蔗糖和甘露醇复合处理对菊花低温离体保存的影响
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摘要
试验在(7±2)℃下,以MS培养基添加不同浓度蔗糖和甘露醇复合处理对菊花‘蒙娜丽莎黄’试管苗进行离体保存,统计试管苗存活率、株高和枯叶率,观察保存结束后试管苗组织结构,并对保存后植株恢复生长能力和再生植株遗传稳定性进行检测。结果表明,保存12个月后,15 g/L蔗糖与15 g/L甘露醇复合处理存活率最高,存活率达93.33%,枯叶率为17.6%;0 g/L蔗糖与5 g/L甘露醇处理存活率达86.67%;30 g/L蔗糖下,随甘露醇浓度增加存活率递增,以与20 g/L甘露醇的处理存活率最高,存活率为80%,枯叶率为35.2%;而高浓度蔗糖(45 g/L, 60 g/L)与甘露醇复合处理对试管苗保存效果不佳。保存12个月后试管苗叶片和茎段细胞变小、细胞密度增加;恢复正常培养后生长良好,形态正常,SSR-PCR扩增图谱与对照无差异,保持了良好的遗传稳定性。该结果为菊花低温离体保存提供了技术支持。
In this study, we conserved the test tube plantlets of chrysanthemum 'Monalisa Yellow' in vitro at(7±2)℃in MS medium, supplemented by a combined treatment of sucrose and mannitol at different concentrations to count the survival rate, plant height, and wilthered leaf rate of the test-tube plantlets and observe the tissure structure of the test-tube plantlets after preservation. The recovery growth ability and genetic stability of regenerated plants after preservation were also investigated. The results showed that the highest survival rate of 93.33% was observed for the combined treatment of 15 g/L sucrose and 15 g/L mannitol after 12 months conservation, with a wilthered leaf rate of 17.6%, and the survival rate was 86.67% for the treatment of 0 g/L sucrose and 5 g/L mannitol. When 30 g/L sucrose was added, the survival rate of chrysanthemum increased with the increase of mannitol concentration, and when 20 g/L mannitol was also added, the highest survival rate of 80% was observed,with the withered leaf rate of 35.2%. However, the combined treatment of high concentration sucrose(45 g/L, 60 g/L)and mannitol had no good effect on the preservation of test-tube plantlets. After the preservation for 12 months, it was found that the cells in leaves and stems of test-tube plantlets became smaller, and the cell density increased.After returning to normal culture, the test tube plantlets grew well with normal morphology, maintaining good genetic stability, and the SSR-PCR amplification map was no different from the control. The results of this study would provide a technical support for the in vitro conservation of chrysanthemum.
In this study, we conserved the test tube plantlets of chrysanthemum 'Monalisa Yellow' in vitro at(7±2)℃in MS medium, supplemented by a combined treatment of sucrose and mannitol at different concentrations to count the survival rate, plant height, and wilthered leaf rate of the test-tube plantlets and observe the tissure structure of the test-tube plantlets after preservation. The recovery growth ability and genetic stability of regenerated plants after preservation were also investigated. The results showed that the highest survival rate of 93.33% was observed for the combined treatment of 15 g/L sucrose and 15 g/L mannitol after 12 months conservation, with a wilthered leaf rate of 17.6%, and the survival rate was 86.67% for the treatment of 0 g/L sucrose and 5 g/L mannitol. When 30 g/L sucrose was added, the survival rate of chrysanthemum increased with the increase of mannitol concentration, and when 20 g/L mannitol was also added, the highest survival rate of 80% was observed,with the withered leaf rate of 35.2%. However, the combined treatment of high concentration sucrose(45 g/L, 60 g/L)and mannitol had no good effect on the preservation of test-tube plantlets. After the preservation for 12 months, it was found that the cells in leaves and stems of test-tube plantlets became smaller, and the cell density increased.After returning to normal culture, the test tube plantlets grew well with normal morphology, maintaining good genetic stability, and the SSR-PCR amplification map was no different from the control. The results of this study would provide a technical support for the in vitro conservation of chrysanthemum.
