无核葡萄胚挽救育种与杂种后代分子标记辅助选择
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摘要
【目的】对无核葡萄胚挽救影响因素与‘美丽无核’幼胚和胚乳的发育及败育过程进行研究,为提高胚挽救效率提供理论依据;通过大田杂交与胚挽救育种技术,从中选出无核杂种优株,为选育无核新品种提供材料。【方法】以8个杂交组合的杂种后代为材料,研究亲本基因型、取样时期和胚发育培养基相态对成苗率的影响,同时通过分子标记辅助选择对杂种后代进行无核性状检测;采用石蜡切片法对‘美丽无核’的幼胚和胚乳的发育与败育进程进行细胞学观察。【结果】利用胚挽救技术从8个杂交组合中获得杂种单株468株。其中‘昆香无核’ב新郁’杂交组合成苗率较高,为9.48%;‘火焰无核’ב昆香无核’、‘昆香无核’ב红宝石无核’、‘红宝石无核’ב克瑞森无核’杂交组合分别在授粉后41、47和55 d取样,进行胚挽救的成苗率最高;‘火焰无核’ב昆香无核’和‘红宝石无核’ב克瑞森无核’在固体培养基上成苗率较高;‘昆香无核’ב北醇’在固体或固液双相培养基上的成苗率无显著差异;利用无核基因探针GLSP1-569、SCAR标记SCF27-2000对4个杂交组合的326个杂交后代进行筛选,初步确定210个株系携带无核基因;‘美丽无核’幼胚发育至球形胚时期开始败育,时间为花后38 d。【结论】‘昆香无核’和‘红宝石无核’适宜作无核葡萄胚挽救育种的母本材料;以‘火焰无核’‘昆香无核’和‘红宝石无核’为母本的杂交组合的最佳取样时期分别为授粉后41、47和55 d;不同母本的杂交组合对胚发育培养基的相态要求有差异;‘美丽无核’作母本材料时适宜取样时期为花后34～36 d。
【Objective】Embryo rescue is one of the efficient breeding methods for seedless grapevine.However,there are many factors limiting a high rate of plantlet generation through embryo rescue like cultivars used as parents,time of sampling of hybrid fruit,medium for culturing,culture methods,addition of plant growth regulators,etc.The objective of our studies were to improve breeding efficiency of seedless grapevine through embryo rescue and to provide the theoretical basis for more accurate sampling of young embryos.【Methods】8 crosses were made among 7 stenospermic cultivars and 2 seeded cultivars.The hybrid ovules were removed from fruits and cultured in the medium for further embryo development.After 10 weeks in dark condition,embryos were picked from ovules and moved to the medium for development and germination.The number of plantlets were calculated after 1 month.Proliferation of plantlets was conducted in the subcultures to guarantee that every strain could be kept.The prime seedlings were trasplanted to the pots and the survived plantlets were then planted in the vineyard after seedling adaptation.GLSP1-569 and SCF27-2000 were utilized to detect seedless gene in filialgenerations.Cytological observation of embryo development and abortion of'Beauty Seedless' was made using paraffin section and Iron-vitrol hematoxylin hematoxylin staining.Sampling started after full-bloom stage which was regard as 0 d.Under the natural pollination condition,fruits in different growth periods of the same cultivar were picked at a sampling frequency of 2 days.At early stages,the whole fruit was collected.When the ovules could be separated from fruit,they were better choice.Materials were transfered into Carnoy's Fluid(glacial acetic acid:anhydrous ethanol =1:3) to fix for 24 h,and then washed with 70% alcohol.After dehydration in different levels of ethanol,clarification in xylol,saturation in paraffin and embedding,materials were kept in paraffin block and then cut at the thickness of 8 to 10 μ m with paraffin slicing machine.After staining the internal construction of the embryos were observed with light microscope.【Results】7 842 hybrid ovules and 468 hybrid strains wrere obtained from 8 crosses through embryo rescue and the average rate of plantlet generation was 5.97%.Crosses with different females had various rate of plantlet generation.The highest plantlet percentage(9.48%) was in the cross between'Kunxiangwuhe'and'Xinyu',while the lowest(0.0%) was in the cross between'Centennial Seedless' and'Crimson Seedless'.There was a significantly high seedling generation rate of'Kunxiangwuhe'×'Xinyu'than the rate of'Sultanina Rose'×'Xinyu'.And'Ruby Seedless' ×'Crimson Seedless' is significantly high than'Centennial Seedless' ×'Crimson Seedless' in seedling generation rate.When'Kunxiangwuhe'was used as female,seedling generation rates varied with crosseswith different male parents('Beichun':7.35%,'Ruby Seedless' :8.37%,'Xinyu':9.48%).The maximum of embryo germination rate and plantlet rate were found:41 DAP(days after pollution) in the