Anthocyanins, catechins, condensed tannins and piceid production in Vitis vinifera cell bioreactor cultures

Summary Suspension cultures of Vitis vinifera in a stirred fermenter showed characteristics of growth and polyphenol metabolism similar to that found in shake flasks. In the induction medium, the cells produced mainly anthocyanins (1200 mg/l), proanthocyanidins (220 mg/l), catechins (8 mg/l) and trans-piceid (30 mg/l).     

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     

Rosmarinic acid production by Lavandula vera MM cell-suspension culture

The time courses of growth and rosmarinic acid production by Lavandula vera MM cell suspension were investigated. The uptake of the main nutrients (sucrose, nitrogen, phosphorus, K, Ca, Mg) was followed during cultivation and the data on the physiology of the L. vera MM cell culture are presented. It was established that the cell culture synthesizes rosmarinic acid during the linear phase of growth for a relatively short period (between the 4th and 8th days of cultivation). The influence of sucrose concentration in the nutrient medium on cell growth and accumulation of rosmarinic acid by L. vera MM cell culture was investigated. The results showed that 7% sucrose in the nutrient medium ensured a steady growth of the cell suspension and increased the yield of rosmarinic acid (29.2 g/l dry biomass and 507.5 mg/l rosmarinic acid compared to 13.0 g/l dry biomass and 68.6 mg/l rosmarinic acid for the control cultivation with 3% sucrose). Received: 17 September 1996 / Received revision: 31 January 1997 / Accepted: 1 February 1997     

Deoxynivalenol and 15-monoacetyl deoxynivalenol production by Fusarium graminearum R6576 in liquid media

Growth and toxigenesis by Fusarium graminearum R6576, were compared in four liquid media. Parameters monitored during the fermentation were deoxynivalenol (DON) and 15-acetyl deoxynivalenol (15-ADON) production, fungal mass, carbohydrate utilization, and pH. Factors which were varied included basal medium composition, corn steep liquor (CSL) concentration, sucrose concentration and ammonium tartrate concentration. Growth in modified Fries medium resulted in only low levels of DON (0.25 mg/ L) and 15-ADON (0.25 mg/ L) after 20 days. Addition of 4% CSL to modified Fries medium raised the 20 day DON yield to 16.5 mg/ l. Increasing the sucrose concentration in modified Fries medium amended with 4% CSL resulted in increased mycelial dry weight but decreased levels of DON. Concentrations of ammonium tartrate greater than 1% in modified Fries amended with 4% CSL greatly reduced DON yield. Use of glucose-yeast extract-peptone (GYEP) for toxin production resulted in higher yields of 15-ADON (14.0 mg/ L) than DON (5.5 mg/ L) after 20 days. However, supplementation of GYEP with 4% CSL resulted primarily in DON production (4.5 mg/ L) after 20 days. In general, qualitative and quantitative production of DON and 15-ADON by Fusarium graminearum R6576 were dependent on the composition of the complex liquid medium.     

Production of diosgenin by hairy root cultures ofTrigonella foenum-graecum L.

Hairy root cultures ofTrigonella foenum-graecum L. were established withAgrobacterium rhizogenes strain A₄. The hairy roots produce diosgenin, an important spirostanol for the semi-synthesis of steroid hormones. Fourteen different liquid media were investigated. The fastest growth was obtained in McCown's woody plant (WP) medium supplemented with 3% sucrose; the highest diosgenin content was observed in half-strength WP medium with 1% sucrose (0.040% dry weight), which represents almost twice the amount detected in the 8-month-old non-transformed roots (0.024%). A time-course study in WP liquid media supplemented with 3% sucrose was undertaken. In these conditions, 17 g diosgenin/g fresh weight were produced. The influence of cholesterol, medium pH and chitosan on diosgenin production was tested. The addition of 40 mg/l chitosan elevated the diosgenin content to three times that found in non-elicited hairy roots.Abbreviations MS Murashige and Skoog (1962) medium - WP McCown's woody plant medium     

Production of 3-acetyldeoxynivalenol by Fusarium graminearum R 2118 in submerged cultures

Summary Production of of 3-acetyldeoxynivalenol (3-ADN) by Fusarium graminearum R 2118 in submerged cultures was characterized for five different media. Toxin production was examined as a function of mycelial growth, sugar utilization, pH and phosphate concentration. In submerged cultures, 3-ADN appeared after 2 days of incubation at 25°C when mycelial growth had slowed down and the pH of the media had dropped to 4.5 or lower. A two stage process was developed for high and rapid production of 3-ADN, in which the biosynthetically active mycelium was grown in a yeast extract-peptone-sucrose medium and the toxin was produced by the mycelium in a sucrose containing minimal medium. Yields of 90–110 mg/l were obtained within 5 days in the production medium. Acidity (low pH) and low phosphate concentration in the minimal medium were both required for 3-ADN production, representing two independent regulating factors for the 3-ADN biosynthesis.     

