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.     

Accumulation of anthocyanins enhanced by a high osmotic potential in grape (Vitis vinifera L.) cell suspensions

A cell suspension of grape, Vitis vinifera L. cv Gamay Fréaux, was grown under different conditions of water stress (high external osmotic potential) induced by an increase of sucrose concentration or by the addition of mannitol to the culture medium. Best growth (cell density) was achieved in the low osmotic potential medium. Increasing the osmotic potential of the medium from –0.5 MPa to –0.9 MPa medium resulted in a significant increase in accumulation of anthocyanins in pigmented cells. Regulation of the osmotic potential of culture medium may be useful in controlling anthocyanin production.     

Manipulating anthocyanin composition in Vitis vinifera suspension cultures by elicitation with jasmonic acid and light irradiation

Jasmonic acid altered the accumulation of major anthocyanins in Vitis vinifera cell culture. Peonidin 3-glucoside content at day three was increased from 0.3 to 1.7 mg g^–1 dry cell wt while other major anthocyanins were increased by smaller increments. By day 14, the content of methylated and acylated anthocyanins (peonidin 3-p-coumaroylglucoside and malvidin 3-p-coumaroylglucoside) was 6.3 mg g^–1 DCW, in response to treatment with jasmonic acid, and comprising 45% (w/w) of total anthocyanins. In comparison, the untreated control culture contained 1.2 mg g^–1 DCW which made up 32% (w/w) of total anthocyanins. Light further enhanced anthocyanin accumulation induced by jasmonic acid elicitation. The content of peonidin 3-glucoside at day 3 was 6.6 mg g^–1 DCW, 22-fold higher than control cultures while the content in response to light irradiation alone was 0.6 mg g^–1 DCW. When a highly pigmented cell line was elicited with jasmonic acid total anthocyanins increased from 9.2 to 20.7 mg g^–1 DCW, but there was no change in the anthocyanin composition.     

Effects of low nitrate and high sugar concentrations on anthocyanin content and composition of grape (Vitis vinifera L.) cell suspension

In pigmented cells of Vitis vinifera suspension cultures, best accumulation of anthocyanins was obtained when nitrate concentration was reduced from 25 mM to 6.25 mM and when sucrose concentration was increased from 88 mM to 132 mM. Under such conditions growth was greatly decreased. However, cell viability was maintained. The increases in anthocyanins in pigmented cells were due largely to increases in peonidin — glucoside. The high sucrose and the low nitrate concentrations can be one of the important culture factors in controlling of anthocyanin production by cell cultures.     

Anthocyanin production in selected cell lines of grape (Vitis vinifera L.)

Summary Anthocyanin production of two lines ofVitis vinifera cell cultures, i.e., 5.4 and 13.1, which were obtained from the same starting material after 20 and 37 mo. of clonal selection, respectively, was investigated. Cell suspension cultures of lines 5.4 and 13.1 maintained an anthocyanin content of 0.44 ± 0.15 and 1.02 ± 0.31 mg·g⁻¹ fresh weight during 50 and 32 weekly maintenance subcultures, respectively. Under anthocyanin-promoting culture conditions, both lines showed an enhancement of their anthocyanin level by approximately fourfold. While line 5.4 accumulated peonidin 3-glucoside and cyanidin 3-glucoside in decreasing order, line 13.1 accumulated primarily peonidin 3-p-coumaroylglucoside with lesser amounts of malvidin monoglucoside. Results show that while the anthocyanin content was improved during the course of repeated selections, the anthocyanin composition was modified markedly favoring the accumulation of more metabolically-advanced anthocyanins.     

Osmotic regulation of starch synthesis in potato tubers?