cross'Flame Seedless' ×'Kunxiangwuhe'(66.67% and 7.35%),47 DAP in th ecross'Kunxiangwuhe'×'Ruby Seedless'(86.11% and 11.89%) and 55 DAP in th ecross'Ruby Seedless' ×'Crimson Seedless'(95.65% and 8.21%).Embryos generated from the combination'Flame Seedless' ×'Kunxiangwuhe'and'Ruby Seedless' ×'Crimson Seedless' produced more seedlings on solid medium,while no significant difference was found between the seedling rates of embryos generated from the combination'Kunxiangwuhe'×'Beichun'regardless of whether they were cultured on solid medium or on solid-liquid medium.GLSP1-569 could be applied to detect seedless gene of hybrids generated from the crosses with'Flame Seedless' as female parent.SCF27-2000 should be used to test the hybrids in the crosses with'Kunxiangwuhe''Rudy Seedless' 'Crimson Seedless' and'Sultanina Rose'as females parents.The process of embryo development of'Beauty Seedless' began at the stages of dormant period,over dyad,tetrad and octant period,and stopped at globular embryo stage.So the embryo of'Beauty Seedless' was aborted on 38 days after flowering.【Conclusion】Female genotype had a more significant effect on the rate of embryos formation and rate of plantlet generation in embryo rescue although male genotype also affected the result to some extent.'Kunxiangwuhe'and'Ruby Seedless' were superior for being used as female parent in seedless grape breeding.The sampling times for highest embryo germination rate and plantlet rate varied with female parents.The media also varied with female parent.When the'Beauty Seedless' was collected as the female parent for the hybridization,the optimum sample time was 34-36 days after flowering.
【Objective】Embryo rescue is one of the efficient breeding methods for seedless grapevine.However,there are many factors limiting a high rate of plantlet generation through embryo rescue like cultivars used as parents,time of sampling of hybrid fruit,medium for culturing,culture methods,addition of plant growth regulators,etc.The objective of our studies were to improve breeding efficiency of seedless grapevine through embryo rescue and to provide the theoretical basis for more accurate sampling of young embryos.【Methods】8 crosses were made among 7 stenospermic cultivars and 2 seeded cultivars.The hybrid ovules were removed from fruits and cultured in the medium for further embryo development.After 10 weeks in dark condition,embryos were picked from ovules and moved to the medium for development and germination.The number of plantlets were calculated after 1 month.Proliferation of plantlets was conducted in the subcultures to guarantee that every strain could be kept.The prime seedlings were trasplanted to the pots and the survived plantlets were then planted in the vineyard after seedling adaptation.GLSP1-569 and SCF27-2000 were utilized to detect seedless gene in filialgenerations.Cytological observation of embryo development and abortion of'Beauty Seedless' was made using paraffin section and Iron-vitrol hematoxylin hematoxylin staining.Sampling started after full-bloom stage which was regard as 0 d.Under the natural pollination condition,fruits in different growth periods of the same cultivar were picked at a sampling frequency of 2 days.At early stages,the whole fruit was collected.When the ovules could be separated from fruit,they were better choice.Materials were transfered into Carnoy's Fluid(glacial acetic acid:anhydrous ethanol =1:3) to fix for 24 h,and then washed with 70% alcohol.After dehydration in different levels of ethanol,clarification in xylol,saturation in paraffin and embedding,materials were kept in paraffin block and then cut at the thickness of 8 to 10 μ m with paraffin slicing machine.After staining the internal construction of the embryos were observed with light microscope.