Establishment of Croton stellatopilosus suspension culture for geranylgeraniol production and diterpenoid biosynthesis

Diterpenoids in higher plants are biosynthesized from isoprene units obtained from two distinct pathways: the mevalonate pathway and the deoxyxylulose phosphate pathway. The metabolic partitioning of both pathways in plant species is dependent upon the type of culture. In order to study the diterpenoid biosynthesis in Croton stellatopilosus cell culture, callus culture was firstly induced from C. stellatopilosus young leaves in Murashige and Skoog (MS) medium in the presence of 1.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), 1.0 mg/l benzyladenine (BA), 3% (w/v) sucrose and 0.8% (w/v) agar. The suspension culture was further induced from its callus in the same medium without gelling agent. Detection of diterpenoid accumulation by gas chromatography-mass spectrometry revealed that a cell culture could accumulate a low amount of geranylgeraniol (GGOH) and a high content of fatty acids and phytosterols. To improve the GGOH production, the culture conditions were optimized by medium manipulation in terms of hormonal factors. The growth rates of cell cultures were similar in all kinds of media. The GGOH production curve indicated that GGOH plays an important role as a primary metabolite in the cell culture. The optimum medium for GGOH production was MS medium supplemented with 2.0 mg/l 2,4-D and 2 mg/l BA that could produce GGOH with a yield of 1.14 mg/g FW.     

Production of glutaminase (E.C.3.2.1.5) from Zygosaccharomyces rouxii: statistical optimization using response surface methodology

A face centered central composite design was employed to investigate the interactive effects of four variables, viz. concentrations of sucrose, yeast extract, sodium chloride, and glutamine, identified earlier by one-factor-at-a-time approach, on glutaminase production by Zygosaccharomyces rouxii. A significant influence of yeast extract on glutaminase production was noted. Response surface methodology (RSM) showed that a medium containing (g/l) sucrose, 17.8; yeast extract, 48.0; glutamine, 5.0 and sodium chloride, 55.6 to be optimum for the production of glutaminase. This medium was projected to produce, theoretically, an enzyme activity of 149.98 U/l and a specific activity of 0.488 U/mg protein. The applied methodology was validated using this optimized media and enzyme activity 155.89+/-1.68 U/l and specific activity of 0.468+/-0.088 U/mg protein was obtained. Further, this optimization strategy combined with an increase in inoculum enhanced the enzyme activity and specific activity by 2.94 and 3.58 fold, respectively, as compared to the unoptimized media.     

An efficient and reproducible method for regeneration of whole plants from mature seeds of a high yielding Indica rice (Oryza sativa L.) variety PAU 201

Tissue culture is one of the tools necessary for genetic engineering and many other breeding programs. Moreover, selection of high regenerating rice varieties is a pre-requisite for success in rice biotechnology. In this report we established a reproducible plant regeneration system through somatic embryogenesis. The explants used for regeneration were embryogenic calli derived from mature seeds cultured on callus induction media. For callus induction mature seeds were cultured on MS medium containing 30 g/l sucrose combined with 560 mg/l proline and 1.5-3.5 mg/l 2,4-D and 0.5-1.5 mg/l Kin. For plant regeneration, embryogenic calli were transferred to MS medium containing 30 g/l sucrose, supplemented with 1.0-3.0 mg/l BAP, 0.5-1.5 mg/l Kin and 0.5-1.5 mg/l NAA. The highest frequency of callus induction (44.4%) was observed on the MS medium supplemented with 2.5 mg/l 2,4-D, 0.5 mg/l Kin, 560 mg/l proline and 30 g/l sucrose. The highest frequency of shoot regeneration (42.5%) was observed on the MS medium supplemented with 2.0 mg/l BAP, 0.5 mg/l NAA and 0.5 mg/l Kin. The plantlets were hardened and transferred to soil in earthen pots. The developed method was highly reproducible. The in vitro developed plants showed normal growth and flowering under glasshouse conditions.     

