黄瓜胚状体途径再生体系的建立
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摘要
【目的】建立黄瓜高频胚状体再生体系,为黄瓜遗传转化体系的完善奠定基础。【方法】以黄瓜品种津研四号、长春密刺、Q8和14-1为材料,研究不同基因型材料愈伤组织的诱导情况;分别取14-1的子叶、子叶节、下胚轴和成熟胚为外植体,研究不同外植体类型的愈伤组织和胚状体产生情况;以14-1子叶节为外植体,研究不同激素及其质量浓度配比对黄瓜胚状体诱导的影响。【结果】4个不同基因型中,欧洲温室型黄瓜种质14-1可诱导出胚状体。在4种外植体类型中,14-1的子叶节可以高效诱导出愈伤组织和胚状体,其诱导率分别为100.0%和34.1%。激素及其质量浓度组合也会影响胚状体的诱导,在愈伤组织诱导阶段,以MS+1.5mg/L 2,4-D为培养基对胚性愈伤组织的诱导效果最好,其对14-1子叶节的诱导率为100%;在分化培养阶段,MS+0.1mg/L 2,4-D+0.5mg/L KT+0.2mg/L IAA有利于体细胞胚的发生,14-1的出胚率为62.5%。显微形态观察发现,黄瓜体细胞胚发育过程包括球形胚、心形胚、鱼雷胚和子叶期胚等时期,这些胚可以发育成正常小植株。【结论】14-1的子叶节经诱导培养基MS+1.5mg/L 2,4-D和分化培养基MS+0.1mg/L 2,4-D+0.5mg/L KT+0.2mg/L IAA培养后,可高效产生胚状体,并可萌发成正常植株。
【Objective】This study established an efficient regeneration system of cucumber via somatic embryogenesis to provide basis for the genetic transformation system.【Method】Four cultivars cucumber such as"Jinyansihao","Changchunmici","Q8"and"14-1"were used to investigate the effection of different genotypes on callus and embryo induction.Different explants types such as cotyledonary node,cotyledon,hypocotyledonary axis and mature embryo from 14-1 cucumber were obtained to study the effection of explants types.Cotyledonary node of 14-1 were used to investigate the callus and embryo induction supplied with different concentration of hormone.【Result】It showed that genotye 14-1 was able to be induced somatic embryos among the four genotypes.The cotyledon,cotyledon nodes and hypocotyls of 14-1 were induced callus efficiently on the first stage,however,only cotyledon nodes in four kind of explants was able to be induced embryogenic callus and embryos,the induction rates were 100.0%and 34.1%,respectively.The different hormone combinations also affected the percentage of embryo induction,and it showed that the suitable hormone combination for embryo induction of 14-1 was on the MS mediums supplying with 1.5 mg/L 2,4-D to induce callus first and then the embryo was induced on MS medium supplying with 0.1 mg/L 2,4-D,0.5 mg/L KT and 0.2 mg/L IAA,and percentage of embryo formation was 62.5%.The globular embryo,heart-shaped embryo,torpedo-shaped embryo and cotyledonary embryo were observed under a stereoscopic microscope.These embryo was able to grow into plants.【Conclusion】The cotyledon nodes of 14-1 can produce somatic embryos efficiently on the optimal embryogenic callus induced medium(MS medium supplemented with 1.5 mg/L 2,4-D)and embryos induced medium(MS medium supplying with 0.1 mg/L 2,4-D,0.5 mg/L KT and 0.2 mg/L IAA).These somatic embryos can germinate and grow into plants.
