西瓜离体雌核发育诱导单倍体植株再生
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摘要
为建立完善的西瓜未授粉子房离体培养技术体系,本研究以西瓜未授粉子房为外植体,在离体条件下研究了激素种类、基因型、取样时间和热激处理时间对胚状体诱导效果的影响,并对再生植株倍性进行了鉴定。结果表明:外植体在MS+3.0 mg/L 2,4-D+1.5 mg/L 6-BA+0.5 mg/L NAA或MS+0.04 mg/L TDZ两种培养基上,胚状体诱导率分别为23%和22.3%,二者之间差异不显著;不同西瓜品种胚状体诱导率差异显著,‘早佳’最高可达23.3%,‘QL-2’最低只有2.3%;开花前12 h取样接种,胚状体诱导率最高可达22.3%;在35℃黑暗条件下热激5 d有利于子房的膨大和出胚,平均每瓶出胚5.3个;流式细胞术是鉴定再生植株倍性快速准确的方法。本研究结果为西瓜单倍体育种技术在育种实践中的应用提供了技术保障。
In order to establish a complete watermelon unfertilized ovary in vitro culture technological system, In this study, using unfertilized ovaries of watermelon as explants, we studied the effects of hormone types, genotypes, sampling time and heat shock treatment time on embryoid induction via in vitro culture, and we identified the ploidy of the regenerated plants. The results showed that media MS+3.0 mg/L 2,4-D+1.5 mg/L 6-BA +0.5 mg/L NAA and MS+0.04 mg/L TDZ achieved different embryoid induction rates, 23% and 22.3%, respectively, without significant difference. There were remarkable differences on embryoid induction rates of different watermelon cultivars. The 'zao jia' was up to 23.3%, and the 'QL-2' was only 2.3%. Sampling and inoculating 12 hours before flowering, and embryoid induction rate was up to 22.3%. Treatments with darkness and heat shock(35℃) for 5 days could help ovaries swelling and induce embryoids. The average of embryo was 5.3 per bottle. Flow cytometry was a rapid and accurate method for identifying the ploidy of regenerated plants The results of this study provide a technical guarantee for the application of watermelon haploid breeding technology in breeding practice.
In order to establish a complete watermelon unfertilized ovary in vitro culture technological system, In this study, using unfertilized ovaries of watermelon as explants, we studied the effects of hormone types, genotypes, sampling time and heat shock treatment time on embryoid induction via in vitro culture, and we identified the ploidy of the regenerated plants. The results showed that media MS+3.0 mg/L 2,4-D+1.5 mg/L 6-BA +0.5 mg/L NAA and MS+0.04 mg/L TDZ achieved different embryoid induction rates, 23% and 22.3%, respectively, without significant difference. There were remarkable differences on embryoid induction rates of different watermelon cultivars. The 'zao jia' was up to 23.3%, and the 'QL-2' was only 2.3%. Sampling and inoculating 12 hours before flowering, and embryoid induction rate was up to 22.3%. Treatments with darkness and heat shock(35℃) for 5 days could help ovaries swelling and induce embryoids. The average of embryo was 5.3 per bottle. Flow cytometry was a rapid and accurate method for identifying the ploidy of regenerated plants The results of this study provide a technical guarantee for the application of watermelon haploid breeding technology in breeding practice.
