黄瓜体细胞胚途径再生体系的建立及优化
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摘要
以不同生态型的黄瓜(Cucumis sativus L.)为材料,利用不同基因型建立黄瓜体细胞胚再生体系,并在此基础上对黄瓜体细胞胚再生体系进行了优化。结果表明:华南型黄瓜9101和D4能够诱导出正常的胚状体。以筛选出的9101为材料,进一步通过不同组织部位、激素配比、暗培养等对体细胞胚再生体系进行优化,认为子叶节是最佳的外植体,其胚性愈伤组织诱导的最适培养基为MS+2.00 mg·L~(-1) 2,4-D,诱导率可达100.00%,出胚的最适培养基为MS+0.10 mg·L~(-1) 2,4-D+0.5mg·L~(-1) KT,其出胚率可达33.33%。此外,在愈伤诱导培养前期给予暗培养条件能提高愈伤诱导率、出胚率及正常再生植株的获得。对体细胞胚的形态学观察发现,黄瓜体细胞胚发源于子叶节表面的胚性愈伤组织,经历球形胚、心形胚、鱼雷形胚和子叶胚等时期,最终发育成完整的黄瓜植株。
Taking different ecotypes of cucumber as material,this study established and optimized somatic embryo regeneration systems of cucumber with different genotypes.The results indicated that '9101' and 'D4' could induce normal embryoid bodies.Then taking the screened '9101'as material,this study carried out optimization on somatic embryogenesis system through different types of explants,hormone concentrations,and dark treatment in early period during callus induction,cotyledonary node was selected as the best explant. And the most suitable medium for callus induction was MS + 2.00 mg·L~(-1) 2,4-D.The callus induction rate could reach 100.00%.The optimum medium for differentiation was MS + 0.10 mg·L~(-1) 2,4-D + 0.5 mg·L~(-1) KT.Its embryogenesis rate could reach 33.33%.In addition,under dark culture condition in early period of callus induction could improve callus induction rate,embryogenesis rate and normal regeneration plants.The morphological observation of somatic embryos revealed that cucumber somatic embryos originated from the embryogenic callus on the surface of cotyledonary nodes,and experienced globular embryos,heart-shaped embryos,torpedo embryos,and cotyledon embryos;and finally developed into complete cucumber plantlets.
Taking different ecotypes of cucumber as material,this study established and optimized somatic embryo regeneration systems of cucumber with different genotypes.The results indicated that '9101' and 'D4' could induce normal embryoid bodies.Then taking the screened '9101'as material,this study carried out optimization on somatic embryogenesis system through different types of explants,hormone concentrations,and dark treatment in early period during callus induction,cotyledonary node was selected as the best explant. And the most suitable medium for callus induction was MS + 2.00 mg·L~(-1) 2,4-D.The callus induction rate could reach 100.00%.The optimum medium for differentiation was MS + 0.10 mg·L~(-1) 2,4-D + 0.5 mg·L~(-1) KT.Its embryogenesis rate could reach 33.33%.In addition,under dark culture condition in early period of callus induction could improve callus induction rate,embryogenesis rate and normal regeneration plants.The morphological observation of somatic embryos revealed that cucumber somatic embryos originated from the embryogenic callus on the surface of cotyledonary nodes,and experienced globular embryos,heart-shaped embryos,torpedo embryos,and cotyledon embryos;and finally developed into complete cucumber plantlets.
引文
安亚伟.2013.黄瓜离体再生与遗传转化体系的研究[硕士论文].扬州:扬州大学.
陈继峰.2007.黄瓜离体器官再生植株研究进展.上海农业学报,23(1):114-118.
陈小鹏,刘栓桃,孙小镭,赵智中,王秀峰.2005.黄瓜未授粉子房的胚状体诱导研究初报.西北农业学报,14(2):148-151.
段莉莉,朱拼玉,李季,陈劲枫.2018.基因工程技术在甜瓜属作物分子育种中的应用与发展.中国瓜菜,31(8):1-8.