【Results】7 842 hybrid ovules and 468 hybrid strains wrere obtained from 8 crosses through embryo rescue and the average rate of plantlet generation was 5.97%.Crosses with different females had various rate of plantlet generation.The highest plantlet percentage(9.48%) was in the cross between'Kunxiangwuhe'and'Xinyu',while the lowest(0.0%) was in the cross between'Centennial Seedless' and'Crimson Seedless'.There was a significantly high seedling generation rate of'Kunxiangwuhe'×'Xinyu'than the rate of'Sultanina Rose'×'Xinyu'.And'Ruby Seedless' ×'Crimson Seedless' is significantly high than'Centennial Seedless' ×'Crimson Seedless' in seedling generation rate.When'Kunxiangwuhe'was used as female,seedling generation rates varied with crosseswith different male parents('Beichun':7.35%,'Ruby Seedless' :8.37%,'Xinyu':9.48%).The maximum of embryo germination rate and plantlet rate were found:41 DAP(days after pollution) in the cross'Flame Seedless' ×'Kunxiangwuhe'(66.67% and 7.35%),47 DAP in th ecross'Kunxiangwuhe'×'Ruby Seedless'(86.11% and 11.89%) and 55 DAP in th ecross'Ruby Seedless' ×'Crimson Seedless'(95.65% and 8.21%).Embryos generated from the combination'Flame Seedless' ×'Kunxiangwuhe'and'Ruby Seedless' ×'Crimson Seedless' produced more seedlings on solid medium,while no significant difference was found between the seedling rates of embryos generated from the combination'Kunxiangwuhe'×'Beichun'regardless of whether they were cultured on solid medium or on solid-liquid medium.GLSP1-569 could be applied to detect seedless gene of hybrids generated from the crosses with'Flame Seedless' as female parent.SCF27-2000 should be used to test the hybrids in the crosses with'Kunxiangwuhe''Rudy Seedless' 'Crimson Seedless' and'Sultanina Rose'as females parents.The process of embryo development of'Beauty Seedless' began at the stages of dormant period,over dyad,tetrad and octant period,and stopped at globular embryo stage.So the embryo of'Beauty Seedless' was aborted on 38 days after flowering.【Conclusion】Female genotype had a more significant effect on the rate of embryos formation and rate of plantlet generation in embryo rescue although male genotype also affected the result to some extent.'Kunxiangwuhe'and'Ruby Seedless' were superior for being used as female parent in seedless grape breeding.The sampling times for highest embryo germination rate and plantlet rate varied with female parents.The media also varied with female parent.When the'Beauty Seedless' was collected as the female parent for the hybridization,the optimum sample time was 34-36 days after flowering.
引文
[1]刘崇怀.无核葡萄品种的无核性来源分析[J].植物遗传资源学报,2003,4(1):58-62.LIU Chonghuai.Analysis on seedless sources of seedless grape breeding[J].Journal of Plant Genetic Rescouses,2003,4(1):58-62.
[2]EMERSHAD R L,RAMMING D W,SERPE M D.In ovulo embryo development and plant formation from stenospermic genotypes of Vitis vinifera[J].American Journal of Botany,1989,76(3):397-402.
[3]张剑侠,牛茹萱.无核葡萄胚挽救技术的研究现状与展望[J]园艺学报,2013,40(9):1645-1655.ZHANG Jianxia,NIU Ruxuan.The present situation and prospect of embryo rescue technique research in seedless grape breeding[J].Acta Horticulturae Sinica,2013,40(9):1645-1655.
[4]LI J,WANG X H,WANG X P,WANG Y J.Embryo rescue technique and its applications for seedless breeding in grape[J].Plant Cell Tissue&Organ Culture,2015,120(3):861-880.
[5]JIAO Y T,LI Z Q,XU K Y,GUO Y R,ZHANG C,LI T M,JIANG Y X,LIU G T,XU Y.Study on improving plantlet development and embryo germination rates in in vitro embryo rescue of seedless grapevine[J].New Zealand Journal of Crop&Horticultural Science,2018,46(1):39-53.
[6]LI Z Q,LI T M,WANG Y J,XU Y.Breeding new seedless grapes using in ovule embryo rescue and marker-assisted selection[J].In Vitro Cellular&Developmental Biology-Plant,2015,51(3):241-248.
