Development of in vitro techniques for the important medicinal plant Veratrum californicum

VERATRUM CALIFORNICUM (Liliaceae) is an important monocotyledonous medicinal plant which is the only source of the anticancer compound cyclopamine. An IN VITRO culture system for somatic embryogenesis and green plant regeneration of VERATRUM CALIFORNICUM was developed. Embryogenic calli were induced from mature embryos on induction medium. Five basal media supplemented with different growth regulators were evaluated for embryogenic callus induction, modified MS medium with 4 mg/L picloram showing the best result for embryogenic callus production. Fine suspension cell lines were established by employing friable embryogenic calli as starting material and AA medium and L2 medium as culture media. The suspension cell lines cultured in AA medium with 4 mg/L NAA appeared to be fresh yellow and fast growing. The suspension cells were cryopreserved successfully and recovered at a high rate. Green plants were regenerated from embryogenic calli maintained on solid medium with 73 % regeneration ability (green plants/100 calli) in 27-month-old culture. The IN VITRO plantlets contained the steroid alkaloids cyclopamine and veratramine. This IN VITRO system will form the basis for metabolic engineering of VERATRUM cells in the context of biotechnological production of pharmaceutically important secondary metabolites. DMSO:dimethyl sulfoxide fw:fresh weight NAA:naphthaleneacetic acid 2,4-D:2,4-dichlorophenoxyacetic acid picloram:4-amino-3,5,6-trichloro-2-pyridinecarboxylic acid dicamba:3,6-dichloro-2-methoxybenzoic acid.     

In vitro plant regeneration from leaf-derived callus of Cimicifuga racemosa

Cimicifuga racemosa (L.) Nutt., also known as Black Cohosh, is among the top 10 selling medicinal herbs in the United States. The rhizomes have been used to relieve menopausal discomfort. This plant is wild crafted and conservationists have expressed concerns with the sustainability of C. racemosa. Excised tissues from young leaves of C. racemosa were cultured on Murashige and Skoog's medium (MS) supplemented with various concentrations of NAA and TDZ for production of callus. The optimum callus growth and maintenance was in 1.0 microM NAA plus 0.5 microM TDZ. Two-month-old calli were sub-cultured on different concentrations of cytokinins (BA, kinetin, 2ip, TDZ) or in combination with GA(3) for shoot induction. The rate of shoot induction and proliferation was higher in MS media supplemented with 2.0 or 4.0 microM of TDZ. Concentrations of TDZ greater than 4.0 microM suppressed shoot growth. Adding 3.5 microM of GA(3) into media containing BA increased shoot growth. The presence of GA(3) with kinetin or TDZ did not affect shoot production. For rooting, shoots were transferred to MS medium with activated charcoal supplemented with various auxins (IAA, IBA and NAA), roots were noticed 20 days after transference. Activated charcoal was an essential component for vigorous rooting formation. Our results suggest that conservation of C. racemosa is possible through in vitro multiplication of leaf-derived callus.     

山葵愈伤组织培养及其风味物质的研究

通过山葵愈伤组织的诱导培养,测定培养物中的风味物质,为通过组织培养的手段来生产药用成分提供依据.选择山葵幼叶和叶柄作为外植体,以MS为基本培养基,配以不同浓度的激素进行愈伤组织的诱导.选择继代几次以后的愈伤组织进行风味物质的测定.确定MS-4-6-BA 1.0+NAA 3.0为愈伤组织诱导的最佳条件,致密型的愈伤组织经...     

