摘要
无核葡萄育种是世界葡萄研究的重要方向之一。无核葡萄胚挽救育种是提高无核葡萄育种效率的有效方法，胚挽救技术也可以培育多倍体葡萄新品种。本研究主要以培育优质、无核的葡萄新品种为育种目标，采用以下两种途径进行无核葡萄新种质的创建：(1)无核葡萄胚挽救育种及分子辅助育种：用无核品种×无核品种、无核品种×有核品种获得杂种群体，通过早期分子辅助筛选获得无核葡萄单株；(2)多倍体葡萄新种质创制及倍性鉴定：用二倍体无核×四倍体进行杂交，通过细胞流式仪对获得的杂种幼苗进行倍性鉴定，筛选出三倍体无核新种质。本实验初步探讨了杂交组合中不同亲本基因型及其组合，取样接种时期，低温预处理，培养基组成等对胚挽救效率的影响，并对胚挽救技术进行优化。同时，提出将葡萄胚挽救过程中出现的畸形苗进行分类研究，初步探讨了胚挽救过程中畸形苗的形成原因，并对其转化利用进行了研究，以期为通过降低畸形苗的出现比例来提高利用胚挽救技术进行无核葡萄育种效率，提供一定的试验依据。得到以下结果：
1、不同的种子败育型无核葡萄品种做母本进行胚挽救时的适宜取样时期不同，本研究获得的各杂交组合的最佳取样时期分别是：“火焰无核×北醇”为花后39天；“红脸无核×双优”为花后54天；“粉红无核×北醇”为花后54天；“DA7×双优”为花后44天；“红脸无核×无核白”为花后54天；“粉红无核×火焰无核”为花后54天；“DA7×红脸无核”为花后44天；“红宝石无核×黑奥林”为花后63天；“DA7×京优”为花后44天；“火焰无核×藤稔”为花后39天；“黑大粒×巨峰”为花后72天。
2、无核葡萄胚挽救过程中的各个培养阶段有其适宜的培养条件。本研究表明，在胚珠发育阶段：MM4作为基本培养基，添加香蕉泥500mg/L的胚形成培养基在胚形成的作用上效果较好；在胚萌发阶段：WPM作为基本培养基，添加BA0.2mg/L的胚萌发培养基在胚萌发的作用上效果较好。
3、胚挽救育种过程中常常产生畸形苗，胚挽救杂种苗的畸形率主要受母本基因型影响。本研究提出根据外观形态的不同，将胚挽救过程中产生的畸形苗分为以下七类：(1)单子叶畸形苗；(2)无叶无根苗；(3)子叶扭曲褶皱状畸形苗；(4)分化过程中形成的白化苗；(5)下胚轴形成短根，但没有子叶；(6)上胚轴形成子叶，但没有根；(7)萌发后早期停止生长的小苗。
4、将胚挽救过程中出现的多胚分开为单一的胚，并将其逐一接种到新鲜的胚萌发培养基上，可以消除畸形苗的产生，并且胚萌发率也可以达到100%。对采摘的幼果进行低温(4°C)预处理20d后再接种其胚珠，不仅可以有效的降低畸形苗的数量，同时，胚的萌发率也得到了显著性增加。从降低畸形苗形成的角度考虑，用MS+香蕉泥500mg/L作为胚形成培养基，WPM+6-BA0.2mg/L+香蕉泥500mg/L作为胚萌发培养基的效果最好。
5、畸形苗转接至转化培养基后，从外观形态上表现出3种转化结果：(1)转化形成具有正常茎和叶的植株；(2)组织周围形成大量簇生芽；(3)退化形成愈伤组织。将转化产生的簇生芽或正常苗转接至生根培养基后，2周左右即可形成健康的小苗。以2MS+6-BA0.4mg/L+IBA0.1mg/L+香蕉泥500mg/L作为转化培养基，在对单子叶畸形苗和子叶扭曲褶皱状畸形苗进行正常植株的转化的成功率较高。胚萌发后4周是转化的适当时期。
6、本研究通过无核特异探针(GSLP-1)对杂种幼苗的DNA分子鉴定，获得了11个可以扩增出569bp特异条带的株系，分别命名为JW-1-1， JW-1-2，JW-1-3，JW-2-1，JW-2-2，JW-3-1，JW-4-1，JW-4-2，JW-4-3，JW-4-4和JW-4-5；用流式细胞仪对杂种苗的染色体倍性水平鉴定，获得了3个三倍体株系JW3-1，JW3-2和JW3-3和2个单倍体株系JW1-1和JW1-2。并对成苗扩繁后进行了炼苗移栽，已成活436个单株。
Breeding for seedless grapes is one of the targets of grape breeders in worldwide. Theembryo rescue technology is an effective measure to improve the efficiency of seedless grapebreeding and could also be used to cultivate new varieties of polyploid grapes. This projectaimed at breeding seedless grape varieties by embryo rescue through two hybridizationmethods:(1) the early molecular assistant breeding technology of seedless grape: obtainingthe hybrids from cross-breeding between seedless V. vinifera cultivars and wild Chinese Vitisspp or crossing with two seedless cultivars, and then screening the seedless grape strains byusing the early molecular assistant breeding technology; and (2) the evaluation of polyploidnew seedless germplasm by flow cytometry: hybridization with diploid seedless×tetraploidgrape, and then identification and selection new triploid seedless seedlings by flow cytometry.At the same time, we optimized the embryo rescue technique and focused on the effects of thedifferent parental genotypes and their combination, media type and composition, andsampling time, low temperature pretreatment on the embryo germination and plantdevelopment rate. Moreover, we classified the abnormal seedlings into seven differentcategories based on morphology and investigated the formation and preliminarytransformation of the abnormal seedlings during embryo rescue in order to improve theefficiency of seedless grape breeding by the embryo rescue technology. The main results werepresented in the following:
1. The efficiency of seedless grape combinations breeding was influenced by thematernal genotype by means of the embryo rescue technology. The differentcross-combinations gained their highest ovule germination rate at different sampling times.The best sampling times for different combinations were showed in following:‘FlameSeedless×Beichun’(DAF, days after flowering,39d);‘Blush Seedless×Shuangyou’(DAF54d);‘Pink Seedless×Beichun’(DAF54d);‘DA7×Shuangyou’(DAF44d);‘BlushSeedless×Thompson Seedless (DAF54d)’;‘Pink Seedless×Flame Seedless’(DAF54d);‘DA7×Blush Seedless’(DAF44d);‘Ruby Seedless×Black Olympia’(DAF63d);‘DA7×Jingyou’(DAF44d);‘Flame Seedless×Fujiminori’(DAF39d); and ‘Big Black×Kyoho’(DAF72d).
2. Each cultivating stage had corresponding appropriate culture conditions in the process of seedless grapes embryo rescue. At the stage of ovule culture, the best results were foundwhen MM4was used as the basic medium and additional of mashed banana500mg/L; at thestage of embryo germination, the best results were detected when WPM was used as the basicmedium and additional of BA0.2mg/L.
3. There were abnormal seedlings produced in the process of embryo rescue breeding.The deformity rate of the hybrid seedlings from embryo rescue was mainly influenced byfemale parent genotype. The abnormal seedlings were classified into seven differentcategories based on morphology:(1) epicotyl appears only one cotyledon;(2) neithercotyledon nor shoot;(3) the cotyledons were distorted fold-like;(4) the formation of thealbino seedlings in the process of differentiation;(5) hypocotyls formed short root, but nocotyledon;(6) epicotyl formed cotyledons, yet no root;(7) the plantlet earlydevelopment-stopped. The occurrence and proportion of each variety of malformationseedlings were random and uncertain.
4. Separating polyembryony occur in the process of embryo rescue into single-embryoand inoculating each one into fresh embryo germination medium could completely preventthe generation of abnormal seedlings, and the embryo germination rate could reach100%.The treatment of pre-chilling (4°C,20d) on the immature fruit before the ovules werecultured could significantly reduce the number of abnormal seedlings and increase the embryogermination rate. In the aspect of formation of abnormal seedlings, MS+mashed banana500mg/L as the best effect of embryo development medium; WPM+6-BA0.2mg/L+mashedbanana500mg/L as the embryo germination medium effect best. As the aspect of reducingdeformity plantlet, the use of MS+mashed banana500mg/L as the embryo developmentmedium and WPM+6-BA0.2mg/L+mashed banana500mg/L as the embryo germinationmedium worked best.
5. There were three kinds of variations after the deformity seedlings were transferred toconversion medium:(1) formation of normal seedlings with healthy stems and leaves;(2)formation of buds clustered around the tissues;(3) dedifferentiation and formation callus.Generally, those abnormal plantlets could develop into normal healthy plants after transferringthe clustered buds or the transformed normal seedlings to the rooting media after two weeks.The results showed that2MS+6-BA0.4mg/L+IBA0.1mg/L+mashed-banana500mg/Las the transformation medium showed the highest success rate in converting the deformityseedlings to normal plantlets. Four weeks after embryo germination was the proper time forthe transformation of abnormal seedlings.
