Pinoresinol and matairesinol accumulation in a Forsythia × intermedia cell suspension culture

A newly established Forsythia × intermedia cell suspension culture was shown to accumulate (+)- and (–)-pinoresinol as well as matairesinol. The influence of the sucrose content of the culture medium and of the cultivation time on pinoresinol and matairesinol accumulation was evaluated. The highest pinoresinol yield was achieved from cells grown in medium containing 6% sucrose for 12 ± 2 days with levels of 0.6–0.8 mg g^–1 dry weight and an average enantiomeric composition of 75 ± 5% (+)-pinoresinol. The highest matairesinol amount was reached in the same medium at the 14th ± 2 culture day with levels of 1.0–2.7 mg g^–1 dry weight. To our knowledge, this is the first report on pinoresinol accumulation in Forsythia × intermedia plants or cell suspension cultures.     

Permeabilization of Cinchona ledgeriana cells by dimethylsulphoxide. effects on alkaloid release and long-term membrane integrity

Dimethyl sulphoxide (DMSO) has been used to permeabilize cells of Cinchona ledgeriana in suspension culture and promote the release of intracellular alkaloids. 5–6% v/v is required before any release is seen, and greater than 20% DMSO is required for full release. Even at these high levels of DMSO release is slow, taking in excess of seven hours to reach completion. Conditions which produce significant release of alkaloids have a deleterious effect on cells. Many of the membranes permeabilized did not recover their ability to selectively exclude compounds such as mannitol when the DMSO was removed. It is concluded that DMSO is not a suitable material for inducing alkaloid release in any biotechnological exploitation of alkaloid production by C. ledgeriana.Abbreviations DMSO Dimethyl sulphoxide - 2,4D 2,4-Dichlorophenoxyacetic acid     

Production of L-DOPA from cell suspension culture of Mucuna pruriens f. pruriens

Summary Production of L-DOPA was studied in cell suspension culture of Mucuna pruriens f. pruriens. Suspension culture was established in MSI medium composed of half concentration of Murashige and Skoog's salts and 2% sucrose. A two-stage cell suspension culture was developed for enhanced accumulation of L-DOPA. In the first stage, the culture system was composed of MSI medium without CaCl, which was suitable for cell growth and in the second stage MSI medium containing 42.5 mg.l^–1 KH₂PO₄ and 4% sucrose favoured L-DOPA production. A discernible higher production of L-DOPA was obtained in this two-stage cell suspension culture in comparison to single stage culture.     

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.     

Effects of Pluronic F-68 on plant cells in suspension culture

Summary The effects of the non-ionic surfactant, Pluronic F-68, on growth and structure ofSolanum dulcamara cells in suspension culture have been studied. Growth of cells, as measured by dry weight, was unaffected by low concentrations (0.01–1.0% w/v) of pluronic, while culture with higher concentrations (2.5–10.0%) resulted in cell death. It is suggested that low concentrations of pluronic may be valuable supplements in plant cell cultures to protect against mechanical damage and to manipulate membrane systems.     

Regeneration of somatic embryos from protoplasts isolated from an embryogenic suspension culture of white spruce (Picea glauca)

Regeneration of white spruce (Picea glauca) somatic embryos from protoplasts derived from an embryogenic suspension culture was accomplished using a culture medium containing 2 mgl^–1 2,4-D and 1 mgl^–1 6-BAP. Divisions within 2 days led to plating efficiencies in the order of 24% after 9 days. A reduction in the osmoticum, necessary for sustained growth, was carried out gradually over 30 days. Embedding in agarose and culture in 5 cm petri dishes prior to transfer of agarose blocks to a bead type culture, led to the formation of somatic embryos as early as 23 days after isolation and yielded plating efficiencies in the order of 5–10% after 35 days culture.     

