无核抗病葡萄胚挽救育种与应用
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摘要
以无核的欧洲葡萄作母本,抗病性强的‘北醇’‘左优红’‘塘尾’作父本,大田杂交后在胚败育前采集幼果,无菌条件下采用离体胚挽救的方法获得杂种后代植株,研究了亲本基因型、胚萌发培养基中蔗糖浓度和GA_3浓度对无核葡萄胚挽救育种效率的影响,并对杂种幼苗进行分子标记辅助选择,以期为无核葡萄胚挽救育种效率的提高提供参考依据,并初步筛选出无核抗病葡萄新材料。结果表明:获得232个杂交株系,练苗成活株系227株。以‘无核白鸡心’‘昆香无核’为母本的杂交组合胚挽救效果较好,适宜在无核葡萄胚挽救中作母本。胚萌发培养基中最适蔗糖浓度为2.0%,胚萌发培养基中最适GA_3浓度为0.5μmol·L~(-1);对获得的杂种后代幼苗,利用2种葡萄无核标记SCF27-2000、SCC8-1018对124株胚挽救杂交子代无核基因进行分子标记检测,初步确定无核株系74株,胚挽救后代无核率为59.7%,利用葡萄抗白粉病标记ScORN3-R对65株胚挽救杂交子代进行抗白粉病基因检测,65个胚挽救杂交后代株系均未扩增出760bp抗白粉病标记特异性条带。
European seedless cultivars were taken as female parent,and some cultivars with high disease resistance were taken as male parent,such as'Beichun''Zuoyouhong'and'Tangwei'.Young berries were gathered before embryo abortion,and hybrid progenies were collected via embryo rescue in vitro under aseptic conditions.The effects of parental genotype,concentration of sucrose and GA_3 in embryo germination medium on the efficiency of breeding seedless grape by embryo rescue were studied,in addition,molecular markers were applied in screening seedlessness or disease resistance of hybrid seedlings in order to provide reference for improving embryo rescue efficiency and screen the new materials of seedless and disease resistance grape preliminarily.The results showed that 232 strains were obtained and 227 of them were domesticated and transplanted;'Centennial Seedless' and'Kunxiang Seedless' were more suitable for female parent,because their embryo rescue efficiency were better.In the medium for embryo germination,the optimal sugar concentration was 2.0% and the optimal GA_3 concentration was 0.5μmol·L~(-1).Two molecular markers for seedless grapevines,such as SCF27-2000 and SCC8-1018 were selected for detecting 124 hybrid progenies.The result showed that 74 hybrid progenies were identified and the seedless rate of hybrid progenies was 59.7%.For resistance to powdery mildew,the molecular marker ScORN3-R was used for 65 hybrid progenies,but the specific brand 760 bp was not detected.
European seedless cultivars were taken as female parent,and some cultivars with high disease resistance were taken as male parent,such as'Beichun''Zuoyouhong'and'Tangwei'.Young berries were gathered before embryo abortion,and hybrid progenies were collected via embryo rescue in vitro under aseptic conditions.The effects of parental genotype,concentration of sucrose and GA_3 in embryo germination medium on the efficiency of breeding seedless grape by embryo rescue were studied,in addition,molecular markers were applied in screening seedlessness or disease resistance of hybrid seedlings in order to provide reference for improving embryo rescue efficiency and screen the new materials of seedless and disease resistance grape preliminarily.The results showed that 232 strains were obtained and 227 of them were domesticated and transplanted;'Centennial Seedless' and'Kunxiang Seedless' were more suitable for female parent,because their embryo rescue efficiency were better.In the medium for embryo germination,the optimal sugar concentration was 2.0% and the optimal GA_3 concentration was 0.5μmol·L~(-1).Two molecular markers for seedless grapevines,such as SCF27-2000 and SCC8-1018 were selected for detecting 124 hybrid progenies.The result showed that 74 hybrid progenies were identified and the seedless rate of hybrid progenies was 59.7%.For resistance to powdery mildew,the molecular marker ScORN3-R was used for 65 hybrid progenies,but the specific brand 760 bp was not detected.
引文
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[7] AMARAL A L D,OLIVEIRA P R D D,CZERMAINSKI A B C,et al.Embryo growth stages on plant obtention from crosses between seedless grape parents[J].Revista Brasileira de Fruticultura,2001,23(3):647-651.
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[12]PONCE M T,AGERO C B,GREGORI M T,et al.Factors affecting the development of stenospermic grape(Vitis vinifera)embryos cultured in vitro[J].Acta Horticulturae,2000(528):667-672.
[13]刘巧.利用胚挽救技术培育抗寒无核葡萄新种质[D].杨凌:西北农林科技大学,2015.
[14]李桂荣.无核葡萄胚挽救育种技术的研究[D].杨凌:西北农林科技大学,2001.
[15]屈田田,张剑侠,骆强伟,等.无核葡萄抗寒抗病胚挽救育种应用研究[J].果树学报,2017(2):157-165.
[16]李志谦.葡萄底莱特×红宝石无核F-1代优系无核性状改良与标记辅助选择[D].杨凌:西北农林科技大学,2013.
[17]RAMMING D W.The use of embryo culture in fruit breeding[J].HortScience,1990,25(4):393-398.
[18]GRIBAUDO I,ZANETTI R,BOTTA R,et al.In ovulo embryo culture of stenospermocarpic grapes[J].Vitis,1993(32):9-14.
[19]PREZ A J L,DABAUZA M,CARREO J.Somatic embryo germination and plant regeneration of three gravepine cvs:Effect of IAA,GA3and embryo morphology[J].Vitis Journal of Grapevine Research,2006,45(3):141-144.
