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 NH4+/NO3?>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/L 6-BA+0.4 mg/L NAA+NH4+/NO3?(15 mmol∶45 mmol)+40 g/L sucrose,which could get the proliferation fresh weight 246.90 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.
作者
焦展
安胜军
邵铁梅
JIAO Zhan;AN Shengjun;SHAO Tiemei(Hebei Chemical&Pharmaceutical College/Center of Bioreactor and Protein Drug Research and Development of Hebei Universities,Shijiazhuang,Hebei 050026,China;Hebei University of Chinese Medicine,Shijiazhuang,Heibei 050091,China)
出处
《热带作物学报》
CSCD
北大核心
2019年第2期314-322,共9页
Chinese Journal of Tropical Crops
基金
河北省高等学校科学技术研究项目(No.Z2015134)
国家自然科学基金项目(No.81473578)
关键词
金线莲
类原球茎
高效体系
诱导
总黄酮
Anoectochilus roxburghii
protocorm-like bodies(PLBs)
efficient system
induction
total flavonoids