Using potato (Solanum tuberosum L.) tuber discs incubated in a range of mannitol concentrations it has been demonstrated that both sucrose uptake and the conversion of sucrose to starch are sensitive to the osmotic environment of the storage cells. Starch synthesis was optimised at 300 mM but declined sharply at both lower and higher osmotic concentrations. The decline in starch synthesis on either side of optimum was not proportional to the change in mannitol concentration, indicating different inhibitory mechanisms under low and high osmotica. The fraction of the total sucrose converted to starch i.e. the partitioning between sucrose and starch, was also influenced by osmotic environment. The amount of soluble material taken up by the storage cells, but not converted to starch, was maintained under mannitol concentrations (300–400 mM) which inhibited starch synthesis, indicating that sucrose uptake continued during declining starch synthesis. At mannitol concentrations above 400 mM, sucrose uptake was greatly enhanced but no significant change in starch synthesis occurred.     

Cyanidin 3-glucoside accumulation in plane tree (Platanus acerifolia) cell-suspension cultures

The accumulation of only one anthocyanin, cyanidin 3-glucoside, in cell-suspension cultures of plane tree (Platanus aceriflia) is reported for the first time. During a time span of 6 years, no new anthocyanin was detected and cyanidin 3-glucoside was maintained at about 35 mg l^–1 cell culture medium. This stable cell culture system could therefore be used for the biotechnological production of cyanidin 3-glucoside.     

Effect of mannitol and glucose-induced osmotic stress on growth,water relations,and solute composition of cell suspension cultures of poplar (Populus deltoides var.Occidentalis) in relation to anthocyanin accumulation

Summary A cell suspension culture of poplar (Populus deltoides (Marsh.) Bartr. var.occidentalis Rydb.), accumulating the anthocyanin pigment, cyanidin 3-glucoside, in the lag phase of culture growth, was subjected to osmotic stress with glucose and mannitol. Osmotic stress treatments resulted in growth suppression and higher anthocyanin accumulation compared with unstressed cells. Both an increase in the proportion of pigmented cells and an increase in the concentration of anthocyanin in the pigmented cells were responsible for high anthocyanin content of cultured cells subjected to osmotic stress. The osmotic stress induced by glucose suppressed growth more than that by mannitol and produced higher anthocyanin levels. Only small amounts of [U-¹⁴C]mannitol were taken up and metabolized by the cells. Stressed cells accumulated sugars and free amino acids to a different extent resulting in altered cell sugar-to-amino acid ratios. The accumulation of osmotically active solutes and cell growth suppression may both be responsible for the accumulation of anthocyanin in stressed cells.     

Anthocyanin production in callus cultures of Cleome rosea: Modulation by culture conditions and characterization of pigments by means of HPLC-DAD/ESIMS

Leaf and stem explants of Cleome rosea formed calluses when cultured on MS medium supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) or 4-amino-3,5,6-trichloropicolinic acid (PIC). The highest biomass accumulation was obtained in the callus cultures initiated from stem explants on medium supplemented with 0.90 μM 2,4-D. Reddish-pink regions were observed on callus surface after 6–7 months in culture and these pigments were identified as anthocyanins. Anthocyanins production was enhanced by reducing temperature and increasing light irradiation. Pigmented calluses transferred to MS1/2 with a 1:4 ratio NH₄⁺/NO₃⁻, 70 g L⁻¹ sucrose and supplementation with 0.90 μM 2,4-D maintained a high biomass accumulation and showed an increase of 150% on anthocyanin production as compared with the initial culture conditions. Qualitative analysis of calluses was performed by high performance liquid chromatography coupled to diode array detector and electrospray ionization mass spectrometry (HPLC-DAD/ESIMS). Eleven anthocyanins were characterized and the majority of them were identified as acylated cyanidins, although two peonidins were also detected. The major peak was composed by two anthocyanins, whose proposed identity were cyanidin 3-(p-coumaroyl) diglucoside-5-glucoside and cyanidin 3-(feruloyl) diglucoside-5-glucoside.     

