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     

Relationship between cell morphology and indole alkaloid production in suspension cultures of Catharanthus roseus

The relationship between the morphology and indole alkaloid production of Catharanthus roseus cells was investigated. Eleven cell lines were randomly selected from protoplast-derived clones. In each line, most of the cells maintained only one of the two shapes, either spherical or cylindrical. The cell aspect ratio (cell length/width) for most isolates was stable for more than two years of subculture. Cell division patterns of spherical and cylindrical cell isolates were different and patterns of division remained stable in each phenotype and were not considerably affected by auxin or cytokinin levels in the culture media. These observations indicate that cell morphology of our isolates is stable and probably internally determined. Production of the indole alkaloids, ajmalicine and catharanthine was significantly greater when the cell aspect ratio was more than 2.8.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BA 6-benzyladenine - CPA p-chlorophenoxyacetic acid - IAA indole-3-acetic acid - MS Murashige and Skoog (1962) medium - SH Schenk and Hildebrandt (1972) medium     

Flow cytometric analyses in embryogenic and non-embryogenic callus lines of Cyclamen persicum Mill.: relation between ploidy level and competence for somatic embryogenesis

Embryogenic and non-embryogenic callus lines derived from the same diploid Cyclamen persicum genotype (`Purple Flamed') were analyzed by flow cytometry and compared to the initial plant material. The DNA content of the diploid plant in the greenhouse was 1.12 pg DNA/2C as estimated in relation to the internal standards tomato nuclei and chicken erythrocytes. In both callus lines the majority of cells contained the same amount of DNA as the initial plant, indicating that no polyploidization has taken place after 5 years of culture on medium containing 2.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.8 mg/l 6-(γ-γ-dimethylallylamino)purine(zip). Thus, our data suggest that in Cyclamen callus lines there was no strict correlation between the ploidy level and the ability to produce somatic embryos. Furthermore, following the proportion of cells in the three phases of the cell cycle (G0/G1, S, G2/M) during one subculture period of 4 weeks revealed high division activity within the first 2 weeks for both callus lines cultured on the 2,4-D-containing medium. However, when transferred to hormone-free medium, the division activity of the embryogenic cell line decreased markedly, corresponding to the differentiation of somatic embryos. In contrast, for the non-embryogenic callus an increase in cells in the G2/M phase was observed. Received: 22 November 1996 / Revision received: 6 January 1997 / Accepted: 20 February 1997     

Selection of Cell Lines with High Productivity of Shikonin Derivatives by Protoplast Culture of Lithospermum erythrorhizon Cells

We selected high-yield cell lines, using protoplast culture of Lithospermum erythrorhizon cells. Three cell lines having different shikonin productivities were used as parent cells for the selection, and cell lines with high productivity were obtained efficiently in every case. The best cell line had 6.45 g shikonin/g inoculum/23 days of production which was almost 1.5 times higher than that of the original cell line. The productivities of protoplast-derived cell lines were distributed widely and their average productivity was similar to the original one. The subculture of such a protoplast-derived cell line for eight months showed that its shikonin productivity was stabler than the original cell line.     

Establishment of hairy root cultures ofDatura stramonium. Characterization and stability of tropane alkaloid production during long periods of subculturing

The aim of this paper was the screening of the variability of growth patterns, biomass and tropane alkaloid production of 500 hairy root lines ofDatura stramonium. Data on the long term stability in alkaloid production of these lines for more than 5 years are also provided. In an effort to obtain high alkaloid-producing root clones, it is demonstrated that systematic selection is necessary. Comparisons are made, mainly concerning alkaloid production and its stability, with normal root cultures initiated from the same mother plants when necessary. Hairy root cultures were found to have a hyoscyamine and scopolamine bioproductivity of 2 orders of magnitude higher than mother plants.     

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.     

Alkaloid formation in cell suspension cultures of Tabernaemontana elegans after feeding of tryptamine and loganin or secologanin

A cell suspension culture of Tabernaemontana elegans lost its ability to produce alkaloids after a prolonged period of subculture. To determine whether it was still capable of performing the later steps of the alkaloid biosynthetic pathway, the culture was fed with tryptamine and loganin. The precursors and alkaloids were determined in the biomass and in the medium during a growth cycle. In this culture, an increase in the amount of serotonin was found in the biomass after feeding of tryptamine and loganin. Secologanin was detected in small amounts but strictosidine was not. Therefore, a limitation in alkaloid formation in this T. elegans cell line occured in the formation of secologanin from loganin. After feeding of secologanin alone, strictosidine, 10-hydroxy strictosidine, strictosidinic acid and two other indole alkaloids, as yet unidentified, were formed. However, the alkaloids originally produced by this cell line were not found. As the biosynthesis is impaired at several steps, it seems that the loss of productivity is more likely to be to a change on the level of the regulation of the pathway, than due to the loss of the capacity to express an individual biosynthetic gene of the pathway.     