引文
Ai P.F.,and Luo Z.R.,2004,Conservation of in vitro shoots of persimmon and date plum by slow growth and genetic stability of recovered plantlets,Yuanyi Xuebao(Acta Horticulturae Sinica),31(4):441-446(艾鹏飞,罗正荣,2004,柿和君迁子试管苗缓慢生长法保存及其遗传稳定性研究,园艺学报,31(4):441-446)
Chauhan R.,Keshavkant S.,Jadhav S.K.,and Quraishi A.,2016,In vitro slow-growth storage of Chlorophytum borivilianum Sant.et Fernand:a critically endangered herb,In Vitro Cell.Dev.Biol.-Plant,52(3):315-321
Ci H.T.,Wang Y.,and Zhou Y.L.,2017,In vitro conservation of tube plantlet of mentha haplocalyx,Anhui Keji Xueyuan Xuebao(Journal of Anhui Science and Technology University),31(4):39-44(慈惠婷,汪阳,周玉丽,2017,薄荷试管苗的离体保存,安徽科技学院学报,31(4):39-44)
Divakaran M.,Babu K.N.,and Peter K.V.,2006,Conservation of Vanilla species,in vitro,Sci.Hortic.,110(2):175-180
Fu Y.Q.,Kong Y.,and Liu Y.,2012,In vitro propagation and restrict growth preservation of Lilium leichtlinii var.maximowiczii Baker,Zhiwu Shengli Xuebao(Plant Physiology Journal),48(3):277-281(傅伊倩,孔滢,刘燕,2012,大花卷丹的组织培养及限制生长保存,植物生理学报,48(3):277-281)
Lan W.,and Chen F.D.,2010,In vitro conservation and genetic stability of Chrysanthemum shiwogiku var.Kinokuniense,Xibei Zhiwu Xuebao(Acta Botanica Boreali-Occidentalia Sinica),30(3):487-494(兰伟,陈发棣,2010,纪伊潮菊离体保存及其遗传稳定性分析,西北植物学报,30(3):487-494)
Lan W.,Chen S.M.,Yin D.M.,and Chen F.D.,2010,Studies on in vitro conservation of Chrysanthemum yoshinaganthum,Yuanyi Xuebao(Acta Horticulturae Sinica),37(12):2007-2016(兰伟,陈素梅,尹冬梅,陈发棣,2010,那贺川野菊的离体保存,园艺学报,37(12):2007-2016)
Li X.L.,and Chen F.D.,2004,Advances of genetic improvement and germplasm resources for chrysanthemum,Zhiwuxue T-ongbao(Chinese Bulletin of Botany),21(4):392-401(李辛雷,陈发棣,2004,菊花种质资源与遗传改良研究进展,植物学通报,21(4):392-401)
Liu J.F.,Yan X.F.,Cheng Y.Q.,Zhou X.M.,and Zhang X.Y.,2007,Conservation of in vitro shoots of Rhodiola sachalinensis by slow growth and its DNA content analysis,Zhejiang Daxue Xuebao(Journal of Zhejiang University),33(4):373-378(刘剑锋,阎秀峰,程云清,周晓梅,张晓燕,2007,高山红景天(Rhodiola sachalinensis)试管苗缓慢生长法保存及试管苗DNA含量分析,浙江大学学报,33(4):373-378)
Lu M.J.,Jiang Y.R.,Lu G.Q.,Chen G.L.,and Mao Q.,2015,Genetic diversity of quinoa germplasm assessed by SSR markers,Henongxue Bao(Journal of Nuclear Agricultural Sciences),29(2):260-269(陆敏佳,蒋玉蓉,陆国权,陈国林,毛前,2015,利用SSR标记分析藜麦品种的遗传多样性,核农学报,29(2):260-269)
Pan X.J.,Li X.,and Zhang W.E.,2013,Effect of conservation in vitro on the growth and leaf antioxidant enzyme activities of Vitis heyneana,Guoshu Xuebao(Journal of Fruit Science),30(4):615-620(潘学军,李霞,张文娥,2013,离体保存对野生毛葡萄试管苗生长及叶片抗氧化酶活性的影响,果树学报,30(4):615-620)