A system in which anthocyanin synthesis is induced in regenerated torenia shoots

A system in which anthocyanin synthesis can be induced under defined conditions was established in regenerated torenia shoots. Leaf discs prepared from torenia plantlets grown under sterile conditions were placed on solidified half-strength MS medium containing 3% sucrose and 4.4×10^–6 M benzyladenine (BA) and cultured under 16 h light/8 h dark (standard light) conditions for 10 days, then in the dark for a further 10 days. The discs were transferred to medium containing 7% sucrose without BA and cultured under standard light conditions. Six days after transfer, anthocyanin synthesis started in the regenerated shoots, and thereafter, anthocyanin accumulation increased while chlorophyll content decreased. Experiments in which either the timing of illumination was altered or shoots were retransferred to medium containing 1.5% sucrose or other sugars as well as sucrose indicated that both osmotic stress and light are required to induce anthocyanin synthesis. Once anthocyanin synthesis was induced in the torenia shoots 6 days after transfer, the shoots were fated to the synthesis of anthocyanins and the degradation of chlorophylls, and could not revert to the developmental pathway of shoot regeneration. This system may provide a good model for the investigation of the mechanisms underlying the induction of anthocyanin synthesis.     

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.     

The effect of osmotic potential on anther culture in spring wheat (Triticum aestivum)

This study was conducted to determine the effect of osmotic potential in a modified 85D12 medium on both callus induction and plant regeneration in the anther culture of two wheat genotypes, cv. Chris and cv. Pavon. Altering the medium osmotic potential by changing the carbohydrate source and concentration or by adding a non-metabolized osmoticum appeared to have the greatest potential for improving anther-derived green plant production. The medium osmotic potentials were varied (-0.67 to –2.30 MPa) by altering sucrose and PEG concentration. Both osmotica affected callus production, with –0.9 to –1.4 MPa media producing the most calluses. Callus production depended on genotype and osmoticum. Only PEG concentration affected green plant regeneration. The greatest number of green plants (11.5 plants per 100 anthers in cv. Chris) was obtained with 0.0125 M of PEG. This experiment suggested that a low level of PEG in the medium was beneficial for producing green plants from wheat anthers.     

Callus Growth and Proline Accumulation in Response to Sorbitol and Sucrose-Induced Osmotic Stress in Rice

This study investigated the influence of osmotic stress, induced by sorbitol and sucrose combinations, on growth and proline accumulation in callus cultures of rice (Oryza sativa L.). Dehusked mature seeds, cv. Hassawi, were induced to callus on MS medium supplemented with 4.52 µM 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.32 µM 6-furfurylaminopurine (kinetin). The medium also contained 29.2, 58.4, 87.6, and 116.8 mM sucrose combined with 0, 54.9, 109.8, and 164.7 mM sorbitol. Callus formation was observed in about 35 % of the cultured seeds irrespective of the sugar treatment. An increase in callus mass was observed as sucrose concentration increased reaching a maximum growth at 87.6 mM. Callus growth was enhanced in response to 54.9 mM sorbitol but at higher concentration it was inhibitory. Best callus growth was obtained on a medium containing 54.9 mM sorbitol combined with 87.6 mM sucrose. Increasing osmotic stress, as a consequence of increasing sucrose and sorbitol concentrations, induced proline accumulation and the highest concentration of proline, 5.8 µmol g^–1(f.m.), was obtained on 164.7 mM sorbitol combined with 116.8 mM sucrose.     