The Effects of Agitated Liquid Medium on in vitro Cultures of Hevea brasiliensis

The initiation and prolonged growth of callus, from stem explants of young plants of Hevea brasilienies on solid medium yielded a heterogeneous callus, with areas which are the result of compact growth interspersed with brown necrotic tissue and soft white tissue formations. Subculturing this callus (O callus) to agitated liquid medium and returning it to solid medium resulted in the production of a homogeneous friable and rapidly growing callus (Rl callus) The two established lines O and Rl have remained stable over one year in culture and differ in gross morphology, anatomy, growth and auxin content. Both were maintained on Murashige and Skoog's medium, with 2 mg/1 2,4-D and 0.5 mg/I kinetin. R 1 but not O showed enhanced growth at the lower 2,4-D level of 0.2 mg/l: both lines failed to continue growing when 2,4-D was omitted. It is suggested that the changes resulting from subculture in agitated liquid medium are related to those undergone by callus cultures which become habituated. Thus the Rl callus line is regarded as partially habituated. Subculture in agitated liquid medium also resulted in the production of large numberr of polyploid cells but these did not persist over the long periods of subsequent growth on agar medium, Enhanced auxin production by the establihed Rl callus line was thus observed in the absence of a detectable level of polyploidy.     

Increased capsaicin content in PFP-resistant cells of chili pepper (Capsicum annuum L.)

Cell suspensions of chili pepper (Capsicum annuum L.) were subjected to a selection process on semisolid medium containing the amino acid analog p-fluorophenylalanine (PFP). Four cell lines with different degrees of resistance were selected and suspension cultures were established from each of them. Resistance was retained even after 75 days of culture in the absence of PFP. PFP-resistant cell lines accumu lated higher levels of capsaicin than sensitive lines even after prolonged culture in PFP-free medium. Capsaicin production in non-selected cells was only 26.8% of that found in one cell line resistant to 500 M PFP. The capsaicin content in the non-selected cell suspension and in one of the resis tant cell lines was 6.7% and 24.9% respectively, that of fruits.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - PFP p-fluorophenylalanine - d. wt. dry weight - f. wt. fresh weight     

Effects of calcium,alginate, and calcium-alginate immobilization on growth and tropane alkaloid levels of a stable suspension cell line of Datura innoxia Mill

Summary A stabilized two-year old suspension of a Datura innoxia cell line, producing small amounts of tropane alkaloids (scopolamine and hyoscyamine) was used in this study. Calcium alginate immobilization has been shown to be able to increase secondary metabolite (i. e. alkaloid) production. The effects of calcium and ungellified alginate were both beneficial for tropane alkaloid synthesis; a 10mM calcium chloride supply gave the best results, with a 10-fold yield increase.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - S standard cell culture medium - HPLC high performance liquid chromatography - FW fresh weight - FDA fluorescein diacetate - FW fresh weight     

Somatic embryogenesis and indole alkaloid production in Catharanthus roseus

ABSTRACT

A new protocol to obtain an embryogenic cell line from cultured seedling explants of Catharanthus roseus is described. In order to assess the relationship between tissue differentiation and secondary metabolite biosynthesis, the biosynthetic capabilities (alkaloid production) of an embryogenic cell line and two non-embryogenic C. roseus strains were comparatively examined. Faster cell growth rate was associated with higher alkaloid production in the embryogenic cell line. The kinetics of ajmalicine and serpentine production by the three cell lines is also reported.     

Instability of anthocyanin accumulation inVitis vinifera L. var. Gamay Fréaux suspension cultures

The inherent instability of metabolite production in plant cell culture-based bioprocessing is a major problem hindering its commercialization. To understand the extent and causes of this instability, this study was aimed at understanding the variability of anthocyanin accumulation during long-term subcultures, as well as within subculture batches, inVitis vinifera cell cultures. Therefore, four cell line suspensions ofVitis vinifera L. var. Gamay Fréaux, A, B, C and D, originated from the same callus by cell-aggregate cloning, were established with starting anthocyanin contents of 2.73±0.15, 1.45±0.04, 0.77±0.024 and 0.27±0.04 CV (Color Value)/g-FCW (fresh cell weight), respectively. During weekly subculturing of 33 batches over 8 months, the anthocyanin biosynthetic capacity was gradually lost at various rates, for all four cell lines, regardless of the significant difference in the starting anthocyanin content. Contrary to this general trend, a significant fluctuation in the anthocyanin content was observed, but with an irregular cyclic pattern. The variabilities in the anthocyanin content between the subcultures for the 33 batches, as represented by the variation coefficient (VC), were 58, 57, 54, and 84% forV. vinifera cell lines A, B, C and D, respectively. Within one subculture, the VCs from 12 replicate flasks for each of 12 independent subcultures were averaged, and found to be 9.7%, ranging from 4 to 17%. High- and low-producing cell lines, VV05 and VV06, with 1.8-fold differences in their basal anthocyanin contents, exhibited different inducibilities tol-phenylalanine feeding, methyl jasmonate and light irradiation. The low-producing cell line showed greater potential in enhanced the anthocyanin production.     

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.     