[7]刘巧,张立华,王跃进,张剑侠.两个无核葡萄品种胚及胚乳败育的细胞学研究[J].北方园艺,2016(3):31-35.LIU Qiao,ZHANG Lihua,WANG Yuejin,ZHANG Jianxia.Cytological study of embryo and endosperm abortion in two seedless grape varieties[J].Northern Horticulture,2016(3):31-35.
[8]GUO Y S,ZHAO Y H,LI K,LIU Z D,GUO X W,LI C X.In vitro embryo rescue culture of F1 progenies from crosses between tetraploid grape and Vitis amurensis Rupr.[J].African Journal of Agricultural Research,2011,6(21):4906-4909.
[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.PAN Xuejun.Innovating in the technique system of embryo rescue of stenospermocarpic grape and breeding new cultivars of both seedless and disease-resistance traits[D].Yangling:Northwest A&F University,2005.
[10]孟新法,张利,张潞生,王素珍.无核葡萄胚发育及早期离体培养的研究:Ⅲ.培养方式对离体胚发育的影响[J].北京农业大学学报,1992,18(4):393-396.MENG Xinfa,ZHANG Li,ZHANG Lusheng,WANG Suzhen.Study on ovule development of seedless grapes and its early culture in vitro:Ⅲ.Effect of culture methods on embryos development in vitro[J].Agricultural University Pekinensis,1992,18(4):393-396.
[11]李铁梅.‘DR’优系软核性状改良及玫瑰香型无核葡萄新种质创制[D].杨凌:西北农林科技大学,2014.LI Tiemei.Soft seed trait improvement of‘DR’lines and novel germplasm innovation of aroma of Muscat Seedless grapes[D].Yangling:Northwest A&F University,2014.
[12]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.LIU Qiao.Breeding new,cold-resistant,seedless grape germplasm using embryo rescue technology[D].Yangling:Northwest A&F University,2015.
[13]刘小宁,王跃进,张剑侠,江淑萍.Flame Seedless葡萄胚珠、胚乳及胚发育与败育的研究[J].西北植物学报,2005,25(10):1947-1953.LIU Xiaoning,WANG Yuejin,ZHANG Jianxia,JIANG Shuping.Development and abortion of the ovules,endosperms and embryos of Flame Seedless grape[J].Acta Botanica Boreali-Occidentalia Sinica,2005,25(10):1947-1953.
[14]PONCE M T,GUINAZU M,TIZIO R.Effect of putrescine on embryo development in the stenospermocarpic grape cvs Emperatriz and Fantasy[J].Vitis,2002,41(1):53-54.
[15]GRAY D J,HANGER L A.Effect of ovule maturity on recovery of zygotic embryos and embryogenic cultures from muscadine grape[J].Hortscience,1993,28(3):227.
[16]LI G R,JI W,WANG G,ZHANG J X,WANG Y J.An improved embryo-rescue protocol for hybrid progeny from seedless Vitis vinifera grapes×wild Chinese Vitis species[J].In Vitro Cellular&Developmental Biology-Plant,2014,50(1):110-120.
[17]张文娥,潘学军,王飞,王跃进.DNA分子标记技术在葡萄遗传育种中的应用[C]//王秀峰,李宪利.园艺学进展.北京:中国农业出版社,2006:108-115.ZHANG Wen’e,PAN Xuejun,WANG Fei,WANG Yuejin.Application of DNA molecular markers on grape genetics and breeding[C]//WANG Xiufeng,LI Xianli.Advances in Horticulture.Beijing:China Agriculturen Press,2006:108-115.
[18]王跃进,Lamikanra O.检测葡萄无核基因DNA探针的合成与应用[J].西北农林科技大学学报(自然科学版),2002,30(3):42-46.WANG Yuejin,Lamikanra O.Application and synthesis on the DNA probe for detecting seedless genes in grapevine[J].Journal of Northwest A&F University(Natural Science Edition),2002,30(3):42-46.
[19]LAHOGUE F,THIS P,BOUQUET A.Identification of a codominant scar marker linked to the seedlessness character in grapevine[J].Theoretical&Applied Genetics,1998,97(5/6):950-959.
[20]MEJIA N,HINRICHSEN P.A new,highly assertive scar marker potentially useful to assist selection for seedlessness in table grape breeding[J].Acta Horticulturae,2003,603(603):559-564.
