Uptake of phosphate and its effect on phenylalanine ammonia-lyase activity and cinnamoyl putrescine accumulation in cell suspension cultures of Nicotiana tabacum

The influence of phosphate on the medium-induced formation of cinnamoyl putrescines in cell cultures of Nicotiana tabcum was investigated. Phosphate added to a phosphate-free production medium was completely accumulated in the cells within 24h after inoculation at initial concentrations up to 2 mM. At higher concentrations phosphate was partly accumulated with an intracellular saturation at approx. 0.65 mmol/g dr. wt. equivalent to approx. 45 mM intracellular concentration. Enhanced activities of phenylalanine ammonialyase and increased product levels of cinnamoyl putrescines, induced by cell transfer into phosphatefree medium were suppressed similarly at initial phosphate concentrations of 0.02–0.5 mM. At the same time growth was stimulated.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - fr. wt. fresh weight - dr. wt. dry weight - MS-medium Murashige-Skoog-medium (Murashige and Skoog 1962)     

Sodium-dependent phosphate transport in primary cultures of renal tubule cells from young and adult rats

The transport of phosphate by primary cultures of renal cells from young (5-6 weeks) and adult (10-12 months) rats was studied. Renal tubule cells isolated from young and adult groups exhibited typical epithelial morphology and similar growth rates. The Na-dependent phosphate uptake was saturable with a Km of 5-7 microM over a substrate range of 1-500 microM. A decrease in Na-dependent phosphate uptake in adult cells (30%) was found compared to that of young cells. The Na-independent component of phosphate uptake did not vary with age. In addition, the inhibition of phosphate uptake by a variety of compounds (ouabain, gramicidin, 2,4-dinitrophenol, KCN, and arsenate) were similar in both age groups. Kinetic analysis showed that a significant reduction in Vmax (4.4 +/- 0.4 vs. 3.1 +/- 0.2 nmol Pi/mg protein/10 min in young and adult cells, respectively), but not Km, resulted in this decreased uptake of phosphate in adult groups. There was no difference in the efflux of phosphate from both age groups. When cells were preincubated in a phosphate-free medium for 24 hours, the uptake of phosphate was increased to 46% and 24% of their corresponding controls in young and adult cells, respectively. The decreased phosphate uptake and limited adaptation to a phosphate-free medium by the adult renal cells may account for the hypophosphatemia and phosphaturia seen in adult and old animals in vivo.     

Alkaloid accumulation in Ca-alginate entrapped cells of Catharanthus roseus: Using a limiting growth medium

The limitation of growth of Catharanthus roseus cells was investigated with a view to their entrapment in a Ca-alginate matrix. An examination of the effects of lowered 2,4-D and phosphate concentrations on cell viability and indole alkaloid biosynthesis enabled a growth limiting and product formation stimulating medium to be designed. Entrapped cells showed a retention of both respiratory activity and biosynthetic capacity over an extended period of time compared with free cells. Evidence is presented which suggests that immobilization in Ca-alginate beads acts to stabilize cells, resulting in enhanced product accumulation.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - d.w. dry weight     

Stability of alkaloid production in cell suspension cultures of Tabernaemontana divaricata during long-term subculture

Two cell lines of Tabernaemontana divaricata cell suspension culture with different growth and alkaloid production profiles were transferred to the same medium. During 30 subcultures the changes in growth and alkaloid production were followed and compared to those of the original cell lines. The presence of NAA and BAP in the medium resulted in an increase of biomass and alkaloid yield. The effect on the growth proved to be stable during these 30 subcultures. Alkaloid production showed a maximum in the 4th subculture after the change of the medium, and stabilized on a higher level than found in the original cell lines. During some growth cycles also the activities of tryptophan decarboxylase (TDC), strictosidine synthase (SSS), and phenylalanineammonia-lyase (PAL) were measured. In both the original cell lines and the derived cell lines, growth and alkaloid production proved to be stable all through the experiment, although the derived cell lines had a period of adaptation to the new medium with increased productivity.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - BAP benzylaminopurine - DW dry weight - TDC tryptophan decarboxylase - SSS strictosidine synthase - PAL phenylalanineammonia-lyase - PAT phenylalanineammonia-transaminase     

Identification and production of flavonoids in a cell suspension culture of Scutellaria baicalensis G.