[20]SINGH N V,SINGH S K,SINGH A K.Standardization of embryo rescue technique and bio-hardening of grape hybrids(Vitis vinifera L.)using arbuscular mycorrhizal fungi(AMF)under sub-tropical conditions[J].Vitis Journal of Grapevine Research,2011,50(3):115-118.
[21]THIS P,LAHOGUEESNAULT F,BLONDON A F A,et al.Usefulness of two SCAR markers for marker-assisted selection of seedless grapevine cultivars[J].Vitis Journal of Grapevine Research,2001,40(3):147-155.
[22]MEJIA N,HINRICHSEN P.New highly assertive scar marker potentially useful to assist selection for seedlessness in table grape breeding[J].Acta Horticulturae,2003,603:559-564.
[23]AKKURT M,WELTER L,MAUL E,et al.Development of SCAR markers linked to powdery mildew(Uncinula necator)resistance in grapevine(Vitis vinifera L.and Vitis sp.)[J].Molecular Breeding,2007,19(2):103-111.
[24]张剑侠,王跃进,张艳艳,等.中国野生葡萄抗黑痘病基因RAPD标记的克隆、序列分析及辅助育种应用[J].果树学报,2009,26(4):456-460.
[25]张艳艳,张剑侠,王跃进.中国野生葡萄抗霜霉病基因RAPD标记的筛选[J].果树学报,2008,25(6):816-820.
[2]杨有龙.美国和日本的葡萄育种[J].北方果树,1992(2):1-5.
[3] EMERSHAD R L,RAMMING D W.In-ovulo embryo culture of Vitis vinifera L.C.V.‘Thompson Seedless’[J].American Journal of Botany,1984,71(6):873-877.
[4] RAMMING D W,EMERSHAD R L.In ovulo embryo culture of seeded and seedless Vitis vinifera L.[J].HortScience,1982:487.
[5]唐冬梅.无核葡萄杂交胚挽救新种质创建与技术完善[D].杨凌:西北农林科技大学,2010.
[6] GOLDY R G,AMBORN U.In vitro culturability of ovules from 10seedless grape clones[J].HortScience,1987,22(5):952.
[7] AMARAL A L D,OLIVEIRA P R D D,CZERMAINSKI A B C,et al.Embryo growth stages on plant obtention from crosses between seedless grape parents[J].Revista Brasileira de Fruticultura,2001,23(3):647-651.
[8]王飞.葡萄无核品种及其杂种胚败育机理与胚挽救技术研究[D].杨凌:西北农林科技大学,2002.
[9]潘学军.无核抗病葡萄胚挽救技术体系优化及新品系培育[D].杨凌:西北农林科技大学,2005.
[10]田莉莉.抗病无核葡萄胚挽救育种及种质创新[D].杨凌:西北农林科技大学,2007.
[11]GARCIA E,MARTINEZ A,GARCIA D L C E,et al.In vitro culture of ovules and embryos of grape for the obtention of new seedless table grape cultivars[J].Acta Horticulturae,2000,25(528):663-666.
[12]PONCE M T,AGERO C B,GREGORI M T,et al.Factors affecting the development of stenospermic grape(Vitis vinifera)embryos cultured in vitro[J].Acta Horticulturae,2000(528):667-672.
[13]刘巧.利用胚挽救技术培育抗寒无核葡萄新种质[D].杨凌:西北农林科技大学,2015.
[14]李桂荣.无核葡萄胚挽救育种技术的研究[D].杨凌:西北农林科技大学,2001.
[15]屈田田,张剑侠,骆强伟,等.无核葡萄抗寒抗病胚挽救育种应用研究[J].果树学报,2017(2):157-165.
[16]李志谦.葡萄底莱特×红宝石无核F-1代优系无核性状改良与标记辅助选择[D].杨凌:西北农林科技大学,2013.
[17]RAMMING D W.The use of embryo culture in fruit breeding[J].HortScience,1990,25(4):393-398.
[18]GRIBAUDO I,ZANETTI R,BOTTA R,et al.In ovulo embryo culture of stenospermocarpic grapes[J].Vitis,1993(32):9-14.
[19]PREZ A J L,DABAUZA M,CARREO J.Somatic embryo germination and plant regeneration of three gravepine cvs:Effect of IAA,GA3and embryo morphology[J].Vitis Journal of Grapevine Research,2006,45(3):141-144.
[20]SINGH N V,SINGH S K,SINGH A K.Standardization of embryo rescue technique and bio-hardening of grape hybrids(Vitis vinifera L.)using arbuscular mycorrhizal fungi(AMF)under sub-tropical conditions[J].Vitis Journal of Grapevine Research,2011,50(3):115-118.
[21]THIS P,LAHOGUEESNAULT F,BLONDON A F A,et al.Usefulness of two SCAR markers for marker-assisted selection of seedless grapevine cultivars[J].Vitis Journal of Grapevine Research,2001,40(3):147-155.
[22]MEJIA N,HINRICHSEN P.New highly assertive scar marker potentially useful to assist selection for seedlessness in table grape breeding[J].Acta Horticulturae,2003,603:559-564.
[23]AKKURT M,WELTER L,MAUL E,et al.Development of SCAR markers linked to powdery mildew(Uncinula necator)resistance in grapevine(Vitis vinifera L.and Vitis sp.)[J].Molecular Breeding,2007,19(2):103-111.
[24]张剑侠,王跃进,张艳艳,等.中国野生葡萄抗黑痘病基因RAPD标记的克隆、序列分析及辅助育种应用[J].果树学报,2009,26(4):456-460.
[25]张艳艳,张剑侠,王跃进.中国野生葡萄抗霜霉病基因RAPD标记的筛选[J].果树学报,2008,25(6):816-820.