[21]CABEZAS J A,CERVERA M T,RUIZGARCIA L,CAARRE-RIO J,MARTINEZZAPATER J M.A genetic analysis of seed and berry weight in grapevine[J].Genome,2006,49(12):1572-1585.
[22]MEJíA N,SOTO B,GUERRERO M,CASANUEVA X,HOUELC,MICCONO M A,RAMOS R,CUNFF L L,BOURSIQUOT JM,HINRICHSEN P,BLONDON A A.Molecular,genetic and transcriptional evidence for a role of VvAGL11 in stenospermocarpic seedlessness in grapevine[J].Bmc Plant Biology,2011,11(1):57.
[23]江淑平.葡萄无核品种胚败育因子及其机制的研究[D].杨凌:西北农林科技大学,2004.JIANG Shuping.Factors and mechanism of embryo abortion in cultivars of seedless grapes[D].Yangling:Northwest A&F University,2004.
[24]张宏明,孟新法,张潞生.无核葡萄胚珠发育及早期离体培养的研究:Ⅰ.激素对胚珠离体培养的影响[J].中国农业大学学报,1990,5(3):277-283.ZHANG Hongming,MENG Xinfa,ZHANG Lusheng.Study on ovule development of seedless grapes and its early culture in vitro:Ⅰ.the effect of hormone on ovules cultured in vitro[J].Journal of China Agricultural University,1990,16(3):277-283.
[25]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.TIAN Lili.Breeding for the disease-resistant seedless grape novel varities and innovation of new germplasms using embryo rescue[D].Yangling:Northwest A&F University,2007.
[26]屈田田,张剑侠,骆强伟,王跃进.无核葡萄抗寒抗病胚挽救育种应用研究[J].果树学报,2017,34(2):157-165.QU Tiantian,ZHANG Jianxia,LUO Qiangwei,WANG Yuejin.A study on the application of seedless grapevine breeding for cold-hardness and disease-resistance using embryo rescue[J].Journal of Fruit Science,2017,34(2):157-165.
[2]EMERSHAD R L,RAMMING D W,SERPE M D.In ovulo embryo development and plant formation from stenospermic genotypes of Vitis vinifera[J].American Journal of Botany,1989,76(3):397-402.
[3]张剑侠,牛茹萱.无核葡萄胚挽救技术的研究现状与展望[J]园艺学报,2013,40(9):1645-1655.ZHANG Jianxia,NIU Ruxuan.The present situation and prospect of embryo rescue technique research in seedless grape breeding[J].Acta Horticulturae Sinica,2013,40(9):1645-1655.
[4]LI J,WANG X H,WANG X P,WANG Y J.Embryo rescue technique and its applications for seedless breeding in grape[J].Plant Cell Tissue&Organ Culture,2015,120(3):861-880.
[5]JIAO Y T,LI Z Q,XU K Y,GUO Y R,ZHANG C,LI T M,JIANG Y X,LIU G T,XU Y.Study on improving plantlet development and embryo germination rates in in vitro embryo rescue of seedless grapevine[J].New Zealand Journal of Crop&Horticultural Science,2018,46(1):39-53.
[6]LI Z Q,LI T M,WANG Y J,XU Y.Breeding new seedless grapes using in ovule embryo rescue and marker-assisted selection[J].In Vitro Cellular&Developmental Biology-Plant,2015,51(3):241-248.
[7]刘巧,张立华,王跃进,张剑侠.两个无核葡萄品种胚及胚乳败育的细胞学研究[J].北方园艺,2016(3):31-35.LIU Qiao,ZHANG Lihua,WANG Yuejin,ZHANG Jianxia.Cytological study of embryo and endosperm abortion in two seedless grape varieties[J].Northern Horticulture,2016(3):31-35.
[8]GUO Y S,ZHAO Y H,LI K,LIU Z D,GUO X W,LI C X.In vitro embryo rescue culture of F1 progenies from crosses between tetraploid grape and Vitis amurensis Rupr.[J].African Journal of Agricultural Research,2011,6(21):4906-4909.
