激光和磁场对滇紫草愈伤组织色素含量的影响

用Ｈｅ－Ｎｅ激光和一定强度的磁场处理滇紫草愈伤组织,发现２～３ｈ激光辐照可提高色素含量;而１．０Ｔ磁场能促进细胞生长和色素形成,硅胶薄层层析比较两种物理因子对紫草色素成分无显著影响。     

滇紫草愈伤组织中的紫草色素

滇紫草(Onosma paniculatum Bur.et Franch)的幼嫩根茎经二步法诱导产生的愈伤组织.含有较原植物较高的紫草色素。经薄层层析鉴定,此种紫草色素由6种单体组成,其 Rf 值与原植物中的紫草色素各类衍生物非常近似。进一步采用硅胶 H 柱层析进行分离,最后得到4种单体。经结构分析证明它们是:去氧紫色素(deoxyshikonin)、β,β-二甲基丙烯酰阿卡宁(β,β-dimethylacrylalkannin)、乙酰阿卡宁(acetylakannin)和β-乙酰氧基异戊酰阿卡宁(β-acetoxyisovalerylalkannin)。     

寡糖素对滇紫草愈伤组织色素合成的影响

从黑节草、红花和人参中提取的寡糖素对滇紫草愈伤组织的色素合成均能起促进作用,当它们单独使用时,分别找到了它们作用于愈伤组织以生产色素的最适浓度.本研究对将来采用组织培养方法进一步工业化生产紫草素提供了依据。     

滇紫草愈伤组织培养的初步研究

滇紫草(Onosma paniculatum)属紫草科滇紫草属植物,其根部皮层富含的紫草素(α—萘醌系列衍生物),可用作治疗外伤与痔疮的药物或高级染料以及化妆品。滇紫草为云南特有,由于人工栽培困难,加之不断采挖,致使这一资源受到严重的破坏,产品供不应求。日本Tabata等对紫草(Lithospermum erythrorhizon)的组织培养进行了一系列的研究,但对滇紫草组织培养以生产紫草素的研究至今未见报道。基于这一现状,我们从1988年开始着手进行滇紫草组织培养的研究,以探讨应用组织培养技术生产紫草素的可能性。     

滇紫草愈伤组织培养与紫草素产生

浓度为10~(-5)Smol/1和10~(-6)mol/l的2,4-D和NAA分别与10~(-5)mol/l的KT组合,能明显抑制滇紫草(Onosma paniculatum Bur. et Fr.)愈伤组织中紫草素的产生,但几乎不受天然生长素IAA和KT组合的影响。葡萄糖较蔗糖能更有效地促进紫草素的产生,它们的最适浓度均为6％。LH和CH能抑制紫草素的产生,CH浓度大于0.02％时能抑制愈伤组织的生长,LH对生长无明显影响。椰乳浓度为10％时,能明显地促进紫草素的产生,紫草素的含量是对照的24倍。     

生产培养基中寡糖素对滇紫草愈伤组织培养的影响

黑节草寡糖素C-7和C-8,人参寡糖素G-7和G-8分别加入到培养基中,均能影响滇紫草愈伤组织中色素的合成。C-7、C-8、G-7和G-8加入到培养基中,促进紫草色素合成的最适浓度分别为1.5ppm、2.5ppm、2.5ppm和2.5ppm,色素浓度分别为21.39mg/gDW、22.68mg/gDW、29.46mg/gDW和36.73mg/gDW,均明显地高于对照(16.73mg/gDW)。并对寡糖素的作用机理进行了讨论。     

滇紫草Onosma paniculatum愈伤组织中主要成分的抑菌作用

从诱导形成的滇紫草愈伤组织中分离得到四种单体.其中两种单体:β、β-二甲基丙烯酰阿卡宁(β,β-dimethyacrylalkannin,)和乙酰阿卡宁(Acetylalkannin,)的愈伤组织混合色素,对金黄色葡萄球菌和假单孢杆菌都有比较明显的抑制作用.尤其是乙酰阿卡宁抑制效果格外显著.然而对大肠杆菌却表现不出抑制效应.     