引文
Diao W.P.,Chen J.F.,Lei C.,Song H.,and Zhang X.Q.,2008,Factors a ffecting embryo formation in unpollinated ovary culture of cucumber,Nanjing Nongye Daxue Xuebao(Journal of Nanjing Agricultural University),31(1):137-140(刁卫平,陈劲枫,雷春,宋慧,张晓青,2008,影响黄瓜未授粉子房培养胚状体发生因素的研究,南京农业大学学报,31(1):137-140)
Gémes-Juhúsz A.,Balogh P.,Ferenczy A.,and Kristóf Z.,2002,Effect of optimal stage of female gametophyte and heat treatment on in vitro gynogenesis induction in cucumber(Cucumis sativus L.),Plant Cell Reports,21(2):105-111
Hu F.F.,2016,Research on improving technology of megaspore culture in vitro and plantlet regeneration in Cucurbita pepo L.,Thesis for M.S.,Northwest A&F University,Supervisor:Chen Y.A.,pp.14-15(胡芳芳,2016,西葫芦双单倍体育种技术优化研究,硕士学位论文,西北农林科技大学,导师:程永安,pp.14-15)
Li J.J.,Sheng P.,and Yue Y.L.,2013,Embryogenesis and development of isolated microspore in Chinese cabbage,Agricultural Science&Technology,14(6):813-815,831
Mei D.S.,Wang H.Z.,Li Y.C.,Hu Q.,Li Y.D.,and Xu Y.S.,2007,Factors influencing microspore embrogenesis and construction of dihaploid population in yellow-seeded Brassica napus L.,Huabei Nongxuebao(Acta Agriculturae Boreali-Sinica),22(1):112-115(梅德圣,王汉中,李云昌,胡琼,李英德,徐育松,2007,油菜小孢子培养影响因素及黄籽油菜双单倍体群体的构建,华北农学报,22(1):112-115)
Min Z.Y.,Li H.,Zou T.,Tong L.,Cheng J.,and Sun X.W.,2016a,Studies of in vitro culture and plant regeneration of unfertilized ovary of pumpkin,Zhiwu Xuebao(Chinese Bulletin of Botany),51(1):74-80(闵子扬,李涵,邹甜,童龙,成娟,孙小武,2016a,南瓜未授粉子房离体培养及植株再生,植物学报,51(1):74-80)
Min Z.Y.,Liu Z.F.,Yang H.B.,Cheng J.,and Sun X.W.,2016b,Ploidy identification methods for regenerated plant from unfertilized ovary of pumpkin,Hunan Nongye Daxue Xuebao(Journal of Hunan Agricultural University(Natural Sciences)),42(2):162-165(闵子扬,刘泽发,杨红波,成娟,孙小武,2016b,南瓜胚囊再生植株倍性鉴定方法的比较,湖南农业大学学报(自然科学版),42(2):162-165)
Niu M.M.,2012,Study of gynogenesis in vitro to induce haploid plants in melon,Thesis for M.S.,Northeast Agricultural U-niversity,Supervisor:Luan F.S.,pp.26-27(牛明明,2012,甜瓜离体雌核发育诱导单倍体研究,硕士学位论文,东北农业大学,导师:栾非时,pp.26-27)
Shalaby T.A.,2007,Factors afecting haploid induction through in vitro gynogenesis in summer squash(Cucurbita pepo L.),Scientia Horticulturae,115(1):1-6
Sun S.R.,Zhang P.,Hu J.B.,Sun L.P.,Zhang M.,and Sun Z.Q.,2013,In vitro culture of pumpkin unfertilized ovules and plant regeneration,Zhiwu Xuebao(Chinese Bulletin of Botany),48(1):79-86(孙守如,章鹏,胡建斌,孙利萍,张曼,孙治强,2013,南瓜未受精胚珠的离体培养及植株再生,植物学报,48(1):79-86)
Tang T.X.,Cheng Y.A.,Zhang E.H.,Dong H.,and Xu Z.M.,2015,Effect of exogenous additions on embryo induction of summer squash(Cucurbita pepo L.),Xibei Nongye Xuebao(Acta Agriculturae Boreali-occidentalis Sinica),24(6):84-89(唐桃霞,程永安,张恩慧,董韩,许忠民,2015,外源添加物对西葫芦胚状体诱导效果研究,西北农业学报,24(6):84-89)
