长尖对齿藓原丝体快速培育的关键影响因子研究
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摘要
为了给苔藓结皮野外恢复的大规模接种提供丰富种源,本文利用0.1%NaClO(10、15、20、30、45、60、120s)和0.1%HgCl_2(10、15、20、30、45、60、120 s)两种消毒液,采用Knop、MS和Hoagland 3种培养基,设置5.5、6、6.5、7、7.5、8、8.5七个水平的p H值,通过单因素试验设计方法,测定长尖对齿藓[Didymodon ditrichoides(Broth.)]茎叶体的成活率、原丝体长度和分枝数等指标,探讨影响长尖对齿藓原丝体扩繁的关键因子。结果表明:(1)消毒方式、培养基及p H均对长尖对齿藓茎叶体的成活率、原丝体长度和分枝数有显著影响(P<0.05),影响大小依次均为消毒方式>培养基>p H;(2)最适合的消毒方式为:0.1%NaClO消毒15~20 s;(3)最适合长尖对齿藓原丝体生长的培养基是Knop和Hoagland培养基;(4)最适合长尖对齿藓原丝体生长的pH是7.5。从而得出快速培育长尖对齿藓原丝体的最佳组合为:0.1%NaCl O消毒15~20 s+Hoagland/Knop+pH=7.5。本文的研究结果可以缩短长尖对齿藓生长周期以进行快速繁殖,为苔藓结皮的快速培育以及工程化应用提供一定的借鉴。
In order to provide a rich source of mass vaccination for the field restoration of moss crust,we used two kinds of disinfection method,0. 1% NaClO(10,15,20,30,45,60,120 s) and 0. 1% HgCl_2(10,15,20,30,45,60,120 s),with Knop,MS and Hoagland as culture media,and set the p H values(5. 5,6,6. 5,7,7. 5,8,8. 5),through the single factor experiment design method,we determined the survival rate of the cormus of [Didymodon ditrichoides( Broth.)],protonema length and number of branches,thereby exploring key factors that affect the propagation of the protonema of D. ditrichoides. The results are as follows:(1) The disinfection method,culture medium and p H value have significant influence upon the survival rate of the cormus of D. ditrichoides,length of protonema and number of branches( P < 0. 05),in a descending order of disinfection method,culture medium,and pH value;(2) The most suitable disinfection method is 0. 1% NaClO with a duration of 15-20 s;( 3) The most suitable culture medium for the growth of the protonema of D.ditrichoides is Knop and Hoagland;(4) The most suitable p H value for the growth of protonema of D. ditrichoidesis 7. 5. Therefore,the best combination of factors for rapid cultivation of the protonema of D. ditrichoides is0. 1% NaClO disinfection 15-20 s + Hoagland/Knop + pH = 7. 5. Our experiment can be determined to shorten the growth cycle of D. ditrichoides and realize rapid propagation,thereby providing experience for the rapid culture and engineering application of moss crust.
In order to provide a rich source of mass vaccination for the field restoration of moss crust,we used two kinds of disinfection method,0. 1% NaClO(10,15,20,30,45,60,120 s) and 0. 1% HgCl_2(10,15,20,30,45,60,120 s),with Knop,MS and Hoagland as culture media,and set the p H values(5. 5,6,6. 5,7,7. 5,8,8. 5),through the single factor experiment design method,we determined the survival rate of the cormus of [Didymodon ditrichoides( Broth.)],protonema length and number of branches,thereby exploring key factors that affect the propagation of the protonema of D. ditrichoides. The results are as follows:(1) The disinfection method,culture medium and p H value have significant influence upon the survival rate of the cormus of D. ditrichoides,length of protonema and number of branches( P < 0. 05),in a descending order of disinfection method,culture medium,and pH value;(2) The most suitable disinfection method is 0. 1% NaClO with a duration of 15-20 s;( 3) The most suitable culture medium for the growth of the protonema of D.ditrichoides is Knop and Hoagland;(4) The most suitable p H value for the growth of protonema of D. ditrichoidesis 7. 5. Therefore,the best combination of factors for rapid cultivation of the protonema of D. ditrichoides is0. 1% NaClO disinfection 15-20 s + Hoagland/Knop + pH = 7. 5. Our experiment can be determined to shorten the growth cycle of D. ditrichoides and realize rapid propagation,thereby providing experience for the rapid culture and engineering application of moss crust.