Factors affecting induction and development of in vitro rooting in apple rootstocks

Shoots of apple rootstocks raised in vitro were transferred to various rooting media to study the effect of different factors on root initiation and development. Various concentrations of indole-3-butyric acid (IBA) initiated rooting but maximum rooting percentage was found with 2.0 and 2.5 mg l(-1) of IBA in M7 and with 1.0 mg l(-1) of IBA in MM106. The drawback was that the roots were thick, short and with profuse callus. The presence of activated charcoal (AC) in the rooting medium improved the rooting quality but reduced the rooting percentage in both the rootstocks. In high auxin dip of 70, 80 and 90 mg l(-1) IBA for 2, 2 and 1 hr showed 75-85 per cent rooting in M7, but lacked reproducibility of the results. Whereas in MM106, 66 - 70 % rooting was achieved with 70 mg l(-1) of IBA dip for 3 h. Root induction in shoots in IBA containing liquid medium (LM) in dark for few days and root elongation in IBA--free medium in light proved most effective. On the other hand, continuous light treatment showed reduced rooting. Reduction of MS salts and sucrose in root elongation medium showed decreased rooting. Plantlets from two--stage rooting procedure showed more rapid growth and satisfactory survival during hardening of plants and on transfer to field.     

Indole alkaloid accumulation and tryptophan decarboxylase activity in Catharanthus roseus cells cultured in three different media

Catharanthus roseus cells were cultured in three types of media. These media were: a low sucrose subculture medium and two high sucrose media, each of which differed in their mineral and hormonal contents. The kinetics of tryptophan decarboxylase activity and the accumulations of tryptophan, tryptamine, ajmalicine and serpentine were different in each series but no correlation between maximum enzyme activity and alkaloid contents was observed. Ajmalicine and serpentine productions were unaffected by addition of Trp to the media, whereas addition of secologanin enhanced alkaloid production. The results seem to imply that the terpenoid pathway is the limiting factor in alkaloid production in C. roseus cells.     

The Growth of Acer pseudoplatanus Cells in a Synthetic Liquid Medium: Response to the Carbohydrate, Nitrogenous and Growth Hormone Constituents

A synthetic culture medium which supports a high level of growth of a scrially propagated cell suspension culture of Acer pseudoplatanus is described. The sucrose of this medium can be effectively replaced by glucose or fructose or a mixture of glucose and fructose or galactose or maltose or soluble starch. When the carbohydrate is glucose or fructose no other sugars appear in the culture medium in significant amounts. Glucose is absorbed in greater quantity than fructose from an equimolar mixture of these sugars. When sucrose is supplied both glucose and fructose appear in the medium. Glucose appears in maltose medium, and maltose and glucose in soluble starch medium. Under the standard conditions of culture, media containing 2 % sucrose or 2 % glucose become depleted of sugar before the 25th day of incubation. Enhanced yield of the cultures can be obtained by raising the initial sucrose concentration to 6 %. – A supply of nitrate is essential for maximum yield and healthy growth. Growth, in the presence of nitrate, is significantly enhanced by a supply of urea. Addition of casein hydrolysate or of a mixture of amino acids enhances growth in the presence of nitrate and urea and particularly when nitrate is omitted. – When kinetin is omitted or incorporated at the standard level (0.25 mg/I), 2,4-dichlorophenoxyacetic acid (2,4-D) at 1.0 mg/l is essential for continuation of growth at a high level. It cannot be replaced by indol-3yl-acetic acid (IAA). 1-naphthaleneacetic acid (NAA) at 10 mg/l permits of a low level of growth with abnormal aggregation. When the level of kinetin is raised to 10 mg/l a high level of growth occurs in the absence of added auxin but the cultures become brown and tend to show increasing aggregation on subculture.     

