Plant regeneration from hordeum spontaneum and hordeum bulbosum immature embryo derived calli

Callus cultures were initiated from isolated immature embryos of Hordeum spontaneum and Hordeum bulbosum on MS or B5 basal medium supplemented with 2 mg/1 2,4-D. Shoot regeneration occurred on transfer of tissue to media containing 1 mg/1 IAA and 1 mg/1 zeatin. The regenerated shoot buds were rooted on basal medium without hormones. The in vitro regenerated plants were transferred to soil and were grown to fertile mature plants. A low percentage of albino plants was observed among the regenerated plants. No major differences were detected between the two species in respect to their potency to form callus or to the regeneration capacity. The regeneration capacity of calli decreased gradually and ended after 6 months in culture.Abbreviations IAA indole-3-acetic acid - 2,4-D 2,4-dichlorophenoxy-acetic acid - MS Murashige and Skoog medium     

Uncharacteristic alkaloid synthesis by suspension cultures of Cinchona pubescens fed with L-tryptophan

The growth and alkaloid production of a liquid suspension culture of Cinchona pubescens has been studied, particularly with attention to the effect on the alkaloid spectrum of feeding cultures with L-tryptophan. This treatment did not enhance the production of any of the known alkaloids of Cinchona. Above 2mmM, however, the presence of the amino acid was toxic, causing extreme acidification of the medium and cell death. Under these conditions a number of indole and quinoline derivatives accumulated. The principal component of the alkaloid fraction proved to be norharman; indole-3-aldehyde was also isolated. Both these products probably occur by uncharacteristic metabolism of L-tryptophan. Furthermore, evidence for the degradation of endogenous alkaloids was obtained, as 4-hydroxymethylquinoline was also isolated. None of the known quinoline alkaloids of Cinchona, which were present in untreated cells, could be detected after L-tryptophan treatment, even when large amounts of culture were analysed. It is concluded that, in this instance, Cinchona alkaloid production cannot be improved by feeding with a precursor.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid - BA benzyladenine     

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     

Alkaloid formation by Catharanthus roseus cells in a packed column biofilm reactor

Summary Catharanthus roseus cells producing indole alkaloids were grown on surfaces of Ca-alginate beads within the interspacial volume of a packed column. Production media was circulated through the packed column in an upflow mode. Growth and indole alkaloid formation were quantified and compared with suspension culture of cells. Final alkaloid concentration and alkaloid yield obtained in the packed bed was superior to those obtained in suspension culture. This is thought to be due to improved cell-cell contact and interaction in the packed column.     

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     

Production of emetic alkaloid by in vitro culture of cephaelis ipecacuanha A. Richard

Callus and adventitious roots were induced on leaf segments from shoot culture of Cephaelis ipecacuanha A. Richard on Murashige-Skoog medium containing 2,4-dichlorophenoxyacetic acid, indole-3-acetic acid, 1-naphthaleneacetic acid and kinetin. The contents of emetic alkaloids in calli, roots and root suspension cultures were quantified by HPLC. Roots cultured in solid and liquid Murashige-Skoog media yielded emetine and cephaeline. The amount of the two alkaloids in the root suspension culture was very similar to that of roots from ipecac mother plant grown in a greenhouse. In contrast, calli subcultured on Murashige-Skoog media containing combinations of 2,4-dichlorophenoxyacetic acid and kinetin produced only trace amounts of emetic alkaloids.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA l-naphthaleneacetic acid - Kin kinetin - MS Murashige-Skoog - EM emetine - CP cephaeline - DW dry weight.     

Influence of various media constituents on the growth of Cinchona ledgeriana tissue cultures and the production of alkaloids and anthraquinones therein

Using the methods reported by De Fossard et al. (11) the influence of various media constituents on the growth and the alkaloid and anthraquinone production in Cinchona ledgeriana callus cultures was studied. Growth and indole alkaloid production (e.g. cinchonamine) was improved by higher auxin levels. The best growth was observed in the light, although many media resulted in no growth at all in the light. Anthraquinone production was highest at lower auxin levels. Quinoline alkaloid levels (e.g. quinidine) were highest in media with low auxin concentrations. Low and medium cytokinin concentration benefited the quinoline alkaloid production.From the results it was concluded that the pathways leading to the various secondary products, anthraquinones, indole alkaloids and quinoline alkaloids are, at least partly, regulated independently.Abbreviations used IAA indol-acetic acid - IBA indol-butyric acid - NAA -naphtaleneacetic acid - NOA 2-naphtoxy-acetic acid - 2,4-D 2,4-dichlorophenoxy-acetic acid - pCPA parachlorophenoxy-acetic acid - BA benzyladenine     