引文
1.娄红祥.苔藓化学与生物学[M].北京:科学出版社,2012.Lou H X.Chemistry and biology of bryophytes[M].Beijing:Science Press,2012.
2 .杨永胜,冯伟,袁方,等.快速培育黄土高原苔藓结皮的关键影响因子[J].水土保持学报,2015,29(4):289-294.Yang Y S,Feng W,Yuan F,et al.Key influential factors of rapid cultivation of moss crusts on Loess Plateau[J].Journal of Soil and Water Conservation,2015,29(4):289-294.
3 .牛玉璐,曹永胜.生物结皮及其在荒漠治理中的作用[J].生物学教学,2005,30(10):5-6.Niu Y L,Cao Y S.Bio-cluster and its role in desert harnessing[J].Biology Teaching,2005,30(10):5-6.
4 .高丽倩,赵允格,秦宁强,等.黄土丘陵区生物结皮对土壤物理属性的影响[J].自然资源学报,2012,27(8):1316-1326.Gao L Q,Zhao Y G,Qin N Q,et al.Impact of biological soil crust on soil physical properties in the Hilly Loess Plateau Region,China[J].Journal of Natural Resources,2012,27(8):1316-1326.
5 .李金峰,孟杰,叶菁,等.陕北水蚀风蚀交错区生物结皮的形成过程与发育特征[J].自然资源学报,2014,29(1):67-79.Li J F,Meng J,Ye J,et al.The development characteristics and formation process of biological soil crusts in Wind-Water erosion crisscross region,Northern Shaanxi Province,China[J].Journal of Natural Resources,2014,29(1):67-79.
6 .Bu C F,Wu S F,Xie Y S,et al.The study of biological soil crusts:hotspots and prospects[J].Clean-Soil,Air,Water,2013,41(9):899-906.
7 .吴鹏程.苔藓植物生物学[M].北京:科学出版社,1998.Wu P C.Bryological biology,introduction and diverse branches[M].Beijing:Science Press,1998.
8 .黄士良.侧蒴藓类植物孢子萌发与原丝体发育研究[D].石家庄:河北师范大学,2009.Huang S L.Study on the spore germination and protonema development of Pleurocarpous Mosses[D].Shijiazhuang:Hebei Normal University,2009.
9 .田桂泉,张萍.干旱区固定沙丘结皮层藓类植物繁殖生物学特性研究[J].河套大学学报,2004,1(1):20-25.Tian G Q,Zhang P.Study on the characteristic of reproductive biology of mosses in the soil crust of fixed dunes in desert regions[J].Journal of Hetao University,2004,1(1):20-25.
10 .崔巍,张海娟,沙伟.毛尖紫萼藓外植体消毒方法及接种培养基的筛选[J].北方园艺,2012(10):138-140.Cui W,Zhang H J,Sha W.Screen of sterilized methods and inoculation culture medium filter for explants of Grimmia pilifera[J].Northern Horticulture,2012(10):138-140.
11 .郎玉卓,阚世超,刘伟才,等.大羽藓组培初代培养适宜消毒方法的筛选[J].安徽农业科学,2008,36(34):14892-14893.Lang Y Z,Kan S C,Liu W C,et al.Screening of appropriate disinfection method of early cultured Thuidium cymbifolium tissue culture[J].Journal of Anhui Agricultural Sciences,2008,36(34):14892-14893.
12 .Kute,Elzbieta.Callus induction and gametophyte regeneration in moss cultures[M].New Yourk:Mc Graw-Hill,1971.