The Effect of Plant Growth Regulators and Sucrose on the Micropropagation and Microtuberization of <Emphasis Type="Italic">Dioscorea nipponica</Emphasis> Makino

The effects of sucrose, plant growth regulators, MS (Murashige and Skoog), and ½MS salt media formulations were investigated for the development of shoot cultures, microtuber induction, and plantlet regeneration in Dioscorea nipponica. The cytokinin N-benzyladenine (BA) in the range of 0.5–2.0 mg/l showed strong enhancing effects on microtuber induction only when used in conjunction with the auxin alpha-naphthalene acetic acid (NAA), with the effect that NAA increased from 0.5 to 2.0 mg/l. Murashige and Skoog salt media supplemented with sucrose at 3% (w/v) gave the highest frequencies of shoot induction (86%) when BA was present at 2.0 mg/l and NAA at 1.0 mg/l. Sucrose at 7% (w/v) was the single most significant medium constituent for microtuber growth. The heaviest microtubers were formed on media containing 1.0 mg/l BA and 2.0 mg/l (0.073 g), especially with 7% sucrose (3.46 g). With media containing ½MS, 2% sucrose, and 0.1% (w/v) activated charcoal, the percentage of rooting was maximal when supplemented with 1.0 mg/l BA and 0.5 mg/l NAA for the in vitro produced shoots (95%) and BA and NAA both at 0.5 mg/l for the microtubers (100%). When removed from culture flasks and transferred into sterilized soil in a greenhouse, most of the hardened plantlets survived (over 91% after 1 week), and they were suitable for field planting after 1 month.     

Plant regeneration through callus cultures derived from immature-cotyledon explants of oleaster (Elaeagnus angustifolia L.)

Efficient plant regeneration was achieved from callus derived from immature-cotyledon explants of oleaster (Elaeagnus angustifolia L.). Calli were obtained on MS media containing 3% sucrose and different concentrations of TDZ. The highest rate of green, compact and nodular callus was formed on MS medium supplemented with 1 mg/l of TDZ. Shoot organogenesis was achieved when the callus was transferred onto MS media containing 3% sucrose and BA alone (05–4 mg/l) or BA (0.5 and 1 mg/l) combined with NAA or IAA (0.5 and 1 mg/l). Maximum organogenesis was obtained with 1 mg/l BA in combination with 0.5 mg/l NAA. Rooting of the shoots was achieved on MS medium supplemented with 0.2 mg/l IBA. Regenerated plantlets were acclimatized and successfully transplanted to soil.     

Photoautotrophic micropropagation of Russet Burbank Potato

The photoautotrophic micropropagation of potato cv. Russet Burbank was investigated. Single node microcuttings were grown for four weeks on Murashige and Skoog (MS) medium with or without sucrose (30 g l^–1) in the growth room at 21/19 °C day/night temperature, with 16-h photoperiod at 150 mol m^–2 s^–1, with or without supplemental CO₂ at 1500 l l^–1. A 20% increase in the number of nodes per stem (from 7.5 to 9.4) and a 50% increase in stem dry weight were observed in cultures grown on media with sucrose and in CO₂ enriched atmosphere comparing to the conventionally micropropagated cultures or the cultures grown photoautotrophically on media without sucrose but in air supplemented with 1500 l l^–1CO₂. Stems of these cultures (from media with sucrose in CO₂ enriched air) almost doubled in length the stems of cultures from the other two treatments. No significant differences were observed between Control (MS medium supplemented with sucrose, 30 g l^–1) and photoautotrophic cultures coming from MS medium with no sucrose grown under 1500 l l^–1 of CO₂. Photoautotrophic cultures produced stems averaging 43.3 mm, with 7 nodes and weighing 9.2 mg (dry weight), similar to conventionally grown in vitro cultures (47.9 mm with 7.5 nodes, 9.7 mg dry weight). Growers may consider photoautotrophic culturing of potato in areas where the high sterility levels are difficult to maintain. Supplementing air in the growth room with 1500 l l^–1 of CO₂ could be beneficial for potato plantlet production even on media containing sucrose since it significantly improved quality, size and biomass of produced plantlets, speeding up the multiplication.     