谷安根,王立军.1997.子叶区理论研究进展.植物学通报,14(3):30-39.
何莉莉,陈阳,陈俊琴,佟贤,韩成玲.2010.黄瓜连作栽培中营养基质的微量元素、病原菌和产量的变化.沈阳农业大学学报,41(1):13-17.
贺丽娜,量银丽,高静,熊亚梅,周茂娟,韦泽秀.2008.连作对设施黄瓜产量和品质及土壤酶活性的影响.西北农林科技大学学报,36(5):155-159.
胡适宜.1982.被子植物胚胎学.北京:高等教育出版社:237-247.
黄东梅,别蓓蓓,潘俊松,何欢乐,蔡润.2012.黄瓜(Cucumis sativus L.)离体再生和转化体系的优化.上海交通大学学报:农业科学版,29(2):41-47.
李建欣.2006.黄瓜组织培养与农杆菌介导的遗传转化体系研究[硕士论文].郑州:河南农业大学.
李蕾,孟永娇,张璐,娄群峰,李季,钱春桃,陈劲枫.2015.黄瓜遗传转化体系优化的研究.华北农学报,30(5):115-121.
李胜男.2016.黄瓜Cs WRKY30基因在霜霉威胁迫中功能分析[硕士论文].哈尔滨:东北农业大学.
李艳华.2016.黄瓜离体再生和农杆菌介导的遗传转化体系的建立与优化[硕士论文].新乡:河南科技学院.
刘丽英,邓莲,任华中.2006.黄瓜遗传与育种研究进展.泰安:中国园艺学会第七届青年学术讨论会.
刘艳.2016.农杆菌介导的谷氧还蛋白基因Cs GRX4对黄瓜遗传转化体系的建立[硕士论文].杨凌:西北农林科技大学.
刘玉霞,叶德友,欧巧明,李敏权.2014.黄瓜离体子叶再生体系的建立.北方园艺,(19):113-117.
秦智伟,王奕童,刘东,辛明,周秀艳.2018.黄瓜Cs CBS克隆及其对霜霉病和棒孢叶斑病抗性功能的初步验证.东北农业大学学报,49(2):39-47.
王璐,陈小燕,张力,陈怀勐,任华中.2008.不同因素对黄瓜未受精子房胚状体诱导的影响.西北农业学报,17(4):267-270.
王烨,顾兴芳,张圣平.2011.暗培养对黄瓜子叶节再生频率及其相关酶活性和可溶性蛋白含量的影响.西北植物学报,31(9):1793-1798.
王烨,顾兴芳,张圣平,苗晗,陈国华,谢丙炎.2014.RNAi载体导入黄瓜的遗传转化体系.植物学报,49(2):183-189.
王烨,顾兴芳,张圣平,苗晗.2015.黄瓜未受精胚珠离体培养及单倍体植株再生.园艺学报,42(11):2174-2182.
魏文霞,刘苗苗,张婷婷,杨玉婷,李许真,郭晓,陈书霞.2019.黄瓜胚状体途径再生体系的建立.西北农林科技大学学报:自然科学版,47(4):1-9.
闻小霞,刘刚,周爱凤,赵荷仙.2014.黄瓜育种工作研究进展.农业科技通讯,(5):12-14.
许娜,储俊.2008.黄瓜遗传转化研究进展.中国蔬菜,(12):38-42.
杨爱馥,朱延明,侯爱菊.2003.几个影响黄瓜子叶体细胞胚胎发生的因素.植物生理学报,39(3):206-208.
杨建霞.2005.日光温室黄瓜连作障碍研究及防治对策.甘肃农业,(11):209.
余阳俊,朱其杰.1992.黄瓜成熟胚离体培养中的胚状体诱导和植株再生.植物生理学通讯,28(1):37-39.