Cytological changes associated with alkaloid production in cultured cells of Coptis japonica and Thalictrum minus

Cell structures were compared between alkaloid-producing and non-producing cell cultures of Coptis japonica and Thalictrum minus by electron microscopic observation. In alkaloid-producing cells of C. japonica, prior to the onset of alkaloid synthesis, the vacuoles showed a greater volume than in non-producing cells. These were characterized by a number of large starch grains in the cytoplasm. Furthermore, alkaloid-producing cells contained stacks of rough endoplasmic reticulum. Comparison of different cell lines suggested that there might be a negative correlation between accumulation of alkaloids and starch. Similar cytological differences were observed with T. minus cell cultures that release berberine into the culture medium. Alkaloid producing cells were found to contain an abundance of cytoplasmic vesicles (0.5 – 1 m in diameter).     

Growth characteristics of Sanguinaria canadensis L. Cell suspensions and immobilized cultures for production of benzophenanthridine alkaloids

Summary Sanguinaria canadensis L. plants were harvested from a local forest and calli were initiated from leaf explants. The production of benzophenanthridine alkaloids (i.e. sanguinarine, sanguilutine, sanguirubine, chelerythrine, chelilutine and chelirubine) by S. canadensis cell grown in modified B5 and IM2 media was compared to the alkaloid content of rhizomes. Sanguinarine accounted for approximately 80% of the total alkaloid content of cultured cells (1.3%,g g^–1) while sanguinarine and sanguirubine accounted for 70% of rhizome alkaloids (9.0%, g g^–1). Sanguinarine, chelirubine and chererythrine were the only known alkaloids detected in cultured S. canadensis cells. Maximum alkaloid production of cultures performed using B5 medium, containing half the original nitrate concentration, was observed following extracellular nitrate and sugar depletion. The scale-up of this culture was successfully performed in a 2-1 immobilization bioreactor. The consumption of sugar and nitrate as well as the oxygen (OTR) and carbon dioxide (CTR) transfer rates of the immobilized cell culture were monitored for 15 days. The maximum sugar and nitrate consumption rates were 1.8 g l^–1 per day and 2.3 mm per day respectively. The maximum OTR and CTR of the immobilized cell culture were 0.8 mmol O₂ l^–1 h^–1 and 0.95 mmol CO₂ l^–1 h^–1 respectively. The sanguinarine yield of this culture reached 1.0% based on biomass dry weight (g g^–1 dw) by day 15.     

Biological studies upon Enchytraeus variatus Bouguenec & Giani 1987 in breeding cultures

The life history of an enchytraeid worm, Enchytraeus variatus, was studied under laboratory conditions at 18–22 °C. This species can reproduce simultaneously by asexual (architomy) or sexual reproduction. The number of ova per cocoon varies from 5 to 20 (x = 10.9). The generation period (from cocoon to next cocoon) varies from 14 to 39 days (x = 26.1) according to the period of the year. The number of generations per year is between 7.3 and 26.1 (x = 14). A mature worm can lay between 23.7 and 25.8 cocoons during its life (254 days as maximum observed) at a mean rate of 0.12 cocoon worm^–1 day^–1. Experimental cultures were carried out to determine the structure, density and biomass of the populations. A maximal density of 1 396 314 worms was recorded after 85 days of culture. Net production reached 21.48 g m^–2 day^–1 after 26 days in a culture initiated from cocoons.     

Establishment of callus and cell suspension cultures from Gypsophila paniculata leaf segments and study of the attachment of host cells by Erwinia herbicola pv. gypsophilae

