【作者】 徐步青；

【导师】 崔永一；

【作者基本信息】 浙江农林大学 ， 园林植物与观赏园艺， 2012， 硕士

【摘要】 本试验以铁皮石斛类原球茎为材料，用不同光照强度、不同培养时间对铁皮石斛类原球茎生物量和主要有效成分多糖、生物碱的积累进行研究；同时探索影响铁皮石斛PLBs生物反应器培养的条件；并在此基础上对反应器内各种理化性质的变化与类原球茎生长量的变化及多糖积累之间的关系进行研究。研究结果如下：1.铁皮石斛类原球茎生物量的增加在光照强度≥2000Lx下培养40～50d均较高，生产上需要类原球茎维持旺盛的分裂，获得最佳生物量的培养条件为光照强度2000Lx下培养40d，生物量达到0.74g；多糖含量以光照强度2000Lx下培养30d最高，达到16.88%；生物碱含量以光照强度500Lx下培养30d最佳，达到0.028%。2.在悬浮培养条件下,利于铁皮石斛类原球茎增殖和多糖积累的培养基组分为：1/2MS基本培养基添加1mg·L^-1NAA、5%椰乳、3%蔗糖，pH值为6.0。接种量为40g﹒L^-1，采用孔径为15μm的多孔喷头，通气量用0.10cc·min^-1和0.05cc·min-1交替使用等培养条件有效地提高类原球茎增殖系数和多糖含量。3.对多糖积累机理的研究发现：培养36～42d铁皮石斛类原球茎的鲜质量和干质量达最大值，多糖及生物碱含量分别在42d和36d的培养时间下达最高，分别为24.03%和0.056%；在36d的培养周期内，液体培养基中的蔗糖浓度随着培养时间的延长开始转化为葡萄糖和果糖，在培养20d之后，培养基中的蔗糖浓度降至2.0mg﹒ml^-1，而葡萄糖和果糖含量在18d达到最高值，随后开始下降；同时对培养基中元素含量的测定发现，N、P、K、Mg、Ca、Zn、Cu、Mn、B等元素含量均下降，其中P、Mg、Ca、Zn、Mn被大量利用。4.在生物反应器培养中，接种量为80g﹒L-1利于铁皮石斛类原球茎鲜质量的净增长和多糖的积累；球形生物反应器较其他形状反应器能够更有效的促进类原球茎增殖和多糖积累；通气量则以2.0cc·min^-1处理效果最佳；培养过程中，由于营养物质的大量消耗，通过中途补料和换料均能及其显著的促进类原球茎干质量和多糖的增加。更多还原

【Abstract】 Protocorm-like bodys of Dendrobium officinale were selected as explants.The factors whichaffect protocorm-like bodys biomass, polysaccharide and alkaloid accumulation including lightintensity and culture time were tested; While exploring the conditions of Dendrobium officinaleprotocorm-like bodys bioreactor culture;the relationship between medium composition changesand protocorm-like bodys growth、polysaccharide accumulation were also made.The results wereas follows:1.The results indicated that light intensity at2000Lx for40days was the most effective inincreasing growth and the maximum biomass was0.74g. In this condition, PLBs can keep thestrong split. the maximum polysaccharide Percentage content was16.88%on incubating30daysand Light intensity2000Lx.Light intensity at500Lx for30days was beneficial to alkaloidpercentage content,the maximum is0.028%.2.Results showed that1/2MS medium supplemented with1.0mg﹒L^-1NAA,50g﹒L^-1coconutmilk, and30g·L^-1sucrose,6.0pH, was benefit for the proliferation and polysaccharideaccumulation of the PLBs. The culture conditions such as Inoculation with40g PLBs in1L liquidmedium, using15μm pore porous nozzle, aeration volumes by0.10cc min-1and0.05cc min-1used alternately could improve the PLBs growth and polysaccharide contents.3.The fresh mass and dry mass of Dendrobium officinale reached the maximum for36⁴2d.and the content of Polysaccharide alkaloid was the highest （reached24.03%and0.056%） in42dand36d; With the prolonged incubation time at the culture period for36d, the sucroseconcentration in the liquid medium was converted into glucose and fructose, and it dropped to2.0mg﹒ml-1after20d, but the content of Glucose and fructose reached the highest on the18d andthen decreased. At the same time, by measuring we could find the content of elements, such as N、P、K、Mg、Ca、Zn、Cu、Mn、B, and so on, decreased, and which P, Mg, Ca, of Zn, Mn has beenwidely used.4. Inoculation with40g PLBs in1liter of liquid medium was most suitable for PLBsproliferation and polysaccharide accumulation. spherical bioreactor can be more effective thanother shape bioreactor to promote PLBs proliferation and polysaccharide accumulation; the best treatment of aeration volumes was2.0cc min-1for bioreactor culture. In cultivation process, due tothe consumption of large quantities of nutrients, feeding and refueling midium can be significantlystimulate PLBs proliferation and polysaccharide contents。更多还原