葡萄细胞悬浮培养生产白藜芦醇

以巨峰葡萄果皮为外植体,在添加2.0 mg/L 6-苄基嘌呤（6-BA）和0.1 mg/L 2,4-二氯苯氧基（2,4-D）的B5培养基上诱导葡萄愈伤组织; 以50 g/L的初始接种量在添加1.0 mg/L 6-BA和0.05 mg/L 2,4-D的B5液体培养基上建立葡萄悬浮培养体系。在25～27 ℃下,摇床振荡暗培养（120～130 r/min）18 d后,葡萄细胞生物量和白藜芦醇含量达到最大值（16.17 g/L、95.69 μg/g干质量）。在培养第12天时,向培养基中添加100 μmol/L茉莉酸甲酯（MeJA）,经过6 d处理,细胞中白藜芦醇含量达235.73 μg/g干质量。     

Anthocyanic vacuolar inclusions (AVIs) selectively bind acylated anthocyanins in Vitis vinifera L. (grapevine) suspension culture

Anthocyanic vacuolar inclusions (AVIs) appear as dark red-to-purple spheres of various sizes in vacuoles of grapevine (Vitis vinifera L.) cell suspension culture due to their interaction with anthocyanins. AVIs were purified and the bound anthocyanins extracted and analysed by HPLC from two lines of V. vinifera isolated from the same callus accumulating anthocyanin in the dark, yet varying in their anthocyanin profiles and accumulation. An intermediate-pigmented line (FU-1) with a 1.3:1 ratio of acylated:non-acylated anthocyanins, a colour value of 0.84 units and cyanidin and peonidin as the dominant species was compared with a high-pigmented line (FU-2) with a 1.2:1 ratio of acylated:non-acylated anthocyanins, a colour value of 3.72 units and malvidin predominating. The profile of AVI-bound anthocyanins showed an increase in acylated anthocyanins in both lines of approx. 28–29%, with no apparent preference for anthocyanin species. This resulted in a ratio of acylated:non-acylated anthocyanins of 6.2:1 for FU-1 and 4.9:1 for FU-2. The reasons for the selectivity of the AVIs for acylated (specifically p-coumaroylated) species compared with the whole cell profile are discussed.     

Production of 13C-labelled anthocyanins by Vitis vinifera cell suspension cultures.

The use of plant cell cultures for producing isotopically (13C) labelled phenolic substances is reported. Vitis vinifera cells synthesize high levels of anthocyanins when they are cultured in a polyphenol synthesis-inducing medium. Three major anthocyanin monoglucosides found in red wine were identified in grape cells: cyanidin-3-O-beta-glucoside, peonidin-3-O-beta-glucoside, and malvidin-3-O-beta-glucoside. Kinetic study of the intracellular level of phenylalanine and its metabolites showed that it is preferable to add this precursor to grape cell suspensions after the 5th day of culture, i.e. at the beginning of the exponential growth phase. After adding phenylalanine to the culture medium, its uptake was complete and the accumulation of anthocyanins in grape cells was stimulated. Incorporation of [1-13C]-phenylalanine into anthocyanins was measured by means of 13C satellites in the proton NMR spectrum. The maximal rate of 13C enrichment anthocyanins obtained with this technique reached 65%. The production of 13C labelled phenolic compounds was undertaken in order to investigate their absorption and metabolism in humans.     

Galloylated catechins and stilbene diglucosides in Vitis vinifera cell suspension cultures

Suspension cultures of Vitis vinifera were found to produce catechins and stilbenes. When cells were grown in a medium inducing polyphenol synthesis, (−)-epicatechin-3-O-gallate, dimeric procyanidin B-2 3′-O-gallate and two resveratrol diglucosides were isolated, together with a new natural compound that was identified as cis-resveratrol-3,4′-O-β-diglucoside by spectroscopical methods.     