【Objective】This study established an efficient regeneration system of cucumber via somatic embryogenesis to provide basis for the genetic transformation system.【Method】Four cultivars cucumber such as"Jinyansihao","Changchunmici","Q8"and"14-1"were used to investigate the effection of different genotypes on callus and embryo induction.Different explants types such as cotyledonary node,cotyledon,hypocotyledonary axis and mature embryo from 14-1 cucumber were obtained to study the effection of explants types.Cotyledonary node of 14-1 were used to investigate the callus and embryo induction supplied with different concentration of hormone.【Result】It showed that genotye 14-1 was able to be induced somatic embryos among the four genotypes.The cotyledon,cotyledon nodes and hypocotyls of 14-1 were induced callus efficiently on the first stage,however,only cotyledon nodes in four kind of explants was able to be induced embryogenic callus and embryos,the induction rates were 100.0%and 34.1%,respectively.The different hormone combinations also affected the percentage of embryo induction,and it showed that the suitable hormone combination for embryo induction of 14-1 was on the MS mediums supplying with 1.5 mg/L 2,4-D to induce callus first and then the embryo was induced on MS medium supplying with 0.1 mg/L 2,4-D,0.5 mg/L KT and 0.2 mg/L IAA,and percentage of embryo formation was 62.5%.The globular embryo,heart-shaped embryo,torpedo-shaped embryo and cotyledonary embryo were observed under a stereoscopic microscope.These embryo was able to grow into plants.【Conclusion】The cotyledon nodes of 14-1 can produce somatic embryos efficiently on the optimal embryogenic callus induced medium(MS medium supplemented with 1.5 mg/L 2,4-D)and embryos induced medium(MS medium supplying with 0.1 mg/L 2,4-D,0.5 mg/L KT and 0.2 mg/L IAA).These somatic embryos can germinate and grow into plants.
引文
[1]Kim S G,Chang J R,Cha H C,et al.Callus growth and plant regeneration in diverse cultivars of cucumber(Cucumis sativus L.)[J].Plant Cell,Tissue and Organ Culture,1988,12(1):67-74.
[2]赵军良,马蓉丽,李昌华,等.黄瓜子叶组织培养再生植株[J].山西农业科学,1996,24(1):39-41.Zhao J L,Ma R L,Li C H,et al.The cotyledon culture of cucumber in vitro and plant regeneration[J].Journal of Shanxi Agricultural Sciences,1996,24(1):39-41.
[3]侯爱菊,朱延明,杨爱馥,等.诱导黄瓜直接器官发生主要影响因素的研究[J].园艺学报,2003,30(1):101-103.Hou A J,Zhu Y M,Yang A F,et al.Main factors influencing the frequency of direct organogenesis of cucumber in vitro[J].Acta Horticulturae Sinica,2003,30(1):101-103.
[4]赵隽,王华,潘俊松,等.黄瓜子叶节离体再生体系的研究[J].上海交通大学学报(农业科学版),2004,22(1):43-47,53.Zhao J,Wang H,Pan J S,et al.In vitro culture and plantlet regeneration from cotyledonary nodes of cucumber(Cucumis sativus L.)[J].Journal of Shanghai Jiaotong University(Agricultural Science Edition),2004,22(1):43-47,53.
[5]贾士荣,罗美中,林云.黄瓜胚性细胞悬浮培养及原生质体的植株再生[J].植物学报,1988,30(5):463-467.Jia S R,Luo M Z,Lin Y.Embryogenic suspension culture and plant regeneration from suspension-derived protoplasts of cucumber(Cucumis sativus L.)[J].Acta Botanica Sinica,1988,30(5):463-467.
[6]陈继峰.黄瓜离体器官再生植株研究进展[J].上海农业学报,2007,23(1):114-118.Chen J F.The advance of researches on the plant regeneration from excised organs of Cucumis sativus[J].Acta Agriculturae Shanghai,2007,23(1):114-118.
[7]Colijn H C M,Bouwer R,Dons J J M.Plant regenerationfrom cucumber protoplasts[J].Plant Cell Tissue and Organ Culture,1988,12(2):147-150.
[8]Punja Z K,Tang F A,Sarmento G G.Isolation,culture and plantlet regeneration from cotyledon and mesophyll protoplasts of two pickling cucumber(C.sativus L.)genotypes[J].Plant Cell Reports,1990,9(2):61-64.