6. Through the detection of the seedlessness using seedless specific probe GSLP1,11grape lines amplified the569bp specific band and were named as JW-1-1, JW-1-2, JW-1-3, JW-2-1, JW-2-2, JW-3-1, JW-4-1, JW-4-2, JW-4-3, JW-4-4, and JW-4-5; through thedetection of chromosome ploidy identification by flow cytometry,3lines were confirmed astriploid, named as JW3-1, JW3-2, and JW3-3; and2lines were confirmed as haploid, namedas JW1-1and JW1-2, respectively. We carried out the subculture and multiplication andobtained436survival seedlings.
引文
曹孜义，康天兰，张金文.1996.葡萄花药三倍体植株倍性完全型的确定.园艺学报，23(2):194~196
常月梅.2000.果树多倍体鉴定进展.山西林业科技，(1):1~4
陈习刚.2009.葡萄、葡萄酒的起源及传入新疆的时代与路线.古今农业，(1):51~61
戴洪义，孙敏，商传明，徐步玲.1990.葡萄的染色体倍性与气孔性状的关系及其判别分析.中外葡萄与葡萄酒，(2):5~7
董晓玲.1990.葡萄胚珠、胚乳及胚的发育.植物学通报，7(1):53~55
郭海江，王跃进，张剑侠，潘学军，唐冬梅，田莉莉.2005.葡萄抗病无核胚挽救育种及分子标记辅助选择.西北植物学报，25(12):2395~2401
郭修武，郭印山，张海娥，李轶晖，李成祥.2007.接种时期和培养基对无核葡萄胚挽救的影响.园艺学报，34(2):329~332
郭印山,郭修武,张海娥,李轶晖,李成祥.2005.利用胚挽救技术获得三倍体葡萄植株研究.沈阳农业大学学报,36(5):606~608
郭印山，张海娥，郭修武，李轶晖，李成祥.2006.早熟葡萄胚抢救技术研究.中外葡萄与葡萄酒，(1):11~15
郭紫娟，赵胜建，赵淑云，张新忠，周利存.2004.三倍体葡萄新品种“红标无核”的选育及栽培技术研究.河北农业科学，8(1):50~53
贺普超,王跃进,王国英.1991.中国葡萄属野生种抗病性的研究.中国农业科学，24(3):50~56
贺普超，牛立新.1989.我国葡萄属野生种抗寒性的研究.园艺学报，16(2):81~88
贺普超.1999.葡萄学.北京:中国农业出版社:13~14；54；289~291；363~369
蒋爱丽，李世诚，金佩芳，骆军.2002.大败育型无核葡萄胚珠培养成苗技术研究.上海交通大学学报(农业科学版),20(1):45~48
蒋爱丽，李世诚，金佩芳，杨天仪，骆军.2007.胚培无核葡萄新品种—沪培1号的选育.果树学报，24(3):402~403
李桂荣，王跃进，唐冬梅，王西平，骆强伟.2001.无核白胚挽救育种技术研究.西北植物学报,21(3):432~436
李世诚，金佩芳，蒋爱丽，骆军.1998.与四倍体杂交的无核葡萄胚珠培养获得三倍体植株.上海农业学报，14(4):13~17
刘崇怀.2003.无核葡萄品种的无核性来源分析.植物遗传资源学报，4(1):58~62
刘小宁，王跃进，张剑侠，江淑平.2005. Flame Seedless葡萄胚珠、胚乳及胚发育与败育的研究.西北植物学报,25(10):1947~1953
孟新法，张利，张潞生，王素珍.1992.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影响.北京农业大学学报，18(4):393~395
潘学军.2005.无核抗病葡萄胚挽救技术体系的优化及新品种培育.[博士学位论文].陕西:西北农林科技大学
乔玉山，房经贵，沈志军，章镇.2004.果树农艺性状基因的分子标记及其应用.果树学报，21(2):158~163
唐冬梅.2010.无核葡萄杂交胚挽救新种质创建与技术完善.[博士学位论文].陕西:西北农林科技大学
田莉莉.2007.抗病无核葡萄胚挽救育种及种质创新.[博士学位论文].陕西:西北农林科技大学
王飞,王跃进,周建锡.2006.无核葡萄与中国野生葡萄杂种的胚挽救技术研究.园艺学报，33(5):1079-1082
王飞，王跃进，万怡震，任杰.2004.无核葡萄与中国野生葡萄杂种胚败育的某些生理生化变化.园艺学报，31(5):651~653