A high yielding cell line of Scutellaria baicalensis G. has successfully been developed to produce flavonoids. Major components of the flavonoids were identified as baicalin and wogonin-7-O-glucuronic acid by a series of instrumental analyses using UV, IR, MASS, and NMR. After 12 days in suspension culture, the cell growth reached 14 g ^DW/l, and baicalin and wogonin-7-O-glucuronic acid were obtained in concentrations of 2.9 g/l and 1.07 g/l, respectively. The culture temperature was found to be an important parameter for improving production yield of the flavonoids. The yield of baicalin was observed to increase to 4.2 g/l by shifting the temperature from 30 °C to 25 °C after 72 h of suspension culture.Abbreviations DW cell dry weight - FW cell fresh weight - 2,4-D 2,4-dichlorophenoxyacetic acid - PSH medium phytohormone added Schenk and Hildebrandt medium - FPM a modified Schenk and Hildebrandt medium for flavonoid production     

Establishment of Orthosiphon stamineus Cell Suspension Culture for Cell Growth

Callus of Orthosiphon stamineus could be induced successfully from petiole, leaf and stem tissues but not roots when cultured on MS medium containing different concentration of NAA (0–4.0 mg l^–1) and 2,4-D (0–2.0 mg l^–1). Highest fresh weight callus production was obtained from leaf explants and those with best friability were obtained on MS medium plus 1.0 mg l^–1 2,4-D plus 1.0 mg l^–1 NAA. Cell suspension cultures were established from these cultures. The appropriate cell inoculum size for the best cell growth was 0.75 g of cells in 20 ml culture medium. Cell suspension culture using MS medium supplemented with 1.0 mg l^–1 2,4-D promoted the best cell growth with maximum biomass of 8.609 g fresh weight and 0.309 g dry weight 24 days after inoculation. Cells that grew in MS medium supplemented with 1.0 mg l^–1 2,4-D reached the stationary growth phase in 15 days as compared to the cells that grew in MS medium supplemented with 1.0 mg l^–1 2,4-D + 1.0 mg l^–1 NAA reached the stationary phase in 24 days. MS medium supplemented with 1.0 mg l^–1 2,4-D was considered as the maintenance medium for maintaining the optimum cell growth of O. stamineus in the cell suspension cultures with 2-week interval subculture.     

Establishment and characterization of Pinus pinaster suspension cell cultures

We report the establishment of a Pinus pinaster (Ait.) cell suspension culture in a modified MS medium supplemented with 2 mg ml⁻¹ 2,4-D and 1 mg ml⁻¹ BA. Calli were obtained from seedling root segments and established a friable isodiametric cell suspension, suitable for in vitro studies of maritime pine at the cellular level. Growth (dry weight), cell viability, pH, and nutrient consumption: carbon source (sucrose, fructose and glucose), nitrogen source (ammonia and nitrate) and phosphate were monitored over 24 h. Suspension cells exhibited a 15-day exponential growth stage, during which a biphasic consumption profile was observed for all nutrients. Phosphate was the first limiting nutrient and preferable consumption was observed for glucose over fructose and nitrate over ammonium.     

Accumulation of anthraquinones in Morinda citrifolia cell suspensions

Cell suspensions of Morinda citrifolia were cultivated in a B5-medium containing 4% sucrose as the sole carbon source and 1 mg l^-1 naphthyl acetic acid (NAA) or 1 mg l^-1 2,4-dichloro-phenoxyacetic acid (2,4-D). Both auxins were able to support growth but only in the presence of NAA anthraquinone production was observed. 2,4-D inhibited the production in NAA cultures. Anthraquinone synthesis took place in the growth and the stationary phase and amounts of 0.2–0.4 mmol (about 100–200 mg) g^-1 dry weight could be reached.Under both growth conditions sucrose was hydrolyzed extracellularly by invertase. From the resulting monosaccharides, glucose was taken up preferentially and an appreciable uptake of fructose only took place when medium glucose was exhausted. Sugar uptake rates were similar when cells were grown in NAA and in 2,4-D medium but the intracellular sugar contents (expressed on a dry weight basis) differed considerably. The presence of sucrose, glucose and fructose was demonstrated under both growth conditions. The amounts of sucrose and glucose were much lower in the 2,4-D cells than in the NAA-cells especially during the growth phase. Fructose contents were low and comparable, while in NAA cells an unknown sugar (possibly the sugar moiety of the glycosylated anthraquinones) was observed especially at the end of the growth phase and in the stationary phase. The differences in sugar concentrations were even larger due to the lower water contents of the NAA cells.Respiration of 2,4-D cells was much higher than that of NAA cells during the growth phase. A sharp increase in sugar contents (mainly sucrose) occurred in the 2,4-D cells at the end of the growth phase and corresponded with the fall in respiratory activity.A possible correlation between the lack of production of anthraquinones in 2,4-D cells and a less efficient growth metabolism in these cells is discussed.Abbreviations AQ anthraquinones - 2,4-D 2,4-dichloro-phenoxy-acetic acid - DW dry weight - FW fresh weight - NAA naphthyl acetic acid - pCPO p-chloro-phenoxy-acetic acid     

