“二加二”法建立金线莲类原球茎培养新体系
|
摘要
本研究以金线莲组培苗茎段为试材,采用"二加二"法,即"两步诱导"(1.利用暗培养诱导出白化茎段;2.由白化茎段诱导类原球茎)加"两步增殖"(1.新诱导的类原球茎在诱导培养基上培养3周;2.利用L16(45)正交试验优选增殖培养方案)的方法,研究外植体来源、外植体处理方式、暗培养时间、培养基及附加成分对类原球茎诱导的影响,以及培养时间、光照强度、紫外光照射时间对类原球茎增殖的影响,并比较"二加二"法与传统单步法的培养效果。结果表明,适宜白化茎段诱导类原球茎的培养基为MS+2.5 mg/L 6-BA+0.2 mg/L NAA+1.0 mg/L S3307。黑暗腋生白化茎段是最佳外植体,最佳处理方式为整个茎段平放,3周暗培养得到的白化茎段能够显著提高类原球茎的诱导率。增殖培养5个因素对结果的影响顺序为:NH_4~+/NO_3~->NAA浓度>6-BA浓度>S3307浓度>蔗糖浓度。最佳配比为MS+0.5 mg/L S3307+4.0 mg/L 6-BA+0.4 mg/L NAA+NH_4~+/NO_3~-(15 mmol∶45 mmol)+蔗糖40 g/L,可获得增殖鲜重为246.90 g/L。增殖培养最佳时间6周。光照强度500 lx和253.7 nm紫外光每天照射6 h连续6周适宜类原球茎的增殖和总黄酮的积累。利用"二加二"法建立了金线莲类原球茎高效培养新体系,该体系诱导率、初始材料扩大倍数、增殖倍数均高于传统单步诱导法。92%的类原球茎能够稳定增殖,不发生分化和愈伤组织化。
In this study, the stem sections of Anoectochilus roxburghii in vitro were used as the material, and the method of 'double two-step' was discussed. The 'two-step-induction' had the etiolation stem sections induced by dark culture and the protocorm-like bodies induced by etiolation stem sections, and the 'two-step-proliferation' had the new PLBs cultured on induction medium for 3 weeks and proliferative culture program optimized by the orthogonal experimental design of L16(45). The effects of source and treatment method of explants, dark culture time, medium and additional elements on induction of PLBs, and the effects of culture time, light intensity, and UV irradiation time on proliferation culture were studied. The results showed that, medium which was suitable for the etiolation stem sections to induce PLBs was MS +2.5 mg/L 6-BA +0.2 mg/L NAA +1.0 mg/L S3307. The best explant was the etiolation stem sections from leaf axil in dark culture, and most suitable treat method was the whole stem sections horizontally onto the medium.Three weeks dark treatment could improve the induction frequency of PLBs from the tiolation stem sections significantly. The order of effects of the five factors was NH_4~+/NO_3~->concentration of NAA>concentration of 6-BA> concentration of S3307>concentration of sucrose. The optional experimental conditions were MS+0.5 mg/L S3307+4.0 mg/L6-BA+0.4 mg/L NAA+ NH_4~+/NO_3~-(15 mmol∶45 mmol)+ 40 g/L sucrose, which could get the proliferation fresh weight246.90 g/L. The optimal proliferation culturing time was 6 weeks. Light intensity 500 lx, and UV irradiation 253.7 nm for 6 h per day for 6 weeks were suitable for PLBs proliferation and accumulation of total flavonoids. New protocorm-like bodies system of A. roxburghii was established by the 'double two-step' method. The inducing rate, amplification of initial materials, and proliferation multiple of the method were higher than the index of the traditional single step method. 92% of the PLBs could keep the state of steady proliferation, and not to differentiate nor to formate callus.