[9]胡适宜.被子植物胚胎学[M].北京:高等教育出版社,1982:237-247.Hu S Y.The embryology of angiosperms[M].Beijing:Higher Education Press,1982:237-247.
[10]Cade R M,Wehner T C,Blazich F A.Effect of explant age and growth regulator concentration on adventious shoot formation from cotyledonary tissue of cucumber[J].Soc Hort Sci,1990,115:691-696.
[11]赵秀娟,吴定华.黄瓜的组织培养[J].华南农业大学学报,1998,19(4):125-126.Zhao X J,Wu D H.Tissue culture of cucumber[J].Journal of South China Agricultural University,1998,19(4):125-126.
[12]贾士荣,付幼英,林云,等.黄瓜子叶原生质体培养获得体细胞胚发生和再生植株[J].生物工程学报,1985,1(4):54-58.Jia S R,Fu Y Y,Lin Y,et al.Somatic embryogenesis and plant regeneration from cotyledon protoplast culture of cucumber(Cucumis sativus L.)[J].Chinese Journal of Biotechnology,1985,1(4):54-58.
[13]Sladky Z,Jandova B.Micropagation of pea,cucumber and potato[C]//Plant tissue and cell culture application to crop improvement.Prague,Czechoslovakia:Czechoslovak Academy of Sciences,1984:509-510.
[14]Kim J W,Han S K,Kwon S Y,et al.High frequency shoot induction and plant regeneration from cotyledonary hypocotyl explants of cucumber(Cucumis sativus L.)seedlings[J].Journal of Plant Physiology,2000,157(1):136-139.
[15]Raharjo S H T,Punja Z K.Plantlet regeneration from petiole explants of the African horned cucumber,Cucumis metuliferus[J].Plant Cell,Tissue and Organ Culture,1993,32(2):169-174.
[16]张兴国,刘佩瑛.黄瓜原生质体培养再生胚状体和植物研究[J].西南农业大学学报,1998,20(4):287-292.Zhang X G,Liu P Y.Embryogenesis and plant regeneration from cucumber protoplasts[J].Journal of Southwest Agricultural University,1998,20(4):287-292.
[17]Custers J B M,Zijlstra S,Jansen J.Somaclonal variation in cucumber(Cucumis sativus L.)plants regenerated via embryogenesis[J].Acta Botanica Neerlandica,1990,39(2):153-161.
[18]Kuijpers A M,Bouman H,Klerk G J.Increase of embryogenic callus formation in cucumber by initial culture on high concentration of 2,4-dichlorophenoxyacetic acid[J].Plant Cell,Tissue and Organ Culture,1996,46(1):81-83.
[19]Lou H,Kako S.Somatic embryogenesis and plant regeneration in cucumber[J].HortScience,1994,29(8):906-909.
[20]Custers J B M,Deelen J E M van,Bergervoet J H W.Development of callus and somatic embryos from zygotic embryos of cucumber(Cucumis sativus L.)[J].Report,Cucurbit Genetics Cooperative,1988,11:1-2.
[21]余阳俊,朱其杰.黄瓜成熟胚离体培养中的胚状体诱导和植株再生(简报)[J].植物生理学通讯,1992,28(1):37-39.Yu Y J,Zhu Q J.Embryoid induction and plantlet regeneration in mature embryos of cucumber invitro[J].Plant Physiology Communications,1992,28(1):37-39.
[22]Trulson A J,Shahin E A.In vitro plant regeneration in the genus cucumis[J].Plant Science,1986,47(1):35-43.
[23]陈小鹏,刘拴桃,孙晓镭,等.黄瓜未受精子房的胚状体诱导研究初报[J].西北农业学报,2005,14(2):148-151.Chen X P,Liu S T,Sun X L,et al.Primary study on embryoids induced from unpollinated ovaries of cucumber[J].Acta Agriculturae Boreali-occidentalis Sinica,2005,14(2):148-151.
