铁皮石斛无菌播种成苗体系优化
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摘要
以铁皮石斛种子为起始材料,研究了其原球茎萌发、壮苗、生根出苗环节中一些关键影响因子,建立了铁皮石斛种子快速成苗组培技术体系。研究发现,培养基中加入6-BA会抑制原球茎的发育,若整个培养阶段用直径为9 cm的培养皿,起始播种密度以8 000粒种子为宜,冻干香蕉粉对于种子的萌发是必需的,以5～10 g·L~(-1)为宜;壮苗阶段,接种密度以100～120株·瓶-1为宜,培养基及光质对铁皮石斛壮苗影响远小于接种密度;生根阶段,利用3 000 K LED白光代替荧光灯照明,在不降低组培苗茎粗的前提下,可有效提高组培苗株高,增加优质苗比例。研究结果还表明,冻干香蕉粉可完全代替剥皮香蕉作为有机添加物用于铁皮石斛组培苗的培养。整个培养过程中,培养温度(24±2)℃,种子萌发阶段光照时间10 h,光照强度25～30μmol·m~(-2)·s~(-1);其余培养阶段均光照时间12 h,光照强度40μmol·m~(-2)·s~(-1)左右。
Some key factors affecting the seed germination,plant growing and rooting of Dendrobium officinale were studied,and the micropropagation system of Dendrobium officinale via seed culture was established. The results showed that 6-benzyladenine( 6-BA) in the medium inhibited the development of the protocorm seriously. The suitable seed density for initial germination was 8 000 in the condition that the culture vessel with a diameter of 9 cm was used,and banana powder as organic additives was necessary for seed germination with the suitable amount of 5-10 g·L~(-1). The inoculation density was suitable for 100-120 plantlets per bottle in plant growth stage,and inoculation density played a more important role than light quality and medium content. In rooting stage,the plantlet height could be improved greatly when cultured in 3 000 K LED white light than those in fluorescent lamp without reducing the stem diameter,and the proportion of high quality seedlings also improved a lot. The results also showed that banana powder could completely replace fresh banana as organic additives for tissue culture of Dendrobium candidum. During the whole culture process,the temperature was( 24 ± 2) ℃. The photoperiod for seed germination was 10 h and the light intensity was 25-30 μmol·m~(-2)·s~(-1),while in the other two stages,the photoperiod kept at 12 h and the light intensity kept around 40 μmol·m~(-2)·s~(-1) were enough.
Some key factors affecting the seed germination,plant growing and rooting of Dendrobium officinale were studied,and the micropropagation system of Dendrobium officinale via seed culture was established. The results showed that 6-benzyladenine( 6-BA) in the medium inhibited the development of the protocorm seriously. The suitable seed density for initial germination was 8 000 in the condition that the culture vessel with a diameter of 9 cm was used,and banana powder as organic additives was necessary for seed germination with the suitable amount of 5-10 g·L~(-1). The inoculation density was suitable for 100-120 plantlets per bottle in plant growth stage,and inoculation density played a more important role than light quality and medium content. In rooting stage,the plantlet height could be improved greatly when cultured in 3 000 K LED white light than those in fluorescent lamp without reducing the stem diameter,and the proportion of high quality seedlings also improved a lot. The results also showed that banana powder could completely replace fresh banana as organic additives for tissue culture of Dendrobium candidum. During the whole culture process,the temperature was( 24 ± 2) ℃. The photoperiod for seed germination was 10 h and the light intensity was 25-30 μmol·m~(-2)·s~(-1),while in the other two stages,the photoperiod kept at 12 h and the light intensity kept around 40 μmol·m~(-2)·s~(-1) were enough.