培养条件对三叶青愈伤组织生长及总黄酮含量的影响

研究培养条件对三叶青愈伤组织生长及黄酮积累的影响,建立用愈伤组织生产三叶青黄酮的方法。以三叶青的叶诱导产生的愈伤组织为材料,分别采用单因素实验、正交实验的方法比较了不同光照周期、不同有机添加物、不同前体、3种基础培养基、不同6-BA与NAA浓度的培养条件下愈伤组织生长的情况及黄酮含量。用愈伤组织生产三叶青黄酮可采用两步培养法来进行:先在促进愈伤组织生长的最佳处理组合条件:12 h/d间断性光照、MS+3.0 mg/L6-BA+2.0 mg/L NAA+2 g/L蛋白胨下培养30 d,再转入最利于黄酮积累的处理组合:连续光照、B5+4.0 mg/L 6-BA+2.0 mg/L NAA+40 mg/L苯丙氨酸下培养20 d。采用该方法培养的愈伤组织中总黄酮最大浓度可以达到28.4±3.9 mg/g DW。采用愈伤组织培养方式生产三叶青黄酮,不仅可以大大地缩短生产周期,而且培养物中的总黄酮含量也明显高于原植株叶片,接近于根茎中的黄酮含量,具有较大的可行性。     

Micropropagation of Hydrastis canadensis: Goldenseal a North American endangered species

An in vitro propagation protocol for rapidly producing Hydrastis canadensis L., Goldenseal, plantlets from disk tissue of young leaves was developed. Leaf explants were inoculated on MS medium supplemented with various concentrations of NAA and TDZ for production of callus. Two-month-old calli were sub-cultured on MS media containing cytokinins (BA, kinetin, TDZ) in different concentrations for shoot initiation. The optimum level of callus induction and maintenance was in 5.3 microM NAA in combination with 2.2 microM of TDZ. Shoot multiplication was achieved on MS medium with 2.2 microM TDZ in combination with 0.5 microM NAA. The alkaloid profile of micropropagated plantlets was similar to the profile of the mother plants. These results suggest that our in vitro propagation protocol will produce a positive impact in the conservation of H. canadensis.     

海南广藿香试管内芽分化与愈伤组织诱导研究

目的选择海南广藿香的最佳芽分化和愈伤组织诱导培养基。方法利用海南广藿香的叶柄、茎尖和带节茎段进行组织培养芽分化试验,并对上述材料进行愈伤组织诱导培养基选择试验。结果在芽分化培养基中,MS＋3-吲哚丁酸（IBA）1．0mg／L＋6-苄基腺嘌呤（6-BA）0．6mg／L培养基能够较好地诱导茎尖和带节茎端长出新芽,诱导率分别为94．3％和94．6％;在愈伤组织诱导培养基中,MS＋萘乙酸（NAA）0．1mg／L＋6-BA2．0mg／L培养基能较好地诱导海南广藿香叶柄和带节茎段产生愈伤组织,诱导率分别为80．0％和100％。结论海南广藿香的最佳芽分化和愈伤组织诱导培养基分别为Ms＋IBA1．0mg／L＋6-BA0．6mg／L和Ms＋NAA0．1mg／L＋6-BA2．0mg／L。     

凹叶厚朴愈伤组织的诱导及其有效成分含量的比较

目的:研究凹叶厚朴愈伤组织的诱导、生长情况并比较愈伤组织内有效成分的含量。方法:采用单因素比较、正交设计试验,研究影响凹叶厚朴愈伤组织诱导和生长的各种因素;采用高效液相色谱法测定不同来源愈伤组织内有效成分的含量。结果:外植体脱分化形成愈伤组织的能力,以顶芽及茎段最好,雌蕊次之;愈伤组织诱导的最适培养基为B5+NAA0.1mg.L-1+6-BA1.0mg.L-1,而B5+NAA0.5mg.L-1+6-BA4.0mg.L-1对愈伤组织生长促进作用明显。以WPM为基本培养基诱导愈伤组织,继代后厚朴酚与和厚朴酚含量的总和可达0.1679%~0.2344%,但愈伤组织诱导率低,为33.08%~48.65%;培养条件为MS+NAA0.5mg.L-1+6-BA1.0mg.L-1时,愈伤组织诱导率为62.12%,继代后2酚总量为0.1686%。结论:本试验结果对凹叶厚朴愈伤组织的诱导及高产细胞系的筛选具有一定的参考价值。     

