唐棣组织培养体系的建立与优化
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摘要
【目的】建立唐棣离体植株组织培养快繁体系,并对该体系进行优化。【方法】以唐棣带腋芽的茎段为外植体获得无菌苗,再以无菌试管苗为材料,以MS为基础培养基,采用正交试验研究6-BA(0.5,1.0,1.5 mg/L)、NAA(0.02,0.04,0.06 mg/L)、IBA(0.05,0.10,0.15 mg/L)配比对唐棣组培苗扩繁的影响,并研究不同基本培养基(WPM,1/4MS,1/8MS)和NAA(0.1,0.3 mg/L)、IBA(0.5,1.0 mg/L)配比对唐棣无菌苗生根的影响;再以已生根的试管苗为材料,研究不同开瓶时间(12,18,24,36,48 h)、生根时间(15,20,25,30 d)、基质配比(国产泥炭、国产水苔、腐殖质体积比分别为1∶1∶1,1∶3∶1,1∶5∶1和1∶4∶2)对炼苗移栽成活率和生长状况的影响。【结果】最适合唐棣带腋芽茎段的消毒方法是体积分数75%酒精消毒30 s,再用1 g/L HgCl_2消毒8 min,芽萌发率达到17.3%;最适合组培苗增殖的激素组合为MS+0.5 mg/L 6-BA+0.02 mg/L NAA+0.05 mg/L IBA,增殖系数达到6.28;最适合无菌苗生根的培养基为1/4MS+0.5 mg/L IBA,生根率达到100%;在生根培养25 d后开瓶适应24 h,然后再进行炼苗,最适合炼苗的基质配比为V(国产泥炭)∶V(国产水苔)∶V(腐殖质)=1∶5∶1,试管苗移栽成活率达到70.00%。【结论】建立并优化了唐棣组织培养体系,明显提高了无菌苗的增殖数量、生长健康指数及生根率,并且炼苗后移栽成活率也显著提高。
【Objective】 This study aimed to establish and optimize a tissue culture system for Amelanchier sinica.In the a large number of seedlings can be bred.【Method】 Taking A. sinica embryo tissue of somatic embryo callus(embryoid) as test materials,using the method of in vitro culture,the orthogonal experiment was used to study the influence of the proportion of 6-BA(0.5,1.0,1.5 mg/L),NAA(0.02,0.04,0.06 mg/L) and IBA(0.05,0.10,0.15 mg/L) on the propagation of tissue culture seedlings of A.sinica,and the influence of the proportion of different basic media(WPM,1/4 MS,1/8 MS),NAA(0.1,0.3 mg/L) and IBA(0.5,1.0 mg/L) on the sterile rooting seedlings of A.sinica was studied.Then the test tube seedlings of rooting were used to study the influence of different bottle opening time(12,18,24,36,48 h),rooting time(15,20,25,30 d) and the proportion of matrix(the volume ratio of domestic peat,domestic water moss,and humus is 1∶1∶1,1∶3∶1,1∶5∶1,1∶4∶2) on the survival rate of transplanted seedlings.【Result】 The best disinfection method was 75% ethanol for 30 s and 1 g/L HgCl_2 for 8 min.Optimal shoot proliferation occurred on MS medium with 0.5 mg/L 6-BA and 0.02 mg/L NAA and 0.05 mg/L IBA.The best rooting was 1/4 MS with 0.5 mg/L IBA,with rooting rating of 100%.The optimal matrix ratio was V(humus)∶V(sphagna)∶V(peat)=1∶5∶1,watered three times a day with a diminishing cycle of 10 days,after opening a bottle of 24 hours,rooting culture lasts 44 days and the survival rate was 70.00% after 30 days.【Conclusion】 The established and optimized tissue culture system for A.sinica improved reproduction rate and the survival rate of transplanting of tissue cultured seedlings increased obviously.
【Objective】 This study aimed to establish and optimize a tissue culture system for Amelanchier sinica.In the a large number of seedlings can be bred.【Method】 Taking A. sinica embryo tissue of somatic embryo callus(embryoid) as test materials,using the method of in vitro culture,the orthogonal experiment was used to study the influence of the proportion of 6-BA(0.5,1.0,1.5 mg/L),NAA(0.02,0.04,0.06 mg/L) and IBA(0.05,0.10,0.15 mg/L) on the propagation of tissue culture seedlings of A.sinica,and the influence of the proportion of different basic media(WPM,1/4 MS,1/8 MS),NAA(0.1,0.3 mg/L) and IBA(0.5,1.0 mg/L) on the sterile rooting seedlings of A.sinica was studied.Then the test tube seedlings of rooting were used to study the influence of different bottle opening time(12,18,24,36,48 h),rooting time(15,20,25,30 d) and the proportion of matrix(the volume ratio of domestic peat,domestic water moss,and humus is 1∶1∶1,1∶3∶1,1∶5∶1,1∶4∶2) on the survival rate of transplanted seedlings.【Result】 The best disinfection method was 75% ethanol for 30 s and 1 g/L HgCl_2 for 8 min.Optimal shoot proliferation occurred on MS medium with 0.5 mg/L 6-BA and 0.02 mg/L NAA and 0.05 mg/L IBA.The best rooting was 1/4 MS with 0.5 mg/L IBA,with rooting rating of 100%.The optimal matrix ratio was V(humus)∶V(sphagna)∶V(peat)=1∶5∶1,watered three times a day with a diminishing cycle of 10 days,after opening a bottle of 24 hours,rooting culture lasts 44 days and the survival rate was 70.00% after 30 days.【Conclusion】 The established and optimized tissue culture system for A.sinica improved reproduction rate and the survival rate of transplanting of tissue cultured seedlings increased obviously.