Tahtamouni R.,Shibli R.,Al-Abdallat A.,and Qudah T.,2016,Analysis of growth,oil yield,and carvacrol in Thymbra spcata L.after slow-growth conservation,Turkish J.Agri.Fore.,40(2):213-221
Wang R.,Chen Y.Z.,Wang X.N.,Chen L.S.,Peng S.F.,Ma L.,and Luo J.,2013,Effects of carbon sources and different concentration of sucrose on induction of Camellia oleifera callus,Xinan Nongye Xuebao(Southwest China Journal of Agricultural Sciences),26(1):223-225(王瑞,陈永忠,王湘南,陈隆升,彭绍峰,马力,罗健,2013,碳源及蔗糖浓度对油茶愈伤组织诱导的影响,西南农业学报,26(1):223-225)
Wang Y.F.,Fang W.M.,Chen F.D.,Zhao H.B.,and Liu Z.C.,2007,In vitro conservation of chrysanthemum'Jinba'and genetic stability of regenerated plantlets,Xibei Zhiwu Xuebao(Acta Botanica Boreali-Occidentalia Sinica),27(7):1341-1348(王艳芳,房伟民,陈发棣,赵宏波,刘灶长,2007,‘神马’菊花的离体保存及遗传稳定性,西北植物学报,27(7):1341-1348)
Yang X.,Liu F.,Zhang Y.,Cheng Y.F.,Xue L.B.,and Chen X.H.,2016,Study on the genetic diversity of eggplant germplasm with SSR markers,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(12):3450-3457(杨旭,刘飞,张宇,成玉富,薛林宝,陈学好,2016,利用SSR标记研究茄子种质资源遗传多样性,基因组学与应用生物学,35(12):3450-3457)
Ye X.X.,Hang M.L.,Wu J.S.,Zhong H.Q.,Lin B.,and Luo Y.H.,2011,Effect of mannitol and growth inhibitor on in vitro preservation of Oncidium,Fujian Nongye Xuebao(Fujian J-ournal of Agricultural Sciences),26(1):76-82(叶秀仙,黄敏玲,吴建设,钟淮钦,林兵,罗远华,2011,甘露醇和生长抑制剂对文心兰离体保存的影响,福建农业学报,26(1):76-82)
Ye X.X.,Huang M.L.,Fan R.H.,Wu J.S.,and Zhong H.Q.,2014,In vitro conservation and genetic stability of Phalaenopsis amabilis,Fujian Nongye Xuebao(Fujian Journal of Agricultural Sciences),29(10):976-981(叶秀仙,黄敏玲,樊荣辉,吴建设,钟淮钦,2014,蝴蝶兰离体保存及遗传稳定性研究,福建农业学报,29(10):976-981)
Yi H.L.,and Deng X.X.,1999,Advances in the techniques for in vitro preservation of plant germplasm,Zhiwuxue Tongbao(Chinese Bulletin of Botany),16(5):574-581(伊华林,邓秀新,1999,植物种质离体保存技术研究进展,植物学通报,16(5):574-581)
Yin M.H.,Zhou Y.Y.,Zeng R.X.,Su A.F.,Zhong Z.L.,Mi J.,Xia H.Y.,and Hong S.R.,2017,RAPD analysis on the genetic stability of regenerated plantlets of early pear in Shangrao after conservation in vitro,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),15(1):230-237(尹明华,周宇瑶,曾日香,苏爱芳,钟宗林,糜俊,夏华炎,洪森荣,2017,上饶早梨离体保存苗遗传稳定性RAPD分析,分子植物育种,15(1):230-237)
Zhao D.L.,Zheng L.T.,Tang J.,Zhou Z.L.,and Cao Q.H.,2011,Genetic stability and diversity of sweetpotato germplasm with SSR,Zhiwu Yichuan Ziyuan Xuebao(Journal of Plant Genetic Resources),12(3):389-395(赵冬兰,郑立涛,唐君,周志林,曹清河,2011,甘薯种质资源遗传稳定性及遗传多样性SSR分析,植物遗传资源学报,12(3):389-395)