胀果甘草愈伤组织诱导培养

试验以甘草的子叶和下胚轴为外植体，分别接种在不同激素组合的MS培养基上，黑暗条件下培养，诱导愈伤组织。结果表明：诱导甘草子叶愈伤组织的最适培养基为MS 2，4-D(1mg／L) BA(1mg／L)，其愈伤组织诱导率达100％，而且其愈伤组织鲜重与接种外植体重的比值达到15．37倍；诱导甘草下胚轴愈伤组织的最佳培养基为MS 2，4-D(0．5mg／L)和MS 2，4-D(0．5) NAA(0．5)，通过在这两种培养基上进行愈伤组织的增殖培养，也得到了十分可观的增殖效果，其相对生长量分别达到了381．52％和498．80％。     

银杏愈伤组织培养及银杏内酯测定的研究(英文)

国内首次采用固体培养的方法对组织培养法生产银杏内酯进行了研究．考察了银杏不同外植体的愈伤组织的诱导情况;对得到的各种愈伤组织进行了继代驯化,并考察了光照、ＭＳ培养基成分等多种理化因子及激素对愈伤组织的诱导及生长的影响;应用生物法（ＰＡＦ）和ＨＰＬＣ对愈伤组织中的银杏内酯Ａ和Ｂ进行了测试．结果显示,愈伤组织中的银杏内酯Ａ和Ｂ的含量在１０×１０－６～５０×１０－６之间     

Production of eurycomanone from cell suspension culture of Eurycoma longifolia

Context: Eurycomanone is found in the Eurycoma longifolia Jack (Simaroubaceae) tree, exhibits significant antimalarial activity, improves spermatogenesis, suppresses expression of lung cancer cell tumour markers and regulates signalling pathways involved in proliferation, cell death and inflammation.

Objectives: Establishment of cell suspension culture of E. longifolia to determine the eurycomanone accumulation during cultures.

Materials and methods: Callus of E. longifolia was cultured in MS medium supplemented with 0.8% agar, 30/L sucrose, 1.25?mg/L NAA and 1?mg/L KIN for biomass production. Cell suspension culture was established by transferring friable calli to the same medium without agar. Eurycomanone content during cell culture was determined by HPLC with a C18 column, flow rate of 0.8?mL/min, run time of 17.5?min, detector wavelength of 254?nm. The stationary phase was silica gel and the mobile phase was acetonitric:H₂O. Roots of 5 year-old trees were used as the control.

Results: The cells from 3?g of inoculum increased in biomass with a maximum value of 16?g fresh weight (0.7?g dry weight) at 14th day of culture. The cell growth then decreased from day 14 to day 20. Eurycomanone was produced during culture from the beginning to 20th day, its highest content (1.7?mg/g dry weight) also obtained at 14th day (the control is 2.1?mg/g dry weight).

Discussion and conclusions: Cell suspension culture of E. longifolia is a suitable procedure to produce eurycomanone. The yield of eurycomanone biosynthesis in 14 days-old cells are relatively high, approximately 0.8 times the control.     

桔梗悬浮培养对细胞天麻素的生物转化

天麻素即对羟基甲基苯 β Ｄ 吡喃葡糖苷 (4 ｈｙｄｒｏｘｙｍｅｔｈｙｌｐｈｅｎｙｌ β Ｄ ｇｌｕｃｏｐｙｒａｎｏｓｉｄｅ) ,为兰科植物天麻 (ＧａｓｔｒｏｄｉａｅｌａｔａＢｌ .)的主要活性成分[1] ,有镇静、抗惊厥、抗炎、镇痛及增强机体免疫功能等作用。用植物细胞悬浮培养物对外源底物进行生物转化从而对其结构进行修饰 ,以获得更有意义的产物的报道[2 ,3 ] 很多 ,也是当今研究的热点。生物转化(ｂｉｏｔｒａｎｓｆｏｒｍａｔｉｏｎ) ,也称生物催化 (ｂｉｏｃａｔａｌｙｓｉｓ) ,是利用植物离体培养细胞或器官、动物、微生物及细…     