引文
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[5]罗绍强.铁皮石斛组织培养研究[J].林业与环境科学,2017,33(6):80-83.LUO S Q. Study on tissue culture of Dendrobium candidum[J]. Forestry and Environmental Science,2017,33(6):80-83.(in Chinese with English abstract)
[6]徐玲,陈自宏,杨晓娜,等.不同有机附加物对铁皮石斛原球茎增殖和组培苗生根壮苗的影响[J].云南农业大学学报(自然科学版),2016,30(2):250-256.XU L,CHEN Z H,YANG X N,et al. Effect of different organic appendices on protocorm multiplication and seedlings rooting and growth-promoting of Dendrobium officinale[J].Journal of Yunnan Agricultural University(Natural Science),2016,30(2):250-256.(in Chinese with English abstract)
[7]彭文书,马莉,赵苗苗,等.响应面试验优化铁皮石斛组培苗生根培养基[J].中药材,2017,40(9):2013-2017.PENG W S,MA L,ZHAO M M,et al. Optimization of rooting medium for tissue culture of Dendrobium candidum by response surface methodology[J]. Journal of Chinese Medicinal Materials,2017,40(9):2013-2017.(in Chinese with English abstract)
[8]常美花,金亚征,王莉.铁皮石斛快繁技术体系研究[J].中草药,2012,43(7):1412-1417.CHANG M H,JIN Y Z,WANG L. Rapid propagation technique system of Dendrobium officinale[J]. Chinese Traditional and Herbal Drugs,2012,43(7):1412-1417.(in Chinese with English abstract)
[9]戴小英,张淑霞,周莉荫,等.铁皮石斛不同外植体组培快繁技术比较研究[J].中国农学通报2011,27(10):122-126.DAI X Y,ZHANG S X,ZHOU L Y,et al. The study of comparative techniques for tissue culture with different explants in Dendrobium officinale[J]. Chinese Agricultural Science Bulletin,2011,27(10):122-126.(in Chinese with English abstract)
[10]黄昌艳,李魁鹏,杨美纯,等.不同培养基对铁皮石斛组培快繁的影响[J].南方农业学报,2011,42(4):349-352.HUANG C Y,LI K P,YANG M C,et al. Effect of different culture media on micropropagation of Dendrobium candidum Wall. ex Lindl[J]. Journal of Southern Agriculture,2011,42(4):349-352.(in Chinese with English abstract)
[11]杨小兵,王增利,史昊,等.光强对悬浮培养下铁皮石斛原球茎生长的影响[J].河南农业科学,2014,43(1):116-119.YANG X B,WANG Z L,SHI H,et al. Effect of light intensity on peotocorm-like bodies growth of Dendrobium candidum under condition of suspension culture[J]. Journal of Henan Agricultural Sciences,2014,43(1):116-119.(in Chinese with English abstract)
[12]蒋林,丁平,郑迎冬.添加剂对铁皮石斛组织培养和快速繁殖的影响[J].中药材,2003,26(8):539-541.JIANG L,DING P,ZHENG Y D. Effects of additives on tissue culture and rapid propagation of Dendrobium canducum[J]. Journal of Chinese Medicinal Materials,2003,26(8):539-541.(in Chinese with English abstract)
[13]周江明.不同有机物对铁皮石斛试管苗生长发育的影响[J].中国农学通报,2005,21(8):49-50+98.ZHOU J M. Effects of different organic matter on regenerated shoots of Dendrobium candium[J]. Chinese Agricultural Science Bulletin,2005,21(8):49-50+98.(in Chinese with English abstract)
[14]刘世平.不同培养基及培养环境对铁皮石斛幼苗生长的影响试验[J].林业调查规划,2012,37(3):39-42.LIU S P. Effects test of different culture media and environment on seedling growth of Dendrobium candidum[J]. Forest Inventory and Planning,2012,37(3):39-42.(in Chinese with English abstract)
[15]宋顺,许奕,王必尊,等.不同培养基成分对铁皮石斛组织培养的影响[J].中国农学通报,2013,29(13):133-139.SONG S,XU Y,WANG B Z,et al. Different media on the tissue culture of Dendrobium officinale[J]. Chinese Agricultural Science Bulletin,2013,29(13):133-139.(in Chinese with English abstract)