引文
[1] 姚德生,姚颖.唐棣育苗技术 [J].林业实用技术,2011(2):29-30. Yao D S,Yao Y.The seedling technique of Amelanchier sinica [J].Forest Science and Technology,2011(2):29-30.
[2] 龙韬.我国观赏植物资源研究现状及发展趋势 [J].北京农业,2011(18):53-55. Long T.Ornamental plant resources in China research and development trends [J].Beijing Agriculture,2011(18):53-55.
[3] 中国科学院中国植物志编辑委员会.中国植物志:第36卷[M].北京:科学出版社,1974:403. Editorial Board of Chinese Flora.Flora of China:Volume 36[M].Beijing:Science Press,1974:403.
[4] Magdalena M,Ryszard M.Effect of processing and storage on the antioxidant activity of frozen and pasteurized shadblow serviceberry(Amelanchier canadensis) [J].International Journal of Food Proerties,2012,6(13):1225-1233.
[5] Pruski K,Nowak J,Grainger G.Mirpropagation og four cultivars of saskatatoon berry(Amelanchier alnifolia Nutt.) [J].Plant Cell,Tissue and Organ Culturw,1990,21(2):103-109.
[6] Baldwin B D,Bandara M S,Tanino K K.Bud scale maturation in saskatoon berry(Amelanchier alnifolia Nutt.) plantlets following in vitro hormonal treatment [J].Acta Horticulture,2000,520:203-208.
[7] 杜保国.桤叶唐棣的组织培养和再生系统的建立 [D].陕西杨凌:西北农林科技大学,2005. Du B G.Tissue culture and regeneration system establishment of Amelanchier alnifolia Nutt. [D].Yangling,Shaanxi:Northwest A&F University,2005.
[8] 林宝山.尼尔森唐棣的组织培养和快速繁殖 [J].植物生理学通讯,2002(6):589. Lin B S.Tissue culture and rapid propagation of Nelson saskatoon [J].Plant Physiology Communications,2002(6):589.
[9] 郑万钧.中国树木志:第2卷 [M].北京:中国林业出版社,1985:1057. Zheng W J.The trees will Chinese:Volume 2 [M].Beijing:China Forestry Publishing House,1985:1057.
[10] 尹鹏先,蔡靖,李厚华,等.东亚唐棣组织培养体系 [J].东北林业大学学报,2015,43(1):54-56. Yin P X,Cai J,Li H H,et al.Tissue culture system of Amelanchier asiatica [J].Journal of Northeast Forestry University,2015,43(1):54-56.
[11] 何志宇,尹鹏先,程林林,等.东亚唐棣增殖培养条件的优化 [J].西北林学院学报,2016,31(1):126-129. He Z Y,Yin P X,Cheng L L,et al.Optimization on shoot proliferation culture media of Amelanchier asiatica [J].Journal of Northwest Forestry University,2016,31(1):126-129.
[12] 高崇辉,吉文丽,张小波,等.唐棣和鞘柄木的组培及消毒技术研究 [J].西北林学院学报,2005,20(2):107-108. Gao C H,Ji W L,Zhang X B,et al.Studies on tissue culture and sterilization of Amelanchier sinica and Torricellia angulate var intermedia [J].Journal of Northwest Forestry University,2005,20(2):107-108.
[13] 张佳平,丁彦芬.中国野生观赏植物资源调查、评价及园林应用研究进展 [J].中国野生植物资源,2012,31(6):18-23,31. Zhang J P,Ding Y F.Research on the investigation, evaluation and application of wild ornamental plants in China [J].Chinese Wild Plant Resources,2012,31(6):18-23,31.
[14] 胡凯,张立军,白雪梅,等.植物组织培养污染原因分析及外植体的消毒 [J].安徽农业科学,2007,35(3):680-681. Hu K,Zhang L J,Bai X M,et al.Analysis of the cause of contamination and explant sterilization in plant tissue culture [J].Journal of Anhui Agricultural Sciences,2007,35(3):680-681.
