葡萄悬浮细胞长期培养过程中不同继代条件下花青素生产的不稳定性

为了深入研究植物细胞培养生产次生代谢产物不稳定性的机制,以葡萄细胞作为模式体系,研究悬浮培养过程中花青素合成的不稳定性。除了用常规的花青素总含量来表征花青素的生物合成之外,还采用HPLC测定花青素不同组分的含量。结果表明,在长期的继代培养过程中,不仅花青素的含量而且花青素的组成也表现出明显的不稳定性。首次采用了不稳定系数 (δ) 和因素得分 (Factor scores) 来表征植物细胞培养过程中次生代谢生产的不稳定性。培养条件对花青素生物合成的影响实验结果表明,继代周期和接种量均能诱发次生代谢的不稳定性表达,其中接种量的影响相对更大。在考察的 (6.5 d,2.00 g),(7 d,2.00 g),(7.5 d,2.00 g),(7 d,1.60 g) 和 (7 d,2.40 g) 五种不同的继代周期和接种量组合条件中,7 d继代周期和1.60 g接种量最有利于保持花青素的稳定生产。     

细胞均一性对葡萄细胞生长和花青素合成的影响

通过色差筛选法建立了一个相对均一的葡萄细胞悬浮系E,其细胞团较小,在长期继代培养过程中花青素合成能力的变异系数为8.7%,重复摇瓶实验的变异系数为5%。以E为实验材料进行的各组前体饲喂、诱导子添加、光照等联合作用实验,其生物量和花青素合成的变异系数均可控制在12%以内,充分说明了培养体系的均一性对维持稳定生产的重要性;黑暗条件下添加30μmol/L苯丙氨酸(Phe)和218μmol/L茉莉酸甲酯(MeJA)可使单位细胞花青素含量达到对照组的5.89倍,花青素产量为对照组的4.30倍,且连续5次继代培养过程中生物量和花青素合成的变异系数均比对照组降低。     

怀牛膝细胞悬浮培养条件的优化

为进一步优化怀牛膝(Achyranthes bidentata)细胞悬浮培养条件,对接种量、继代周期、pH、光照及Cu2+等多种影响因子的作用效果进行了研究,以提高怀牛膝细胞生长量及牛膝多糖含量。结果显示,接种量50 g·L^–1、继代周期14天,pH5–6和光照培养可以使细胞保持良好的生长状态及多糖合成能力;添加50μmol·L^–1 Cu^2+,细胞的干重最大,可达44.63 g·L^–1,多糖含量也最高,为4.02 mg·g^–1。     

刺葡萄幼胚愈伤组织诱导及其高产原花青素细胞系筛选

以刺葡萄幼胚为材料,研究不同培养方式、培养基配方和培养条件对其愈伤组织诱导的影响,采用正交试验设计法筛选刺葡萄愈伤组织继代增殖的培养基配方,并对继代保持的培养条件和方式进行优化,同时进行了高产原花青素刺葡萄愈伤组织细胞系的筛选研究。结果表明,刺葡萄幼胚以平放的方式接种到MS＋1.0 mg·L^-1 2,4-D或MS＋1.0 mg·L^-1 2,4-D＋0.5mg·L^-1 KT的固体培养基上,在黑暗的条件下,能有效的诱导出愈伤组织,诱导效率为80%;刺葡萄愈伤组织继代增殖以MS＋1.5 mg·L^-1 2,4-D或MS＋1.5 mg·L^-1 2,4-D＋0.5 mg·L^-1 KT的固体培养基为佳,并且采用此两种培养基交替继代培养,在光照条件下能长期保持旺盛且生长一致的刺葡萄愈伤组织;筛选出了紫红色松脆状的高产原花青素的刺葡萄愈伤组织细胞系,培养35 d后每克鲜样的原花青素含量可达1 671.16μg。     