Callus and cell suspension cultures were initiated from leaf segments of G. paniculata. Fresh and dry weights measurements of callus showed that callus growth was optimal on MS medium supplemented with 1.0 mg l^–1 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.2 mg l^–1 benzyladenin (BA). Calli cultured on this medium, showed a two-fold increase in fresh weight by the fourth week of incubation. The initiated hard green callus was repeatedly subcultured on MS medium containing increasing concentrations of 2,4-D in order to increase its friability. The friable callus was then used for establishment of a cell suspension culture. Maximum growth of the suspension culture was on medium supplemented with 1.0 mg l^–1 2,4-D and 0.2 mg l^–1 BA.The suspension culture was used for studying plant host attachment in both electron and light microscopy. Upon infection with E. herbicola, plant cells showed aggregate formation within 24 h of infection. In the presence of the pathogenic Ehg,the number of aggregates formed was 342 aggregates ml^–1, in the presence of the non-pathogenic Ehg154 aggregates ml^–1 and in the control 115 aggregates ml^–1. These results show that the pathogenic strain causes formation of cell aggregates 5.8 times greater than the non-pathogenic one. Based on these results, it can be hypothesized that bacterial cells of the pathogenic strains bind to the plant cells and may form a bridge for attachment of plant cells to one another. Observations by electron microscope show that bacterial cells do attach to plant cells and that this attachment might be via formation of a bridge between the bacteria and the plant cell.     

Cultivation of cell cultures of Berberis wilsonae in 20-l airlift bioreactors

Suspension cultures of Berberis wilsonae produce 4 berberine-type alkaloids: berberine, palmatine, columbamine and jatrorrhizine. In particular the formation of the phenolic alkaloids columbamine and jatrorrhizine and of berberine proves to be dependent on the concentration of dissolved oxygen. With higher aeration rates, berberine and jatrorrhizine yields can be increased considerably. Thus we reached an alkaloid yield of more than 3 g × 1^–1 with 50% dissolved oxygen tension in the medium. As far as we know this is one of the best results in fermenting of alkaloid-producing cell cultures.Abbreviations pO₂ dissolved oxygen concentration in % saturation (using air) - HPLC high-performance liquid chromatography - vvm volume air × volume medium^–1 × minute^–1 - rpm revolutions per minute - IAA indole-3-acetic acid - 2,4-D 2,4-dichlorophenoxy acetic acid     

Improvement of indole alkaloid production in Catharanthus roseus cell cultures by osmotic shock

Catharanthine production in Catharanthus roseussuspension cell cultures was increased by about 4-fold to 28 mg l^–1, 23 mg l^–1and 24 mg l^–1by adding sodium alginate, mannitol or polyvinyl pyrrolidone, respectively. Sodium alginate and polyvinyl pyrrolidone also enhanced ajmalicine production to 28 mg l^–1and 31 mg l^–1, respectively. Up to 55–70% of the total alkaloids were released into the medium. These treatments could stimulate higher alkaloid production in C. roseuscell cultures than NaCl and KCl stresses. The possible mechanisms for these treatment effects are discussed.     

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.     

In situ adsorption for enhanced alkaloid production byCatharanthus roseus

Summary A significant fraction (10–40%) of the indole alkaloids produced byCatharanthus roseus was observed to be secreted into the medium. When a neutral polymeric resin, known to adsorb these alkaloids, was added to the cultivation medium, the accumulation of total indole alkaloids and the specific alkaloids, ajmalicine and serpentine were stimulated. Sorbent addition was also observed to result in increased ratios of ajmalicine to serpentine, which suggests the potential of using in situ adsorption to direct metabolism toward a specific product or intermediate within a given pathway.     

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.     

Elicitation of alkaloids in in vitro PLB (protocorm-like body) cultures of Pinellia ternata

The objective of this study was to determine the effect of two endophytic bacterial elicitors (Pseudomonas sp. and Enterobacter sp.) on the production of alkaloids in protocorm-like bodies (PLBs) of Pinellia ternata Breit. Both bacterial strains increased the growth rate of P. ternata PLBs. Pseudomonas sp. promoted the differentiation of the PLBs, whereas Enterobacter sp. inhibited PLB differentiation. The bacterial strains increased guanosine production in PLBs by 9–166%, inosine production by 2–33%, and trigonelline production by 114–1140% compared to the control. For Pseudomonas sp., guanosine and trigonelline production was greater when bacterial extracts were added to the PLB suspension cultures rather than living cells (co-culture treatment). Inosine production was similar in both the bacterial extract and co-culture treatments. For the Enterobacter sp., guanosine, inosine, and trigonelline production tended to be greatest when living cells were added to the PLB suspension cultures rather than bacterial extracts. These results suggest that Pseudomonas sp. and Enterobacter sp. could increase alkaloid yield from P. ternata under field or tissue culture conditions. We also observed that Pseudomonas sp. and Enterobacter sp. produced some of the same alkaloids as their host plants. Additional study needs to be done to determine if these endophytic bacteria could be used to produce alkaloids in the fermentation industry.     