袁澍,贾勇炯,林宏辉.2003.诱导植物体细胞胚发生的几个生理因素.植物生理学报,39(5):508-512.
张若纬,顾兴芳,王烨,张圣平,张宝玺.2009.基因型和6-BA对黄瓜子叶节再生频率的影响.中国蔬菜,(22):45-48.
张圣平,顾兴芳,王烨,苗晗.2010.“十一五”我国黄瓜遗传育种研究进展.中国蔬菜,(22):1-10.
张兴国,刘佩瑛.1998.黄瓜原生质体培养再生胚状体和植物研究.西南农业大学学报,20(4):288-292.
赵子瑶.2018.与黄瓜株型性状相关的两个候选基因(长下胚轴Cs NABP和圆叶Cs PID)的功能初步分析[硕士论文].杨凌:西北农林科技大学.
朱宗源,吴世昌,匡开源,王冬生,袁永达,张繁琴.2000.现代温室黄瓜病虫害发生与防治.上海农业学报,16(s):1-5.
Cade R M,Wehner T C,Blazich F A.1990.Somatic embryos derived from cotyledons of cucumber.Journal of the American Society for Horticultural Science,115(4):691-696.
Chee P P.1990.High frequency of somatic embryogenesis and recovery of fertile cucumber plants.Hort Science,25(7):792-793.
Chee P P,Tricoli D M.1988.Somatic embryogenesis and plant regeneration from cell suspension cultures of Cucumis sativus L.Plant Cell Reports,7(4):274-277.
Colijn-Hooymans C M,Hakkert J C,Jansen J,Custers J B M.1994.Competence for regeneration of cucumber cotyledons is restricted to specific developmental stages.Plant Cell Tissue and Organ Culture,39(3):211-217.
Kantharajah A S,Dodd W A.1990.Factors that influence the yield and viability of cucumber(Cucumis sativus L.)cotyledon protoplasts.Australian Journal of Botany,38:169-175.
Kim Y H,Janik J.1989.Origin of somatic embryos in celery tissue culture.Hortscience,24:671-673.
Kim H A,Lee B Y,Jeon J J,Choi D W,Choi P S,Utomo S D,Lee J H,Kang T H,Lee Y J.2008.Gus gene expression and plant regeneration via somatic embryogenesis in cucumber(Cucumis sativus L.).Journal of Plant Biotechnology,35(4):275-280.
Kuijpers A M,Bouman H,Klerk G J.1996.Increase of embryogenic callus formation in cucumber by initial culture on high concentration of 2,4-Dichlorophenoxyacetic acid.Plant Cell Tissue and Organ Culture,46(1):81-83.
Masuda H,Oohashi S I,Tokuji Y,Mizue Y.1995.Direct embryo formation from epidermal cells of carrot hypocotyls.Journal of Plant Physiology,145(4):531-534.
Oláh R,Zok A,Pedryc A,Howard S,Kovács L G.2009.Somatic embryogenesis in a broad spectrum of grape genotypes.Scientia Horticulturae,120(1):134-137.
Punja Z K,Abbas N,Sarmento G G,Tang F A.1990.Regeneration of Cucumis sativus var.sativus and C.sativus var.hardwickii,C.melo,and C.metuliferus from explants through somatic embryogenesis and organogenesis.Plant Cell Tissue&Organ Culture,21(2):93-102.
Staub J E,Chung S M,Fazio G.2005.Conformity and genetic relatedness estimation in crop species having a narrow genetic base:the case of cucumber.Plant Breeding,124(1):44-53.
Ziv M,Gadasi G.1986.Enhanced embryogenesis and plant regeneration from cucumber(Cucumis sativus L.)callus by activated charcoal in solid/liquid double-layer cultures.Plant Science,47(2):115-122.
陈继峰.2007.黄瓜离体器官再生植株研究进展.上海农业学报,23(1):114-118.
