Production of shikonin derivatives by cell suspension cultures of Lithospermum erythrorhizon

Differences in the production of shikonin derivatives by callus and suspension cultures of Lithospermum erythrorhizon Sieb. et Zucc. were examined. When Linsmaier and Skoog medium was used in suspension cultures, cell growth was not accompanied by the production of shikonin compounds. Shikonin derivatives were produced, however, when this medium was used in callus cultures. Differences in shikonin production were examined in terms of the nutrient supply, the effect of the agar itself, and the oxygen supply. Shikonin derivatives could be produced without agar by keeping the cells exposed to air while providing an adequate supply of nutrients. In callus cultures, the production of shikonin compounds was reduced remarkedly when the oxygen concentration in the atmosphere was lowered, evidence that shikonin production during L. erythrorhizon cell growth on Linsmaier and Skoog agar medium is enhanced by an abundant supply of oxygen.     

The effect of dipicolinic acid on maize tissue culture growth is not solely due to inhibition of lysine biosynthesis

Dipicolinic acid, a known inhibitor of an enzyme (dihydrodipicolinic acid reductase) in the maize (Zea mays L.) lysine biosynthetic pathway, inhibits the growth of maize suspension and callus cultures. Inhibited cultures contain somewhat lower free lysine levels, but the inhibition of suspension culture growth was not reversible with simultaneous addition of L-lysine to the culture medium. It is concluded that dipicolinic acid does not act solely as an analog blocking lysine production. Dipicolinic acid thus appears to be unsuitable as a selection for maize tissue culture mutants with lysine overproduction.Abbreviations FW fresh weight - I₅₀ inhibitor concentration at which cell growth is inhibited by 50% - MS Murashige and Skoog (1962) culture medium - ZM Black Mexican Zea mays suspension culture of Chourey and Zurawski (1981)     

Solasodine production by immobilized cells and suspension cultures of Solanum surattense

Summary Immobilized cells ofSolanum surattense Burm release far more solasodine into the medium than free cell suspension cultures. This enhancement is probably due to stabilization of cells after immobilization as well as the effect of growth hormones in the medium.     

Specific differences in tolerance to exogenous berberine among plant cell cultures

The tolerance of plant cells to exogenously administered berberine, an antimicrobial isoquinoline alkaloid, was studied using berberine-producing and nonproducing cell suspension cultures. Both Coptis japonica and Thalictrum flavum cells, which have an intrinsic ability to synthesize berberine, took up exogenous berberine from the culture medium by an energy-requiring active transport to accumulate it exclusively in vacuoles. By contrast, T. minus cells, which excrete indigenous berberine mostly into the medium, did not take up exogenously supplied berberine, indicating that the alkaloid transport in this species is unidirectional. No inhibition of cell growth by exogenous berberine was observed in the three berberine-producing cell cultures. On the other hand, a small amount of exogenous berberine strongly inhibited cell growth in the berberine-free cultures of Datura innoxia, Catharanthus roseus, and Paeonia albiflora. The berberine taken up actively by Datura cells could not be transported into vacuoles but was dispersed in the cytoplasm, causing a severe inhibition of cell growth.     

Extracellular accumulation of high specific-activity peroxidase by cell suspension cultures of cowpea

Cell suspension cultures of cowpea (Vigna sp.) were able to produce extracellular peroxidase. Different growth regulator concentrations induced different peroxidase activity in callus. The crude extracellular medium after four weeks of culture showed higher (6 times) specific peroxidase activity and higher thermo stability than commercial horse-radish peroxidase. The commercial production of peroxidase enzyme from cowpea by utilizing plant cell cultures is discussed.     

Plant regeneration from cell suspension cultures ofArabidopsis thaliana heynh

A technique is described for the establishment, maintenance, and regeneration of plants from cell suspension cultures ofArabidopsis thaliana (L) Heynh. Friable, rapidly growing cell suspension cultures were initiated from leaf or hypocotyl callus cultures and these have been maintained in liquid culture for 24 months. The cells grown in liquid culture were used to study the effects of growth regulators, medium salts composition, culture temperature, sucrose concentration and medium solidifying agents on morphogenesis. The most important parameters for plant regeneration were culture temperatures lower than 25°C, the medium solidification agent gelrite at 0.2% (w/v) and zeatin or thidiazuron as the choice of cytokinin. These cell suspensions continue to regenerate fertile plants with a total of over 200 plants having been rooted to date and they also serve as convenient sources of cells for protoplast isolation, biochemical, and molecular assays.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - IAA indoleacetic acid - BAP 6-benzylaminopurine     

Chromosome stability of cell suspension cultures of Nicotiana spp.

Cell suspension cultures of Nicotiana were initiated using conditions designed to selectively favor stable chromosome number. These conditions included use of leaf explants to initiate cultures, growth of cells in culture medium containing 2,4-D, and transfer of cells with short subculture intervals. Four cell lines derived from Nicotiana tissue with 2n=24, 48, or 72 were established and retain stable chromosome number. Each line could be regenerated to recover plants that retained the somatic chromosome number during culture. Establishment of haploid and diploid cell lines with stable chromosome number is important for mutant isolation and protoplast fusion.     