[24]李建欣,葛桂民,庞淑敏,等.三个基因型黄瓜品种未授粉子房胚状体诱导及植株再生研究[J].北方园艺,2012(23):131-134.Li J X,Ge G M,Pang S M,et al.Study on in vitro culture and plantlet regeneration from unpollinated ovary of Cucumis sativus L.[J].Northern Horticulture,2012(23):131-134.
[25]王烨,顾兴芳,张圣平.预处理和外源激素对黄瓜未授粉子房的胚状体诱导的影响[J].华北农学报,2008,23(S1):50-53.Wang Y,Gu X F,Zhang S P.Study the effect of pretreatment and exogenous hormones on embryoids induced from unpollinated ovaries of cucumber[J].Acta Agriculturae Boreali-sinica,2008,23(S1):50-53.
[26]王璐,陈小燕,张力,等.不同因素对黄瓜未受精子房胚状体诱导的影响[J].西北农业学报,2008,17(4):267-270.Wang L,Chen X Y,Zhang L,et al.Effect of different factors on induction of embryoids in unfertilized ovaries of cucumber[J].Acta Agriculturae Boreali-occidentalis Sinica,2008,17(4):267-270.
[27]薛丹丹,张凤生,王保菊,等.黄瓜再生体系的建立[J].北方园艺,2010(7):119-121.Xue D D,Zhang F S,Wang B J.The regeneration system of cucumber[J].Northern Horticulture,2010(7):119-121.
[28]杨宏,刘小俊,梁根云,等.农杆菌介导的华南型黄瓜遗传转化体系的初步研究[J].西南农业学报,2014,27(4):1656-1660.Yang H,Liu X J,Liang G Y,et al.Establishment of genetic transformation system by Agrobacterium tumefaciens of cucumber[J].Southwest China Journal of Agricultural Sciences,2014,27(4):1656-1660.
[29]何业华,方少秋,马均,等.菠萝愈伤组织中体细胞胚起源过程的组织细胞学观察[J].园艺学报,2010,37(5):689-696.He Y H,Fang S Q,Ma J,et al.Histocytology observation on the somatic embryogenesis in Ananas comosus callus[J].Acta Horticulturae Sinica,2010,37(5):689-696.
[30]崔凯荣,陈克明,王晓哲,等.植物体细胞胚胎发生研究的某些现状[J].植物学通报,1993,10(3):14-20.Cui K R,Chen K M,Wang X Z,et al.Current reseach on plant somatic embryogenesis[J].Chinese Bulletin of Botany,1993,10(3):14-20.
[31]谷安根,王立军.子叶节区理论研究进展[J].植物学通报,1997,14(3):30-90.Gu A G,Wang L J.Advances in cotyledon node zone theory[J].Chinese Bulletin of Botany,1997,14(3):30-90.
[2]赵军良,马蓉丽,李昌华,等.黄瓜子叶组织培养再生植株[J].山西农业科学,1996,24(1):39-41.Zhao J L,Ma R L,Li C H,et al.The cotyledon culture of cucumber in vitro and plant regeneration[J].Journal of Shanxi Agricultural Sciences,1996,24(1):39-41.
[3]侯爱菊,朱延明,杨爱馥,等.诱导黄瓜直接器官发生主要影响因素的研究[J].园艺学报,2003,30(1):101-103.Hou A J,Zhu Y M,Yang A F,et al.Main factors influencing the frequency of direct organogenesis of cucumber in vitro[J].Acta Horticulturae Sinica,2003,30(1):101-103.
[4]赵隽,王华,潘俊松,等.黄瓜子叶节离体再生体系的研究[J].上海交通大学学报(农业科学版),2004,22(1):43-47,53.Zhao J,Wang H,Pan J S,et al.In vitro culture and plantlet regeneration from cotyledonary nodes of cucumber(Cucumis sativus L.)[J].Journal of Shanghai Jiaotong University(Agricultural Science Edition),2004,22(1):43-47,53.