[16]尚文倩,王政,侯甲男,等.不同红蓝光质比LED光源对铁皮石斛试管苗生长的影响[J].西北农林科技大学学报(自然科学版),2013,41(5):155-159.SHANG W Q,WANG Z,HOU J N,et al. Effects of light emitting diode(LED)with different red/blue quality ratios on the growth of Dendrobium officinale plantlet in vitro[J]. Journal of Northwest A&F University(Natural Science Edition),2013,41(5):155-159.(in Chinese with English abstract)
[17]杨维杰,梁君,杨林林.不同LED光质对铁皮石斛生长及多糖含量的影响[J].浙江农业科学,2015,56(6):1188-1190.YANG W J,LIANG J,YANG L L. Effects of different LED light on the growth and polysaccharide content of Dendrobium candidum[J]. Journal of Zhejiang Agricultural Sciences,2015,56(6):1188-1190.(in Chinese)
[18]高亭亭,斯金平,朱玉球,等.光质与种质对铁皮石斛种苗生长和有效成分的影响[J].中国中药杂志,2012,37(2):198-201.GAO T T,SI J P,ZHU Y Q,et al. Effects of light quality and germplasm on growth and effective ingredients of Dendrobium officinale germchit[J]. China Journal of Chinese Materia Medica,2012,37(2):198-201.(in Chinese with English abstract)
[2]叶秀粦,程式君,王伏雄,等.黑节草未成熟种子的形态发育及其在离体培养时的表现[J].云南植物研究,1988,10(3):285-290.YE X L,CHENG S J,WANG F X,et al. Morphological development of immature seeds of Dendrobium candidum root and germination in-vitro culture[J]. Acta Botanica Yunnanica,1988,10(3):285-290.(in Chinese with English abstract)
[3]张治国,王黎,刘烨,等.铁皮石解原球茎分化适宜培养基研究[J].中国中药杂志,1993,18(1):16-19.ZHANG Z G,WANG L,LIU Y,et al. Study on culture medium for protocorn differentiation in Dendrobium candidum Wall.ex Lindl.[J]. China Journal of Chinese Materia Medica,1993,18(1):16-19.(in Chinese with English abstract)
[4]饶宝蓉,陈泳和,江文清,等.铁皮石斛不同外植体组培快繁技术比较[J].安徽农业科学,2017,45(4):138-141+195.RAO B R,CHEN Y H,JIANG W Q,et al. Comparison of tissue culture and rapid propagation techniquesof different explants in Dendrobium officinale[J]. Journal of Anhui Agricultural Sciences,2017,45(4):138-141+195.(in Chinese with English abstract)
[5]罗绍强.铁皮石斛组织培养研究[J].林业与环境科学,2017,33(6):80-83.LUO S Q. Study on tissue culture of Dendrobium candidum[J]. Forestry and Environmental Science,2017,33(6):80-83.(in Chinese with English abstract)
[6]徐玲,陈自宏,杨晓娜,等.不同有机附加物对铁皮石斛原球茎增殖和组培苗生根壮苗的影响[J].云南农业大学学报(自然科学版),2016,30(2):250-256.XU L,CHEN Z H,YANG X N,et al. Effect of different organic appendices on protocorm multiplication and seedlings rooting and growth-promoting of Dendrobium officinale[J].Journal of Yunnan Agricultural University(Natural Science),2016,30(2):250-256.(in Chinese with English abstract)
[7]彭文书,马莉,赵苗苗,等.响应面试验优化铁皮石斛组培苗生根培养基[J].中药材,2017,40(9):2013-2017.PENG W S,MA L,ZHAO M M,et al. Optimization of rooting medium for tissue culture of Dendrobium candidum by response surface methodology[J]. Journal of Chinese Medicinal Materials,2017,40(9):2013-2017.(in Chinese with English abstract)
[8]常美花,金亚征,王莉.铁皮石斛快繁技术体系研究[J].中草药,2012,43(7):1412-1417.CHANG M H,JIN Y Z,WANG L. Rapid propagation technique system of Dendrobium officinale[J]. Chinese Traditional and Herbal Drugs,2012,43(7):1412-1417.(in Chinese with English abstract)