Polysaccharide production in liquid cell suspension cultures of Phleum pratense L.

The production of carbohydrates by cell suspension cultures of Phleum pratense (timothy grass) is described. Extracellular polysaccharides similar in monosaccharide composition to native cell wall polymers were accumulated, together with polymers of fructose (fructans). The fructans had similar properties to the intracellular reserve polymers found in intact plants, and were found in both cells and media of young, slow-growing cultures.Production of extracellular polysaccharides differed in cultures grown on sucrose or equimolar glucose/fructose as carbon source. These differences were observed only when autoclaved media were used, and were not related to changes in either pH or osmolarity. Autoclaving medium containing radioactive glucose and fructose produced a novel, unidentified labelled compound which was absent in medium containing labelled sucrose.     

Studies on Ailanthus altissima cell suspension cultures. The effect of basal media on growth and alkaloid production

Time-course experiments have been carried out to investigate the relationship between growth and alkaloid accumulation in A. altissima cell suspension cultures. Results indicate that the type of basal medium, viz. Murashige and Skoog, Linsmaier and Skoog, Schenk and Hildebrandt or Gamborg's B5, has a significant influence on both growth and alkaloid production.     

Alkaloid production in cell suspension cultures of Thalictrum flavum and T. dipterocarpum

Both cell suspension cultures of Thalictrum flavum and T. dipterocarpum were found to produce berberine (0.3 and 0.4 g/l, respectively) as a main alkaloid. Berberine production in the latter was markedly stimulated by 1-naphthaleneacetic acid in combination with 6-benzylaminopurine, whereas it was rather suppressed by the same auxin in the former. T. flavum cultures accumulated berberine and columbamine in the cells without releasing them into medium. On the other hand, T. dipterocarpum cultures released berberine into medium during the logarithmic growth phase, but thereafter accumulated all the berberine synthesized in the cells.Abbreviations LS Linsmaier and Skoog - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - BA 6-benzylaminopurine - TFG a culture strain of T. flavum ssp. glaucum - TDP a culture strain of T. dipterocarpum     

Growth of Dioscorea deltoidea at high sugar concentrations

Dioscorea deltoidea cell suspension cultures were grown at initial sucrose concentrations of 35 to 200 g/L. The growth rates were similar (about 0.50 day^–1) with all of the initial sugar concentrations examined. The ratio of fresh weight to dry weight of cells was dependent on the initial sugar concentration, however, it remained fairly constant as long as the sugar was present in the growth medium. These results are different from results recently published, claiming that the growth rate of D. deltoidea cells is dependent on sugar concentration and the fresh weight to dry weight ratio increases throughout growth.     

Acetylene reduction by transfilter suspension cultures of Rhizobium japonicum.

Cultures of Rhizobium japonicum were grown in a defined medium and then placed in a transfilter-apparatus. Suspension cultures of soybean root cells were grown in Gamborg's B-5 defined medium and then were placed in a second chamber of this apparatus. The plant-cell medium was renewed under conditions shown to give partial synchrony in soybean cell cultures. Sampling of rhizobia showed that acetylene reduction activity could be obtained after approximately four days in the transfilter-apparatus. Criteria for precluding contaminations have been listed. This is the first report on the activation of Rhizobium japonicum in transfilter suspension cultures using defined media.     

Gallotannin production in cell cultures ofCornus officinalis Sieb. et Zucc.

Gallotannin mixtures composed of tri-, tetra- and pentagalloylglucoses were produced by callus and suspension cultures ofCornus officinalis Sieb. et Zucc. The content of the main tannin, 1,2,3,6-tetragalloylglucose, was 36 times that of the intact fruits. The other three tannins, 1,2,6-trigalloyl-glucose, 1,2,3,4,6-pentagalloyl-glucose, and 6-digalloyl-1,2,3-trigalloyl-glucose, were isolated and identified with the authentic specimens. The ratios of the amounts among these tannins were not changed much during the culture period, and by the differences in the combination of plant growth regulators in the medium. Tannin production was stimulated by 6-benzyladenine, whereas cell growth required 2,4-dichlorophenoxyacetic acid or 1-naphthaleneacetic acid. Light irradiation appears to have inhibited tannin production in the cell cultures.     

Effect of auxin on cytodifferentiation and production of quinoline alkaloids in compact globular structures of Cinchona ledgeriana

Fine cell suspension cultures of Cinchona ledgeriana produce only very low amounts of quinoline alkaloids. These cultures formed self-propagating compact globular structures (CGS) on medium containing 2,4-D and BAP. These CGS could be induced to produce significant amounts of quinoline alkaloids by replacing 2,4-D by low amounts of 1-NAA, which was accompanied by histological changes of the CGS. A few high producing CGS clones could be selected. The stability of this trait was studied over a period of about one year of culture in maintenance medium.Abbreviations BAP Benzylaminopurine - 2,4-D 2,4-Dichlorophenoxyacetic acid - 1-NAA 1-Naphthylacetic acid - CGS compact globular structures     

Antiplasmodial activity of Artemisia annua plant cell cultures

Extracts of Artemisia annua cultures have been assessed for in vitro activity against the malarial parasite Plasmodium falciparum. Callus and suspension cells and medium were analysed and examined for their activity at different stages of growth and development. Time-course experiments were carried out to investigate the influence of various basal media, plant growth regulators and light on both growth and possible artemisinin production. Two active fractions were obtained but artemisinin was not detected.     