怀牛膝细胞悬浮培养条件的优化

为进一步优化怀牛膝(Achyranthes bidentata)细胞悬浮培养条件,对接种量、继代周期、pH、光照及Cu~(2+)等多种影响因子的作用效果进行了研究,以提高怀牛膝细胞生长量及牛膝多糖含量。结果显示,接种量50 g·L~(–1)、继代周期14天,pH5–6和光照培养可以使细胞保持良好的生长状态及多糖合成能力;添加50μmol·L~(–1 )Cu~(2+),细胞的干重最大,可达44.63 g·L~(–1),多糖含量也最高,为4.02 mg·g~(–1)。     

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。     

生物学因子对紫苏悬浮培养细胞生长和花色素形成的影响

应用摇瓶培养研究了生物学因子,即：细胞聚集体大小、继代周期和接种量,对紫苏悬浮细胞生长和次生代谢物花色素产生的影响。结果表明：与未经筛选的或细胞聚集体小于250μm的细胞团块相比,大于250μm的细胞团块作为接种细胞时,培养所得的花色素含量较低。7一10天为合适的继代周期,在长时期的继代过程中,细胞生长良好、并且色素含量也高。实验还表明,每升接种50克湿细胞最适合于细胞增殖与色素积累。     

玫瑰茄县浮细胞合成花青素的光效应研究

光照对悬浮培养的玫瑰茄细胞生物量无影响。随着光照强度增大,玫瑰茄细胞合成花青素的量增加,光照强度３１．０ｗ／ｍ＾２为饱和光照强度,超过该强度,玫瑰茄细胞合成花青素的量不再进一步增加;可见光中蓝光（４２０￣５３０ｎｍ）是促进玫瑰茄细胞合成花青素最有效单色光,光强为３０．０ｗ／ｍ＾２,接种量为０．２ｇ湿细胞的５０ｍｌ培养液经１６ｄ培养,花青素产量为８．９７ｍｇ／５０ｍｌ,高出相同光照强度全色光下的６．     

玫瑰茄悬浮细胞合成花青素的光效应研究

光照对悬浮培养的玫瑰茄细胞生物量无影响。随着光照强度增大,玫瑰茄细胞合成花青素的量增加,光照强度３１．０ｗ／ｍ２为饱和光照强度,超过该强度,玫瑰茄细胞合成花青素的量不再进一步增加;可见光中蓝光（４２０～５３０ｎｍ）是促进玫瑰茄细胞合成花青素最有效单色光,光强为３０．０ｗ／ｍ２,接种量为０．２ｇ湿细胞的５０ｍｌ培养液经１６ｄ培养,花青素产量为８．９７ｍｇ／５０ｍｌ,高出相同光照强度全色光下的６．５３ｍｇ／ｍｌ;黄光和绿光分别有一定的促进作用。当黑暗下的培养时间不超过８ｄ,后期经过不少于８ｄ的光照可以诱导出和全程光照相当的花青素产量,分别为６．６４和６．７２ｍｇ／５０ｍｌ（总培养时间不少于１６ｄ）。当黑暗下培养时间超过１２ｄ,由于营养成分消耗,光照延长,花青素产量也无法提高,添加１０ｍｌ新鲜培养基再进行光诱导,花青素产量可以提高（６．７５ｍｇ／５０ｍｌ）。     

甲基营养菌MP688胞外多糖合成的影响因素研究

目的：考察培养基组分和发酵条件对甲基营养菌MP688合成胞外多糖的影响,确定最主要的影响因素。方法：将甲基营养菌MP688接种到基础培养基中,通过改变基础培养基的氮源、培养温度、初始pH值和培养时摇床转速等条件,检测在每种条件下培养5 d后发酵液中的多糖含量,确定每种因素的最适范围;进而选取8个因素,通过Plackett-Burman实验设计12组实验,通过检测每种组合条件下的多糖产量和结果统计分析,确定影响多糖合成的最主要因素。结果：甲基营养菌合成胞外多糖的最适氮源为硝酸钠,最适温度为30-37°C,最适pH值为6.5-7.0,最适摇床转速为200-250 r/min;甲醇、硝酸钠、初始pH值和接种量是MP688合成多糖的主要影响因子。结论：运用Plack-ett-Burman实验设计筛选到甲基营养菌MP688胞外多糖合成的主要影响因子,MP688是具有多糖生产潜力的菌株。     