人参愈伤组织培养的初步研究

本工作初步研究了人参愈伤组织培养的适宜培养基和培养条件、各种生长物质和补充物质对其生长及其人参皂甙含量的影响。此外,还对不同的无性繁殖系与不同年龄的人参愈伤组织的生长特性,进行了初步的观察。应用薄层层析-光电比色法测定证明:愈伤组织中人参皂甙的成分和含量和栽培的人参根相近;所获得的愈伤组织具有合成人参皂甙的能力。     

地丁草组织培养及无性系建立的研究

目的:为满足人们栽培对种苗的需要,并保护野生资源.方法:以地丁革不同外植体为材料,进行了愈伤组织诱导与分化、不定芽生根和试管苗生根继代培养研究,建立起地丁革的无性系.结果:MS+ NH4H2PO420 mg·L-1+BA0.2 mg·L-1+IBA0.1mg·L-1+ NAA 0.8～1.0mg·L-1是愈伤组织继代扩...     

杜仲带腋芽茎段组织培养的研究

目的研究不同培养条件下杜仲带腋芽茎段愈伤组织的诱导和生长情况,以及腋芽的生长情况。方法以带腋芽的杜仲茎段为外植体,设计9种不同的培养基配方,为了防止褐化,在23.4℃下培养箱中暗培养,30 d后调查出愈率及腋芽的生长情况。结果 NAA浓度从0.05~0.5 mg.L-1,随着浓度增加,腋芽生长受抑制;6-BA浓度从0.3~1.0 mg.L-1,随着浓度增加,愈伤组织块增大,达到1.0 mg.L-1时愈伤组织的生长受到抑制;本实验中最佳的杜仲愈伤组织诱导和生长培养基配方为6号培养基:B5+0.5 mg.L-1NAA+0.3 mg.L-16-BA。腋芽生长最佳培养基配方为4号培养基:B5+0.05 mg.L-1NAA+0.5 mg.L-16-BA。结论本试验的结果对杜仲的生物技术利用有一定的参考价值。     

银杏悬浮细胞对酯蟾毒配基3-羟基的选择性异构化反应

目的　利用银杏悬浮细胞对酯蟾毒配基进行结构修饰。方法　利用只含生长素 2 ,4 D的MS培养基诱导银杏嫩叶 ,使细胞脱分化形成愈伤组织 ,然后将愈伤组织转移至含一定浓度 6 BA ,NAA ,和 2 ,4 D的液体MS培养基中以形成悬浮细胞。把酯蟾毒配基加入生长状态良好的悬浮细胞中转化四天。提取出溶解于液体相的转化产物 ,采用硅胶吸附柱层析法 ,以石油醚和丙酮为展开体系进行梯度洗脱 ,然后对转化产物进行分离纯化。结果　经过四天转化 ,得到一个转化产物 ,转化率达 4 0 % ,通过对转化产物的质谱 ,核磁共振氢谱和碳谱等波谱数据进行分析 ,并与有关文献进行对比 ,证明转化产物为 3 表 酯蟾毒配基。结论以银杏悬浮细胞作为一种生物酶体系 ,可以把来源于动物的蟾蜍甾烯类化合物酯蟾毒配基转化成 3 表 酯蟾毒配基。     

Production of lignans in calluses of <Emphasis Type="Italic">Schisandra chinensis</Emphasis>

Calluses were induced from leaves of Schisandra chinensis Baillon (Schisandraceae). Murashige–Skoog (MS) and Woody Plant (WP) media were used for the induction, in full and half strength (1/2 MS or 1/2 WP) salt formulations. Test media were solidified with 0.25% gelrite and supplemented with 2% sucrose and various concentrations and combinations of 2,4-dichlorophenoxyacetic acid (2,4-D), kinetin (Kin), 3-indolebutyric acid (IBA), and 6-benzylaminopurine (BAP). Optimal conditions for callus induction and growth were found to be 1/2 MS medium containing 0.02 mg/l Kin and 0.2 mg/l 2,4-D. Chloroform extracts of all induced calluses contained gomisin A and F as major components. Gomisin A and F contents of calluses that were cultured under the optimal conditions mentioned above were highest compared to the calluses incubated with other combinations of plant hormones and media. Subculture, by repeated transfer of cultured calluses to fresh medium, caused no decrease in the production of gomisin A and F. Optimal conditions for lignan production were found to be 1/2 MS medium supplemented with 0.05 mg/1 Kin and 0.2 mg/l 2,4-D. Under these conditions, gomisin A and gomisin F contents were 0.05 and 0.04% of callus dry weight, respectively.     