13 .Gonzalez M L,Mallon R,Reinoso J,et al.In vitro micropropagation and long-term conservation of the endangered moss Splachnum ampullaceum[J].Biologia Plantarum,2006,50(3):339-345.
14 .张楠,杜宝明,季梦成.细叶小羽藓(Haplocladium microphyllum)配子体组织培养的消毒方法及蔗糖浓度筛选[J].浙江大学学报:农业与生命科学版,2012,38(3):288-292.Zhang N,Du B M,Ji M C.Sterilization method and sucrose concentration selection for initial culture of Haplocladium microphyllum gametophyte[J],Journal of Zhejiang University:Agriculture&Life Sciences,2012,38(3):288-292.
15 .李敏,赵建成,黄士良,等.中华缩叶藓孢子萌发与原丝体发育特征研究[J].西北植物学报,2006,26(8):1521-1525.Li M,Zhao J C,Huang S L,et al.Characteristics of spore germination and protonemal development in Ptychomitrium sinense[J].Acta Botanica Boreali-Occidentalia Sinica,2006,26(8):1521-1525.
16 .刘保东,丛迎芝.波叶仙鹤藓的孢子培养及发育生物学研究[J].植物研究,2003,23(2):159-163.Liu B D,Cong Y Z.Studies on spores culture and development al biology of Atrichum undulatum[J].Bulletin of Botanical Research,2003,23(2):159-163.
17 .范庆书,赵建成,黄士良.金灰藓(Pylaisiella polyantha)配子体发生的实验观察[J].植物科学学报,2004,22(2):140-144.Fan Q S,Zhao J C,Huang S L.Experimental observations on the gametophyte development of Pylaisiella polyantha[J].Journal of Wuhan Botanical Research,2004,22(2):140-144.
18 .于淑玲,商艳艳,赵建成.3种藓类植物的原丝体发育特征[J].河北师范大学学报:自然科学版,2010,34(3):343-347.Yu S L,Shang Y Y,Zhao J C.Study on characteristics of protonema development in three species of mosses[J].Journal of Hebei Normal University:Natural Science Edition,2010,34(3):343-347.
19 .Duckett J G,Burch J,Fletcher P W,et al.In vitro cultivation of bryophytes:a review of practicalities,problems,progress and promise[J].Journal of Bryology,2004,26(1):3-20.
20 .Christianson M L.ABA prevents the second cytokinin-mediated event during the induction of shoot buds in the moss Funaria hygrometrica[J].American Journal of Botany,2000,87(10):1540-1545.
21 .Bopp M.50 years of the moss story[M].//Esser K,Kadereit J W,Lüttge U,et al.Progress in Botany.Berlin Heidelberg:Springer,2000:3-34.
22 .Cvetic T,Sabovljevic A,Sabovljevic M,et al.Development of the moss Pogonatum urnigerum(Hedw.)P.Beauv.under in vitro culture conditions[J].Archives of Biological Sciences,2007,59(1):57-61.
23 .刘丽,胡光珍,王幼芳,等.萘乙酸(NAA)对葫芦藓(Funaria hygrometrica)孢子萌发及原丝体生长的影响[J].华东师范大学学报:自然科学版,2004(4):138-141.Liu L,Hu G Z,Wang Y F,et al.Effect of NAA on spore germination and protonema growth of Funaria hygrometrica[J].Journal of east China normal university:Natural Science,2004(4):138-141.
24 .于淑玲.观赏苔藓植物的组织培养[J].生物学通报,2009,44(4):56-57.Yu S L.Tissue culture of watching bryophyte[J].Bulletin of Biology,2009,44(4):56-57.
25 .刘世彪,陈军,李菁,等.光照和温度对尖叶拟船叶藓孢子萌发及原丝体发育的影响[J].西北植物学报,2003,23(1):101-106.Liu S B,Chen J,Li J,et al.Effects of light and temperatures on spore germination and protonema development of Dolichomitriopsis diversiformis[J].Acta Botanica BorealiOccidentalia Sinica,2003,23(1):101-106.
