银叶真藓外植体组织培养消毒方法的筛选
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摘要
为给银叶真藓全基因组序列测定提供无污染材料,本研究对银叶真藓外植体组织培养的消毒方法进行筛选。分别使用酒精、次氯酸钠溶液和青霉素溶液作为消毒剂,在不同浓度和不同时间下处理银叶真藓外植体,然后分别将处理后的材料接种在改良的Knop培养基上进行培养。一定时间后,通过形态观察银叶真藓的生长状况。实验结果表明,酒精和次氯酸钠溶液对银叶真藓的消毒效果欠佳,而青霉素溶液对银叶真藓的消毒效果较好,由此筛选出银叶真藓外植体组织培养的最佳消毒方法为:用浓度为300 mg/L青霉素溶液浸泡1.5 h。利用本研究筛选出的消毒方法可以获得形态观察上无杂菌污染的银叶真藓材料,为后续全基因组序列测定前银叶真藓DNA的获取提供物质基础。
The disinfection methods for tissue culture using Bryum argenteum as explants were screened in this study to provide non-polluting materials for the whole genome sequencing of B. argenteum. Using ethanol, sodium hypochlorite solution and penicillin solution as disinfection reagents, B. argenteum explants were treated under different concentrations and time, and then the treated explants were inoculated on the modified Knop medium for culture. After a certain time, the growth status of B. argenteum was observed through morphology. The results showed that the disinfection effect of penicillin solution on B. argenteum was better than that of ethanol and sodium hypochlorite solution. Thus, the best disinfection method for B. argenteum explant tissue culture was that samples were soaked in 300 mg/L penicillin solution for 1.5 h. B. argenteum material without microbial contamination on morphological observation could be obtained by using the disinfection method screened in this experiment, which could provide a material basis for further acquisition of B. argentium DNA before whole genome sequencing.
The disinfection methods for tissue culture using Bryum argenteum as explants were screened in this study to provide non-polluting materials for the whole genome sequencing of B. argenteum. Using ethanol, sodium hypochlorite solution and penicillin solution as disinfection reagents, B. argenteum explants were treated under different concentrations and time, and then the treated explants were inoculated on the modified Knop medium for culture. After a certain time, the growth status of B. argenteum was observed through morphology. The results showed that the disinfection effect of penicillin solution on B. argenteum was better than that of ethanol and sodium hypochlorite solution. Thus, the best disinfection method for B. argenteum explant tissue culture was that samples were soaked in 300 mg/L penicillin solution for 1.5 h. B. argenteum material without microbial contamination on morphological observation could be obtained by using the disinfection method screened in this experiment, which could provide a material basis for further acquisition of B. argentium DNA before whole genome sequencing.
引文
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Gao B.,Zhang D.Y.,Li X.S.,Yang H.L.,Liang Y.Q.,Chen M.X.,Zhang Y.M.,Zhang J.H.,and Andrew W.,2018,Desiccation tolerance in bryophytes:the rehydration proteomes of Bryum argenteum provide insights into the resuscitation mechanism,J.Arid Land,10(1):152-167
Hui R.,Li X.R.,Jia R.L.,Zhao X.,Liu Y.M.,and Chen C.Y.,2012,Effects of enhanced UV-B radiation on physiological characteristics of Bryum argenteum,Shengtaixue ZaZhi(Chinese Journal of Ecology),31(1):38-43(回嵘,李新荣,贾荣亮,赵昕,刘艳梅,陈翠云,2012,增强UV-B辐射对真藓结皮生理特性的影响,生态学杂志,31(1):38-43)