In this study, the stem sections of Anoectochilus roxburghii in vitro were used as the material, and the method of 'double two-step' was discussed. The 'two-step-induction' had the etiolation stem sections induced by dark culture and the protocorm-like bodies induced by etiolation stem sections, and the 'two-step-proliferation' had the new PLBs cultured on induction medium for 3 weeks and proliferative culture program optimized by the orthogonal experimental design of L16(45). The effects of source and treatment method of explants, dark culture time, medium and additional elements on induction of PLBs, and the effects of culture time, light intensity, and UV irradiation time on proliferation culture were studied. The results showed that, medium which was suitable for the etiolation stem sections to induce PLBs was MS +2.5 mg/L 6-BA +0.2 mg/L NAA +1.0 mg/L S3307. The best explant was the etiolation stem sections from leaf axil in dark culture, and most suitable treat method was the whole stem sections horizontally onto the medium.Three weeks dark treatment could improve the induction frequency of PLBs from the tiolation stem sections significantly. The order of effects of the five factors was NH_4~+/NO_3~->concentration of NAA>concentration of 6-BA> concentration of S3307>concentration of sucrose. The optional experimental conditions were MS+0.5 mg/L S3307+4.0 mg/L6-BA+0.4 mg/L NAA+ NH_4~+/NO_3~-(15 mmol∶45 mmol)+ 40 g/L sucrose, which could get the proliferation fresh weight246.90 g/L. The optimal proliferation culturing time was 6 weeks. Light intensity 500 lx, and UV irradiation 253.7 nm for 6 h per day for 6 weeks were suitable for PLBs proliferation and accumulation of total flavonoids. New protocorm-like bodies system of A. roxburghii was established by the 'double two-step' method. The inducing rate, amplification of initial materials, and proliferation multiple of the method were higher than the index of the traditional single step method. 92% of the PLBs could keep the state of steady proliferation, and not to differentiate nor to formate callus.
引文
[1]Ye S Y,Shao Q S,Zhang A L.Anoectochilus roxburghii:Areview of its phytochemistry,pharmacology,and clinical applications[J].Journal of Ethnopharmacology,2017,209(14):184-202.
[2]Refish N M R,Fu C H,Wang M G.Comparative study of the chemical components of Anoectochilus roxburghii and Anoectochilus formosanus tissue culture[J].International Journal of Life Sciences Research,2015,3(2):81-87.
[3]Giap D D,Thai T D,Thang D D,et al.Effects of several organic extracts on the growth,yield and quality of Anoectochilus formosanus biomass[J].International Journal of Agricultural Technology,2018,14(2):171-182.
[4]杨成行,李晓婷,袁建振,等.金线莲组织培养技术研究进展[J].草业科学,2018,35(5):1047-1056.
[5]何碧珠,何官榕,黄铭星,等.福建野生金线莲快速繁育技术[J].农业工程,2013,3(2):72-76,39.
[6]王建明,王松良,詹巧杰,等.金线莲组织培养的条件优化研究[J].中国现代中药,2013,15(1):45-49.
[7]韩晓红,罗明英,欧阳志成,等.金线莲原球茎增殖研究[J].北方园艺,2012,36(24):144-146.
[8]封应丽,余元涛,杨春桃,等.植物生长物质对金线莲离体再生的影响[J].北方园艺,2010,34(7):136-138.
[9]黄敏,梁春辉,李秀平,等.应用正交设计优化金线莲继代增殖和生根培养的研究[J].北方园艺,2015,39(1):96-98.
[10]吕志伟,吴文平,毕丽伟,等.建立金线莲原球茎悬浮体系生产次生代谢产物[J].中国生物工程杂志,2012,32(5):43-50.
[11]陈钢,林兰英,王建勤.药用金线莲组培类原球茎研究初报[J].福建中医药,1994,25(4):23.
[12]王建勤,林兰英,陈钢.金线莲原球茎诱导与植株再生[J].植物学通报,1996,13(4):54-55.
[13]韩晓红,王春龙,段春红.不同激素水平对金线莲组织培养的影响[J].广东农业科学,2012,39(18):94-97.
[14]邱玥,龚宁,张奎一.金线兰种子萌发及其原球茎的增殖培养[J].广西植物,2010,30(4):555-559.
[15]崔仕超.金线莲活性物质的提取及生理活性的研究[D].汕头:汕头大学,2010.
[16]王贵章,徐凌飞,马锋旺,等.富士苹果黄化茎段再生不定芽的研究[J].西北农林科技大学学报(自然科学版),2006,34(6):79-81.
[17]师校欣,杜国强,高仪,等.黑暗培养对苹果组培快繁及叶片再生的影响[J].河北农业大学学报,2004,27(4):18-21.