Condensed tannin and anthocyanin production in Vitis vinifera cell suspension cultures

Suspension cultures of Vitis vinifera were cultured in different media in order to establish a model system for promoting high levels of phenolic substances identical with those found in wine. These media were: a low sucrose maintenance medium (MM) and four high sucrose media (differing mainly in sucrose and mineral contents) which were shown to induce secondary metabolism. In MM medium, polyphenol accumulation in the cells was low, and concentrations of 0.1 mg/gfw for condensed tannins and 0.3 mg/gfw for anthocyanins were reached within two weeks of cultivation. Values of 1.4 and 6.4 mg/gfw, respectively, were obtained with a low nitrate and high sucrose medium (HM1), but cell proliferation was reduced. To obtain a maximal production of polyphenols, we investigated the most effective conditions for cell growth and polyphenol production (a high mineral and high sucrose medium, IM1; inoculum dilution of 1.25:10). Under these conditions, the cells produced mainly anthocyanins (1100 mg/l), proanthocyanidins (300 mg/l) and catechins (25 mg/l).Abbreviations BuOH n-butanol - dw dry weight - fw fresh weight     

Regulation of polyphenol production in Vitis vinifera cell suspension cultures by sugars

Sucrose was found to modulate polyphenol accumulation in Vitis vinifera cell cultures. The production of anthocyanins increased 12-fold after addition of 0.15 m sucrose, while that of stilbenes was only slightly affected. Sucrose did not play a physical role because metabolic sugars were required for the induction of polyphenol accumulation. Indeed, the polyols, mannitol and sorbitol, had no effect on this accumulation. We established a model system to investigate the mechanism of sucrose regulation of polyphenol production without inhibition of grape cell growth. After addition of sucrose to the culture medium, the major sugars accumulated in grape cells were glucose and fructose, reaching 40% of the dry weight. The increase in the level of these hexoses closely coincided with the increase in anthocyanin accumulation in grape cells. Received: 18 August 1997 / Revision received: 6 November 1997 / Accepted: 5 January 1998     

Carbon-14 biolabelling of wine polyphenols in Vitis vinifera cell suspension cultures

14C-L-phenylalanine is incorporated into a range of polyphenolic compounds when fed to grape cell cultures. Optimisation of several parameters such as the quantity of precursor applied and the duration of metabolism led to incorporation yields of 15% and to specific activities of 875 mu Ci g(-1) in stilbenes. Purification of the products by several chromatographic steps is reported. Both trans- and cis-resveratrols were easily obtained by enzymatic hydrolysis of their corresponding glucosides, with specific activity of 1200-1400 mu Ci g(-1). The specific radioactivity obtained for all the compounds is suitable for in vivo feeding trials to trace their metabolic fate when consumed by animals and for in vitro activity mechanism studies. Indeed, these polyphenols seem to be implicated in the health benefits associated with regular and moderate wine consumption but little is known about their pharmacokinetics and cellular uptake.     

The effects of cadmium chloride on secondary metabolite production in Vitis vinifera cv. cell suspension cultures

Background

Plant secondary metabolites are possess several biological activities such as anti-mutagenic, anti-carcinogenic, anti-aging, etc. Cell suspension culture is one of the most effective systems to produce secondary metabolites. It is possible to increase the phenolic compounds and tocopherols by using cell suspensions. Studies on tocopherols production by cell suspension cultures are seldom and generally focused on seed oil plants. Although fresh grape, grape seed, pomace and grape seed oil had tocopherols, with our best knowledge, there is no research on tocopherol accumulation in the grape cell suspension cultures. In this study, it was aimed to determine the effects of cadmium chloride treatments on secondary metabolite production in cell suspension cultures of grapevine. Cell suspensions initiated from callus belonging to petiole tissue was used as a plant material. Cadmium chloride was applied to cell suspension cultures in different concentration (1.0 mM and 1.5 mM) to enhance secondary metabolite (total phenolics, total flavanols, total flavonols, trans-resveratrol, and α-, β-, γ- δ-tocopherols) production. Cells were harvested at two days intervals until the 6^th day of cultures. Amounts of total phenolics, total flavanols and total flavonols; trans-resveratrol and tocopherols (α-, β-, γ- and δ-tocopherols) and dry cell weights were determined in the harvested cells.

Results

Phenolic contents were significantly affected by the sampling time and cadmium concentrations. The highest values of total phenolic (168.82 mg/100 g), total flavanol (15.94 mg/100 g), total flavonol (14.73 mg/100 g) and trans-resveratrol (490.76 μg/100 g) were found in cells treated with 1.0 mM CdCl₂ and harvested at day 2. Contents of tocopherols in the cells cultured in the presence of 1.0 mM CdCl₂ gradually increased during the culture period and the highest values of α, β and γ tocopherols (145.61, 25.52 and 18.56 μg/100 g) were detected in the cell cultures collected at day 6.

Conclusions

As a conclusion, secondary metabolite contents were increased by cadmium chloride application and sampling time, while dry cell weights was reduced by cadmium chloride treatments.     