Zhao M.Z.,Wang Z.W.,Qian Y.M.,Su J.L.,and Yuan J.,2006,Effects of different media on the conservation in vitro of strawberry germplasm,Guoshu Xuebao(Journal of Fruit Science),23(1):27-30(赵密珍,王壮伟,钱亚明,苏家乐,袁骥,2006,不同培养基对草莓种质离体保存的影响,果树学报,23(1):27-30)
Chauhan R.,Keshavkant S.,Jadhav S.K.,and Quraishi A.,2016,In vitro slow-growth storage of Chlorophytum borivilianum Sant.et Fernand:a critically endangered herb,In Vitro Cell.Dev.Biol.-Plant,52(3):315-321
Ci H.T.,Wang Y.,and Zhou Y.L.,2017,In vitro conservation of tube plantlet of mentha haplocalyx,Anhui Keji Xueyuan Xuebao(Journal of Anhui Science and Technology University),31(4):39-44(慈惠婷,汪阳,周玉丽,2017,薄荷试管苗的离体保存,安徽科技学院学报,31(4):39-44)
Divakaran M.,Babu K.N.,and Peter K.V.,2006,Conservation of Vanilla species,in vitro,Sci.Hortic.,110(2):175-180
Fu Y.Q.,Kong Y.,and Liu Y.,2012,In vitro propagation and restrict growth preservation of Lilium leichtlinii var.maximowiczii Baker,Zhiwu Shengli Xuebao(Plant Physiology Journal),48(3):277-281(傅伊倩,孔滢,刘燕,2012,大花卷丹的组织培养及限制生长保存,植物生理学报,48(3):277-281)
Lan W.,and Chen F.D.,2010,In vitro conservation and genetic stability of Chrysanthemum shiwogiku var.Kinokuniense,Xibei Zhiwu Xuebao(Acta Botanica Boreali-Occidentalia Sinica),30(3):487-494(兰伟,陈发棣,2010,纪伊潮菊离体保存及其遗传稳定性分析,西北植物学报,30(3):487-494)
Lan W.,Chen S.M.,Yin D.M.,and Chen F.D.,2010,Studies on in vitro conservation of Chrysanthemum yoshinaganthum,Yuanyi Xuebao(Acta Horticulturae Sinica),37(12):2007-2016(兰伟,陈素梅,尹冬梅,陈发棣,2010,那贺川野菊的离体保存,园艺学报,37(12):2007-2016)
Li X.L.,and Chen F.D.,2004,Advances of genetic improvement and germplasm resources for chrysanthemum,Zhiwuxue T-ongbao(Chinese Bulletin of Botany),21(4):392-401(李辛雷,陈发棣,2004,菊花种质资源与遗传改良研究进展,植物学通报,21(4):392-401)
Liu J.F.,Yan X.F.,Cheng Y.Q.,Zhou X.M.,and Zhang X.Y.,2007,Conservation of in vitro shoots of Rhodiola sachalinensis by slow growth and its DNA content analysis,Zhejiang Daxue Xuebao(Journal of Zhejiang University),33(4):373-378(刘剑锋,阎秀峰,程云清,周晓梅,张晓燕,2007,高山红景天(Rhodiola sachalinensis)试管苗缓慢生长法保存及试管苗DNA含量分析,浙江大学学报,33(4):373-378)
Lu M.J.,Jiang Y.R.,Lu G.Q.,Chen G.L.,and Mao Q.,2015,Genetic diversity of quinoa germplasm assessed by SSR markers,Henongxue Bao(Journal of Nuclear Agricultural Sciences),29(2):260-269(陆敏佳,蒋玉蓉,陆国权,陈国林,毛前,2015,利用SSR标记分析藜麦品种的遗传多样性,核农学报,29(2):260-269)
Pan X.J.,Li X.,and Zhang W.E.,2013,Effect of conservation in vitro on the growth and leaf antioxidant enzyme activities of Vitis heyneana,Guoshu Xuebao(Journal of Fruit Science),30(4):615-620(潘学军,李霞,张文娥,2013,离体保存对野生毛葡萄试管苗生长及叶片抗氧化酶活性的影响,果树学报,30(4):615-620)
Tahtamouni R.,Shibli R.,Al-Abdallat A.,and Qudah T.,2016,Analysis of growth,oil yield,and carvacrol in Thymbra spcata L.after slow-growth conservation,Turkish J.Agri.Fore.,40(2):213-221