[15] 袁柳祥.绣线梅组织培养技术研究及遗传多样性分析 [D].陕西杨凌:西北农林科技大学,2016. Yuan L X.Study on tissue culture and genetic diversity of Neillia thyrsiflora [D].Yangling,Shaanxi:Northwest A&F University,2006.
[16] Beck E,Scheibe R.Senescence and ageing in plants and cyanobacteria [J].Physiologia Plantarum,2003,119(1):1-4.
[17] Xu W J,Kalima-N K M,Cui K N.Programmed cell death during terminal bud senescence in a sympodial branching tree,Eucommia ulmoides [J].Progress in Nature Science,2004,14(8):694-699.
[18] Wang D Y,Hu S,Li Q,et al.Photoperiod control of apical bud and leaf senescence in pumpkin(Cucurbita pepo)stain 185 [J].Acta Bot Sin,2002,44(1):55-62.
[19] 李秋玲,李青,刘燕,等.春石斛继代培养主要影响因素 [J].东北林业大学学报,2014,42(7):69-73. Li Q L,Li Q,Liu Y,et al.Main influence factors of dendrobium nobile subculture [J].Journal of Northeast Forestry University,2014,42(7):69-73.
[20] 包振华,郭军战,周玮,等.枸杞组织培养再生体系优化 [J].西北林学院学报,2010,25(5):73-76. Bao Z H,Guo J Z,Zhou W,et al.Optimization of tissue culture regeneration system of Lycium barbarum [J].Journal of Northwest Forestry University,2010,25(5):73-76.
[21] Al Malki A A H S,Elmeer K M S.Influence of auxin and cytokinine on in vitro multiplication of Ficus anastasia [J].Afr J Biotechnol,2010,5:635-639.
[22] 刘忠荣,陈屏昭.植物组织培养技术在观赏植物中的应用 [J].昭通师范高等专科学校学报,2003,25(5):41-43,46. Liu Z R,Chen P Z.The application of plant tissue culture technology on the ornamental plants [J].Journal of Zhaotong Teacher's College,2003,25(5):41-43,46.
[23] Druart P.Optimization of culture media for in vitro rooting ofMalus domestica Borkh.cv.compact Spartan [J].Biologia Plantarum,1997,39(1):67-77.
[24] 燕丽萍,夏阳,毛秀红,等.邓恩桉的组织培养 [J].林业科学,2011,47(5):157-161. Yan L P,Xia Y,Mao X H,et al.Tissue culture of Eucalyptus dunnii [J].Scientia Silvae Sinicae,2011,47(5):157-161.
[25] 吴雅琼,刘婧,汪贵斌,等.美国红枫的组织培养与快繁技术 [J].北方园艺,2016(20):97-102. Wu Y Q,Liu J,Wang G B et al.Tissue culture and rapid propagation of Acer rubrum [J].Northern Horticulture,2016(20):97-102.
[2] 龙韬.我国观赏植物资源研究现状及发展趋势 [J].北京农业,2011(18):53-55. Long T.Ornamental plant resources in China research and development trends [J].Beijing Agriculture,2011(18):53-55.
[3] 中国科学院中国植物志编辑委员会.中国植物志:第36卷[M].北京:科学出版社,1974:403. Editorial Board of Chinese Flora.Flora of China:Volume 36[M].Beijing:Science Press,1974:403.
[4] Magdalena M,Ryszard M.Effect of processing and storage on the antioxidant activity of frozen and pasteurized shadblow serviceberry(Amelanchier canadensis) [J].International Journal of Food Proerties,2012,6(13):1225-1233.
[5] Pruski K,Nowak J,Grainger G.Mirpropagation og four cultivars of saskatatoon berry(Amelanchier alnifolia Nutt.) [J].Plant Cell,Tissue and Organ Culturw,1990,21(2):103-109.
[6] Baldwin B D,Bandara M S,Tanino K K.Bud scale maturation in saskatoon berry(Amelanchier alnifolia Nutt.) plantlets following in vitro hormonal treatment [J].Acta Horticulture,2000,520:203-208.
[7] 杜保国.桤叶唐棣的组织培养和再生系统的建立 [D].陕西杨凌:西北农林科技大学,2005. Du B G.Tissue culture and regeneration system establishment of Amelanchier alnifolia Nutt. [D].Yangling,Shaanxi:Northwest A&F University,2005.
[8] 林宝山.尼尔森唐棣的组织培养和快速繁殖 [J].植物生理学通讯,2002(6):589. Lin B S.Tissue culture and rapid propagation of Nelson saskatoon [J].Plant Physiology Communications,2002(6):589.