[18]郑连金,马增强,肖玉兰.不同光照强度对台湾金线莲生长发育和次生代谢物合成的影响[J].安徽农学通报,2016,22(16):25-26,39.
[19]魏翠华,谢宇,秦建彬,等.光照强度对金线莲生长及产量的影响[J].北方园艺,2015,39(12):139-141.
[20]李杰,王再花,刘海林,等.不同光质的LED对2种金线莲组培苗增殖、生根及生长的影响[J].热带作物学报,2017,38(9):1666-1670.
[21]王伟苏,明华,李惠华,等.蓝光补光对福建金线莲生长及药用成分的影响[J].热带作物学报,2018,39(5):894-899.
[22]崔琳琳,周一鸣,季红斌,等.不同光质对苦荞萌发过程中的总黄酮类化合物及其抗氧化活性的影响[J].食品工业科技,2016,37(6):104-108.
[2]Refish N M R,Fu C H,Wang M G.Comparative study of the chemical components of Anoectochilus roxburghii and Anoectochilus formosanus tissue culture[J].International Journal of Life Sciences Research,2015,3(2):81-87.
[3]Giap D D,Thai T D,Thang D D,et al.Effects of several organic extracts on the growth,yield and quality of Anoectochilus formosanus biomass[J].International Journal of Agricultural Technology,2018,14(2):171-182.
[4]杨成行,李晓婷,袁建振,等.金线莲组织培养技术研究进展[J].草业科学,2018,35(5):1047-1056.
[5]何碧珠,何官榕,黄铭星,等.福建野生金线莲快速繁育技术[J].农业工程,2013,3(2):72-76,39.
[6]王建明,王松良,詹巧杰,等.金线莲组织培养的条件优化研究[J].中国现代中药,2013,15(1):45-49.
[7]韩晓红,罗明英,欧阳志成,等.金线莲原球茎增殖研究[J].北方园艺,2012,36(24):144-146.
[8]封应丽,余元涛,杨春桃,等.植物生长物质对金线莲离体再生的影响[J].北方园艺,2010,34(7):136-138.
[9]黄敏,梁春辉,李秀平,等.应用正交设计优化金线莲继代增殖和生根培养的研究[J].北方园艺,2015,39(1):96-98.
[10]吕志伟,吴文平,毕丽伟,等.建立金线莲原球茎悬浮体系生产次生代谢产物[J].中国生物工程杂志,2012,32(5):43-50.
[11]陈钢,林兰英,王建勤.药用金线莲组培类原球茎研究初报[J].福建中医药,1994,25(4):23.
[12]王建勤,林兰英,陈钢.金线莲原球茎诱导与植株再生[J].植物学通报,1996,13(4):54-55.
[13]韩晓红,王春龙,段春红.不同激素水平对金线莲组织培养的影响[J].广东农业科学,2012,39(18):94-97.
[14]邱玥,龚宁,张奎一.金线兰种子萌发及其原球茎的增殖培养[J].广西植物,2010,30(4):555-559.
[15]崔仕超.金线莲活性物质的提取及生理活性的研究[D].汕头:汕头大学,2010.
[16]王贵章,徐凌飞,马锋旺,等.富士苹果黄化茎段再生不定芽的研究[J].西北农林科技大学学报(自然科学版),2006,34(6):79-81.
[17]师校欣,杜国强,高仪,等.黑暗培养对苹果组培快繁及叶片再生的影响[J].河北农业大学学报,2004,27(4):18-21.
[18]郑连金,马增强,肖玉兰.不同光照强度对台湾金线莲生长发育和次生代谢物合成的影响[J].安徽农学通报,2016,22(16):25-26,39.
[19]魏翠华,谢宇,秦建彬,等.光照强度对金线莲生长及产量的影响[J].北方园艺,2015,39(12):139-141.
[20]李杰,王再花,刘海林,等.不同光质的LED对2种金线莲组培苗增殖、生根及生长的影响[J].热带作物学报,2017,38(9):1666-1670.
[21]王伟苏,明华,李惠华,等.蓝光补光对福建金线莲生长及药用成分的影响[J].热带作物学报,2018,39(5):894-899.
[22]崔琳琳,周一鸣,季红斌,等.不同光质对苦荞萌发过程中的总黄酮类化合物及其抗氧化活性的影响[J].食品工业科技,2016,37(6):104-108.