Wang R.,Chen Y.Z.,Wang X.N.,Chen L.S.,Peng S.F.,Ma L.,and Luo J.,2013,Effects of carbon sources and different concentration of sucrose on induction of Camellia oleifera callus,Xinan Nongye Xuebao(Southwest China Journal of Agricultural Sciences),26(1):223-225(王瑞,陈永忠,王湘南,陈隆升,彭绍峰,马力,罗健,2013,碳源及蔗糖浓度对油茶愈伤组织诱导的影响,西南农业学报,26(1):223-225)
Wang Y.F.,Fang W.M.,Chen F.D.,Zhao H.B.,and Liu Z.C.,2007,In vitro conservation of chrysanthemum'Jinba'and genetic stability of regenerated plantlets,Xibei Zhiwu Xuebao(Acta Botanica Boreali-Occidentalia Sinica),27(7):1341-1348(王艳芳,房伟民,陈发棣,赵宏波,刘灶长,2007,‘神马’菊花的离体保存及遗传稳定性,西北植物学报,27(7):1341-1348)
Yang X.,Liu F.,Zhang Y.,Cheng Y.F.,Xue L.B.,and Chen X.H.,2016,Study on the genetic diversity of eggplant germplasm with SSR markers,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(12):3450-3457(杨旭,刘飞,张宇,成玉富,薛林宝,陈学好,2016,利用SSR标记研究茄子种质资源遗传多样性,基因组学与应用生物学,35(12):3450-3457)
Ye X.X.,Hang M.L.,Wu J.S.,Zhong H.Q.,Lin B.,and Luo Y.H.,2011,Effect of mannitol and growth inhibitor on in vitro preservation of Oncidium,Fujian Nongye Xuebao(Fujian J-ournal of Agricultural Sciences),26(1):76-82(叶秀仙,黄敏玲,吴建设,钟淮钦,林兵,罗远华,2011,甘露醇和生长抑制剂对文心兰离体保存的影响,福建农业学报,26(1):76-82)
Ye X.X.,Huang M.L.,Fan R.H.,Wu J.S.,and Zhong H.Q.,2014,In vitro conservation and genetic stability of Phalaenopsis amabilis,Fujian Nongye Xuebao(Fujian Journal of Agricultural Sciences),29(10):976-981(叶秀仙,黄敏玲,樊荣辉,吴建设,钟淮钦,2014,蝴蝶兰离体保存及遗传稳定性研究,福建农业学报,29(10):976-981)
Yi H.L.,and Deng X.X.,1999,Advances in the techniques for in vitro preservation of plant germplasm,Zhiwuxue Tongbao(Chinese Bulletin of Botany),16(5):574-581(伊华林,邓秀新,1999,植物种质离体保存技术研究进展,植物学通报,16(5):574-581)
Yin M.H.,Zhou Y.Y.,Zeng R.X.,Su A.F.,Zhong Z.L.,Mi J.,Xia H.Y.,and Hong S.R.,2017,RAPD analysis on the genetic stability of regenerated plantlets of early pear in Shangrao after conservation in vitro,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),15(1):230-237(尹明华,周宇瑶,曾日香,苏爱芳,钟宗林,糜俊,夏华炎,洪森荣,2017,上饶早梨离体保存苗遗传稳定性RAPD分析,分子植物育种,15(1):230-237)
Zhao D.L.,Zheng L.T.,Tang J.,Zhou Z.L.,and Cao Q.H.,2011,Genetic stability and diversity of sweetpotato germplasm with SSR,Zhiwu Yichuan Ziyuan Xuebao(Journal of Plant Genetic Resources),12(3):389-395(赵冬兰,郑立涛,唐君,周志林,曹清河,2011,甘薯种质资源遗传稳定性及遗传多样性SSR分析,植物遗传资源学报,12(3):389-395)
Zhao M.Z.,Wang Z.W.,Qian Y.M.,Su J.L.,and Yuan J.,2006,Effects of different media on the conservation in vitro of strawberry germplasm,Guoshu Xuebao(Journal of Fruit Science),23(1):27-30(赵密珍,王壮伟,钱亚明,苏家乐,袁骥,2006,不同培养基对草莓种质离体保存的影响,果树学报,23(1):27-30)