西藏野生金钗石斛高效植株再生体系的建立
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摘要
以西藏野生金钗石斛的种子为外植体,通过研究不同基本培养基、植物激素配比和有机添加物对原球茎诱导、增殖和分化的影响,筛选适宜金钗石斛植株再生的条件,建立金钗石斛组培快繁技术体系。结果表明:适宜金钗石斛生长的基本培养基为MS;种子萌发和原球茎诱导的最适培养基为MS+NAA 1.0mg/L,种子萌发率为98.00%,培养50d,有95.00%的原球茎分化;原球茎增殖和分化的最适培养基为MS+NAA 0.5 mg/L+蛋白胨2.0 g/L+土豆泥60 g/L+活性炭0.5 g/L,培养20 d,增殖倍数为8.65,将此培养基上获得的芽苗继代培养60 d,分蘖数为25.33株,生根率为98.52%;以腐殖土和松树树皮(1﹕1)作为栽培基质,试管苗的移栽成活率为96.67%。该研究结果为保护金钗石斛野生资源和工厂化育苗提供了科学依据和技术支持。
In order to establish an high-efficiency plant regeneration system of Dendrobium nobile Lindl., the seeds obtained from Tibetan wild D. nobile used as explants, the effects of basic medium, hormone combination and organic additive on the protocorm induction, proliferation and differentiation were studied. The results showed that the optimum basic medium for D. nobile growth was MS. The optimal medium for seed germination and protocorm induction was MS+NAA 1.0 mg/L, with seed germination rate of 98.00% and the protocorm differentiated cotyledon rate of 95.00% after cultured for 50 d. The optimal medium for protocorm proliferation and differentiation was MS+NAA 0.5 mg/L+peptone 2.0 g/L+mashed potato 60 g/L+acticarbon0.5 g/L, and the maximum mutuplication factor of protocorm reached 8.65 after cultured for 20 d.Then bud seedlings subcultured in this medium, the tiller number was 25.33 after cultured for 60 d, and the rooting rate was 98.52%. Using humus and pine bark(1﹕1)as cultivation substrate,the transplanting survival rate of plantlets was 96.67%. This study provides an effective way for keeping good varieties of features and rapid propagation of D. nobile and at the same time helps to solve some theoretical problems in factory production of D. nobile.
In order to establish an high-efficiency plant regeneration system of Dendrobium nobile Lindl., the seeds obtained from Tibetan wild D. nobile used as explants, the effects of basic medium, hormone combination and organic additive on the protocorm induction, proliferation and differentiation were studied. The results showed that the optimum basic medium for D. nobile growth was MS. The optimal medium for seed germination and protocorm induction was MS+NAA 1.0 mg/L, with seed germination rate of 98.00% and the protocorm differentiated cotyledon rate of 95.00% after cultured for 50 d. The optimal medium for protocorm proliferation and differentiation was MS+NAA 0.5 mg/L+peptone 2.0 g/L+mashed potato 60 g/L+acticarbon0.5 g/L, and the maximum mutuplication factor of protocorm reached 8.65 after cultured for 20 d.Then bud seedlings subcultured in this medium, the tiller number was 25.33 after cultured for 60 d, and the rooting rate was 98.52%. Using humus and pine bark(1﹕1)as cultivation substrate,the transplanting survival rate of plantlets was 96.67%. This study provides an effective way for keeping good varieties of features and rapid propagation of D. nobile and at the same time helps to solve some theoretical problems in factory production of D. nobile.
引文
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[2]杨澜,刘鹏飞,王爱华等.金钗石斛叶艺性状保持及丛生芽增殖研究[J].北方园艺,2016(12):166-170.
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[11]徐德林,蔡振锋,张林等.金钗石斛种子萌发率提高及可诱导愈伤组织培养基的研究[J].遵义医学院学报,2014,37(5):541-545.
[12]中国科学院植物研究所,2013.中国珍稀濒危植物名录[DB/OL].http://rep.iplant.cn/protlist.
[13]漆小雪,韦霄,陈宗游等.不同肥料对金钗石斛生物学性状,SPAD值和石斛碱含量的影响[J].北方园艺,2014(5):143-146.
[14]郑世刚,颜寿,胡亚东等.川北金钗石斛引种及仿野生栽培试验[J].四川大学学报(自然科学版),2018,55(3):649-654.
[15]许莉,王江瑞,郭力等.金钗石斛化学成分的研究[J].中成药,2018,40(5):1110-1112.