Melatonin and phenolics biosynthesis-related genes in Vitis vinifera cell suspension cultures are regulated by temperature and copper stress

Plant Cell, Tissue and Organ Culture (PCTOC) - Temperature and copper stress are two common abiotic stresses in viticulture. To better understand biosynthesis of melatonin and phenolics in response...     

Growth kinetics of vitis vinifera cell suspension cultures: I. Shake flask cultures

Vitis vinifera cell suspension cultures carried out in shake flasks were closely examined for biomass growth and cell division in relation to carbohydrate, NH(4), NO(3)PO(4), and dissolved oxygen (DO)consumption. After inoculation, the oxygen uptake rate of the cultures measured on-tine was observed to increase continuously to a maximum value of 3.8 mmol O(2)L(-1)h(-1) at day 7 when cell division ceased and dissolved oxygen reached its lowest level of 17% air saturation. During this first phase of growth, the specific oxygen uptake rate remained constant at approximately 0.6 mmol 02 O(2) g(-1) dw h(-1)or approximately 2.2 mumol O(2), (10(6) cells)(-1) h(-1) whereas dry biomass concentration increased exponentially from 1.5 to 6.0 g dw L(-1). Thereafter, dry biomass concentration increased linearly to approximately 14 g dw L(-1) at day 14 following nitrate and carbohydrate uptake. During this second phase of growth, the biomass wet-to-dry weight ratio was found to increase in an inverse relationship with the estimated osmotic pressure of the culture medium. This corresponded to inflection points in the dry and wet biomass concentration and packed cell volume curves. Furthermore, growth and nutrient uptake results suggest that extracellular ammonium or phosphate ion availability may limit cell division. These findings indicate that cell division and biomass production of plant cell cultures may not always be completely associated, which suggests important new avenues to improve their productivity. (c) 1995 John Wiley & Sons, Inc.     

Glucose Transport in Vitis vinifera L. Protoplasts

Transport of glucose and its analogues was studied in grapevine(Viris vinfera L. cv. Soultanina) leaf protoplasts. The transportsystem was hexose specific and the stereospecificity was closelyrelated to carbon-1 of the glucose molecule. Glucose structuralanalogues were not metabolized beyond the stage of phosphorylationand differences between these compounds and glucose were observedin their transport rates and in their specificity for the carrier.Concentration-dependent uptake of labelled glucose by grapevineprotoplasts was linear for concentrations higher than 1?5 molm^–3 at lower concentrations a saturating pattern was observed.The carrier was driven by the proton motive force and the substrateentered the cell probably in an unchanged form. Efflux studieswere not useful as an indication of the rate of metabolism orassimilation of transported compounds in grapevine protoplasts. Key words: Sugar transport, protoplasts, grapevine     

Production of highly 13C-labeled polyphenols in Vitis vinifera cell bioreactor cultures

The use of Vitis vinifera cells grown in a 2 l-stirred tank bioreactor for producing isotopically 13C-labeled phenolic substances is presented. Several culture parameters were optimized to achieve characteristics of growth and polyphenol metabolism similar to that recorded in shake flasks. Administration of [1-13C]L-phenylalanine (3 mM) to grape cell suspension cultures led to the production of 13C-labeled stilbenes (trans- and cis-piceids), catechins (catechin and epicatechin) and anthocyanins (delphinidin-, cyanidin-, petunidin-, peonidin- and malvidin-3-O-beta-glucosides). Incorporation of [1-13C]L-phenylalanine into polyphenols was measured by means of 13C satellites in the proton NMR spectrum and EA-IRMS. The enrichment of labeling obtained for all the compounds (between 40 and 65%) is sufficient to investigate their absorption and metabolism in humans.     

Jasmonates and Na-orthovanadate promote resveratrol production in Vitis vinifera cv. Barbera cell cultures

Here the effect of jasmonic acid, methyljasmonate and Na-orthovanadate on the production of resveratrol was studied in Vitis vinifera cv. Barbera cell suspension cultures. Na-orthovanadate at 0.1 mm and 1 mm concentration was efficient in promoting the production and/or accumulation and release in the culture medium of cis-resveratrol while trans-resveratrol levels were not affected by this treatment. Methyljasmonate was highly effective in stimulating both trans- and cis-resveratrol endogenous accumulation, as well as their release into the culture medium. Cis-resveratrol was absent or detected in very low amounts in the controls. Jasmonic acid was less efficient than methyljasmonate in promoting endogenous resveratrol accumulation, but it stimulated the release in the culture medium especially of cis-resveratrol. Gel analysis was performed on control and 10 microm MeJA treated cell suspensions. Results showed an up-regulation of the stilbene synthase demonstrating that MeJA stimulated the synthesis ex-novo of this protein.     