软紫草愈伤组织的初步培养

外植体来源不同的软紫草愈伤组织产生紫草色素的能力各异。６０Ｃｏ－ｒ射线辐照处理后的Ｈ２无性系愈伤组织生长量和色素产生均属上乘。改良ＭＳ基本培养基添加１毫克／升ＫＴ,０．５毫克／升ＩＡＡ,５％（Ｗ／Ｖ）蔗糖对愈伤组织培养较为适宜。马铃薯提取液对愈伤组织生长有明显的促进作用。     

Effects of abscisic acid on the secondary metabolism of cultured Onosma paniculatum cells

ABA addition to B5 or M9 medium at the concentrations from 0.1 to 5.0 mg/l suppressed growth of Onosma paniculatum cells. The addition of these ABA concentrations to M9 medium also significantly decreased the formation of shikonin and its derivatives in the cultured cells during the entire course of culturing. The enzyme activity assay showed that, on the 4th day after inoculation, 0.1 mg/l ABA significantly decreased the activities of phenylalanine ammonia-lyase, the first enzyme, and p-hydroxybenzoic acid-geranyltransferase, a key enzyme involved in shikonin biosynthesis. However, no significant change in these two enzyme activities was found during the following days for testing (8, 12, and 16 days). Interestingly, RT-PCR analysis of the PAL1 and LePGT1 gene expression showed no significant changes on the 4th day after inoculation. Furthermore, we found that the inhibitory effect of ABA on the secondary metabolism could be alleviated significantly by the addition of 2-aminoethyl diphenyl borate (an inhibitor of inositol triphosphate-receptor) or nicotinamide (an inhibitor of ADP-ribose cyclase), which functions by decreasing the intracellular calcium concentration. Published in Russian in Fiziologiya Rastenii, 2007, Vol. 54, No. 4, pp. 597–603. The text was submitted by the authors in English.     

γ—射线对滇紫草细胞产生色素的影响

应用５００～２５００伦琴（Ｒ）的６０钴γ射线照射滇紫草（ＯｎｏｓｍａｐａｎｉｃｕｌａｔｕｍＢｕｒ．ｅｔＦｒａｎｃｈ．）细胞系,确定了应用γ射线处理促进紫草素合成的最适辐照剂量为１５００Ｒ,处理后的细胞系在生产培养基上培养２１ｄ后紫草素含量（以干重计）达到９４．７９ｍｇ／ｇ,较对照提高了１４４６％,通过小团块选种法从中筛选到了色素含量（以干重计）高达１０３．４２ｍｇ／ｇ的高产细胞系Ｍｕｔ１,其营养生长与对照相比没有差别。     

真菌诱导物在滇紫草细胞培养中对紫草色素形成的影响

在滇紫草细胞悬浮培养中,真菌诱导物可抑制细胞生长,促进紫草色素的合成。将培养６ｄ的曲霉菌丝体的粗提物以６００μｇ碳水化合物／５０ｍｌ培养液的浓度加入到处于指数生长初期的滇紫草细胞悬浮培养物中,诱导物促进紫草色素合成的作用最大,紫草色素含量为对照的两倍。经高压锅处理２０ｍｉｎ到２ｈ不影响诱导物的活性。真菌诱导物还影响了紫草色素各衍生物的相对含量。     

米曲霉中的紫草色素诱导物对滇紫草细胞代谢的影响

在滇紫草细胞培养第８天时加入来源于米曲霉的紫草色素诱导物,１２ｈ后滇紫草细胞中紫草色素含量增加,其衍生物之一的乙酰紫草素的相对含量增加,胞内可溶蛋白合成量上升,细胞对碳源消耗增多,而细胞生长受到抑制。处理１２ｈ后,培养液电导率下降,ｐＨ上升,Ｋ＋外泄和Ｎａ＋大量内流,表明离子跨膜运输发生改变。     

CTAB法提取中草药材滇紫草细胞的总RNA

介绍一种适合富含次生产物的中草药材滇紫草细胞总RNA的提取方法——CTAB法。采用CTAB作为去污剂,分别用氨仿和水饱和酚反复抽提以及LiCl沉淀以去除蛋白质、碳水化合物和次生代谢物等杂质,最后用纯乙醇沉淀获得RNA。该方法不但能获得完整性好和纯度高的总RNA,而且操作简单、快速、成本低廉,对其它富含次生物质的植物特别是中草药材组织总RNA的提取具有借鉴意义。     

Effect of Brassinolide on Growth and Shikonin Formation in Cultured Onosma paniculatum Cells