[16]Li Q,Ding G,Li B,et al.Transcriptome analysis of genes involved in dendrobine biosynthesis in Dendrobium nobile Lindl.infected with mycorrhizal fungus MF23(Mycena sp.)[J].Scientific Reports,2017,doi:10.1038/s41598-017-00445-9.
[17]牛俊奇,苏建睦,王小敏等.基于高通量测序的金钗石斛叶转录组数据分析[J].分子植物育种,2018,16(3):747-756.
[18]Paromik B,Suman K,Pramod T.Applicability of ISSR and DAMD markers for phyto-molecular characterization and association with some important biochemical traits of Dendrobium nobile,an endangered medicinal orchid[J].Phytochemistry,2015,117(306):306-316.
[19]李清,李标,郭顺星.金钗石斛转录组SSR位点信息分析[J].中国中药杂志,2017,42(1):63-69.
[20]Paromik B,Suman K,Reemavareen D,et al.Genetic stability and phytochemical analysis of the in vitro regenerated plants of Dendrobium nobile Lindl.,an endangered medicinal orchid[J].Meta Gene,2014,2(4):489-504.
[2]杨澜,刘鹏飞,王爱华等.金钗石斛叶艺性状保持及丛生芽增殖研究[J].北方园艺,2016(12):166-170.
[3]国家药典委员会.中华人民共和国药典:一部[S].北京:中国医药科技出版社,2015:92-93.
[4]张晓敏,孙志蓉,陈龙等.金钗石斛的化学成分和药理作用研究进展[J].中国现代应用药学,2014,31(7):895-899.
[5]Meng C W,He Y L,Peng C,et al.Picrotoxane sesquiterpenoids from the stems of Dendrobium nobile and their absolute configurations and angiogenesis effect[J].Fitoterapia,2017,121:206-211.
[6]Zhang Y,Wang H X,Mei N N,et al.Protective effects of polysaccharide from Dendrobium nobile against ethanolinduced gastric damage in rats[J].International Journal of Biological Macromolecules,2018,107(Pt A):230-235.
[7]和磊,罗婧,王亚芸等.金钗石斛脂溶性生物碱提取物诱导人结肠癌HT-29细胞凋亡[J].食品工业科技,2017,38(3):170-174,191.
[8]李庆宏,宋常美.金钗石斛愈伤组织培养[J].贵阳学院学报(自然科学版),2011,6(3):76-79.
[9]李静婷,牛磊,侯玉杰.金钗石斛的快速繁殖研究[J].湖北农业科学,2014,53(9):2181-2183.
[10]罗林会,王勤.金钗石斛的离体快繁体系研究[J].安徽农业科学,2012,40(15):8476-8479.
[11]徐德林,蔡振锋,张林等.金钗石斛种子萌发率提高及可诱导愈伤组织培养基的研究[J].遵义医学院学报,2014,37(5):541-545.
[12]中国科学院植物研究所,2013.中国珍稀濒危植物名录[DB/OL].http://rep.iplant.cn/protlist.
[13]漆小雪,韦霄,陈宗游等.不同肥料对金钗石斛生物学性状,SPAD值和石斛碱含量的影响[J].北方园艺,2014(5):143-146.
[14]郑世刚,颜寿,胡亚东等.川北金钗石斛引种及仿野生栽培试验[J].四川大学学报(自然科学版),2018,55(3):649-654.
[15]许莉,王江瑞,郭力等.金钗石斛化学成分的研究[J].中成药,2018,40(5):1110-1112.
[16]Li Q,Ding G,Li B,et al.Transcriptome analysis of genes involved in dendrobine biosynthesis in Dendrobium nobile Lindl.infected with mycorrhizal fungus MF23(Mycena sp.)[J].Scientific Reports,2017,doi:10.1038/s41598-017-00445-9.
[17]牛俊奇,苏建睦,王小敏等.基于高通量测序的金钗石斛叶转录组数据分析[J].分子植物育种,2018,16(3):747-756.
[18]Paromik B,Suman K,Pramod T.Applicability of ISSR and DAMD markers for phyto-molecular characterization and association with some important biochemical traits of Dendrobium nobile,an endangered medicinal orchid[J].Phytochemistry,2015,117(306):306-316.
[19]李清,李标,郭顺星.金钗石斛转录组SSR位点信息分析[J].中国中药杂志,2017,42(1):63-69.
[20]Paromik B,Suman K,Reemavareen D,et al.Genetic stability and phytochemical analysis of the in vitro regenerated plants of Dendrobium nobile Lindl.,an endangered medicinal orchid[J].Meta Gene,2014,2(4):489-504.