Influence of cell turgor on sucrose partitioning in potato tuber storage tissues

Sucrose uptake and partitioning in potato (Solanum tuberosum L.) tuber discs were examined under a range of mannitol and ethylene-glycol concentrations. Mannitol caused the same changes in turgor over a wide range of incubation periods (90 min-6 h), indicating that it did not penetrate the tissue. In comparison, ethylene glycol reduced turgor losses but did not eliminate them, even after 6 h. Between 100 mM and 300 mM mannitol, turgor fell by 350 kPa, compared with 35 kPa in ethylene glycol. Uptake experiments in mannitol alone showed that total sucrose uptake was strongly correlated with both osmotic potential and with turgor potential. In subsequent experiments sucrose uptake and partitioning were examined after 3 h equilibration in 100 mM and 300 mM concentrations of mannitol and ethylene glycol. Total sucrose uptake and the conversion of sucrose to starch were enhanced greatly only at 300 mM mannitol, indicating an effect of turgor, rather than osmotic potential on sucrose partitioning. The inhibitors p-chloromercuribenzenesulfonic acid and carbonylcyanide m-chlorophenylhydrazone (CCCP) both reduced sucrose uptake, but in quite different ways. p-Chloromercuribenzenesulfonic acid reduced total sucrose uptake but did not affect the partitioning of sucrose to starch. By contrast, CCCP inhibited total uptake and virtually eliminated the conversion of sucrose to starch. Despite this, sucrose uptake in the presence of CCCP continued to increase as the mannitol concentration increased, indicating an increase in passive transport at higher mannitol concentrations. Increased sucrose uptake above 400 mM mannitol was shown to be the result of uptake into the free space. The data show that starch synthesis is optimised at low but positive turgors and the relation between sucrose partitioning and the changing diurnal water relations of the tuber are discussed.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - PCMBS p-chloromercuribenzenesulfonic acid     

Development of callus and suspension cultures of potato resistant to NaCl and mannitol and their response to stress

Callus and suspension cultures adapted to various concentrations of NaCl or mannitol were developed from the cultivated potato Solanum tuberosum cv. Desire. Growth of the calli was less inhibited by mannitol than by iso-osmotic concentrations of NaCl. Reduction of growth by both NaCl and mannitol was considerably lower in osmotically adapted calli than in non-adapted ones. Salt-adapted suspension cultures that grew in the medium to which they had been originally adapted had a shorter lag in growth as well as a shorter time required to achieve the maximum growth, as compared with non-adapted cells. Suspension cultures adapted to NaCl concentrations higher than 150 mM were obtained only after preadaptation to osmotic stress. Adaptation of these cells was found to be stable. Accumulation of Na⁺ was lower and level of K⁺ was more stable in osmotically adapted than in non-adapted calli, when both were exposed to salt. Potassium level in NaCl-adapted calli exposed to saline medium was lower than that in non-adapted calli in standard medium. The maximum of Cl^– and Na⁺ accumulation was reached at higher external salt concentration in salt-adapted than in non-adapted suspension cultures. In both callus and suspension cultures, Cl^– accumulated more than Na⁺. Potassium level decreased more in non-adapted than in NaCl-adapted suspension cultures. The decrease of osmotic potential in osmotically adapted calli exposed to mannitol and in salt-adapted calli and suspension cultures exposed to salt was correlated to the increase of the external concentration. Such a correlation was not found in osmotically adapted calli exposed to salt. Non-electrolytes were found to be the main contributors to the decrease is osmotic potential in both callus and suspension cultures.     

主要营养成分对悬浮培养玫瑰茄细胞生长和花青素合成的影响

本文研究了培养基中碳源和氮源变化对悬浮培养玫瑰茄细胞生长和花青素合成的影响。在８种不同的碳源中,麦芽糖有利于花青素的积累,而蔗糖和葡萄糖适合细胞生长,并有较高的花青素产率。在１％～１０％蔗糖浓度范围内,４％浓度下细胞生长和花青素产率最高,而６％浓度下细胞花青素含量最高,高渗环境较有利于细胞花青素的积累。１３５ｍＭ的氮源总量已足够维持玫瑰茄细胞生长和花青素合成,氮源总量增加对细胞代谢有抑制作用。ＮＨ＋４对细胞有显著抑制作用。总量１３５ｍＭ,ＮＯ－３与ＮＨ＋４比例２５∶２和２３∶４时细胞生长和花青素合成最佳。     

Enhancement of phenylethanoid glycosides biosynthesis in cell cultures of <Emphasis Type="Italic">Cistanche deserticola</Emphasis> by osmotic stress