王晶，罗国光.1996.巨峰葡萄胚和胚乳的发育.园艺学报，23(2):191~193
王军.2009.生物技术与葡萄遗传育种.中国农业科学，42(8):2862~2874
王跃进，Lamikanra O，卢江，Ramming D W.1996. Identification of genetic marker linker seedless genesin grapes using RAPD.西北农业大学学报，24(5):1~10
王跃进，Lamikanra O.2002.检测葡萄无核基因DNA探针的合成与应用.西北农林科技大学学报（自然科学版），30(3):42~46
王跃进，江淑平，刘小宁，张剑侠.2007.假单性结实无核葡萄胚败育机理研究.西北植物学报,27(10):1987~1993
王跃进，万怡震.2002.美国加州的葡萄生产与科研.西北农林科技大学（自然科学版），30(1):134~140
王跃进，徐炎，张剑侠，周鹏.2002b.中国野生葡萄果实抗炭疽病基因的RAPD标记.中国农业科学，35(5):536~540
王跃进，杨英军，周鹏，张剑侠，王西平.2002a.用DNA探针检测我国栽培的无核葡萄与辅助育种初探.园艺学报，29(2):105~108
王跃进，张剑侠，李桂荣，唐冬梅，骆强伟.2001.采用胚挽救技术获得抗病无核葡萄新材料的研究.甘肃农业大学学报，36(专辑):105~109
吴雅琴，赵艳华，程和禾，赵胜建，郭紫娟，马爱红.2006.流式细胞术检测葡萄倍性的研究.见：王秀峰，李宪利（主编）.园艺学进展—中国园艺学会第七届青年学术讨论会论文集.北京:中国农业出版社出版社:182~185
徐海英，闫爱玲，张国军.2005.葡萄二倍体与四倍体品种间杂交胚挽救取样时期的确定.中国农业科学，38:629~633
徐海英，张国军，闫爱玲.2001.无核葡萄育种及杂交亲本的选择.中外葡萄与葡萄酒，3:30~32
闫爱玲，张国军，徐海英.2008.葡萄不同倍性品种间杂交胚挽救及鉴定.西北农业学报，17(3):223~226
杨承时.2003.中国葡萄栽培的起始及演化.中外葡萄与葡萄酒,(3):4~7
杨克强，王跃进，张今今，王西平，万怡震，张剑侠.2005.葡萄无核基因定位与作图的研究.遗传学报，32(3):297~302
杨英军，王跃进，周鹏，王西平，张剑侠.2002.葡萄无核基因的SCAR标记及Southern blot分析.西北农林科技大学(自然科学版)，80(6):77~80
杨有龙.1992.美国和日本的葡萄育种.北方果树,2:1~2
张利，孟新法，张潞生，张彦明，王素珍.1991.无核葡萄胚珠发育及早期离体培养的研究.Ⅱ.无核葡萄胚发育的特点.北京农业大学学报,17(4):55~59
张宗勤，纪薇，丁家华，孙锋，于建娜，王跃进.2011.‘无核白’葡萄及其营养变异系的光合作用与果实特性.西北植物学报,31(8):1657~1664
朱林，李佩芬，卢炳芝，于向荣.1992.无核葡萄品种的胚珠养和胚分化（简报）.植物生理学通讯,28(4):273~274
Adam-Blondon A F, Lahogue-Esnault F, Bouquet A, Boursiquot J M., This P.2001. Usefulness of twoSCAR markers for marker-assisted selection of seedless grapevine cultivars. Vitis,40:147~155.
Amaral A L, Oliveira P R, Czerainski A B, Camargo U A.2001. Embryo growth stages on plant obtentionfrom crosses between seedless grape parents. Rev Bras Frutic,23:647~651
Agüero C, Riquelme C, Tizio R.1995. Embryo rescue from seedless grapevines (Vitis vinifera L.) treatedwith growth retardants. Vitis,34:73~76
Agüero C, Gregori M T, Ponce M T, Iandolino A, Tizio R.1996. Improved germination of stenospermicgrape fertilized ovules by low temperatures. Biocell,20:123~126
Badenes M L, unquan Lin S, Zhang Z, Liang G L, Wang W.2013. Breeding Loquat. Plant Breeding Rev:259.