[5]贾士荣,罗美中,林云.黄瓜胚性细胞悬浮培养及原生质体的植株再生[J].植物学报,1988,30(5):463-467.Jia S R,Luo M Z,Lin Y.Embryogenic suspension culture and plant regeneration from suspension-derived protoplasts of cucumber(Cucumis sativus L.)[J].Acta Botanica Sinica,1988,30(5):463-467.
[6]陈继峰.黄瓜离体器官再生植株研究进展[J].上海农业学报,2007,23(1):114-118.Chen J F.The advance of researches on the plant regeneration from excised organs of Cucumis sativus[J].Acta Agriculturae Shanghai,2007,23(1):114-118.
[7]Colijn H C M,Bouwer R,Dons J J M.Plant regenerationfrom cucumber protoplasts[J].Plant Cell Tissue and Organ Culture,1988,12(2):147-150.
[8]Punja Z K,Tang F A,Sarmento G G.Isolation,culture and plantlet regeneration from cotyledon and mesophyll protoplasts of two pickling cucumber(C.sativus L.)genotypes[J].Plant Cell Reports,1990,9(2):61-64.
[9]胡适宜.被子植物胚胎学[M].北京:高等教育出版社,1982:237-247.Hu S Y.The embryology of angiosperms[M].Beijing:Higher Education Press,1982:237-247.
[10]Cade R M,Wehner T C,Blazich F A.Effect of explant age and growth regulator concentration on adventious shoot formation from cotyledonary tissue of cucumber[J].Soc Hort Sci,1990,115:691-696.
[11]赵秀娟,吴定华.黄瓜的组织培养[J].华南农业大学学报,1998,19(4):125-126.Zhao X J,Wu D H.Tissue culture of cucumber[J].Journal of South China Agricultural University,1998,19(4):125-126.
[12]贾士荣,付幼英,林云,等.黄瓜子叶原生质体培养获得体细胞胚发生和再生植株[J].生物工程学报,1985,1(4):54-58.Jia S R,Fu Y Y,Lin Y,et al.Somatic embryogenesis and plant regeneration from cotyledon protoplast culture of cucumber(Cucumis sativus L.)[J].Chinese Journal of Biotechnology,1985,1(4):54-58.
[13]Sladky Z,Jandova B.Micropagation of pea,cucumber and potato[C]//Plant tissue and cell culture application to crop improvement.Prague,Czechoslovakia:Czechoslovak Academy of Sciences,1984:509-510.
[14]Kim J W,Han S K,Kwon S Y,et al.High frequency shoot induction and plant regeneration from cotyledonary hypocotyl explants of cucumber(Cucumis sativus L.)seedlings[J].Journal of Plant Physiology,2000,157(1):136-139.
[15]Raharjo S H T,Punja Z K.Plantlet regeneration from petiole explants of the African horned cucumber,Cucumis metuliferus[J].Plant Cell,Tissue and Organ Culture,1993,32(2):169-174.
[16]张兴国,刘佩瑛.黄瓜原生质体培养再生胚状体和植物研究[J].西南农业大学学报,1998,20(4):287-292.Zhang X G,Liu P Y.Embryogenesis and plant regeneration from cucumber protoplasts[J].Journal of Southwest Agricultural University,1998,20(4):287-292.
[17]Custers J B M,Zijlstra S,Jansen J.Somaclonal variation in cucumber(Cucumis sativus L.)plants regenerated via embryogenesis[J].Acta Botanica Neerlandica,1990,39(2):153-161.
[18]Kuijpers A M,Bouman H,Klerk G J.Increase of embryogenic callus formation in cucumber by initial culture on high concentration of 2,4-dichlorophenoxyacetic acid[J].Plant Cell,Tissue and Organ Culture,1996,46(1):81-83.
[19]Lou H,Kako S.Somatic embryogenesis and plant regeneration in cucumber[J].HortScience,1994,29(8):906-909.