Relation between auxin autotrophy and tryptophan accumulation in cultured plant cells

Auxin autotrophy was studied in cultured carrot (Daucus carota L.), tobacco (Nicotiana tabacum L.), and potato (Solanum tuberosum L.) cell lines. Of 10 carrot lines resistant to 5-methyltryptophan (5MT), which accumulate free tryptophan (trp) because of an altered control enzyme, 5 were auxinautotrophic while the normal parent line was not. Carrot lines selected from the same parent line as resistant to other amino-acid analogs were not auxinautotrophic, like the parent. The only 5MT-resistant potato line studied was also auxin-autotrophic while the normal line was only partially auxin-autotrophic. The tobacco lines which accumulated free trp were not auxin-autotrophic, and no auxin-autotrophic tobacco lines were selected by screening for growth in medium lacking 2,4-dichlorophenoxyacetic acid (2,4-D). Several auxin-autotrophic carrot and potato lines were selected from the normal lines and none of these lines were resistant to 5MT. Length of time in culture and difficulty in selecting auxin-autotrophic lines were correlated on the 3 normal carrot lines studied. The addition of trp or indole to the culture medium would partially alleviate the auxin requirement of the normal lines studied. 2,4-D (0.4 mg/l) stimulated the growth of all auxin-autotrophic carrot lines.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - PEP DL-p-fluorophenylalanine - IAA indole-3-acetic acid - 5MT DL-5-methyltryptophan - trp L-tryptophan     

Regulation of anthocyanin and lignin synthesis in Petunia hybrida cell suspensions

In Petunia hybrida cv. Violet 30 cell suspensions the phenylpropanoid pathway can be induced to produce lignin and anthocyanins. Orthovanadate addition leads to lignin accumulation, subculturing the cells using small inoculum sizes (<2 g fresh weight l^-1) gives rise to both anthocyanin and lignin production. Orthovanadate has a negative effect on cell growth. By replacing the medium, one day after orthovanadate addition, by medium without elicitor, we were able to restore growth without disturbing the lignin accumulation. The activity of phenylalanine ammonia-lyase (PAL) increased immediately after orthovanadate addition; this increase stopped upon medium replacement without affecting the lignin production. Reduction of the NAA concentration from 2 mg l^-1 to 0.1 mg l^-1, subsequent to the elicitation by orthovanadate or dilution stress, gave rise to a further increase in the production of lignin and anthocyanins respectively. Decreasing the NAA concentration without a prior elicitation, didn't have any effect on either PAL activity or product formation.Abbreviations 2,4-D 2,4 dichlorophenoxyacetic acid - BSA bovine serum albumine - FW fresh weight - NAA naphthaleneacetic acid - PAL phenylalanine ammonia-lyase - PPP phenyl propanoid pathway     

Callus production from willow (Salix viminalis L.) protoplasts

Protoplasts were isolated from cell suspensions of Salix viminalis (basket willow) clone 78-0-90 and S. schwerinii clone 77-0-77, using cellulysin and macerase in modified Woody Plant medium. For clone 78-0-90, 6.3 · 10⁶ ± 1.9 · 10⁶ protoplasts were obtained per gram fresh weight. Cell divisions started two days after protoplast isolation and gave rise to callus which has been maintained in culture for up to four years. Protoplast yield from the clone 77-0-77 was lower (less than 10⁶ protoplasts per gram cells), cell division was infrequent and no callus was obtained. Protoplasts were also isolated from the leaves of willow shoot cultures using cellulysin and pectolyase, but these did not show cell divisions.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - MS medium Murashige & Skoog (1962) medium - WP medium Woody Plant medium (Lloyd & McCown 1981)     

Improved Growth of Tissue Cultures of the Onion, Allium cepa

A forty-fold increase in tissue fresh weight has been achieved for Allium cepa stem tissue callus grown on a modified B5 medium with increased ammonium, phosphate and nitrogen levels; and decreased level of 2,4-dichlorophenoxyacetic acid. Growth was examined on several standard media, using fresh weight, dry weight and cell number as parameters over an 8-week growth period. Further, tissue grown upon this medium is more friable than on other media. The value of such friable callus for suspension culture initiation and for single cell cloning experiments is discussed.     