陈小鹏,刘栓桃,孙小镭,赵智中,王秀峰.2005.黄瓜未授粉子房的胚状体诱导研究初报.西北农业学报,14(2):148-151.
段莉莉,朱拼玉,李季,陈劲枫.2018.基因工程技术在甜瓜属作物分子育种中的应用与发展.中国瓜菜,31(8):1-8.
谷安根,王立军.1997.子叶区理论研究进展.植物学通报,14(3):30-39.
何莉莉,陈阳,陈俊琴,佟贤,韩成玲.2010.黄瓜连作栽培中营养基质的微量元素、病原菌和产量的变化.沈阳农业大学学报,41(1):13-17.
贺丽娜,量银丽,高静,熊亚梅,周茂娟,韦泽秀.2008.连作对设施黄瓜产量和品质及土壤酶活性的影响.西北农林科技大学学报,36(5):155-159.
胡适宜.1982.被子植物胚胎学.北京:高等教育出版社:237-247.
黄东梅,别蓓蓓,潘俊松,何欢乐,蔡润.2012.黄瓜(Cucumis sativus L.)离体再生和转化体系的优化.上海交通大学学报:农业科学版,29(2):41-47.
李建欣.2006.黄瓜组织培养与农杆菌介导的遗传转化体系研究[硕士论文].郑州:河南农业大学.
李蕾,孟永娇,张璐,娄群峰,李季,钱春桃,陈劲枫.2015.黄瓜遗传转化体系优化的研究.华北农学报,30(5):115-121.
李胜男.2016.黄瓜Cs WRKY30基因在霜霉威胁迫中功能分析[硕士论文].哈尔滨:东北农业大学.
李艳华.2016.黄瓜离体再生和农杆菌介导的遗传转化体系的建立与优化[硕士论文].新乡:河南科技学院.
刘丽英,邓莲,任华中.2006.黄瓜遗传与育种研究进展.泰安:中国园艺学会第七届青年学术讨论会.
刘艳.2016.农杆菌介导的谷氧还蛋白基因Cs GRX4对黄瓜遗传转化体系的建立[硕士论文].杨凌:西北农林科技大学.
刘玉霞,叶德友,欧巧明,李敏权.2014.黄瓜离体子叶再生体系的建立.北方园艺,(19):113-117.
秦智伟,王奕童,刘东,辛明,周秀艳.2018.黄瓜Cs CBS克隆及其对霜霉病和棒孢叶斑病抗性功能的初步验证.东北农业大学学报,49(2):39-47.
王璐,陈小燕,张力,陈怀勐,任华中.2008.不同因素对黄瓜未受精子房胚状体诱导的影响.西北农业学报,17(4):267-270.
王烨,顾兴芳,张圣平.2011.暗培养对黄瓜子叶节再生频率及其相关酶活性和可溶性蛋白含量的影响.西北植物学报,31(9):1793-1798.
王烨,顾兴芳,张圣平,苗晗,陈国华,谢丙炎.2014.RNAi载体导入黄瓜的遗传转化体系.植物学报,49(2):183-189.
王烨,顾兴芳,张圣平,苗晗.2015.黄瓜未受精胚珠离体培养及单倍体植株再生.园艺学报,42(11):2174-2182.
魏文霞,刘苗苗,张婷婷,杨玉婷,李许真,郭晓,陈书霞.2019.黄瓜胚状体途径再生体系的建立.西北农林科技大学学报:自然科学版,47(4):1-9.
闻小霞,刘刚,周爱凤,赵荷仙.2014.黄瓜育种工作研究进展.农业科技通讯,(5):12-14.
许娜,储俊.2008.黄瓜遗传转化研究进展.中国蔬菜,(12):38-42.
杨爱馥,朱延明,侯爱菊.2003.几个影响黄瓜子叶体细胞胚胎发生的因素.植物生理学报,39(3):206-208.
杨建霞.2005.日光温室黄瓜连作障碍研究及防治对策.甘肃农业,(11):209.