Jiang S.,and Tian X.W.,2012,A study on tissue culture media of the medical mosses Bryum argenteum and Sphagnum palustre L.,Xinan Daxue Xuebao(Journal of Southwest University(Natural Science Edition)),34(12):61-67(姜山,田学武,2012,药用苔藓植物银叶真藓和泥炭藓组织培养基配方研究,西南大学学报(自然科学版),34(12):61-67)
Lang Y.Z.,Kan S.C.,Liu W.C.,Zhou J.C.,and Xiong Y.X.,2008,Screening of appropriate disinfection method of cultured Thuidium cymbifolium tissue culture,Anhui Nongye Kexue(Journal of Anhui Agricultural Sciences),36(34):14892-14893(郎玉卓,阚世超,刘伟才,周金川,熊源新,2008,大羽藓组培初代培养适宜消毒方法的筛选,安徽农业科学,36(34):14892-14893)
Li D.H.,Ma J.H.,Peng T.,Wen H.B.,Mo X.M.,and Zhou J.Z.,2017,The explant disinfection and culture medium selection of the Sphagnum multifibrosum,Fenzi Zhiwu Yuzhong(Molecluar Plant Breeding),15(4):1569-1574(李大华,麻俊虎,彭涛,文浩斌,莫秀模,周建忠,2017,多纹泥炭藓外植体消毒及其培养基筛选,分子植物育种,15(4):1569-1574)
Li L.B.,and Zhao J.C.,2009,Peristome morphology of Bryum Hedw.(Musci Bryaceae)and its taxonomic significance,Zhiwuyanjiu(Bulletin of Botanical Research),29(6):651-658(李利博,赵建成,2009,真藓属(Bryum Hedw.)蒴齿形态特征及其分类学意义,植物研究,29(6):651-658)
Liu R.X.,Wang Z.H.,and Zhang Z.H.,2011,Ecological monitoring of bryophytes for mercury pollution in Danzhai Mercury Mine Area,Guizhou Province,China,Shengtai Xuebao(Acta Ecologica Sinica),31(6):1558-1566(刘荣相,王智慧,朝晖,2011,苔藓植物对贵州丹寨汞矿区汞污染的生态监测,生态学报,31(6):1558-1566)
Shi Y.,Zhao X.,Jia R.L.,and Li X.R.,2012,The reactive oxygen species scavenging mechanism of Bryum argenteum and Didymodon vinealis in biological soil crusts under gradual drought stress,Zhongguo Shamo(Journal of Desert Research),32(3):683-690(石勇,赵昕,贾荣亮,李新荣,2012,逐渐干旱胁迫下生物土壤结皮中真藓和生对齿藓的活性氧清除机制,中国沙漠,32(3):683-690)
Wang C.Y.,2012,Influences of in vitro factors on the photosynthetic pigment content in the process of protonema development in Bryum argenteum,Anhui Nongye Kexue(Journal of Anhui Agricultural Sciences),40(22):11167-11168(汪琛颖,2012,组培条件对银叶真藓原丝体发育阶段光合色素含量的影响,安徽农业科学,40(22):11167-11168)
Xie M.,Hui R.,Liu L.C.,Yang H.T.,Wang Y.L.,Wei W.F.,and Wang X.Q.,2017,Effects of snowfall on physiological characteristics of Bryum argenteum and Didymodon vinealis Distributed in biological soil crusts,Zhongguo Shamo(Journal of Desert Research),37(1):116-123(谢敏,回嵘,刘立超,杨昊天,王艳莉,魏文斐,王雪芹,2017,沙漠生物土壤结皮中真藓(Bryum argenteum)和土生对齿藓(Didymodon vinealis)对降雪的生理响应,中国沙漠,37(1):116-123)
Yu J.,2012,Analysis of the polymorphism and the dominant population of endophytic batteria in Dendrobium officinale,Thesis for M.S.,Zhejiang Sci-Tech University,Supervisor:Hu X.F.,pp.56-58(俞婕,2012,铁皮石斛的内生细菌多样性与优势种群分析,浙江理工大学,导师:胡秀芳,pp.56-58)
Zhang X.T.,2016,Adaptability of bryophytes to acidicsoil environment,Anhui Nongxue Tongbao(Anhui Agriculural Science Bulletin),22(5):18-19(张啸天,2016,苔藓植物对酸性恶劣土壤环境的适应性研究,安徽农学通报,22(5):18-19)
Zheng Y.P.,Zhao J.C.,Zhang B.C.,Li L.,and Zhang Y.M.,2009,Advances on ecological studies of algae and mosses in biological soil crust,Zhiwu Xuebao(Chinese Bulletin of Botany),44(3):371-378(郑云普,赵建成,张丙昌,李琳和张元明,2009,荒漠生物结皮中藻类和苔藓植物研究进展,植物学报,44(3):371-378)
Gao B.,Zhang D.Y.,Li X.S.,Yang H.L.,Liang Y.Q.,Chen M.X.,Zhang Y.M.,Zhang J.H.,and Andrew W.,2018,Desiccation tolerance in bryophytes:the rehydration proteomes of Bryum argenteum provide insights into the resuscitation mechanism,J.Arid Land,10(1):152-167