Wang Y.,Gu X.F.,and Zhang S.P.,2008,Study the effect of pretreatment and exogenous hormones on embryoids induced from unpollinated ovaries of cucumber,Huabei Nongxuebao(Acta Agriculturae Boreali-Sinica),(23):50-53(王烨,顾兴芳,张圣平,2008,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,华北农学报,(23):50-53)
Xie B.,Wang X.F.,and Fan Z.C.,2006,Analysis of the main factors effecting on plantlet regenerating from embryoid via in vitro gynogenesis in summer squash(Cucurbita pepo L.),Xibei Nongye Xuebao(Acta Agriculturae Boreali-occidentalis Sinica),15(5):182-186(谢冰,王秀峰,樊治成,2006,西葫芦离体雌核发育胚状体成苗影响因子分析,西北农业学报,15(5):182-186)
Xu X.F.,and Huang X.L.,2003,TDZ:An efficacious plant growth regulato,Zhiwuxue Tongbao(Chinese Bulletin of Botany),20(2):227-237(徐晓峰,黄学林,2003,TDZ:一种有效的植物生长调节剂,植物学通报,20(2):227-237)
Yang J.Y.,and Li X.F.,2002,In vitro induction of plant genogenetic haploids,Zhiwuxue Tongbao(Chinese Bulletin of Botany),19(5):552-559(杨江义,李旭锋,2002,植物雌性单倍体的离体诱导,植物学通报,19(5):552-559)
Zeng A.S.,Gao B.,Song L.X.,Zhang Y.X.,Li J.Q.,Li Y.,Hou X.L.,and Yan J.Y.,2014,Cytological analysis of microspore embryogenesis in white cabbage(Brassica oleracea L.var.capitate)isolated microspore culture,Nanjing Nongye Daxue Xuebao(Journal of Nanjing Agricultural University),37(5):47-54(曾爱松,高兵,宋立晓,张云霞,李健绮,李英,侯喜林,严继勇,2014,结球甘蓝小孢子胚胎发生的细胞学研究,南京农业大学学报,37(5):47-54)
Zeng W.D.,Lu L.Y.,Xie X.Y.,and Yan H.B.,2015,Research on somatic embryogenesis and plant regeneration of cassava,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(7):1522-1526(曾文丹,陆柳英,谢向誉,严华兵,2105,木薯体细胞胚的发生及植株再生的研究,基因组学与应用生物学,34(7):1522-1526)
Gémes-Juhúsz A.,Balogh P.,Ferenczy A.,and Kristóf Z.,2002,Effect of optimal stage of female gametophyte and heat treatment on in vitro gynogenesis induction in cucumber(Cucumis sativus L.),Plant Cell Reports,21(2):105-111
Hu F.F.,2016,Research on improving technology of megaspore culture in vitro and plantlet regeneration in Cucurbita pepo L.,Thesis for M.S.,Northwest A&F University,Supervisor:Chen Y.A.,pp.14-15(胡芳芳,2016,西葫芦双单倍体育种技术优化研究,硕士学位论文,西北农林科技大学,导师:程永安,pp.14-15)
Li J.J.,Sheng P.,and Yue Y.L.,2013,Embryogenesis and development of isolated microspore in Chinese cabbage,Agricultural Science&Technology,14(6):813-815,831
Mei D.S.,Wang H.Z.,Li Y.C.,Hu Q.,Li Y.D.,and Xu Y.S.,2007,Factors influencing microspore embrogenesis and construction of dihaploid population in yellow-seeded Brassica napus L.,Huabei Nongxuebao(Acta Agriculturae Boreali-Sinica),22(1):112-115(梅德圣,王汉中,李云昌,胡琼,李英德,徐育松,2007,油菜小孢子培养影响因素及黄籽油菜双单倍体群体的构建,华北农学报,22(1):112-115)
Min Z.Y.,Li H.,Zou T.,Tong L.,Cheng J.,and Sun X.W.,2016a,Studies of in vitro culture and plant regeneration of unfertilized ovary of pumpkin,Zhiwu Xuebao(Chinese Bulletin of Botany),51(1):74-80(闵子扬,李涵,邹甜,童龙,成娟,孙小武,2016a,南瓜未授粉子房离体培养及植株再生,植物学报,51(1):74-80)