26 .李敏,赵建成,黄士良,等.垂蒴真藓原丝体发育特征的研究[J].西北植物学报,2005,25(2):363-367.Li M,Zhao J C,Huang S L,et al.Study on the characteristics of protonema development of Bryum uliginosum[J].Acta Botanica Boreali-Occidentalia Sinica,2005,25(2):363-367.
27 .张梅娟,沙伟.东亚砂藓组织培养技术方法研究[J].植物科学学报,2013,31(6):616-622.Zhang M J,Sha W.Research on tissue culture technology of Racomitrium japonicum[J].Plant Science Journal,2013,31(6):616-622.
28 .李晓毓,吴翠珍,熊源新,等.尖叶匍灯藓的组织培养及显微观察[J].山地农业生物学报,2006,25(3):217-222.Li X Y,Wu C Z,Xiong Y X,et al.The tissue culture and microscopic observations of Plagiomnium cuspidatum[J].Journal of Mountain Agriculture and Biology,2006,25(3):217-222.
29 .Schween G,Hohe A,Koprivova A,et al.Effects of nutrients,cell density and culture techniques on protoplast regeneration and early protonema development in a moss,Physcomitrella patens[J].Journal of Plant Physiology,2003,160(2):209-212.
30 .杭璐璐,张楠,季梦成.不同培养基和蔗糖质量浓度对细叶小羽藓孢子萌发的影响[J].浙江农林大学学报,2012,29(3):383-387.Hang L L,Zhang N,Ji M C.Haplocladium microphyllum spore germination with different culture mediums and sucrose concentrations[J].Journal of Zhejiang A&F University,2012,29(3):383-387.
31 .Reutter K,Atzorn R,Hadeler B,et al.Expression of the bacterial ipt gene in Physcomitrella rescues mutations in budding and in plastid division[J].Planta,1998,206(2):196-203.
32 .Schulz P,Reski R,Maldiney R,et al.Kinetics of cytokinin production and bud formation in Physcomitrella:analysis of wild type,a developmental mutant and two of its ipt transgenics[J].Journal of Plant Physiology,2000,156(5-6):768-774.
33 .王振杰,黄士良,李志,等.不同p H值和培养基对金灰藓(Pylaisiella polyantha)孢子萌发的影响[J].河北师范大学学报:自然科学版,2008,32(6):813-816.Wang Z J,Huang S L,Li Z,et al.The effect of different p H and cultures on the Pylaisiella polyantha spore germination[J].Journal of Hebei Normal University:Natural Science Edition,2008,32(6):813-816.
2 .杨永胜,冯伟,袁方,等.快速培育黄土高原苔藓结皮的关键影响因子[J].水土保持学报,2015,29(4):289-294.Yang Y S,Feng W,Yuan F,et al.Key influential factors of rapid cultivation of moss crusts on Loess Plateau[J].Journal of Soil and Water Conservation,2015,29(4):289-294.
3 .牛玉璐,曹永胜.生物结皮及其在荒漠治理中的作用[J].生物学教学,2005,30(10):5-6.Niu Y L,Cao Y S.Bio-cluster and its role in desert harnessing[J].Biology Teaching,2005,30(10):5-6.
4 .高丽倩,赵允格,秦宁强,等.黄土丘陵区生物结皮对土壤物理属性的影响[J].自然资源学报,2012,27(8):1316-1326.Gao L Q,Zhao Y G,Qin N Q,et al.Impact of biological soil crust on soil physical properties in the Hilly Loess Plateau Region,China[J].Journal of Natural Resources,2012,27(8):1316-1326.
5 .李金峰,孟杰,叶菁,等.陕北水蚀风蚀交错区生物结皮的形成过程与发育特征[J].自然资源学报,2014,29(1):67-79.Li J F,Meng J,Ye J,et al.The development characteristics and formation process of biological soil crusts in Wind-Water erosion crisscross region,Northern Shaanxi Province,China[J].Journal of Natural Resources,2014,29(1):67-79.
