedless and self-pollinated Sultanina;For embryo germination, basic medium with the addition of l.Smg·L/-1BA+1.0mg·L--6-BA +0.2mg·L/-GA3 was suitable for 6 crosses including Changsui Seedless X Beichun, Sultanina X Taishan-1, Dalihong Seedless X Sultanina, open-pollinated Dawn Seedless, Olmo Seedless X Zhuoshan-1, Zhenzhou Seedless X Heilongjinag Seedling; 1.0mg·I/-1BA +1.0 mg·L-16 BA +0.2 mg·L-1GA for Flame Seedless X Helongjiang Seedling, self-pollinated Sultanina, Ruby Seedless X Beichun, Ruby Seedless X Olmo Seedless, Jingzaojin Seedless X Heilongjiang Seedling, Jingzaojin Seedless X Yingyu.For establishment of embryo rescue plants, basic medium with the addition of O.lmg ·L-1IBA+0.02mg ·L--6-BA was suitable for Flame SeedlessX Helongjiang Seedling, Changsui Seedless X Beichun, Dalihong Seedless X Sultanina, self-pollinated Sultanina, open-pollinated Dawn Seedless, SultaninaX Yingyu, Olmo Seedless X Zhuoshan-1.For tissue culture of embryo rescue plants, 1/2MS更多还原