Effects of inoculum size and age on biomass growth and paclitaxel production of elicitor-treated Taxus yunnanensis cell cultures

Suspension cultures of Taxus yunnanensis cells were inoculated with cells of different culture ages (12-24 days) at various densities [50-250 g fresh weight (fw)/l], and treated (on day 7) with a mixture of elicitors, including Ag(+), chitosan and methyl jasmonate. The biomass productivity (during the production stage) increased dramatically with inoculum size, but decreased with inoculum age over 16 days. The volumetric yield and productivity of taxol (paclitaxel) also increased with inoculum size, while the specific taxol yield (per cell) was mainly dependent on inoculum age, with an optimum of 20 days, during the early stationary phase. The highest taxol yield and productivity, 39.8 mg/l and 1.9 mg/l per day, respectively, were obtained with a 20-day-old inoculum at 200 g fw/l. Taxol excretion by the cells increased with inoculum age but decreased with inoculum size. The elicitor-induced activities of catalase (CAT) and phenylalanine ammonia-lyase (PAL) also depended mainly on inoculum age; higher PAL activity and lower CAT activity were obtained with an older inoculum, corresponding to a higher taxol yield. The results show that both inoculum size and age are important variables for taxol production, though the latter more profoundly influences elicitor-induced taxol biosynthesis of the cells. Inoculum size and age are also interrelated and should be optimized together in a two-stage culture process.     

High-density cultivation of Perilla frutescens cell suspensions for anthocyanin production: Effects of sucrose concentration and inoculum size

High-density cultivation of Perilla frutescens cells for anthocyanin production was carried out in both batch and fed-batch modes in a 500-ml shake flask. In fed-batch cultures, a high cell density of 27.7 g dry cells l⁻¹ and a total anthocyanin production of 3.87 g l⁻¹ by intermittent feeding of all medium components except hormones were obtained. In batch cultures, both initial sucrose concentration and inoculum size showed a conspicuous effect on the kinetics of cell growth, sugar consumption, and secondary metabolite (anthocyanins) production by suspended P. frutescens cells. At an inoculum size of 50 g wet cells l⁻¹, the maximum cell density of 38.3 g dry cells l⁻¹ was obtained after 11 days of cultivation at an initial sucrose concentration of 60 g l⁻¹, the highest pigment production of>5.8 g l⁻¹ was attained after 10 days of cultivation at an initial sucrose concentration of 45 g l⁻¹. These amounts of cell mass and anthocyanin pigments were 3.3 and 24 times higher than those at an initial sucrose concentration of 15 g l⁻¹ and inoculum size of 15 g wet cells l⁻¹, respectively.     

Increase in anthocyanin yield from wild-carrot cell cultures by a selection system based on cell-aggregate size

Wild-carrot (Daucus carota L.) cell cultures were screened to yield small (less than 63 m) or large (greater than 170 m) cell aggregates which were then subcultured. Cultures of the small-size class had a higher, those of the large-size class a lower anthocyanin yield than the unscreened culture. This relationship became accentuated with an increasing number of passages with screening prior to subculture. At the end of six months (12 passages), the pigment yield of the small-size class was triple that of the unscreened cells. Following this selection period, the tendency of the small-size fraction to increase in clump size when subcultured without screening was much less than that of freshly isolated cell aggregates of the same size. These observations may be explainable on the basis of a differential distribution of cytokinin between aggregates of different sizes. High levels of cytokinin inhibit anthocyanin accumulation and inhibit cell separation; these effects result in large cell aggregates having low levels of anthocyanin. In support of this hypothesis, it is shown that addition of kinetin to cultures of small cell aggregates causes an increase in the size of cell aggregates and a parallel decrease in anthocyanin yield.     

