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。     

栀子组织和细胞培养生产天然食用色素的研究：Ⅰ.愈务组织…

在诱导出愈伤组织的基础上,对各种不同的培养条件进行分析研究,考察它们对愈伤组织生长和栀子黄色素产生的影响,筛选出适宜的生长培养基：Ｂ５＋ＴＢＡ １ｍｇ／１＋ｋＴ０．２３ｍｇ／、ＭＧ－５＋ＩＢＡ １ｍｇ／＋ＫＴ０．２３ｍｇ／、Ｂ５＋ＩＡＡ１．５ｍｇ／ｌ和生产培养基;Ｍ－９＋ＩＡＡ １ｍｇ／ｌ、Ｍ－９＋ＩＡＡ １．５ｍｇ／ｌ。并且,获得了几个色素含量较高的愈伤组织系。另外,还研究了含色素和不含色素的愈     

提高籼稻愈伤组织再生频率的研究

为了提高籼稻愈伤组织的植株再生频率,研究了影响再生的各种因素,如：在诱导培养基或继代培养基中加细胞分裂素和萘乙酸（ＫＴ、ＢＡＰ、玉米素或Ｚｉｐ１毫克／升）,或加Ｔｈｉｄｉａｚｕｒｏｎ（０．５毫克／升）,以及愈及组织的部分干燥处理等。这些措施明显地提高籼稻愈伤组织的再生频率。结合使用这些处理可使ＴＮ１、ＩＲ７２和ＩＲ６４的愈伤组织再生植株频率较对照提高５－１４倍。     

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。     

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)。     

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。     

愈伤组织继代接种量、继代周期和年龄对软紫草原生质体脱壁的影响

本文的实验结果表明：软紫草愈伤组织脱壁所需的适宜酶浓度为：０．１％果胶酶十０．２５％纤维素酶;酶解处理的适宜时间与愈伤组织年龄有关：愈伤组织的适宜年龄随其继代周期、愈伤组织继代接种量而有变化。当接种量,１克／瓶,转代后７天进行原生质体分离：接种量３克／瓶,酶解材料则以培养５天愈伤组织为宜。继代周期１３天和１５天的比１５天和１５天的,适宜脱壁的愈伤组织当代培养天数要提前１天。     

滇紫草培养细胞中色素合成的代谢调节

培养基中分别加入浓度为10^-5mol／L的铜离子,滇紫草愈伤组织中色素含量提高了5．5倍,悬浮细胞中色素含量提高8．1倍。细胞培养第21天,加入浓度为10^-5mol／L的L-Phe,色素的合成量最大。浓度为10^-6mol／L的抗坏血酸,能明显地促进培养细胞中色素的合成。     

新疆紫草的组织培养及其染色体分析

新疆紫草(Arnebia euchroma)的胚轴、子叶、根都产生愈伤组织。不同来源的外植体产生苗的能力不同,胚轴来源的愈伤组织分化苗最多,而且苗生长旺盛。根的愈伤组织只产生不定根。新疆紫草的核型是 K(2n)=14=2m 8sm 2sm(SAT) 2st。随着继代培养代数增多,染色体变异的种类和频率以及染色体数目显著增加。如果继代培养时间较长,染色体数目变异的范围更广,而且出现染色体结构变异。     

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。     

Effects of Physical and Chemical Factors on Callus Growth and Shikonin Derivative Formation in the Callus Cultures of Arnebia euchroma

The effects of some physical and chemical factors on callus growth and shikonin derivative formation in the callus cultures of Arnebla euchroma were discussed. According to experiments, the optimum temperature for callus growth and shikonin derivative formation was 25℃, and the favorable initial pH of media was in the range of 5.3–5.8. Authors also found that both callus growth and shikonin derivative formation were strongly inhibited by white light. Callus growth was promoted when 0.2 mg/L IAA and 0.5 mg/L KT were added to the media, but IAA and KT did not promote shikonin derivative formation. Furthermore, the content and yield of shikonin derivatives in cultures decreased in company with the increase of IAA and KT concentration in the media.     

Production of shikonin derivatives by cell suspension cultures of Lithospermum erythrorhizon

An excellent new medium was developed for the production of shikonin derivatives by suspension cultures of Lithospermum erythrorhizon. We investigated the effects of all the components of White's medium on the production of these derivatives. Nitrate, phosphate, copper, sulfate and sucrose had especially marked effects. With the new, M-9, medium produced from these studies the yield of shikonin derivatives was 1400 mg/l and the yield for dried cells was about 12%, whereas it was 120 mg/l, or about 2% with White's medium.     

pH值、激素对新疆紫草悬浮培养细胞生长及紫草宁衍生物合成的影响

报道了不同ｐＨ值、激素对新疆紫草悬浮培养细胞生长及紫草宁衍生物合成的影响。结果表明,新疆紫草细胞具有自我调节其培养液ｐＨ值的功能。适合于细胞生长及紫草宁衍生物形成的ｐＨ值为５．６±０．４０。ＢＡＰ、２,４－Ｄ、ＮＡＡ或ＩＢＡ对细胞生长无显著的促进作用,且都会抑制紫草宁衍生物的形成。在生长培养基中添加１．０ｍｇ／ｌ　ＩＡＡ和０．５ｍｇ／ｌＫＴ可促进细胞生长,而在生产培养基中附加０．１ｍｇ／ｌＫＴ和０．７５─１．０ｍｇ／ｌＩＡＡ则有利于紫草宁衍生物含量及产量的提高。     