Hui R.,Li X.R.,Jia R.L.,Zhao X.,Liu Y.M.,and Chen C.Y.,2012,Effects of enhanced UV-B radiation on physiological characteristics of Bryum argenteum,Shengtaixue ZaZhi(Chinese Journal of Ecology),31(1):38-43(回嵘,李新荣,贾荣亮,赵昕,刘艳梅,陈翠云,2012,增强UV-B辐射对真藓结皮生理特性的影响,生态学杂志,31(1):38-43)
Jiang S.,and Tian X.W.,2012,A study on tissue culture media of the medical mosses Bryum argenteum and Sphagnum palustre L.,Xinan Daxue Xuebao(Journal of Southwest University(Natural Science Edition)),34(12):61-67(姜山,田学武,2012,药用苔藓植物银叶真藓和泥炭藓组织培养基配方研究,西南大学学报(自然科学版),34(12):61-67)
Lang Y.Z.,Kan S.C.,Liu W.C.,Zhou J.C.,and Xiong Y.X.,2008,Screening of appropriate disinfection method of cultured Thuidium cymbifolium tissue culture,Anhui Nongye Kexue(Journal of Anhui Agricultural Sciences),36(34):14892-14893(郎玉卓,阚世超,刘伟才,周金川,熊源新,2008,大羽藓组培初代培养适宜消毒方法的筛选,安徽农业科学,36(34):14892-14893)
Li D.H.,Ma J.H.,Peng T.,Wen H.B.,Mo X.M.,and Zhou J.Z.,2017,The explant disinfection and culture medium selection of the Sphagnum multifibrosum,Fenzi Zhiwu Yuzhong(Molecluar Plant Breeding),15(4):1569-1574(李大华,麻俊虎,彭涛,文浩斌,莫秀模,周建忠,2017,多纹泥炭藓外植体消毒及其培养基筛选,分子植物育种,15(4):1569-1574)
Li L.B.,and Zhao J.C.,2009,Peristome morphology of Bryum Hedw.(Musci Bryaceae)and its taxonomic significance,Zhiwuyanjiu(Bulletin of Botanical Research),29(6):651-658(李利博,赵建成,2009,真藓属(Bryum Hedw.)蒴齿形态特征及其分类学意义,植物研究,29(6):651-658)
Liu R.X.,Wang Z.H.,and Zhang Z.H.,2011,Ecological monitoring of bryophytes for mercury pollution in Danzhai Mercury Mine Area,Guizhou Province,China,Shengtai Xuebao(Acta Ecologica Sinica),31(6):1558-1566(刘荣相,王智慧,朝晖,2011,苔藓植物对贵州丹寨汞矿区汞污染的生态监测,生态学报,31(6):1558-1566)
Shi Y.,Zhao X.,Jia R.L.,and Li X.R.,2012,The reactive oxygen species scavenging mechanism of Bryum argenteum and Didymodon vinealis in biological soil crusts under gradual drought stress,Zhongguo Shamo(Journal of Desert Research),32(3):683-690(石勇,赵昕,贾荣亮,李新荣,2012,逐渐干旱胁迫下生物土壤结皮中真藓和生对齿藓的活性氧清除机制,中国沙漠,32(3):683-690)
Wang C.Y.,2012,Influences of in vitro factors on the photosynthetic pigment content in the process of protonema development in Bryum argenteum,Anhui Nongye Kexue(Journal of Anhui Agricultural Sciences),40(22):11167-11168(汪琛颖,2012,组培条件对银叶真藓原丝体发育阶段光合色素含量的影响,安徽农业科学,40(22):11167-11168)
Xie M.,Hui R.,Liu L.C.,Yang H.T.,Wang Y.L.,Wei W.F.,and Wang X.Q.,2017,Effects of snowfall on physiological characteristics of Bryum argenteum and Didymodon vinealis Distributed in biological soil crusts,Zhongguo Shamo(Journal of Desert Research),37(1):116-123(谢敏,回嵘,刘立超,杨昊天,王艳莉,魏文斐,王雪芹,2017,沙漠生物土壤结皮中真藓(Bryum argenteum)和土生对齿藓(Didymodon vinealis)对降雪的生理响应,中国沙漠,37(1):116-123)
Yu J.,2012,Analysis of the polymorphism and the dominant population of endophytic batteria in Dendrobium officinale,Thesis for M.S.,Zhejiang Sci-Tech University,Supervisor:Hu X.F.,pp.56-58(俞婕,2012,铁皮石斛的内生细菌多样性与优势种群分析,浙江理工大学,导师:胡秀芳,pp.56-58)
Zhang X.T.,2016,Adaptability of bryophytes to acidicsoil environment,Anhui Nongxue Tongbao(Anhui Agriculural Science Bulletin),22(5):18-19(张啸天,2016,苔藓植物对酸性恶劣土壤环境的适应性研究,安徽农学通报,22(5):18-19)
Zheng Y.P.,Zhao J.C.,Zhang B.C.,Li L.,and Zhang Y.M.,2009,Advances on ecological studies of algae and mosses in biological soil crust,Zhiwu Xuebao(Chinese Bulletin of Botany),44(3):371-378(郑云普,赵建成,张丙昌,李琳和张元明,2009,荒漠生物结皮中藻类和苔藓植物研究进展,植物学报,44(3):371-378)