[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.PAN Xuejun.Innovating in the technique system of embryo rescue of stenospermocarpic grape and breeding new cultivars of both seedless and disease-resistance traits[D].Yangling:Northwest A&F University,2005.
[10]孟新法,张利,张潞生,王素珍.无核葡萄胚发育及早期离体培养的研究:Ⅲ.培养方式对离体胚发育的影响[J].北京农业大学学报,1992,18(4):393-396.MENG Xinfa,ZHANG Li,ZHANG Lusheng,WANG Suzhen.Study on ovule development of seedless grapes and its early culture in vitro:Ⅲ.Effect of culture methods on embryos development in vitro[J].Agricultural University Pekinensis,1992,18(4):393-396.
[11]李铁梅.‘DR’优系软核性状改良及玫瑰香型无核葡萄新种质创制[D].杨凌:西北农林科技大学,2014.LI Tiemei.Soft seed trait improvement of‘DR’lines and novel germplasm innovation of aroma of Muscat Seedless grapes[D].Yangling:Northwest A&F University,2014.
[12]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.LIU Qiao.Breeding new,cold-resistant,seedless grape germplasm using embryo rescue technology[D].Yangling:Northwest A&F University,2015.
[13]刘小宁,王跃进,张剑侠,江淑萍.Flame Seedless葡萄胚珠、胚乳及胚发育与败育的研究[J].西北植物学报,2005,25(10):1947-1953.LIU Xiaoning,WANG Yuejin,ZHANG Jianxia,JIANG Shuping.Development and abortion of the ovules,endosperms and embryos of Flame Seedless grape[J].Acta Botanica Boreali-Occidentalia Sinica,2005,25(10):1947-1953.
[14]PONCE M T,GUINAZU M,TIZIO R.Effect of putrescine on embryo development in the stenospermocarpic grape cvs Emperatriz and Fantasy[J].Vitis,2002,41(1):53-54.
[15]GRAY D J,HANGER L A.Effect of ovule maturity on recovery of zygotic embryos and embryogenic cultures from muscadine grape[J].Hortscience,1993,28(3):227.
[16]LI G R,JI W,WANG G,ZHANG J X,WANG Y J.An improved embryo-rescue protocol for hybrid progeny from seedless Vitis vinifera grapes×wild Chinese Vitis species[J].In Vitro Cellular&Developmental Biology-Plant,2014,50(1):110-120.
[17]张文娥,潘学军,王飞,王跃进.DNA分子标记技术在葡萄遗传育种中的应用[C]//王秀峰,李宪利.园艺学进展.北京:中国农业出版社,2006:108-115.ZHANG Wen’e,PAN Xuejun,WANG Fei,WANG Yuejin.Application of DNA molecular markers on grape genetics and breeding[C]//WANG Xiufeng,LI Xianli.Advances in Horticulture.Beijing:China Agriculturen Press,2006:108-115.
[18]王跃进,Lamikanra O.检测葡萄无核基因DNA探针的合成与应用[J].西北农林科技大学学报(自然科学版),2002,30(3):42-46.WANG Yuejin,Lamikanra O.Application and synthesis on the DNA probe for detecting seedless genes in grapevine[J].Journal of Northwest A&F University(Natural Science Edition),2002,30(3):42-46.
[19]LAHOGUE F,THIS P,BOUQUET A.Identification of a codominant scar marker linked to the seedlessness character in grapevine[J].Theoretical&Applied Genetics,1998,97(5/6):950-959.
[20]MEJIA N,HINRICHSEN P.A new,highly assertive scar marker potentially useful to assist selection for seedlessness in table grape breeding[J].Acta Horticulturae,2003,603(603):559-564.
[21]CABEZAS J A,CERVERA M T,RUIZGARCIA L,CAARRE-RIO J,MARTINEZZAPATER J M.A genetic analysis of seed and berry weight in grapevine[J].Genome,2006,49(12):1572-1585.
[22]MEJíA N,SOTO B,GUERRERO M,CASANUEVA X,HOUELC,MICCONO M A,RAMOS R,CUNFF L L,BOURSIQUOT JM,HINRICHSEN P,BLONDON A A.Molecular,genetic and transcriptional evidence for a role of VvAGL11 in stenospermocarpic seedlessness in grapevine[J].Bmc Plant Biology,2011,11(1):57.
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