Tannin inclusions in cell suspension cultures of white spruce

Summary Tannins were detected cytochemically in cell suspension cultures of white spruce (Picea glauca [Moench] Voss) and were studied by electron microscopy. Tannin inclusions originated within cytoplasmic vacuoles, possibly derived from the endoplasmic reticulum, and accumulated in the central vacuole through enlargement and coalescence of those cytoplasmic vacuoles. Structural information supported the suggested metabolic relationship between starch and tannin, although tannins did not develop within plastids. Membranous material, resembling myelinlike bodies, was often observed in close association with tannins.     

Characterization of Vitis vinifera L. somatic variants exhibiting abnormal flower development patterns

Mutants have proven to be a key resource for functional genomic studies in model annual plant species. In perennial plant species where mutants are difficult to generate and to screen, spontaneous somatic variants represent a unique resource to understand the genetic control of complex developmental patterns. The morphological and histological characterization of six Vitis vinifera L. somatic variants that display four different abnormal phenotypes of flower development are described here. A phenotype of reiterated reproductive meristems (RRM), with both flower and petal reiteration, was observed in a somatic variant of the cultivar Carignan. An abnormal development of reproductive organs was displayed by the unfused carpels (UFC) somatic variant of cv. Bouchalès, while a somatic variant of cv. Mourvèdre named carpel-less (CLS) developed abnormal ovules in the absence of carpels. Finally, three independent somatic variants in cvs Gamay, Morrastel, and Pinot displayed a phenotype of multiple perianth whorls (MPW). Gene expression studies showed that the expression profiles of VvMADS-box 1, 2, and 3 (putative orthologues of Arabidopsis flowering genes AG, SEP, and AGL13), were altered during grapevine flower development in the somatic variants, whereas the corresponding original cultivars displayed similar VvMADS-box gene expression profiles. Phenotypic and molecular characterization of these variants allowed the development of hypotheses on genetic functions that might be altered in most of the variants in light of the current ABCDE flower model.     

New insight into the structures and formation of anthocyanic vacuolar inclusions in flower petals

Background  

Although the biosynthetic pathways for anthocyanins and their regulation have been well studied, the mechanism of anthocyanin accumulation in the cell is still poorly understood. Different models have been proposed to explain the transport of anthocyanins from biosynthetic sites to the central vacuole, but cellular and subcellular information is still lacking for reconciliation of different lines of evidence in various anthocyanin sequestration studies. Here, we used light and electron microscopy to investigate the structures and the formation of anthocyanic vacuolar inclusions (AVIs) in lisianthus (Eustoma grandiflorum) petals.     

Accumulation of peonidin 3-glucoside enhanced by osmotic stress in grape (Vitis vinifera L.) cell suspension

Cell cultures of grapes, Vitis vinifera L. cv Gamay Fréaux were grown under different conditions of external osmotic potential induced by an increase of sucrose concentration or by the addition of mannitol to the culture medium. Addition of 82 mM mannitol or increasing sucrose concentration to 132 mM had similar effects on repressing growth. Cyanidin 3-glucoside, peonidin 3-glucoside and peonidin 3-p-coumaroylglucoside are three main anthocyanins of Vitis cells. Increasing osmotic potential from –0.43 MPa to –0.8 MPa in the medium resulted in a significant intracellular accumulation of anthocyanin especially peonidin 3-glucoside in the pigmented cells. High osmotic potential appears to stimulate the methylation of anthocyanins. Osmotic potential is an important culture factor and may be useful in the controlling of anthocyanin production and composition.     

Characterization of buffelgrass (Cenchrus Ciliaris L.) cell suspension cultures

Summary Cell suspension cultures of buffelgrass were established from two types of callus, a friable tan callus and a brown gelatinous callus, using Murashige and Skoog medium containing 13.6 μM 2,4-dichlorophenoxyacetic acid (2,4-D). The friable callus formed a rapidly growing suspension culture, designated BG, which had a doubling time of 2.5 days. The gelatinous callus formed a very slow-growing suspension culture, designated BGG, which had a doubling time of 1 mo. During growth, the medium of the BGG line slowly increased in viscosity, becoming a thickened gel by the end of the subculture period. Both lines had high cell viability. Embryogenesis could be induced in both lines by culturing on charcoal-containing, 2,4-D-free medium. No embryos formed in the absence of charcoal.