[9] 郑万钧.中国树木志:第2卷 [M].北京:中国林业出版社,1985:1057. Zheng W J.The trees will Chinese:Volume 2 [M].Beijing:China Forestry Publishing House,1985:1057.
[10] 尹鹏先,蔡靖,李厚华,等.东亚唐棣组织培养体系 [J].东北林业大学学报,2015,43(1):54-56. Yin P X,Cai J,Li H H,et al.Tissue culture system of Amelanchier asiatica [J].Journal of Northeast Forestry University,2015,43(1):54-56.
[11] 何志宇,尹鹏先,程林林,等.东亚唐棣增殖培养条件的优化 [J].西北林学院学报,2016,31(1):126-129. He Z Y,Yin P X,Cheng L L,et al.Optimization on shoot proliferation culture media of Amelanchier asiatica [J].Journal of Northwest Forestry University,2016,31(1):126-129.
[12] 高崇辉,吉文丽,张小波,等.唐棣和鞘柄木的组培及消毒技术研究 [J].西北林学院学报,2005,20(2):107-108. Gao C H,Ji W L,Zhang X B,et al.Studies on tissue culture and sterilization of Amelanchier sinica and Torricellia angulate var intermedia [J].Journal of Northwest Forestry University,2005,20(2):107-108.
[13] 张佳平,丁彦芬.中国野生观赏植物资源调查、评价及园林应用研究进展 [J].中国野生植物资源,2012,31(6):18-23,31. Zhang J P,Ding Y F.Research on the investigation, evaluation and application of wild ornamental plants in China [J].Chinese Wild Plant Resources,2012,31(6):18-23,31.
[14] 胡凯,张立军,白雪梅,等.植物组织培养污染原因分析及外植体的消毒 [J].安徽农业科学,2007,35(3):680-681. Hu K,Zhang L J,Bai X M,et al.Analysis of the cause of contamination and explant sterilization in plant tissue culture [J].Journal of Anhui Agricultural Sciences,2007,35(3):680-681.
[15] 袁柳祥.绣线梅组织培养技术研究及遗传多样性分析 [D].陕西杨凌:西北农林科技大学,2016. Yuan L X.Study on tissue culture and genetic diversity of Neillia thyrsiflora [D].Yangling,Shaanxi:Northwest A&F University,2006.
[16] Beck E,Scheibe R.Senescence and ageing in plants and cyanobacteria [J].Physiologia Plantarum,2003,119(1):1-4.
[17] Xu W J,Kalima-N K M,Cui K N.Programmed cell death during terminal bud senescence in a sympodial branching tree,Eucommia ulmoides [J].Progress in Nature Science,2004,14(8):694-699.
[18] Wang D Y,Hu S,Li Q,et al.Photoperiod control of apical bud and leaf senescence in pumpkin(Cucurbita pepo)stain 185 [J].Acta Bot Sin,2002,44(1):55-62.
[19] 李秋玲,李青,刘燕,等.春石斛继代培养主要影响因素 [J].东北林业大学学报,2014,42(7):69-73. Li Q L,Li Q,Liu Y,et al.Main influence factors of dendrobium nobile subculture [J].Journal of Northeast Forestry University,2014,42(7):69-73.
[20] 包振华,郭军战,周玮,等.枸杞组织培养再生体系优化 [J].西北林学院学报,2010,25(5):73-76. Bao Z H,Guo J Z,Zhou W,et al.Optimization of tissue culture regeneration system of Lycium barbarum [J].Journal of Northwest Forestry University,2010,25(5):73-76.
[21] Al Malki A A H S,Elmeer K M S.Influence of auxin and cytokinine on in vitro multiplication of Ficus anastasia [J].Afr J Biotechnol,2010,5:635-639.
[22] 刘忠荣,陈屏昭.植物组织培养技术在观赏植物中的应用 [J].昭通师范高等专科学校学报,2003,25(5):41-43,46. Liu Z R,Chen P Z.The application of plant tissue culture technology on the ornamental plants [J].Journal of Zhaotong Teacher's College,2003,25(5):41-43,46.
[23] Druart P.Optimization of culture media for in vitro rooting ofMalus domestica Borkh.cv.compact Spartan [J].Biologia Plantarum,1997,39(1):67-77.
[24] 燕丽萍,夏阳,毛秀红,等.邓恩桉的组织培养 [J].林业科学,2011,47(5):157-161. Yan L P,Xia Y,Mao X H,et al.Tissue culture of Eucalyptus dunnii [J].Scientia Silvae Sinicae,2011,47(5):157-161.
[25] 吴雅琼,刘婧,汪贵斌,等.美国红枫的组织培养与快繁技术 [J].北方园艺,2016(20):97-102. Wu Y Q,Liu J,Wang G B et al.Tissue culture and rapid propagation of Acer rubrum [J].Northern Horticulture,2016(20):97-102.
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