Bharathy P V, Karibasappa G S, Biradar A B, Kulkarni D D, Solanke A U, Patil S G, Agrawal D C.2003.Influence of pre-bloom sprays of benzyladenine on in vitro recovery of hybrid embryos fromcrosses of Thompson seedless and8seeded varieties of grape (Vitis spp.). Vitis,42:199~202
Bharathy P V, Karibasappa G S, Patil S G, Agrawal.2005. In ovulo rescue of hybrid embryos in FlameSeedless grapes—Influence of pre-bloom sprays of benzyladenine. Sci Hort-Amsterdam,106:353~359
Bouquet A.1980. Vitis×Muscadinia hybridization as a method of introducing resistance characters intocultivated vine by introgression, and the cytogenetic and taxonomic problems in parents. Annales De LAmelioration des Plantes,30:213~214
Bouquet A and Davis H P.1989. In vitro ovule and embryo culture for breeding seedless table grapes (Vitisvinifera L.). Agronomie,9:565~574
Bouquet A, Danglot Y.1996. Inheritance of seedlessness in grapevine (Vitis vinifera L.). Vitis,35:35~42
Bozhinova-Boneva I.1978. Inheritance of seedlessness in grapes. Genet Sel,11:399~405
Burger P and Trautmann I A.2000. Manipulations of ovules to improve in vitro development of Vitisvinifera L. embryos. Acta Hort,528:613~619
Burger P, Gerber C A, Gerber A, Ellis P J L.2003. Breeding seedless grapes in South African by means ofembryo rescue. Acta Hort,603:565~569
Cabezas J A, Cervera M T, Ruiz-Garcia L, Carreno J, Martinez-Zapater J M.2006. A genetic analysis ofseed and berry weight in grapevine. Genome,49:1572~1585
Cain D W, Emershad R L, Tarailo R E.1983. In ovule embryo culture and seedling development of seededand seedless grape (Vitis vinifera L). Vitis,22:9~14
Constantinescu G, Pena A, Indreas A.1975. Inheritance of some qualitative and quantitative characters inthe progeny of crosses between functionally female (gynodynamic) and apyrene (androdynamic)varieties. Plobleme de Genetica Teoretica, SiAplicata,7:213~241
Deg irmenci D, Marasali B.2001. Effects of growth regulators on induction of seed trace development andgermination rate of stenospermic Sultani C ekirdeksiz and Perlette grape cultivars. J Agric Sci,7:148~152(in Turkish).
Doligez A, Bouquet A, Danglot Y, Lahogue F, Riaz S, Meredith C P, Edwards K J, This P.2002. Geneticmapping of grapevine (Vitis vinifera L.) applied to the detection of QTLs for seedless and berry weight.Theor Appl Genet,105:780~795
Druart P H.2006. Aneuploidy, a source of genetic diversity for fruit species. Acta hortic,725
Dudnik N A, Moliver M G.1976. Inheritance of seedlessness in grape in south of the UkrainianSSR.Referativnyi Zhurnal5.55.118:105~113
Durham R E, Moore G A, Gray D J, Mortensen J A.1989. The use of leaf GPI and IDH isozymes toexamine the origin of polyembryony in cultured ovules of seedless grape. Plant Cell Rep,7:669~672
Emershad R L and Ramming D W.1984. In ovulo embryo culture of Vitis vinifera L.c.v.‘Thompsonseedless’. Am J Bot,71:873~877
Emershad R L, Ramming D W, Serpe M D.1989. In ovulo embryo development and plant formation fromstenospermic genotypes of Vitis vinifera. Am J Bot,76:397~402
Emershad R L and Ramming D W.1994. Somatic embryogenesis and plant development from immaturezygotic embryos of seedless grapes (Vitis vinifera L.). Plant Cell Rep,14:6~12
Fernandez G E, Clark J R, Moore J N.1991. Effect of seedcoat manipulation on the germination ofstenospermocarpic grape embryos cultured in ovule. Hortscience,26:1220
Garcia E, Martinez A, Garcia de la Calera E, Perez L J, Cenis J L, Carreno J.2000. In vitro culture ofovules and embryos of grape for the obtain of new seedless table grape cultivars. Acta Hort,528:663~666
Gaspero G D,Cipriani G.2003. Nucleotide binding site/leucine-rich repeats, pto-like and receptor-likekinases related to disease resistance in grapevine. Mo Gen Genomics,269:612-623
Glaser N.2012. Influence of natural food compounds on DNA stability. URL:http://opus.bibliothek.uni-wuerzburg.de/volltexte/2012/7287/
Glimelius K.2006. High growth rate and regeneration capacity of hypocotyl protoplasts in someBrassicaceae. Physiolo. Plantarum,61:38~44.