余阳俊,朱其杰.1992.黄瓜成熟胚离体培养中的胚状体诱导和植株再生.植物生理学通讯,28(1):37-39.
袁澍,贾勇炯,林宏辉.2003.诱导植物体细胞胚发生的几个生理因素.植物生理学报,39(5):508-512.
张若纬,顾兴芳,王烨,张圣平,张宝玺.2009.基因型和6-BA对黄瓜子叶节再生频率的影响.中国蔬菜,(22):45-48.
张圣平,顾兴芳,王烨,苗晗.2010.“十一五”我国黄瓜遗传育种研究进展.中国蔬菜,(22):1-10.
张兴国,刘佩瑛.1998.黄瓜原生质体培养再生胚状体和植物研究.西南农业大学学报,20(4):288-292.
赵子瑶.2018.与黄瓜株型性状相关的两个候选基因(长下胚轴Cs NABP和圆叶Cs PID)的功能初步分析[硕士论文].杨凌:西北农林科技大学.
朱宗源,吴世昌,匡开源,王冬生,袁永达,张繁琴.2000.现代温室黄瓜病虫害发生与防治.上海农业学报,16(s):1-5.
Cade R M,Wehner T C,Blazich F A.1990.Somatic embryos derived from cotyledons of cucumber.Journal of the American Society for Horticultural Science,115(4):691-696.
Chee P P.1990.High frequency of somatic embryogenesis and recovery of fertile cucumber plants.Hort Science,25(7):792-793.
Chee P P,Tricoli D M.1988.Somatic embryogenesis and plant regeneration from cell suspension cultures of Cucumis sativus L.Plant Cell Reports,7(4):274-277.
Colijn-Hooymans C M,Hakkert J C,Jansen J,Custers J B M.1994.Competence for regeneration of cucumber cotyledons is restricted to specific developmental stages.Plant Cell Tissue and Organ Culture,39(3):211-217.
Kantharajah A S,Dodd W A.1990.Factors that influence the yield and viability of cucumber(Cucumis sativus L.)cotyledon protoplasts.Australian Journal of Botany,38:169-175.
Kim Y H,Janik J.1989.Origin of somatic embryos in celery tissue culture.Hortscience,24:671-673.
Kim H A,Lee B Y,Jeon J J,Choi D W,Choi P S,Utomo S D,Lee J H,Kang T H,Lee Y J.2008.Gus gene expression and plant regeneration via somatic embryogenesis in cucumber(Cucumis sativus L.).Journal of Plant Biotechnology,35(4):275-280.
Kuijpers A M,Bouman H,Klerk G J.1996.Increase of embryogenic callus formation in cucumber by initial culture on high concentration of 2,4-Dichlorophenoxyacetic acid.Plant Cell Tissue and Organ Culture,46(1):81-83.
Masuda H,Oohashi S I,Tokuji Y,Mizue Y.1995.Direct embryo formation from epidermal cells of carrot hypocotyls.Journal of Plant Physiology,145(4):531-534.
Oláh R,Zok A,Pedryc A,Howard S,Kovács L G.2009.Somatic embryogenesis in a broad spectrum of grape genotypes.Scientia Horticulturae,120(1):134-137.
Punja Z K,Abbas N,Sarmento G G,Tang F A.1990.Regeneration of Cucumis sativus var.sativus and C.sativus var.hardwickii,C.melo,and C.metuliferus from explants through somatic embryogenesis and organogenesis.Plant Cell Tissue&Organ Culture,21(2):93-102.
Staub J E,Chung S M,Fazio G.2005.Conformity and genetic relatedness estimation in crop species having a narrow genetic base:the case of cucumber.Plant Breeding,124(1):44-53.
Ziv M,Gadasi G.1986.Enhanced embryogenesis and plant regeneration from cucumber(Cucumis sativus L.)callus by activated charcoal in solid/liquid double-layer cultures.Plant Science,47(2):115-122.