Tissue cultures of Artemisia pallens: Organogenesis,terpenoid production

Germinated seedlings of Artemisia pallens gave three types of cultures on MS medium supplemented with different plant growth hormones. Medium containing BA+2,4-D stimulated unorganized callus; BA+IAA medium, semi-organized tissues interspersed with shoot buds; and BA+NAA+IAA medium, multiple shoot cultures. The in vitro shoots developed roots in medium devoid of growth hormones. TLC and GLC analysis of the tissue extracts showed that linalool was present in the cultured tissues, with maximum concentration in the unorganized tissue. Although the TLC profiles of the three culture extracts were similar, the extracts did not contain the major polar compounds of the plant. The plant extracts contained more polar compounds and gave the characteristic fragrance of davana.Abbreviations MS Murashige & Skoog's basal medium - BA benzyladenine - Kn kinetin - NAA naphthaleneacetic acid - IAA indoleacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - PCV packed cell volume     

Ethanol production and toxicity in suspension-cultured carrot cells and embryos

The process of carrot (Daucus carota L.) somatic embryogenesis is highly sensitive to exogenously added ethanol, since 5 mM ethanol inhibits this process by 50%, whereas the growth of proliferating carrot cells is inhibited to the same extent by 20 mM ethanol. This is consistent with the fact that proliferating cultures produce ethanol and release it into the medium at concentrations up to 20 mM, whereas embryogenic culture medium contains less than 1 mM ethanol. Data are presented showing the influence of cell density and 2,4-dichlorophenoxyacetic acid on ethanol production and on the presence of an alcohol-dehydrogenase (EC 1.1.1.1.) inactivator in carrot embryos.Abbreviations ADH alcohol dehydrogenase - 6-BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - DTT dithiothreitol - FW fresh weight     

In suspension cultures of Catharanthus roseus the cyanide-resistant pathway is engaged in respiration by excess sugar in combination with phosphate or nitrogen starvation

Suspension cultured cells of Catharanthus roseus were used to study the effects of excess sugar, and phosphate or nitrogen starvation on growth and respiration. Excess sugar resulted in a higher dry weight but the cell number was not significantly higher than in the controls. After inoculation in phosphate-free medium almost no growth was observed, whereas in medium without nitrogen some growth still occurred, indicating that the medium contains more than enough nitrogen and only a limited amount of phosphate. The maintenance respiration in the nitrogen-free medium was lower than in the control, probably as a result of a lower protein production. In phosphate-free medium, however, the maintenance respiration was higher, because the cyanide-resistant pathway was engaged and the P/O ratio was decreased. In Catharanthus roseus cells the cyanide-resistant pathway is engaged upon phosphate or nitrogen starvation in combination with excess sugar, while excess sugar, as such, does not engage the cyanide-resistant pathway. In the presence of excess sugar the rate of glycolysis is adenylate-controlled. It is only in combination with mineral starvation that the rate of glycolysis does not match the capacity of the cytochrome pathway, resulting in an overflow via the alternative pathway. After combination of mineral starvation with a low sugar supply the rate of glycolysis is controlled by both the substrate supply and the adenylates, and the cyanide-resistant pathway is not engaged.     

Selection of cell lines for the production of rosmarinic acid from cell suspension cultures of Orthosihon stamineus benth

Summary Cell suspension cultures of Orthosiphon stamineus were established from friable calluses produced from leaf pieces of in vitro plantlets that were derived from nodal segments of the mother plants collected from three different geographical locations. Eight lines were eventually selected after seven subculture cycles based on the growth characteristic (plant height) of the plantlets from the three locations: two fast-growing lines (>5.1 cm tall), three intermediate-growing lines (3.1–5.0 cm tall), and three slow-growing lines (<3.0 cm tall). All eight lines grew well in liquid Murashige and Skoog medium supplemented with 4.5 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 5.4 μM 1-naphthaleneacetic acid (NAA). All cell lines exhibited the same growth pattern but produced different maximum cell biomass when cultured in this medium. The time of harvesting the plant cells from the culture medium and the geographical source of the original plant material were both found to affect the production of rosmarinic acid (RA) in cell cultures. Two cell lines were successfully selected and identified to produce high amounts of RA. These cell lines were a fast-growing cell line from Air Itam, Penang and an intermediate-growing cell line from Relau Agriculture Research Centre, Penang which could produce 5% [(w/w) dry weight] and 4.5% [(w/w) dry weight] of RA, respectively.     