Characterization of Coptis japonica cells with different alkaloid productivities

Several cell lines of Coptis japonica with different alkaloid productivities were characterized to obtain information on how a high metabolite production is established. High and low metabolite producing cells, except those from one cell line, showed similar growth kinetics and a similar pattern of nutrient uptake. Amino acid contents, especially that of tyrosine, differed between cell lines, but no correlation was found between the amino acid or tyrosine levels and alkaloid production. Since the addition of tyrosine did not increase the production of berberine, this primary substrate is apparently not the limiting factor for high production in cultured Coptis cells. The addition of berberine to the medium revealed that low-producing cells also have the ability to store alkaloid, and that low productivity is not due to decomposition of alkaloids which have been produced. The direct measurement of the biosynthesis of berberine using ¹⁴C-tyrosine clearly showed that high-producing cells had a higher biosynthetic activity of berberine from tyrosine than low-producing cells. The measurement of enzyme activities in berberine biosynthesis indicated that the early steps of berberine biosynthesis are important in the increased production of berberine.     

Recovery and evaluation of soybean plants transgenic for aBacillus thuringiensis var.Kurstaki insecticidal gene

Summary Lepidopteran insects are major defoliating pests of soybean in the southeastern United States. Soybean plants transgenic for a nativecryIA(b) gene fromBacillus thuringiensis var.kurstaki HD-1 were obtained. Embryogenic cultures were induced by plating cotyledons on a Murashige and Skoog-based medium supplemented with 40 mg/liter of 2,4-dichlorophenoxyacetic acid (2,4-D). The embryogenic cultures were maintained in liquid medium containing 5 mg/liter 2,4-D. These cultures were subjected to microprojectile bombardment, followed by selection on 50 mg/liter hygromycin. Resistant embryogenic cell lines were transferred to growth regulator-free medium to permit recovery of mature somatic embryos. After a desiccation period, the somatic embryos were returned to growth regulator-free medium for conversion into plants. Southern hybridization analysis verified transformation. Feeding assays of T₁ plants from one cell line deterred feeding, development, and survival of velvetbean caterpillar at a level comparable to that of GatIR81-296, a soybean breeding line with a high level of insect resistance. Reduced feeding on T₁ plants correlated with the presence of the transgene.     

Induction of catharanthine synthesis and stimulation of major indole alkaloids production by Catharanthus roseus cells under non-growth-altering treatment with Pythium vexans extracts

A Catharanthus roseus cell line was selected that synthesised catharanthine exclusively under elicitation.From the first day of culture, treatment with very low concentrations of a Pythium extract did not alter the growth of the suspension but, within 24 hours, induced the synthesis of catharanthine and stimulated the production of ajmalicine. Kinetic analysis showed that serpentine then began to accumulate and that all of these effects lasted more than 7 days. Elicitation also induced changes in the cell/medium distribution of the alkaloids. Higher, although non-lethal, concentrations of the fungal elicitor were shown to impair alkaloid production. This cell line will serve as a model to study the conditions for the expression of catharanthine synthesis at the molecular level.Abbreviations gE glucose-equivalent - MS Murashige and Skoog medium - 2,4-D 2,4-Dichlorophenoxyacetic 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.     

Some Accounts on Culture Conditions of Callus Tissues of Solanum laciniatum Ait. for Producing Solasodine

Production of solasodine in callus cultures of Solanum laciniatum Ait. was examined under several culture conditions. The steroidal alkaloid was produced more actively in rapidly proliferating callus tissues cultured on PN medium. The alkaloid concentration in the tissue was about 0.05% (dry weight basis) during the first 5 weeks’ culture. The highest accumulation of the alkaloid per culture was obtained with 2,4-d concentration in the medium at 1~2 ppm. It is noteworthy that the alkaloid production was not inhibited by such high concentration of 2,4-d as up to 10 ppm in the medium. Supplementation of kinetin slightly increased the alkaloid production.     

Transgenic periwinkle tissues overproducing cytokinins do not accumulate enhanced levels of indole alkaloids

Cytokinins play a critical role in several aspects of plant growth, metabolism and development. We previously reported that adding cytokinins to the culture medium of a suspension-cultured cell line of periwinkle increased the accumulation of indole alkaloids, and our aim was to compare the effect of exogenously-applied cytokinins with that of elevated levels of endogenous cytokinins on indole alkaloid production. We used an Agrobacterium tumefaciens strain yielding a plasmid with the isopentenyl transferase gene under control of its own promoter. Co-culture of suspension cells with the bacteria caused a severe stress response leading to cell necrosis. Therefore, we failed to transform this material but we succeeded in transforming periwinkle cotyledons. We verified that callus cultures generated from the isopentenyl transferase-transgenic cotyledons accumulated high cytokinin concentrations. Treating normal callus cultures (generated from untransformed cotyledons) with cytokinins enhanced their alkaloid production. By contrast, the enhanced concentration of endogenous cytokinins in transgenic calli did not increase indole alkaloid production, and thus did not mimic the effect of exogenously-applied cytokinins. Hypothesis to explain this discrepancy are discussed.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - DW dry cell mass - ipt isopentenyl transferase gene