The effect of osmotic stress on cell growth and phenylethanoid glycosides (PeGs) biosynthesis was investigated in cell suspension cultures of Cistanche deserticola Y. C. Ma, a desert medicinal plant grown in west region of China. Various initial sucrose concentrations significantly affected cell growth and PeGs biosynthesis in the suspension cultures, and the highest dry weight and PeGs accumulation reached 15.9 g l⁻¹-DW and 20.7 mg g⁻¹-DW respectively at the initial osmotic stress of 300 mOsm kg⁻¹ where the sucrose concentration was 175.3 mM. Stoichiometric analysis with different combinations of sucrose and non-metabolic sugar (mannitol) or non-sugar osmotic agents (PEG and NaCl) revealed that osmotic stress itself was an important factor for enhancing PeGs biosynthesis in cell suspension cultures of C. deserticola. The maximum PeGs contents of 26.9 and 23.8 mg g⁻¹-DW were obtained after 21 days at the combinations of 87.6 mM sucrose with 164.7 mM mannitol (303 mOsm kg⁻¹) or 20 mM PEG respectively, which was higher than that of C. deserticola cell cultures grown under an initial sucrose concentration of 175.3 mM after 30 days. The stimulated PeGs accumulation in the cell suspension cultures was correlated to the increase of phenylalanine ammonium lyase (PAL) activity induced by osmotic stress.     

Effect of sucrose on polyploidization in early callus cultures of Solanum tuberosum

Leaf segments of a monohaploid, dihaploid and tetraploid genotype of the potato (Solanum tuberosum; x = 12) were cultured on callus-inducing medium with 10, 20, 30 or 40 gl^–1 sucrose. After 5 and 7 days of culture, metaphases contained the somatic or polyploidized number of mono- or diplochromosomes. The percentages of polyploidized metaphases were inversely correlated with the number of chromosome sets of the genotypes. In monohaploid leaf segments the percentages of polyploidized metaphases and of metaphases with diplochromosomes increased when the sucrose concentration was raised from 10 or 20 to 30 gl^–1 and remained constant or decreased from 30 to 40 gl^–1. Higher concentrations of sucrose but not higher osmolalities of the medium due to mannitol induced endoreduplication in more cells. The frequency of polyploidized metaphases and metaphases with diplochromosomes in dihaploid and tetraploid leaf segments remained constant through increases in sucrose concentrations.     

Maturation of black spruce somatic embryos: Sucrose hydrolysis and resulting osmotic pressure of the medium

The physiological and osmotic roles of sucrose during black spruce (Picea mariana (Mill.) B.S.P.) embryo maturation were investigated. The results showed that when both sucrose and mannitol were present in the medium, the optimum sucrose concentration varied between 4% and 6%. From these data, mannitol does not apparently replace sucrose during the maturation of somatic embryos and therefore it might not be a suitable osmoticum. For the media supplemented with 4% to 12% sucrose and various concentrations of mannitol, the osmotic pressure of the medium rose during maturation, particularly for the highest sucrose concentrations (7% to 12%). Medium containing 3% each of fructose and glucose produced fewer mature embryos compared to the medium with 6% sucrose. An increment in the osmotic potential was observed in medium with 6% sucrose in contrast to that containing 3% each of fructose and glucose. Sugar analysis revealed that the sucrose hydrolysis in the medium was detectable within 1 week of incubation and continued throughout the maturation period. Moreover, no significant uptake of the sugars was detected, since the total amount of fructose, glucose and sucrose remained constant. Our results indicate that the action of sucrose on embryo maturation is mostly achieved through an osmotic control.     

Effects of carbohydrates and nitrogen on the development of anthocyanins of a red leaf peach (Prunus persica (L.) Batsch) in vitro

Heterozygous red leaf peach (Prunus persica (L.) Batsch) shoots were implanted on media with varying nitrogen and carbohydrate regimes to identify a combination which elicited maximum anthocyanin production in explants. A medium with relatively low nitrogen (5 mM NH₄₊ and 10 mM NO_3-) and high sucrose (234 mM) was most effective in stimulating anthocyanin production. Sucrose was more effective as a carbon source than glucose, fructose, or starch under given nitrogen levels. The major anthocyanin in red leaf peach was tentatively identified as cyanidin 3-glucoside based on PC and HPLC analysis.