Production of shikonin derivatives by cell suspension cultures of Lithospermum erythrorhizon

Differences in the production of shikonin derivatives by callus and suspension cultures of Lithospermum erythrorhizon Sieb. et Zucc. were examined. When Linsmaier and Skoog medium was used in suspension cultures, cell growth was not accompanied by the production of shikonin compounds. Shikonin derivatives were produced, however, when this medium was used in callus cultures. Differences in shikonin production were examined in terms of the nutrient supply, the effect of the agar itself, and the oxygen supply. Shikonin derivatives could be produced without agar by keeping the cells exposed to air while providing an adequate supply of nutrients. In callus cultures, the production of shikonin compounds was reduced remarkedly when the oxygen concentration in the atmosphere was lowered, evidence that shikonin production during L. erythrorhizon cell growth on Linsmaier and Skoog agar medium is enhanced by an abundant supply of oxygen.     

Biosynthesis of shikonin in callus cultures of Lithospermum erythrorhizon

Administration of various supposed precursors to the callus cultures of Lithospermum erythrorhizon grown on the Linsmaier—Skoog medium supplemented with IAA and kinetin established that the constituent shikonin is formed via shikimic acid, p-hydroxybenzoic acid, m-geranyl-p-hydroxybenzoic acid and geranylhydroquinone. In a strain of callus culture lacking the capacity to synthesize shikonin and in callus cultures which have had this capacity but lost it due to cultivation on a medium supplemented with 2,4-D, substances up to m-geranyl-p-hydroxybenzoic acid in the biosynthetic sequence have been detected. Although illumination with white light also arrested shikonin production, traces of pigment were still formed presumably because light did not reach the innermost part of the callus cultures.     

Physico-chemical factors influencing the shikonin derivatives production in cell suspension cultures of Arnebia euchroma (Royle) Johnston, a medicinally important plant species

Cell suspension cultures of Arnebia euchroma were raised from in vitro leaf-derived friable callus on liquid MS [Murashige and Skoog] medium supplemented with BAP (6-benzylaminopurine) (10.0 μM) and IBA (indole-3-butyric acid) (5.0 μM). A two-stage culture system was employed using growth and production medium for cell biomass and shikonin derivatives, respectively. Factors such as light, temperature, sucrose and pH (hydrogen ion concentration) were studied to observe their effect on the shikonin derivative production. Light conditions completely inhibited shikonin derivative production. Out of different temperature regimes tested, the highest yield (586.17 μg/g FW) was found at 25°C. Maximum production (656.14 μg/g FW) was observed in 6% sucrose. An alkaline pH (7.25-9.50) favoured shikonin derivative production. The results showed that physical and chemical factors greatly influence the production of shikonin derivatives in cell suspension cultures of A. euchroma. Therefore, by employing optimum culture conditions, it is possible to enhance the production of secondary compounds from the cells. The factors optimized for in vitro production of shikonin derivatives during the present study can successfully be employed for their large-scale production in bioreactors.     

固定化培养和产物释放促进剂对硬紫草细胞代谢的影响

固定化培养的硬紫草细胞生长缓慢,仅包埋球外层的细胞生长明显。其蛋白质合成的量也低。培养３０ｄ的细胞色素产量达到４．２ｍｇ／ｇＦＷ,相对色素分泌量达到７０％,而色素的组成成分及各组分的比例也与悬浮细胞的不同。以正十六烷处理固定化细胞可促进产物释放,其不同的处理时间对细胞没有显著影响。连续培养的固定细胞保持其色素形成能力达８０ｄ之久,色素总产量达２０ｍｇ／ｇＦＷ。     

The Effective Production of Shikonin by Cultures with an Increased Cell Population

We have studied the efficient production of shikonin derivatives by suspension cultures of Lithospermum erythrorhizon with an increased cell population. The yield of shikonin derivatives was highest (800 mg/liter) when 2.8 g dry wt/liter of the cells was inoculated into the M-2 medium which we had developed for the production, but the excess inoculum lowered the yield.

We investigated suitable conditions for production with the increased cell population. The optimum amount of inoculum rose to 4.9 g dry wt/liter when the concentrations of all the components contained in the M-8 medium, which we developed for increasing the productivity by modification of the M-2 medium, were increased in proportion to the amount of inoculum, and consequently we could increase the yield of the shikonin derivatives from 1400 mg/liter to 1900 mg/liter. Moreover, the increased rate of oxygen supply in addition to the enrichment of the medium made it possible to produce 2300 mg/liter of the shikonin derivatives from a culture for which 5.6 g dry wt/liter of the cells was inoculated.