荷花玉兰组织培养的研究

采用正交设计法考察了４种植物激素对诱导荷花玉兰愈伤组织产生的影响,筛选出最佳的植物激素配比为：ＭＳ培养基＋２,４－Ｄ（２,４－二氯苯氧乙酸,４ｍｇ／Ｌ）＋６－ＢＡ（６－苄基氨基嘌呤,１ｍｇ／Ｌ）＋ＮＡＡ（ａ－萘乙酸,４ｍｇ／Ｌ）;找到最适外植体为花托和叶芽,对比了不同外植体形成愈伤组织的时间、形态和质地的差异。     

刺五加愈伤组织的诱导及愈伤组织状态调控

目的获得适宜悬浮培养的刺五加愈伤组织。方法利用植物组织和细胞培养技术,考察3种诱导培养基及9种继代培养基对刺五加愈伤组织的诱导及愈伤组织状态的影响。结果以α-萘乙酸(α-Naphthaleneacetic acid)2.0 mg.L-1+6-苄氨基嘌呤[N-(phenylmethyl)-1H-purin-6-amine,6-BA]2.0 mg.L-1诱导出的愈伤组织适于悬浮培养,叶和叶柄的诱导率分别为93.3%和100%;筛选出最佳固体继代培养的外源激素配比为NAA1.0 mg.L-1+6-BA1.0 mg.L-1,在此激素配比下,愈伤组织生长代谢迅速,性状稳定,呈易分散的颗粒状,易于分散,适合下一步的悬浮培养。结论以刺五加嫩叶为外植体,NAA2.0 mg.L-1+6-BA2.0 mg.L-1为诱导培养基,NAA1.0 mg.L-1+6-BA1.0 mg.L-1为继代培养基可以获得适宜悬浮培养的愈伤组织。     

川麦冬脱病毒和组织培养技术的研究

采用茎尖分生组织培养技术，获得了川麦冬的脱病毒试管苗。通过川麦冬组织培养技术的正交试验及优化筛选，筛选出最佳的培养基组成，用于脱病毒苗的快速繁殖。建立了川麦冬根尖染色体鉴定的最佳条件。结果表明，川麦冬最适繁殖培养基MS＋BA（6-卞基腺嘌呤）2．0mg／L＋NAA（萘乙酸）0．5mg／L；川麦冬最佳诱导愈伤培养基：MS＋BA1．5mg／L＋IAA（吲哚乙酸）0．1mg／L＋2，4-D（2，4-二氯苯氧乙酸）1．0mg／L；通过在培养基中添加不同浓度生长素，得到适合川麦冬的生根培养基：1／2MS＋IAA0．5mg／L＋ABT0．5mg／L。染色体鉴定结果表明：川麦冬的染色体为2n=68。     

细辛组织培养技术研究

目的研究细辛组织培养技术,优化细辛组织培养体系。方法以野生细辛越冬芽为外植体,在不同激素浓度配比的培养基上进行初代培养、不定芽增殖及生根培养,并进行生根苗的移栽。结果细辛组织培养过程中适宜的培养基为：（1）初代培养基,MS＋6-BA1．0mg／L＋NAA0．1rag／L;（2）不定芽增殖培养基．MS＋6-BA1．5rag／L＋NAA0．2mg／L;（3）生根培养基,1／2MS＋IBA1．0mg／L。结论优化的培养体系适宜细辛的组织培养,可在短期内获得大量优质种苗。