[20]Custers J B M,Deelen J E M van,Bergervoet J H W.Development of callus and somatic embryos from zygotic embryos of cucumber(Cucumis sativus L.)[J].Report,Cucurbit Genetics Cooperative,1988,11:1-2.
[21]余阳俊,朱其杰.黄瓜成熟胚离体培养中的胚状体诱导和植株再生(简报)[J].植物生理学通讯,1992,28(1):37-39.Yu Y J,Zhu Q J.Embryoid induction and plantlet regeneration in mature embryos of cucumber invitro[J].Plant Physiology Communications,1992,28(1):37-39.
[22]Trulson A J,Shahin E A.In vitro plant regeneration in the genus cucumis[J].Plant Science,1986,47(1):35-43.
[23]陈小鹏,刘拴桃,孙晓镭,等.黄瓜未受精子房的胚状体诱导研究初报[J].西北农业学报,2005,14(2):148-151.Chen X P,Liu S T,Sun X L,et al.Primary study on embryoids induced from unpollinated ovaries of cucumber[J].Acta Agriculturae Boreali-occidentalis Sinica,2005,14(2):148-151.
[24]李建欣,葛桂民,庞淑敏,等.三个基因型黄瓜品种未授粉子房胚状体诱导及植株再生研究[J].北方园艺,2012(23):131-134.Li J X,Ge G M,Pang S M,et al.Study on in vitro culture and plantlet regeneration from unpollinated ovary of Cucumis sativus L.[J].Northern Horticulture,2012(23):131-134.
[25]王烨,顾兴芳,张圣平.预处理和外源激素对黄瓜未授粉子房的胚状体诱导的影响[J].华北农学报,2008,23(S1):50-53.Wang Y,Gu X F,Zhang S P.Study the effect of pretreatment and exogenous hormones on embryoids induced from unpollinated ovaries of cucumber[J].Acta Agriculturae Boreali-sinica,2008,23(S1):50-53.
[26]王璐,陈小燕,张力,等.不同因素对黄瓜未受精子房胚状体诱导的影响[J].西北农业学报,2008,17(4):267-270.Wang L,Chen X Y,Zhang L,et al.Effect of different factors on induction of embryoids in unfertilized ovaries of cucumber[J].Acta Agriculturae Boreali-occidentalis Sinica,2008,17(4):267-270.
[27]薛丹丹,张凤生,王保菊,等.黄瓜再生体系的建立[J].北方园艺,2010(7):119-121.Xue D D,Zhang F S,Wang B J.The regeneration system of cucumber[J].Northern Horticulture,2010(7):119-121.
[28]杨宏,刘小俊,梁根云,等.农杆菌介导的华南型黄瓜遗传转化体系的初步研究[J].西南农业学报,2014,27(4):1656-1660.Yang H,Liu X J,Liang G Y,et al.Establishment of genetic transformation system by Agrobacterium tumefaciens of cucumber[J].Southwest China Journal of Agricultural Sciences,2014,27(4):1656-1660.
[29]何业华,方少秋,马均,等.菠萝愈伤组织中体细胞胚起源过程的组织细胞学观察[J].园艺学报,2010,37(5):689-696.He Y H,Fang S Q,Ma J,et al.Histocytology observation on the somatic embryogenesis in Ananas comosus callus[J].Acta Horticulturae Sinica,2010,37(5):689-696.
[30]崔凯荣,陈克明,王晓哲,等.植物体细胞胚胎发生研究的某些现状[J].植物学通报,1993,10(3):14-20.Cui K R,Chen K M,Wang X Z,et al.Current reseach on plant somatic embryogenesis[J].Chinese Bulletin of Botany,1993,10(3):14-20.
[31]谷安根,王立军.子叶节区理论研究进展[J].植物学通报,1997,14(3):30-90.Gu A G,Wang L J.Advances in cotyledon node zone theory[J].Chinese Bulletin of Botany,1997,14(3):30-90.
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