Goldy R G.1987. In vitro cultivability of ovules from10seedless grape clones. Hortscience,22:952
Goldy R, Emershad R, Ramming D, Chaparro J.1988. Embryo culture as a means of introgressoryseedlessness from Vitis vinifera to Vitis rotundifolia. Hortscience,23:886~889
Goldy R G, Ramming D W, Emershad R L,Chaparro J X.1989. Increasing production of Vitisvinifera×V.rotundifolia hybrids through embryo rescue. Hortscience,24:820~822
Golodriga P Y, Troshin L P, Frolava L I.1986. Inheritance of character of seedlessness in the hybridgeneration of V. vinifera. Tsitologyia Genetika,19:372~376
Gray D J, Fisher L C, Mortensen J A.1987. Comparison of methodologies for in ovule embryo rescue ofseedless grapes. HortScience,22:1334~1335
Gribaudo I, Zanetti R., Botta R, Vallania R, Eynard I.1993. In ovule embryo culture of stenospermocarpicgrapes.Vitis,32:9~14
Grosser J W, Gmitter Jr F G.2011. Protoplast fusion for production of tetraploids and triploids: applicationsfor scion and rootstock breeding in citrus. Plant Cell Tiss Org,104:343~357.
Harris-Shultz K R, Schwartz B M, Brady J A.2011. Identification of Simple Sequence Repeat Markers thatDifferentiate Bermudagrass Cultivars Derived from ‘Tifgreen’. J Am Soc Hortic Sci,136:211~218.
Hazarika R R, Mishra V K, Chaturvedi R.2013. In vitro haploid production-A fast and reliable approachfor crop improvement.Crop improvement under adverse conditions. Springer New York:171~212.
Horiuchi S, Kurooka H, Furuta T.1991. Studies on the embryo dormancy in grape. J Jpn Soc Hortic Sci,60:1~7
Ji W, Li Z Q, Zhou Q, Yao W K, Wang Y J.2013. Breeding new seedless grape by means of in vitroembryo rescue. Genet Mol Res,12:859~869.
Johnston S A, Nijs T P M den, Peloquin S J, Hanneman R E Jr.1980. The significance of genic balance toendosperm development in interspecific crosses. Theor Appl Genet,57:5~9
Kebeli N, Boz Y, Ozer C.2003. Studies on the applying of embryo culture in breeding new hybrids bycrossing seedless grape cultivars. Acta Hort,625:279~281
Lahogue F, This P, Bouquet A.1998. Identification of a codominant scar marker linked to the seedlessnesscharacter in grapevine. Theor Appl Gent,97:950~959
Ledbetter C A, Ramming D W.1989. Seedlessness in grapes. Horticultural Reviews,11:159~184
Ledbetter C A, Shonnard C B.1990. Improved seed development and germination of stenosermic grapes byplant growth regulators. J Hortic Sci,65:269~274
Ledbetter C A, Burgos L.1994. Inheritance of stenospermocarpic seedlessness in Vitis Vinifera L. J Hered,85:157~160
Liu S M, Sykes S R, Clingeleffer P R.2003. Improved in ovulo embryo culture for stenospermocarpicgrapes (Vitis vinifera L.). Auts J Agr Res,54:869~876
Lloyd G B, McCown, BH.1980. Commercially-feasible micropropagation of mountain laurel, Kalmialatifolia, by use of shoot-tip culture. Proc Int Plant Propag Soc,30:421~437.
Loomis N H, Weinberger J H.1979. Inheritance studies of seedlessness in grape. J Amer Soc Hort Sci,104:181~184
Lu J, Lamikanra O.1996. Barriers to inter subgeneric crosses between Muscadinia and Euvitis.Hortscience,31:269~271
Midani A R, Sharma H C, Singh S K.2002. Effect of ovule age on ovulo-embryo culture in seeded andseedless grape genotypes. Indian Journal of Horticulture,59:359~362
Minernura M, Izumi K, Yamashita H, Tsukahara K.2009. Breeding a new grape cultivar 'Nagano Purple'and its characteristics. Hortic Res-Japan,8:115~122
Moreau L, Charcosset A, Hospital F, Gallais A.1998. Marker-assisted selection efficiency in populations offinite size. Genetics,148:1353~1365
Palmer J L, Lawn R J, Adkins S W.2002. An embryo-rescue protocol for Vigna interspecific hybrids.Aust JBot,50:331~338
Park S M, Hiramatsu M, Wakana A.1999. Aneuploid plants derived from crosses with triploid grapesthrough immature seed culture and subsequent embryo culture. Plant Cell Tiss Org,59:125~133
Perl A N, Sahar P, Spiegel-Roy S, Gavish R, Elyasi E, Orr E, Bazak H.2000. Conventional andbiotechnological approaches in Breeding seedless table grapes. Acta Hort,528:607~612
Perl A, Sahar N, Eliassi R, Baron I, Spiegel-Roy P, Bazak H.2003. Breeding of new seedless table grapesinIsreal conventional and biotechnological approach. Acta Hort,603:185~187
Pinto D L P, de Almeida Barros B, Viccini L F, de Campos, J. M. S., da Silva, M. L.,&Otoni, W. C.2010.Ploidy stability of somatic embryogenesis-derived Passiflora cincinnata Mast. plants as assessed byflow cytometry. Plant Cell Tiss Org,103,71~79.