[9]戴小英,张淑霞,周莉荫,等.铁皮石斛不同外植体组培快繁技术比较研究[J].中国农学通报2011,27(10):122-126.DAI X Y,ZHANG S X,ZHOU L Y,et al. The study of comparative techniques for tissue culture with different explants in Dendrobium officinale[J]. Chinese Agricultural Science Bulletin,2011,27(10):122-126.(in Chinese with English abstract)
[10]黄昌艳,李魁鹏,杨美纯,等.不同培养基对铁皮石斛组培快繁的影响[J].南方农业学报,2011,42(4):349-352.HUANG C Y,LI K P,YANG M C,et al. Effect of different culture media on micropropagation of Dendrobium candidum Wall. ex Lindl[J]. Journal of Southern Agriculture,2011,42(4):349-352.(in Chinese with English abstract)
[11]杨小兵,王增利,史昊,等.光强对悬浮培养下铁皮石斛原球茎生长的影响[J].河南农业科学,2014,43(1):116-119.YANG X B,WANG Z L,SHI H,et al. Effect of light intensity on peotocorm-like bodies growth of Dendrobium candidum under condition of suspension culture[J]. Journal of Henan Agricultural Sciences,2014,43(1):116-119.(in Chinese with English abstract)
[12]蒋林,丁平,郑迎冬.添加剂对铁皮石斛组织培养和快速繁殖的影响[J].中药材,2003,26(8):539-541.JIANG L,DING P,ZHENG Y D. Effects of additives on tissue culture and rapid propagation of Dendrobium canducum[J]. Journal of Chinese Medicinal Materials,2003,26(8):539-541.(in Chinese with English abstract)
[13]周江明.不同有机物对铁皮石斛试管苗生长发育的影响[J].中国农学通报,2005,21(8):49-50+98.ZHOU J M. Effects of different organic matter on regenerated shoots of Dendrobium candium[J]. Chinese Agricultural Science Bulletin,2005,21(8):49-50+98.(in Chinese with English abstract)
[14]刘世平.不同培养基及培养环境对铁皮石斛幼苗生长的影响试验[J].林业调查规划,2012,37(3):39-42.LIU S P. Effects test of different culture media and environment on seedling growth of Dendrobium candidum[J]. Forest Inventory and Planning,2012,37(3):39-42.(in Chinese with English abstract)
[15]宋顺,许奕,王必尊,等.不同培养基成分对铁皮石斛组织培养的影响[J].中国农学通报,2013,29(13):133-139.SONG S,XU Y,WANG B Z,et al. Different media on the tissue culture of Dendrobium officinale[J]. Chinese Agricultural Science Bulletin,2013,29(13):133-139.(in Chinese with English abstract)
[16]尚文倩,王政,侯甲男,等.不同红蓝光质比LED光源对铁皮石斛试管苗生长的影响[J].西北农林科技大学学报(自然科学版),2013,41(5):155-159.SHANG W Q,WANG Z,HOU J N,et al. Effects of light emitting diode(LED)with different red/blue quality ratios on the growth of Dendrobium officinale plantlet in vitro[J]. Journal of Northwest A&F University(Natural Science Edition),2013,41(5):155-159.(in Chinese with English abstract)
[17]杨维杰,梁君,杨林林.不同LED光质对铁皮石斛生长及多糖含量的影响[J].浙江农业科学,2015,56(6):1188-1190.YANG W J,LIANG J,YANG L L. Effects of different LED light on the growth and polysaccharide content of Dendrobium candidum[J]. Journal of Zhejiang Agricultural Sciences,2015,56(6):1188-1190.(in Chinese)
[18]高亭亭,斯金平,朱玉球,等.光质与种质对铁皮石斛种苗生长和有效成分的影响[J].中国中药杂志,2012,37(2):198-201.GAO T T,SI J P,ZHU Y Q,et al. Effects of light quality and germplasm on growth and effective ingredients of Dendrobium officinale germchit[J]. China Journal of Chinese Materia Medica,2012,37(2):198-201.(in Chinese with English abstract)