The effect of brassinolide (BR) on cell growth and shikonin and its derivative formation in Onosma paniculatum cell culture was studied. BR addition with IAA and BAP (+BR/+IAA/+BAP) in B₅ medium slightly increased the cell growth at 0.01–0.1 ppb concentration compared with a growth control (−BR/+IAA/+BAP). Only BR addition (+BR/−IAA/−BAP) at 0.001–100 ppb in B₅ medium significantly increased the cell fresh weight compared with a growth control (−BR/−IAA/−BAP). The same concentration of BR tested at 0–1000 ppb increased the cell fresh weight of +IAA/+BAP significantly more than that of −IAA/−BAP. BR at 0.001–0.1 ppb with IAA and BAP added (+BR/+IAA/+BAP) in M₉ medium increased shikonin and its derivative content markedly by 31–87%, compared with its control (−BR/+IAA/+BAP). BR at 0.001–1000 ppb without IAA and BAP added to M₉ medium (+BR/−IAA/−BAP) also increased shikonin and its derivative content compared with its control (−BR/−IAA/−BAP). However, the amount of shikonin and derivative formed of +IAA/+BAP was greater than that of −IAA/−BAP only at the same concentration of BR at 0–1 ppb. These combined results show that BR at 0.01 ppb with IAA and BAP added was the best for cell growth and shikonin formation. Formation of shikonin and its derivative by adding BR at 0.01 ppb with IAA and BAP (+BR/+IAA/+BAP) in M₉ medium was significantly enhanced 4 days after BR addition compared with a production control (−BR/+IAA/+BAP). In contrast, +BR/−IAA/−BAP vs. −BR/−IAA/−BAP was not as effective as +BR/+IAA/+BAP vs. −BR/+IAA/+BAP for the shikonin formation. The time course study for shikonin formation also showed that +BR/+IAA/+BAP and −BP/+IAA/+BAP only slightly increased cell growth in M₉ medium. Similarly, soluble protein content in the cells treated by BR at 0.01 ppb with IAA and BAP (+BR/+IAA/+BAP) exceeded that of the control (−BR/+IAA/+BAP) 4 days after BR addition. And +BR/−IAA/−BAP only slightly increased the soluble protein content over that of −BR/−IAA/−BAP. Received November 2, 1998; accepted August 25, 1999     

徐长卿子叶组织培养及再生体系的建立

以徐长卿子叶为材料,进行愈伤组织诱导、愈伤组织和不定芽分化、试管苗生根、移栽及移植等研究。结果表明,MS+6-BA 0.3 mg/L+NAA 0.2 mg/L+2,4-D 0.4 mg/L是子叶愈伤组织诱导和继代培养的理想培养基;MS+6-BA 0.8 mg/L+NAA 0.2 mg/L是愈伤组织分化培养的理想培养基;MS+6-BA 0.6 mg/L+NAA 0.2 mg/L是不定芽分化继代培养的理想培养基;炉灰渣是试管苗移栽的理想基质,移栽成活率可达97%。试管苗移植后长势旺盛,根系发达,当年开花。根据本试验结果可建立徐长卿子叶诱导再生体系。     

Variation in pigment production in Lithospermum erythrorhizon callus cultures

Different strains of callus cultures of Lithospermum erythrorhizon showed wide variations in the production of shikonin derivatives. From these cultures, two high pigment-producing strains, whose content of shikonin derivatives are stable and similar to that of intact plant root, have been established by repeated selection.     

Effect of temperature on carbohydrate content,ADP glucose pyrophosphorylase,and ATP- and PPi-dependent phosphofructokinase activity of potato tuber callus tissue

Callus derived from Lemhi Russet and Russet Burbank tuber tissue was incubated at 20°C and 30°C on a high sucrose medium for starch-formation and subjected to simulated storage and reconditioning treatments at 5°C and 25°C after transfer of the callus to a medium without a carbon source. Percent dry weight of callus from both cultivars averaged about 21% after starch formation and 5% after storage and reconditioning treatments. Total sugars were higher in callus incubated on the starch forming treatment. Lemhi Russet callus contained predominantly reducing sugars, while Russet Burbank callus contained mostly non-reducing sugars. Total sugar content was generally lower for both cultivars after the storage and reconditioning treatment in callus after starch formation at 20°C. Starch content was generally higher in Lemhi Russet tissue. After starch formation at 20°C Lemhi Russet had higher starch after the storage and reconditioning treatment than tissue from 30°C, while the opposite trend was found in Russet Burbank tissue. Total protein declined in Russet Burbank tissue during the storage and reconditioning treatment regardless of prior incubation conditions, while this decline only occurred in Lemhi Russet tissue initially incubated at 30°C during the starch formation treatment. In tissue of both cultivars, ATP- and PPi-dependent phosphofructokinase activities were inversely proportional to total sugar concentrations, while in RB callus ADP glucose pyrophosphorylase activities were proportional to starch content.Research Paper 91B1 of the Idaho Agricultural Experiment Station.