Min Z.Y.,Liu Z.F.,Yang H.B.,Cheng J.,and Sun X.W.,2016b,Ploidy identification methods for regenerated plant from unfertilized ovary of pumpkin,Hunan Nongye Daxue Xuebao(Journal of Hunan Agricultural University(Natural Sciences)),42(2):162-165(闵子扬,刘泽发,杨红波,成娟,孙小武,2016b,南瓜胚囊再生植株倍性鉴定方法的比较,湖南农业大学学报(自然科学版),42(2):162-165)
Niu M.M.,2012,Study of gynogenesis in vitro to induce haploid plants in melon,Thesis for M.S.,Northeast Agricultural U-niversity,Supervisor:Luan F.S.,pp.26-27(牛明明,2012,甜瓜离体雌核发育诱导单倍体研究,硕士学位论文,东北农业大学,导师:栾非时,pp.26-27)
Shalaby T.A.,2007,Factors afecting haploid induction through in vitro gynogenesis in summer squash(Cucurbita pepo L.),Scientia Horticulturae,115(1):1-6
Sun S.R.,Zhang P.,Hu J.B.,Sun L.P.,Zhang M.,and Sun Z.Q.,2013,In vitro culture of pumpkin unfertilized ovules and plant regeneration,Zhiwu Xuebao(Chinese Bulletin of Botany),48(1):79-86(孙守如,章鹏,胡建斌,孙利萍,张曼,孙治强,2013,南瓜未受精胚珠的离体培养及植株再生,植物学报,48(1):79-86)
Tang T.X.,Cheng Y.A.,Zhang E.H.,Dong H.,and Xu Z.M.,2015,Effect of exogenous additions on embryo induction of summer squash(Cucurbita pepo L.),Xibei Nongye Xuebao(Acta Agriculturae Boreali-occidentalis Sinica),24(6):84-89(唐桃霞,程永安,张恩慧,董韩,许忠民,2015,外源添加物对西葫芦胚状体诱导效果研究,西北农业学报,24(6):84-89)
Wang Y.,Gu X.F.,and Zhang S.P.,2008,Study the effect of pretreatment and exogenous hormones on embryoids induced from unpollinated ovaries of cucumber,Huabei Nongxuebao(Acta Agriculturae Boreali-Sinica),(23):50-53(王烨,顾兴芳,张圣平,2008,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,华北农学报,(23):50-53)
Xie B.,Wang X.F.,and Fan Z.C.,2006,Analysis of the main factors effecting on plantlet regenerating from embryoid via in vitro gynogenesis in summer squash(Cucurbita pepo L.),Xibei Nongye Xuebao(Acta Agriculturae Boreali-occidentalis Sinica),15(5):182-186(谢冰,王秀峰,樊治成,2006,西葫芦离体雌核发育胚状体成苗影响因子分析,西北农业学报,15(5):182-186)
Xu X.F.,and Huang X.L.,2003,TDZ:An efficacious plant growth regulato,Zhiwuxue Tongbao(Chinese Bulletin of Botany),20(2):227-237(徐晓峰,黄学林,2003,TDZ:一种有效的植物生长调节剂,植物学通报,20(2):227-237)
Yang J.Y.,and Li X.F.,2002,In vitro induction of plant genogenetic haploids,Zhiwuxue Tongbao(Chinese Bulletin of Botany),19(5):552-559(杨江义,李旭锋,2002,植物雌性单倍体的离体诱导,植物学通报,19(5):552-559)
Zeng A.S.,Gao B.,Song L.X.,Zhang Y.X.,Li J.Q.,Li Y.,Hou X.L.,and Yan J.Y.,2014,Cytological analysis of microspore embryogenesis in white cabbage(Brassica oleracea L.var.capitate)isolated microspore culture,Nanjing Nongye Daxue Xuebao(Journal of Nanjing Agricultural University),37(5):47-54(曾爱松,高兵,宋立晓,张云霞,李健绮,李英,侯喜林,严继勇,2014,结球甘蓝小孢子胚胎发生的细胞学研究,南京农业大学学报,37(5):47-54)
Zeng W.D.,Lu L.Y.,Xie X.Y.,and Yan H.B.,2015,Research on somatic embryogenesis and plant regeneration of cassava,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(7):1522-1526(曾文丹,陆柳英,谢向誉,严华兵,2105,木薯体细胞胚的发生及植株再生的研究,基因组学与应用生物学,34(7):1522-1526)