Propagation of Musa textilis Neé plants from apical meristem slices in vitro

Cultures were propagated from apical meristem slices of Musa textilis plants. They were cultured in vitro in light on either MS medium containing BAP (10 mg/l), but without edamin or MS mineral salts supplemented with 100 mg/l each of inositol, tyrosine, ascorbic acid; 150 mg/l citric acid; 2 mg/l cysteine; 0.4 mg/l thiamine HCl; 3% sucrose and 0.5–0.8% agar. Shoot initials were induced using media containing 5–10 mg/l BAP. Further promotion of shoot induction was achieved when BAP (1–3 mg/l) was supplemented with either NAA (1 mg/l) or adenine sulphate (80–160 mg/l). Shoot initials were multiplied on media containing 3–5 mg/l BAP, 0.1 mg/l IBA and 160–200 mg/l adenine sulphate. Plantlets generated roots on media without adenine sulphate but containing 1–1.5% sucrose and either NAA (0.1–1 mg/l) or IBA (2–10 mg/l). Plantlets were transferred into pots in the greenhouse 7 days after rooting.     

Acetaldehyde production in Saccharomyces cerevisiae wine yeasts

Abstract Eighty-six strains of Saccharomyces cerevisiae were investigated for their ability to produce acetaldehyde in synthetic medium and in grape must. Acetaldehyde production did not differ significantly between the two media, ranging from a few mg/l to about 60 mg/l, and was found to be a strain characteristic. The fermentation temperature of 30°C considerably increased the acetaldehyde produced. This study allowed us to assign the strains to different phenotypes: low, medium and high acetaldehyde producers. The low and high phenotypes differed considerably also in the production of acetic acid, acetoin and higher alcohols and can be useful for studying acetaldehyde production in S. cerevisiae , both from the technological and genetic point of view.     

Soybean somatic embryogenesis: interactions between sucrose and auxin

The interaction between sucrose and auxin in soybean (Glycine max L. Merr.) somatic embryogenesis was investigated by culturing immature cotyledon explants on factorial combinations of NAA (6.25, 12.5, 25 and 50 mg/l) and sucrose (0.5, 1, 2 and 4%). A significant interaction between sugar and auxin was observed; balanced concentrations of the two components were required for optimal embryo production and normality. The highest numbers of normal somatic embryos were produced on media which contained combinations of low to intermediate levels of sucrose (1 or 2%) and NAA (6.25 or 12.5 mg/l). Cotyledon explants from induction media having a low (0.5%) sucrose content showed the most efficient embryogenesis in secondary culture. The highest frequencies of germination (32 to 41%) were seen among embryos induced on media containing 0.5% sucrose.Abbreviations BAP 6-benzylaminopurine - NAA 1-naphthaleneacetic acid This paper (No. 88-3-161) is published with the approval of the Director of the Kentucky Agricultural Experiment Station.     

Effects of high ammonium concentrations on growth and anthocyanin formation in grape (Vitis vinifera L.) cell suspension cultured in a production medium

Cultivating Vitis vinifera cell suspensions in a production medium which is characterized by high sucrose and low nitrate concentrations (132 mM and 6.25 mM respectively) repressed growth but enhanced the intracellular accumulation of anthocyanins, especially peonidin 3-glucoside. Increasing the ammonium concentration of the production medium from 2 to 8–16 mM increased growth and decreased the accumulation of anthocyanins and peonidin 3-glucoside specifically. Instead, peonidin 3-p-coumaroylglucoside accumulated. At 24 mM ammonium concentration, growth was inhibited and accumulation of peonidin 3-p-coumaroylglucoside was significant (p<0.05) and represented 42% of total anthocyanins after 12 days of culture compared with 19% in the production medium with 2 mM ammonium.Contribution Number 217.     

Factors influencing somatic embryo maturation, high frequency germination and plantlet formation in Terminalia chebula Retz.

The factors influencing somatic embryo maturation, high frequency somatic embryo germination, and plantlet formation were studied in Terminalia chebula Retz. Maturation of somatic embryo were influenced by a number of factors such as in vitro culture passage, concentrations of sucrose, levels of abscisic acid (ABA), basal media and media additive combinations. Maximum frequency of somatic embryo maturation (57.22 ± 2.02), was obtained on MS medium supplemented with 50 g/l sucrose. Different factors such as strengths of MS nutrients, plant growth regulators, media additives and their combinations controlling somatic embryo germination and plantlet formation were studied. High frequency of germination and plantlet formation (58.80 ± 1.47) were achieved by subsequent subculture of mature somatic embryos on MS medium containing 30 g/l sucrose and 0.5 mg/l benzyladenine (BA). However, although duration of in vitro passage of the callus tissue was critical, contribution of the combinations of plant growth regulators and media additives showed nugatory effect on somatic embryo maturation and germination as evident from variable responses.