Influence of Fusarium wilt toxin(s) on carnation cells

The influence of culture filtrates of Fusarium oxysporum f.sp. dianthi which causes Fusarium wilt was investigated on growth and viability of carnation tissue cultures and leaf segments. Culture filtrates of avirulent race 1 of this fungus did not affect calli and leaf segments of cultivars both susceptible and resistant to Fusarium wilt. However, culture filtrates of virulent race 2 decreased viability and suppressed growth of callus of the susceptible cultivar. In contrast, callus of the resistant cultivar showed resistance to the culture filtrates. The results of these experiments may provide information on methods of selection of new wilt resistant carnation varieties.Abbreviations A270 absorbance at 270 nm - 2,4-d 2,4-dichlorophenoxyacetic acid - CF-MCD culture filtrate of 16064 grown in MCD medium - MCD medium modified Czapeck-Dox medium - MS medium basal medium of Murashige and Skoog - MW molecular weight - PD medium potato dextrose medium - TTC 2,3,5-triphenyl tetrazolium chloride     

Plant regeneration from mesophyll protoplasts of Lactuca perennis

Cultured protoplasts of young, unexpanded leaves of the wild lettuce, Lactuca perennis, divided to produce cell colonies in an agarose-solidified, modified MS medium with reduced levels of inorganic salts, together with 2,4-d, NAA and zeatin at 0.2, 0.1 and 0.5 mg 1^-1 respectively. Organogenesis followed the initial transfer of protoplastderived colonies to modified MS medium with 2,4-d, NAA and zeatin (0.1, 1.0 and 0.2 mg 1^-1 respectively) and then to full-strength MS medium with 6-BA and NAA (0.4 and 0.05 mg 1^-1). Shoots were rooted on agar-solidified MS medium lacking growth regulators. Regenerated shoots were established ex vitro, 21 weeks after protoplast isolation.Abbreviations 6-BA 6-benzyladenine - BSA bovine serum albumin - d days - 2.4-d 2,4-dichlorophenoxyacetic acid - f. wt. fresh weight - IAA indoleacetic acid - MES 2 [N-morpholino]ethane sulphonic acid - MS Murashige & Skoog (1962)     

Somatic embryogenesis and fertile green plant regeneration from suspension cell-derived protoplasts of rye (<Emphasis Type="Italic">Secale cereale</Emphasis> L.)

A method for somatic embryogenesis and fertile green plant regeneration from suspension cell-derived protoplasts of rye (Secale cereale L. cv. Auvinen) was developed. Fast-growing and friable embryogenic calli with a high regeneration capacity were induced from immature rye inflorescences using modified MS medium. These friable embryogenic calli were used for suspension culture initiation in liquid AA medium. A high yield of protoplasts was obtained from suspension cell clumps after 3–5 days of subculture. Isolated protoplasts were cultured in KM8p medium. The frequency of protoplast cell divisions and colony formations in liquid culture medium were similar to those on agarose-solidified medium. Compact embryogenic calli were developed from protoplast-derived microcalli in growth medium mMS. Approximately 7% of the transferred embryogenic calli produced green shoots on N₆ regeneration medium. Of 33 green plants, 28 were fertile with normal flowering and seed set. The ratio of green and albino plantlets was 1:4. Rye protoplast-derived green plants showed normal diploid characters as determined by flow cytometer analysis and chromosome counting.Abbreviations 2,4-D 2,4-Dichorophenoxyacetic acid - FDA Fluorescein diacetate - FW Fresh weight - GA₃ Gibberellic acid - Kinetin 6-Furfurylaminopurine - IAA Indole-3-acetic acid - NAA -Naphthaleneacetic acid     

Alkaloid production by plant cell suspension cultures of Holarrhena antidysenterica: I. Effect of major nutrients

The effect of major nutrients on growth and alkaloid production by plant cell culture of Holarrhena antidysenterica was studied with a view to increasing the yield of the alkaloid conessine, a therapeutic drug used for treatment of dysentery and helminthic disorders. The studies resulted in development of a modified Murashige and Skoog (MS) medium that contained 60 mM total nitrogen with a NH(4) (+)-to-NO(3) (-) ratio of 5:1, 0.25 mM phosphate, and 40 g/L sucrose. The growth regulators 2,4-dichlorophenoxy acetic acid (2,4-D) and kinetin (Kn) were also found to affect the synthesis of alkaloid. Using an optimal level of inoculum (3 g/L), the modified medium resulted in alkaloid synthesis of 0.66 g/100 g dry cell weight, which represented a 4.25-fold increase over that obtained in standard MS medium.