Induction and in vitro culture of soybean crown gall tumors

Induction of crown galls on 4–6 week old soybean (Glycine max (L.) Merr.) plants cultured in growth chambers was obtained with Agrobacterium tumefaciens strains C58, T37 and ACH5. The crown galls were isolated and cultured in vitro as sterile callus and liquid suspension cultures. Transformation was tested by opine tests and by molecular hybridization of restricted cell DNA with T-DNA fragments. Protoplasts were isolated from suspension cultures. Transformed protoplasts regenerate cell walls, divide and form calli without an exogenous supply of hormones.     

Improved diosgenin production in Dioscorea deltoidea cell cultures by immobilization in polyurethane foam

Dioscorea deltoidea Wall (Dioscoreaceae) cell cultures were entrapped by passive invasion into reticulate polyurethane foam cubes. Immobilization of cells grown in medium containing 3% sucrose reduced the lag phase in growth and thereby reduced the time required to reach maximum diosgenin concentration by 36% compared to cells in suspension culture. Immobilization also increased the total diosgenin produced by 40%. Increased efficiency in diosgenin production was greatest in 3% sucrose; higher concentrations inhibited diosgenin production.     

Benomyl: A broad spectrum fungicide for use in plant cell and protoplast culture

The systemic fungicide methyl-1-(butylcarbamoyl)-2-benzimidazole carbamate (benomyl), is a broad spectrum fungicide. Benomyl at concentrations up to 50 mg/l does not inhibit the growth of suspension cultures ofNicotiana tabacum, Datura innoxia, Daucus carota, Glycine canescens, andSolanum tuberosum nor growth ofN. tabacum orN. plumbaginifolia protoplasts if benomyl is dissolved by autoclaving or boiling. Addition of benomyl dissolved in dimethyl sulfoxide results in a visible toxicity. Benomyl, at 6.25–50 mg/l preventsPenicillium spp. growth in both protoplast and cell cultures and can be used to remove fungal contaminates after one to three transfers without visibly retarding plant cell growth. Due to the broad spectrum of fungicidal activity, and nontoxicity at high concentrations when dissolved by boiling or autoclaving, benomyl can be used effectively to control or prevent fungal contamination in plant cell and protoplast cultures.     

Production of reserpine and its optimization in cultured Rauwolfia serpentina Benth. cells

Cultured R. serpentina cells have been maintained on modified Linsmaier-Skoog medium for over 13 years. These cultured cells produced much more ajmaline (0.005–0.012% dW) than reserpine (0–0.003% dW). Selection of callus which survived the stress induced by alteration of the medium composition including hormones, was repeated over several generations. Surviving callus was then transferred back to the original liquid growth medium and subculture continued, during which time the cells exhibited a return to their pre- stress rate of growth, enhanced reserpine production, and a decrease in ajmaline production. R. serpentina cell suspension cultures selected as described and serially subcultured in fresh growth medium every 3 weeks consistently produce reserpine at a yield of approximately 0.03–0.06% dW.Abbreviations LS Linsmaier Skoog(1965)medium - ML Modified Linsmaier Skoog medium - B5 Gamborg(1968)medium - 2,4-D 2,4-Dichlorophenoxyacetic acid - NAA 1-Naphthaleneacetic acid - IAA Indole-3-acetic acid - BA Benzyl adenine     

Somatic embryogenesis and plant regeneration from cell suspension cultures of Cucumis sativus L.

A procedure for the regeneration of whole cucumber plants (Cucumis sativus L. cv. Poinsett 76) by embryogenesis from cell suspension cultures is described. Embryogenic callus was initiated from the primary leaves of 14–17 day old plants. Suspension cultures of embryogenic cells were grown in liquid Murashige and Skoog basal medium containing 5 uM 2,4,5-trichlorophenoxyacetic acid and 4 uM 6-benzylaminopurine. Suspension cultures were composed of a population of cells that were densely cytoplasmic and potentially embryogenic. Differentiation of embryos was enhanced by washing the suspension culture cells with MS basal medium containing 0.5% activated charcoal and twice with MS basal medium followed by liquid shake cultures in MS basal medium. Sixty to 70 percent of the embryos prewashed with activated charcoal germinated into plantlets with normal morphology. Embryos obtained from suspension cultured cells without prewashing with activated charcoal organized into plantlets with abnormal primary leaves. Morphologically normal plantlets were obtained by excising the shoot tips and transferring them to fresh medium.Abbreviation BAP 6-benzylaminopurine - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid - MS Murashige and Skoog