Analysis of pigmentation in individual cultured plant cells using an image processing system

Anthocyanin accumulation in strawberry (Fragaria ananassa) cells cultured on a solid medium was monitored using an image-processing system that did not require direct sampling or destruction of the cells. Because of the intercellular heterogeneity of secondary metabolite production in plant cell cultures, the maximum metabolite concentration in individual cells is often more than 10 times higher than that of the average concentration. An image-processing based method enabled the growth and the pigmentation behavior of individual cells to be traced. Changes in the time courses of the anthocyanin content of individual cells differed from each other, although the average anthocyanin contents increased gradually with time in a batch culture. However, these various changing patterns in the anthocyanin content of each cell were independent of the cell cycle. In addition, image analysis revealed that the two cells just after cell division were almost identical to each other both in size and anthocyanin content. The proposed method which uses an image-processing system provides a useful tool for analyzing the secondary metabolism in individual cultured plant cells.     

Instability of anthocyanin accumulation inVitis vinifera L. var. Gamay Fréaux suspension cultures

The inherent instability of metabolite production in plant cell culture-based bioprocessing is a major problem hindering its commercialization. To understand the extent and causes of this instability, this study was aimed at understanding the variability of anthocyanin accumulation during long-term subcultures, as well as within subculture batches, inVitis vinifera cell cultures. Therefore, four cell line suspensions ofVitis vinifera L. var. Gamay Fréaux, A, B, C and D, originated from the same callus by cell-aggregate cloning, were established with starting anthocyanin contents of 2.73±0.15, 1.45±0.04, 0.77±0.024 and 0.27±0.04 CV (Color Value)/g-FCW (fresh cell weight), respectively. During weekly subculturing of 33 batches over 8 months, the anthocyanin biosynthetic capacity was gradually lost at various rates, for all four cell lines, regardless of the significant difference in the starting anthocyanin content. Contrary to this general trend, a significant fluctuation in the anthocyanin content was observed, but with an irregular cyclic pattern. The variabilities in the anthocyanin content between the subcultures for the 33 batches, as represented by the variation coefficient (VC), were 58, 57, 54, and 84% forV. vinifera cell lines A, B, C and D, respectively. Within one subculture, the VCs from 12 replicate flasks for each of 12 independent subcultures were averaged, and found to be 9.7%, ranging from 4 to 17%. High- and low-producing cell lines, VV05 and VV06, with 1.8-fold differences in their basal anthocyanin contents, exhibited different inducibilities tol-phenylalanine feeding, methyl jasmonate and light irradiation. The low-producing cell line showed greater potential in enhanced the anthocyanin production.     

Nitrogen repression of fumonisin B1 biosynthesis in Gibberella fujikuroi

Fumonisins are a group of structurally related mycotoxins produced by Gibberella fujikuroi. The fungus produced fumonisin B1 (FB1) as early as 18 hour in a defined medium containing 1.25 mM or 2.5 mM ammonium phosphate, whereas fumonisin B1 production was repressed for 75 hour and 125 hour when mycelia were resuspended in media containing ammonium phosphate at 10 mM or 20 mM, respectively. Although total fumonisin B1 production was greater in resuspension cultures grown in higher concentrations of ammonium phosphate, the accumulation was independent of the inoculum size and carbon/nitrogen ratio. The addition of ammonium phosphate to cracked corn cultures also repressed fumonisin B1 production by 97%, and persisted for at least three weeks. Thus, biosynthesis of fumonisin B1 is regulated by a mechanism involving nitrogen metabolite repression, suggesting that control strategies that target the regulatory elements of nitrogen metabolism may be effective at reducing the risk of fumonisin contamination in food.