Transformation of menthane monoterpenes by Mentha piperita cell culture

(+)-Isopiperitenone (100 mg l^–1) was converted into (4S,6R)-6-hydroxy- and (4S,8R)-8,9-epoxyisopiperitenone, aside from the already known (+)-7-hydroxyisopiperitenone, by suspension cell culture of Mentha piperita. As (–)-isopiperitenone was hydroxylated similarly, this implies that the hydroxylating enzyme(s) have a broad substrate stereospecificity in regards to the stereochemistry at C4. (–)-(4R)-Carvone was reduced by the Mentha cells both at carbonyl and C1-C6 double bond to give (1R,2S,4R)-neodihydrocarveol and (1R,2R,4R)-dihydrocarveol with the former being the major product. (+)-(4S)-Carvone had a similar reduction pattern, producing (1S,2R,4S)-neodihydrocarveol and (1S,4S)-dihydrocarvone. Formation of these compounds indicates that the peppermint cell culture cannot only hydroxylate the allylic position but also reduce the ,-unsaturated carbonyl system.     

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.     

Induction of freezing tolerance in alfalfa cell suspension cultures

The effects of ABA, 2,4-D, kinetin and cold exposure on the cold hardiness of Medicago sativa L. cell suspensions were investigated. Cultures treated with 5×10^–5 M ABA at 2°C for 4 weeks in the absence of kinetin showed a 50% survival after freezing to –12.5°C, whereas cultures grown at 25°C under normal conditions tolerated freezing to only –3°C. The optimum ABA treatment of 5×10^–5 M for 4 weeks was effective only in combination with cold exposure. Of six cell lines tested, all showed different degrees of induced cold hardiness. The results suggest that ABA alone cannot induce freezing tolerance on alfalfa cell suspension cultures and that the deletion of kinetin and combination of low temperature and ABA is critical for the induction of cold hardiness in alfalfa cell suspension cultures.Abbreviations ABA abscisic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - LT₅₀ 50% killing temperature     

Oligosaccharide Inhibits Ethylene Synthesis and Stimulates Somatic Embryogenesis in a Cotton Cell Culture

The effect of a three-component oligosaccharide fragment of xyloglucan FucGalXyl (XG3) on callus-tissue growth and somatic embryogenesis was investigated in a cotton (Gossypium hirsutumL.) cell suspension culture. The oligosaccharide introduced into an induction medium at 10^–8and 10^–7M concentrations did not affect the frequency of callus formation from hypocotyl segments; however, it enhanced the monthly increment of callus-tissue weight 1.5- and 3-fold, respectively. Induction and culturing of the callus on an XG3-containing medium adversely affected its morphogenetic potential. Addition of XG3 to the culture medium during the cell suspension preparation stimulated cell division resulting, after 40 days, in a 3.4-fold (at 10^–8M XG3) and a 1.7-fold (at 10^–7M XG3) increase in the cell number as compared to the control. Exclusion of 2,4-D, kinetin, and oligosaccharide from the culture medium caused, after two weeks, a 3.8-fold increase in the number of embryos in the 10^–7M XG3-treated suspension culture as compared to the control. The stimulation of somatic embryogenesis by the oligosaccharide was accompanied by a 12-fold decrease in ethylene emission. The morphogenetic effect of oligosaccharide is suggested to result from its anti-auxin action, which, in particular, inhibited the auxin-dependent ethylene synthesis.