Pommer C V, Ramming David W, Emershad, R L.1995. Influence of grape genotype, ripening season, seedtrace size, and culture date on in ovule embryo development and plant formation. Bragantia,54:237~249
Ponce M T, Agüero C B, Gregori M T, Tizio R.2000. Factors affecting the development of stenospermicgrape (Vitis vinifera) embryos cultured in vitro. Acta Hort,528:667~671
Ponce M T, Guinazu M, Tizio R.2002a. Effect of putrescine on embryo development in thestenoepermocarpic grape cvs Emperatriz and Fantasy. Vitis,41:53~54
Ponce M T, Guinazu M E, Tizio R.2002b. Improved in vitro embryo development of stenospermic grapeby putrescine. Biocell,26:263~266
Ramming D W, Emershad R L.1982. In-ovule embryo culture of seeded and seedless Vitis viniferaL.(Abst.). Hortscience,17:487
Ramming D W.1990. The use of embryo culture in fruit breeding. HortScience,25:393~398
Reisch B I, Owens C L, Cousins PS.2012. Grape. Springer US:225~262
Roytchev V.1998. Inheritance of Grape Seedlessness in Seeded and Seedless Hybrid Combinations ofGrape Cultivars with Complex Genealogy. Am J Enol Vitic,49:302~305
Sahijram L, Kanamadi V C.2004. In ovulo hybrid embryo culture in controlled grape crosses involvingstenospermocarpic parents. Acta Hort,662:281~288
Sandhu A S, Jawanda J S, Uppal D K.1984. Inheritance of seed characters in hybrid populations ofintercultivar crosses of grapes (Vitis vinifera L.). J Res Punjab Agric Univ,21:39~44
Singh N V, Singh S K, Singh A K.2011. Standardization of embryo rescue technique and bio-hardening ofgrape hybrids (Vitis vinifera L.) using Arbuscular mycorrhizal fungi (AMF) under sub-tropicalconditions. Vitis50:115-118.
Siwach P, Chanana S, Gill A R, Dhanda P, Rani J, Sharma K, Kumari D.2012. Effects of adenine sulphate,glutamine and casein hydrolysate on in vitro shoot multiplication and rooting of Kinnow mandarin(Citrus reticulata Blanco). Afr J Biotechnol,11:15852-15862.
Spiegel-Roy P, Sahar N, Baron J, Lavi U.1985. In vitro culture and plant formation from grape cultivarswith abortic ovules and Seeds, J Amer Soc Hort Sci,1l0:109~112
Stout A B.1936. Seedlessness in grapes. New York State Agric Expt. Sta.(Geneva) Tech. Bull.238
Stout A B.1937. Breeding for hardy seedless grapes. Proc Amer Soc Hort Sci,34:416~420
Striem M J, Spiegel-Roy P, Sahar N.1992. The degree of development of the seed coat and the endospermas separate subtraits of stenospermocarpic seedlessness in grapes. Vitis,31:149~155
Striem M J, Benhayyim G, Spiegel-Roy P.1994. Developing molecular genetic markers for grape breedingusing polymerase chain reaction procedures. Vitis,33:53~54
Striem M J, Ben-Hayyim G, Spiegel-Roy P.1996. Identifying molecular genetic markers associated withseedlessness in grape. J Amer Soc Hort Sci,121:758~763
Sun L, Zhang G J, Yan A L, Xu H Y.2011.The study of triploid progenies crossed between differentploidy grapes. Afr J Biotechnol,10:5967~5971.
Tang D M, Wang Y J, Cai J S, Zhao R H,2009. Effects of exogenous application of plant growthregulators on the development of ovule and subsequent embryo rescue of stenospermic grape (Vitisvinifera L.).Sci Hortic,120:51~57.