6 .Bu C F,Wu S F,Xie Y S,et al.The study of biological soil crusts:hotspots and prospects[J].Clean-Soil,Air,Water,2013,41(9):899-906.
7 .吴鹏程.苔藓植物生物学[M].北京:科学出版社,1998.Wu P C.Bryological biology,introduction and diverse branches[M].Beijing:Science Press,1998.
8 .黄士良.侧蒴藓类植物孢子萌发与原丝体发育研究[D].石家庄:河北师范大学,2009.Huang S L.Study on the spore germination and protonema development of Pleurocarpous Mosses[D].Shijiazhuang:Hebei Normal University,2009.
9 .田桂泉,张萍.干旱区固定沙丘结皮层藓类植物繁殖生物学特性研究[J].河套大学学报,2004,1(1):20-25.Tian G Q,Zhang P.Study on the characteristic of reproductive biology of mosses in the soil crust of fixed dunes in desert regions[J].Journal of Hetao University,2004,1(1):20-25.
10 .崔巍,张海娟,沙伟.毛尖紫萼藓外植体消毒方法及接种培养基的筛选[J].北方园艺,2012(10):138-140.Cui W,Zhang H J,Sha W.Screen of sterilized methods and inoculation culture medium filter for explants of Grimmia pilifera[J].Northern Horticulture,2012(10):138-140.
11 .郎玉卓,阚世超,刘伟才,等.大羽藓组培初代培养适宜消毒方法的筛选[J].安徽农业科学,2008,36(34):14892-14893.Lang Y Z,Kan S C,Liu W C,et al.Screening of appropriate disinfection method of early cultured Thuidium cymbifolium tissue culture[J].Journal of Anhui Agricultural Sciences,2008,36(34):14892-14893.
12 .Kute,Elzbieta.Callus induction and gametophyte regeneration in moss cultures[M].New Yourk:Mc Graw-Hill,1971.
13 .Gonzalez M L,Mallon R,Reinoso J,et al.In vitro micropropagation and long-term conservation of the endangered moss Splachnum ampullaceum[J].Biologia Plantarum,2006,50(3):339-345.
14 .张楠,杜宝明,季梦成.细叶小羽藓(Haplocladium microphyllum)配子体组织培养的消毒方法及蔗糖浓度筛选[J].浙江大学学报:农业与生命科学版,2012,38(3):288-292.Zhang N,Du B M,Ji M C.Sterilization method and sucrose concentration selection for initial culture of Haplocladium microphyllum gametophyte[J],Journal of Zhejiang University:Agriculture&Life Sciences,2012,38(3):288-292.
15 .李敏,赵建成,黄士良,等.中华缩叶藓孢子萌发与原丝体发育特征研究[J].西北植物学报,2006,26(8):1521-1525.Li M,Zhao J C,Huang S L,et al.Characteristics of spore germination and protonemal development in Ptychomitrium sinense[J].Acta Botanica Boreali-Occidentalia Sinica,2006,26(8):1521-1525.
16 .刘保东,丛迎芝.波叶仙鹤藓的孢子培养及发育生物学研究[J].植物研究,2003,23(2):159-163.Liu B D,Cong Y Z.Studies on spores culture and development al biology of Atrichum undulatum[J].Bulletin of Botanical Research,2003,23(2):159-163.
17 .范庆书,赵建成,黄士良.金灰藓(Pylaisiella polyantha)配子体发生的实验观察[J].植物科学学报,2004,22(2):140-144.Fan Q S,Zhao J C,Huang S L.Experimental observations on the gametophyte development of Pylaisiella polyantha[J].Journal of Wuhan Botanical Research,2004,22(2):140-144.
18 .于淑玲,商艳艳,赵建成.3种藓类植物的原丝体发育特征[J].河北师范大学学报:自然科学版,2010,34(3):343-347.Yu S L,Shang Y Y,Zhao J C.Study on characteristics of protonema development in three species of mosses[J].Journal of Hebei Normal University:Natural Science Edition,2010,34(3):343-347.
