【作者】 卢文芸；

【导师】 乙引； 严志坚；

【作者基本信息】 贵州师范大学 ， 植物学， 2005， 硕士

【摘要】 本论文以贵州产环草石斛为实验材料,研究了其组织培养过程、栽培过程以及多糖和石斛碱等次生代谢产物积累的规律。结果如下: 1、建立了环草石斛种子及其茎段的离体快繁体系。结果表明:环草石斛种子在1/2MS +3%蔗糖的无激素培养基上萌发率可达99%,原 球 茎 或 拟 原 球 茎 分 化 增 殖 的 适 宜 培 养 基 为1/2MS+NAA0.2mg/L+6-BA2.0mg/L , 种 子 苗 壮 苗 的 适 宜 培 养 基 为1/2MS+NAA0.2 mg/L+6-BA1.0mg/L,种子苗生根的适宜培养基为1/2MS+NAA0.7mg/L ; 茎 段 外 植 体 芽 诱 导 的 适 宜 培 养 基 为MS+NAA0.1mg/L+6-BA1.0mg/L , 芽 增 殖 与 继 代 的 适 宜 培 养 基 为MS+NAA0.1mg/L+6-BA3.0mg/L , 壮 苗 与 生 根 适 宜 培 养 基 为1/2MS+NAA0.7mg/L;移栽最适基质均以碳酸盐小石子:木屑=1:1,上面覆盖1cm厚的苔藓为最好,幼苗的成活率达85～95%。 2、首次建立了环草石斛愈伤组织悬浮培养体系,并获得了通过悬浮培养规模化提供可供移栽试管苗的技术。结果表明:环草石斛愈伤组织诱导的适宜外植体为茎尖和茎段上部的茎节,愈伤组织诱导增殖的最适固体培养基为MS1+2,4-D(0.1～2.0)mg/L+3%蔗糖;愈伤组织悬浮培养的最适培养条件为: MS1+2,4-D 0.1mg/L+3%蔗糖的液体培养基,pH值( 5.8～6.2),摇床转速100 rpm,培养温度(25±2)℃,光周期12h/12h,光照强度(500～1000)lx,60d继代一次;悬浮培养的愈伤组织分化获得的试管苗的增殖、壮苗和生根的适宜培养基与茎段外植体的相同,移栽最适基质也与环草石斛种子苗及其茎段更多还原

【Abstract】 This dissertation studied the tissue culture, fieldcultivation and metabolite accumulation of Dendrobiumloddigesii Rolfe, a Chinese traditional medicine. The resultswere as follows: Firstly, a method was established on solid and suspentiontissue culture for D. loddigesii. The optimal mediums forpropagating protocorm, strengthing seed-originated seedlingand rooting seed-originated seedling were 1/2MS + NAA 0.2mg/L+ 6-BA2.0mg/L, 1/2MS + NAA 0.2mg/L + 6-BA 1.0mg/L and 1/2MS +NAA 0.7mg/L, respectively. The optimal medium for inducing buds,propagating, rooting from stem explants were MS + NAA 0.1mg/L+ 6-BA 1.0mg/L, MS + NAA 0.1mg/L + 6-BA 3.0mg/L, 1/2MS + NAA0.7mg/L, respectively. In addition, the optimal medium forcultivating was limestone : wood power = 1:2 with 1cm of mossabove. In this case, about 85～95% of survival rate was found. Secondly, the suspension culture lines of callus of D.loddigesii. were firstly reported, which opens an alternativeroad to large-scale culture this herb. The explants exertedstrong influence on the induction of callus of D. loddigesii.It is very difficult for mature stems to dedifferentiate. Thecallus induction rate of young stem peak tissue were higher thanmature stem. The callus had the best inductional rate in theMS1+2,4-D(0.1～2.0)mg/L +3% sugar solid medium . The callus ofD. loddigesii had best multiplication rate in liquid MS1 mediumand it grows better in lightness than dark. In addition, Thebest suspension culture medium of callus of Dendrobiumloddigesii Rolfe. is MS1 + 2,4-D 0.1 mg/L + 3% sugar, pH(5.8～6.2),100 rpm, (23～2)℃, 12h/12h, (500～1000)lx, the mediumis changed every 60 days. The optimal medium for propagation,strengthing seedling, rooting and cultivating of the tubingshoots which comes from the callus of suspension culture is sameto stem explants seedling and seed-originated seedling. Thirdly, the manual cultivated system which imitates wildwas established. It effectively resolved the problem of lowsurvival rate of transplanting about tissue culturedmaterials. Finally, the colorimetry method of bromocresol green andphenol-vitriol which studied total alkaloids andpolysaccharides was established. The dissertation probed intothe relation between the growth and the secondary metaboliteof D. loddigesii.. The results indicated the growth curve ofcallus in suspension culture was “S” type. On the culturingcondition of light, the secondary metabolite accumulation wasassociated with its growth. But on the condition of dark, thecallus grows slowly and the accumulation of polysaccharides wasnot associated with its growth. At the same time, theaccumulation of total alkaloids was enhanced greatly. Theeffective component in tubing shoots, callus, PLBs approachedor exceeded that in stem of wild D. loddigesii. These factsillustrate that tubing shoots, callus and PLBs have thepossibility which extract directly original effectivecomponents. The studied results provided basic datum for germ plasmconservation, medicinal materials scale cultivation and theindustrialization of secondary metabolite of D. loddigesii..更多还原