Tsolova V.1990. Obtaining plants from crosses of seedless grapevine varieties by means of in vitroembryo culture. Vitis29:1~4
Tucker A, Yilmazer-Musa M, Frei B.2010. In vitro Inhibition of Alpha-Amylase and Alpha-Glucosidaseby Bioflavonoids. An Oregon State University Publication Volume VI,10:315~31.
Valdez J G.2005. Immature embryo rescue of grapevine (Vitis vinifera L) after an extended period of seedtrace culture. Vitis,44:17~23
Wang J, Gao W, Zhang J, Huang T, Wen T, Zhang L, Huang L.2011. Production of saponions andpolysaccharide in the presence of lactoalbumin hydrolysate in Panax quinquefolium L. cells cultures.Plant Growth Regul,63:217~223.
Weinberger J H, Harmon F N.1964. Seedlessness in vinifera grapes. Proc Amer Soc Hort Sci,85:270~271
Weinberger J H, Harmon F N.1974.‘Flame seedless’ grape. Hortscience,9:63.
Wu J H.2012. Manipulation of ploidy for kiwifruit breeding and the study of actinidia genomics. ActaHortic,961:539.
Yamane H.1997. Studies on the breeding in grapes with reference to large berry and seedlessness.[Ph.Dthesis]. Japan: Kyoto University
Yamashita H, Horiuchi S, Taira T.1993. Development of seeds and the growth of triploid seedlingsobtained from reciprocal crosses between diploids and triploid grapes. J Jap Soc Hortic Sci,62:249~255
Yamashita H, Shigehara I, Haniuda T.1998. Production of triploid grapes by in ovulo embryo culture.Vitis,37:113~117
Negral A.M.1934.Contribution to the question of parthenocarpy and apomixes in the grape.Tr Prikl BotSelem Ser.3:229~268
Gray D J, Mortensen J A, Benton C M.1990. Ovule culture to obtain progeny from hybrid seedless bunchgrapes. J Am Soc for Hort Sci,115:1019~1024
Mariscalco G., Crespan M.1995. Polyembryony and somatic embryogenesis of in vitro cultured in ovuloembryos of seedless grapes. Die Weinwissenschaft,50:39-43
Valdez J G and Ulanovsky S M.1997. In vitro germination of stenospermic seeds from reciprocal crosses(Vitis vinifera L.) applying different techniques. Vitis,36:105~107
Bessho H, Miyake M, Kondo M.2000. Grape breeding in Yamanashi, Japan present and future. ActaHortic538:493-496.
Scott K D, Ablett E M.2000. AFLP markers distinguishing an early mutant of Flame Seedless grape.Eupytica,113:245-249
Valdez J G, Andreoni M A, Castro P, Ulanovsky S M.2000. Variety response to direct germination ofstenospermic seeds classified according to the hardness of their seed coats. Acta Hort,528:659~662
Notsuka K, Tsuru T, Shiraishi M.2001. Seedless-seedless grape hybridization via in ovule embryo culture.JJap Soc Hortic Sci,70:7~15
Ander N, Byrne D H, Sinclair J, Burrell A M.2002. Cooler temperature during germination improves thesurvival of embryo cultured peach seed. Hortscience,37:402~403
Park S M, Wakana A, Kim J H, Jeong C S.2002. Male and female fertility in triploid grapes (Vitis complex)with special reference to the production of aneuploid plants.Vitis,41:11~19
Ebadi A, Sarikhani H, Zamani Z, Babalar M.2004. Effect of male parent and application of boric acid onembryo rescue in some seedless grapevine (Vitis vinifera) cultivars. Acta Hort,640:255~260
Heo J Y, Park K S, Yun H K, Park S M.2007. Degree of abortion and germination percentage in seedsderived from interploid crosses between diploid and tetraploid grape cultivars. HorticultureEnvironment and Biotechnology,48:115~121
Tian L L, Wang Y J, Niu L, Tang D M.2008. Breeding of disease-resistant seedless grapes using Chinesewild Vitis spp. I. In vitro embryo rescue and plant development. Sci Hort-Amsterdam,117:136~141
Al-Khayri J M.2011. Influence of yeast extract and casein hydrolysate on callus multiplication and somaticembryogenesis of date palm ( Phoenix dactylifera L.). Sci Hortic,130:531~535.
Zhao Y H, Cheng H H, Wu Y Q, Wu Y J, Li Y S.2011. Germplas development of triploid grapevine andrapid identification of chromosomes ploidy. Acta Hortic Sinica,6:21