19 .Duckett J G,Burch J,Fletcher P W,et al.In vitro cultivation of bryophytes:a review of practicalities,problems,progress and promise[J].Journal of Bryology,2004,26(1):3-20.
20 .Christianson M L.ABA prevents the second cytokinin-mediated event during the induction of shoot buds in the moss Funaria hygrometrica[J].American Journal of Botany,2000,87(10):1540-1545.
21 .Bopp M.50 years of the moss story[M].//Esser K,Kadereit J W,Lüttge U,et al.Progress in Botany.Berlin Heidelberg:Springer,2000:3-34.
22 .Cvetic T,Sabovljevic A,Sabovljevic M,et al.Development of the moss Pogonatum urnigerum(Hedw.)P.Beauv.under in vitro culture conditions[J].Archives of Biological Sciences,2007,59(1):57-61.
23 .刘丽,胡光珍,王幼芳,等.萘乙酸(NAA)对葫芦藓(Funaria hygrometrica)孢子萌发及原丝体生长的影响[J].华东师范大学学报:自然科学版,2004(4):138-141.Liu L,Hu G Z,Wang Y F,et al.Effect of NAA on spore germination and protonema growth of Funaria hygrometrica[J].Journal of east China normal university:Natural Science,2004(4):138-141.
24 .于淑玲.观赏苔藓植物的组织培养[J].生物学通报,2009,44(4):56-57.Yu S L.Tissue culture of watching bryophyte[J].Bulletin of Biology,2009,44(4):56-57.
25 .刘世彪,陈军,李菁,等.光照和温度对尖叶拟船叶藓孢子萌发及原丝体发育的影响[J].西北植物学报,2003,23(1):101-106.Liu S B,Chen J,Li J,et al.Effects of light and temperatures on spore germination and protonema development of Dolichomitriopsis diversiformis[J].Acta Botanica BorealiOccidentalia Sinica,2003,23(1):101-106.
26 .李敏,赵建成,黄士良,等.垂蒴真藓原丝体发育特征的研究[J].西北植物学报,2005,25(2):363-367.Li M,Zhao J C,Huang S L,et al.Study on the characteristics of protonema development of Bryum uliginosum[J].Acta Botanica Boreali-Occidentalia Sinica,2005,25(2):363-367.
27 .张梅娟,沙伟.东亚砂藓组织培养技术方法研究[J].植物科学学报,2013,31(6):616-622.Zhang M J,Sha W.Research on tissue culture technology of Racomitrium japonicum[J].Plant Science Journal,2013,31(6):616-622.
28 .李晓毓,吴翠珍,熊源新,等.尖叶匍灯藓的组织培养及显微观察[J].山地农业生物学报,2006,25(3):217-222.Li X Y,Wu C Z,Xiong Y X,et al.The tissue culture and microscopic observations of Plagiomnium cuspidatum[J].Journal of Mountain Agriculture and Biology,2006,25(3):217-222.
29 .Schween G,Hohe A,Koprivova A,et al.Effects of nutrients,cell density and culture techniques on protoplast regeneration and early protonema development in a moss,Physcomitrella patens[J].Journal of Plant Physiology,2003,160(2):209-212.
30 .杭璐璐,张楠,季梦成.不同培养基和蔗糖质量浓度对细叶小羽藓孢子萌发的影响[J].浙江农林大学学报,2012,29(3):383-387.Hang L L,Zhang N,Ji M C.Haplocladium microphyllum spore germination with different culture mediums and sucrose concentrations[J].Journal of Zhejiang A&F University,2012,29(3):383-387.
31 .Reutter K,Atzorn R,Hadeler B,et al.Expression of the bacterial ipt gene in Physcomitrella rescues mutations in budding and in plastid division[J].Planta,1998,206(2):196-203.
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