【作者】 张建佳；

【导师】 何凤发；

【作者基本信息】 西南大学 ， 药用植物栽培， 2011， 硕士

【摘要】 黄连为毛莨科(Ranuculaceae)的黄连(Coptis chinensis Franch)、三角叶黄连(Copt is deltoidea C. Y. Cheng et Hsiao)或云连(Coptis teeta Wall)的干燥根茎。黄连根茎含有大量生物碱,对多种疾病有显著疗效,已在临床上广泛应用。黄连根茎生长极为缓慢,药源紧缺。应用植物细胞培养以生产次生代谢产物能够克服化学合成的不足,以及人工栽培的困难。而由于在植物细胞的培养过程中,细胞的生长与其次生代谢产物的合成之间又存在矛盾,而不同培养基对于促进细胞的生长和次生代谢产物的合成有着不同的作用。因此,本实验在前人研究基础上,对毛茛科植物黄连采用二步培养法进行深入探索,为黄连获取药用次生代谢产物、大规模工业化生产、以及加速植物细胞培养技术商业化生产进程等提供有效的途径和方法。对两步培养法中的基本培养基更改为6,7-V1和6,7-V2,为两种培养基分别设置不同浓度的激素,分别进行细胞生长和小檗碱含量测定,以期得到最佳的激素浓度组合。结果如下：1黄连外植体的消毒：选取黄连的叶片作为外植体,首先用三年生的黄连叶片对黄连愈伤组织诱导培养的消毒方法进行了预备实验,从而得到最适消毒方法和消毒时间。具体为：先用软毛刷将尘埃刷除,绒毛较多的黄连叶片用皂液浸泡5min左右,用清水洗去皂液,清水冲洗2h左右,用灭菌消毒过的吸水纸吸干表面水分,再用75%酒精浸泡30s,取出后用加有1～2滴吐温-80的0.1%HgCl2溶液浸泡12min,无菌水冲洗6次。2初代培养：诱导黄连叶片愈伤组织的最适激素浓度配比均为6,7-V+2.4-D 1.0mg/L+KT0.2mg/L,接种后黄连叶片愈伤组织在第32d开始启动,出愈率为89.5%。3继代培养：从愈伤组织开始出现50d左右在固体培养基上进行继代培养。为了防止褐变以及材料渗出过多水分的影响,每隔15d左右转接一次。4愈伤组织状态的调控：灰白色水渍状的愈伤组织,可以单独加入玉米素(ZT),或者是配以6-BA和NAA等激素继代4次左右,愈伤组织的状态可以得到明显改善。对于浅黄色、黄色或者浅绿色、绿色类愈伤组织,加入ZT 0.05 mg/L+6-BA 0.5 mg/L,或者配以水解酪蛋白(CH500),继代2到3次以后,愈伤组织的状态也能得到较好的改善。5二步法细胞悬浮培养：用一步法培养黄连叶片的悬浮细胞时,最佳激素浓度配比为6.7-V1+2.4-D1.5 mg/L+KT0.2 mg/L;收获的细胞鲜重为182.92 g/L,干重为21.58 g/L,其总生物碱的含量为8.3%。用二步法培养黄连叶片的悬浮细胞时,最佳激素浓度配比为6,7-V2+NAA 1.5 mg/L+KT 0.2 mg/L；收获的细胞鲜重为129.01 g/L,干重为17.89g/L,其总生物碱的含量为13.8%。两种方法培养出的细胞对于细胞生长而言,一步法的细胞生长指标高于二步法；而对于生物碱的含量而言,虽然各生物碱的含量不是成比例增加的,但是二步法的生物碱含量显然高于一步法。更多还原

【Abstract】 As medicine, coptis is the dry roots of the Coptis chinensis Franch. Coptis deltoidea C.Y. Cheng et Hsiao,and Coptis teeta Wall in Ranuculaceae. Coptis roots contains a lot of alkaloids, and there is remarkable curative effect on various diseases. already widely used in clinical Growth of coptis dry roots is extremely slow, pharmaceutical is very lack. Plus coptis belong to yin born plants, artificially cultivated is difficult.To Production secondary metabolites by Plant cell culture can overcome the chemical synthesis shortage and the difficult of Artificially cultivated.Due to the process of training in plant cells.there is contradiction between cell growth and the synthesis of secondary metabolites. And different medium to promote cells to grow and secondary metabolite synthesis has different effect.So In the previous research foundation, this experiment deep-explor the buttercup family plant coptis by two-step culture method. The basic medium were change for set up for 6,7-V1 and 6,7-V2.And set different concentrations of hormone for the two kinds of culture media, with the cell growth and Berberine content determination, expecting to get the best hormone concentration combination. The results are as follows:1、Explant disinfection:coptis leaves as selected explant, fisrt prepare the experiment on the disinfection methods of rhizome coptis blade callus induction training with three years borned.optimal disinfection method and time is:first use soft-brush brush dust, Soak fluffy coptis blade about 5min with soap, And remove soap. Then flush water 2h or so. After washing, use the disinfected paper absorb surface moisture, soaking in 75% ethanol for 30s, At last. remove it and timely use HgCl2 added 1～2 drops 0.1% of polysorbate-80 soaking 12min, and flushed 6 times by sterile water.2、Primary training:the optimal hormone concentration ratio in inducing coptidis blade callus is 6,7-V+2.4-D 1.0 mg/L +KT 0.2 mg/L. In this hormone concentration, coptis blade callus is started in the 32 day,and the callus induction rate is 89.5%.3、Subculture:subculture on the solid medium when callus appeared about 50d.and transfer once about every 15day.This is to prevent the impact of browning, and materials leak too much fluid.4、Callus state regulation:hoar, waterlogging shape callus can be improved in suculturing 4 times within ZT, or match with 6-BA and NAA.For light yellow, yellow, light green, green callus, state also can be better improved in suculturing 2 or 3 times alone within ZT 0.05 mg/L+6-BA 0.5 mg/L. or match with casein acids hydrolysate (CH500).5、Two-step method culture cell:Best hormone concentration ratio is 6.7-V1+2.4-D 1.5 mg/L+KT 0.2 mg/L in suspending of one-step method.dry weight of harvest cell is 21.58 g/L.fresh weight is 182.92 g/L,and the content of total alkaloid is 8.3%; Best hormone concentration ratio is 6,7-V:+NAA 1.5+KT 0.2 in suspending of two-step method.dry weight of harvest cell is 17.89 g/L.fresh weight is 129.01 g/L.and the content of total alkaloid is 13.8%.;In concerned of cell growth, cell growth index determinated by one-step method is higher than two-step method. And in concerned of alkaloid content, alkaloid content is obviously higher than determinated by one-step method, although each alkaloid content is not proportional increase. Keywords:Coptis, Callus, Cell Suspension Culture, Alkaloid更多还原