Photoautotrophic cell suspension cultures of periwinkle (Catharanthus roseus (L.) G. Don): Transition from heterotrophic to photoautotrophic growth

Chlorophyllous, heterotrophic periwinkle (Catharanthus roseus (L.) G. Don) cells were capable of sustained photoautotrophic growth in sugar-free B5 medium containing naphthaleneacetic acid and kinetin when provided with a CO₂-enriched atmosphere. An increase in cell fresh weight, first observed approximately 2 weeks after transfer from heterotrophic to photoautotrophic conditions, coincided with the development of maximum chlorophyll content and photosynthetic activity. Electron micrographs revealed that chloroplasts of cells cultured photoautotrophically in continuous light contained large starch granules and exhibited a less extensive thylakoid system than did periwinkle mesophyll chloroplasts. Photoautotrophic cells did not accumulate vindoline or dimeric alkaloids.Abbreviations Chl chlorophyll - dry wt dry weight - fr wt fresh weight - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid     

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     

Production of first and second generations aposporous gametophytes fromPyrrosia piloselloides (L.) Price frond strips cultured in vitro

Summary Second generation aposporous gametophytes were obtained from sporophytes derived from first generation aposporous gametophytes, which in turn came from the mature fronds grown from spores in the laboratory. Murashige and Skoog modified medium in 1% agar supplemented with sugar alcohols (sorbitol, mannitol), auxins (NAA, 2,4-D) and cytokinin (BA) promoted a higher percentage of aposporous development from mature fronds ofPyrrosia piloselloides derived from aseptically cultured spores as compared with those obtained from plants in the field. A method using 46-diamidino-2-phenyl indole and fluorescence microscopy correlated the deoxyribonucleic acid contents of the aposporous gametophytes and sporophytes derived from them with their ploidy level.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - DAPI 46-diamidino-2-phenyl indole - DNA deoxyribonucleic acid - MS Murashige and Skoog medium - BA benzyladenine - NAA 1-naphthaleneacetic acid     

Putative sites of cytokinin action during their enhancing effect on indole alkaloid accumulation in periwinkle cell suspensions

Summary The effects of cytokinins on the different branches of the indole alkaloid pathway were investigated in Catharanthus roseus cell cultures. Addition of zeatin to a 2,4-dichlorophenoxyacetic acid-containing medium decreased tryptamine levels and increased the bioconversion of secologanin to ajmalicine. Zeatin also enhanced the geraniol-10 hydroxylase activities and modified the indole alkaloid pattern. The results are discussed in the light of previous works showing that cytokinins have a positive effect on indole alkaloid accumulation in some lines of C. roseus.Abbreviations BSTFA bis(trimethylsilyl) trifluoroacetamide - CK cytokinin - 2,4-D 2,4-dichlorophenoxyacetic acid - dw dry weight - G-10H geraniol-10 hydroxylase - NAA naphthaleneacetic acid - SE standard error - TDC tryptophan decarboxylase - Z zeatin     

Secondary metabolites produced by callus cultures of various Ruta species

Callus cultures were established from hypocotyl explants of R. bracteosa, R. chalepensis and R. macrophylla. Calli were maintained for more than three years on MS-medium supplemented with 1 mg l^-1 of each 2,4-D and kinetin. Acridone and furoquinoline alkaloids and coumarins have been isolated from four week old calli grown on a hormone containing and hormone-free medium. A new chlorinated acridone alkaloid has been detected.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MS medium after Murashige & Skoog [6]     

Pravastatin: A tool for investigating the availability of mevalonate metabolites for primary and secondary metabolism in Catharanthus roseus cell suspensions

In the C20 strain of Catharanthus roseus , 2,4-dichlorophenoxyacetic acid (2,4-D) reduces alkaloid accumulation by inhibiting the synthesis of precursors of alkaloid terpenoids. However, the presence of this growth regulator is necessary to promote growth, as measured in terms of dry weight and sterol content. The terpenoid metabolism implicated in the accumulation of alkaloids would therefore be the target of 2,4-D inhibition and not the metabolism leading to sterol biosynthesis. The specific inhibition by pravastatin of 3-hydroxy-3-methylglutaryl CoA reductase (EC 1.1.1.34), the enzyme that catalyses mevalonate synthesis, enables the limitation of mevalonate on sterol content and on alkaloid accumulation to be studied. In presence of pravastatin cells are supplied with labelled mevalonate. Under these conditions the mevalonate is incorporated into sterols but not into alkaloids accumulated in the absence of 2,4-D. The inhibition of sterol biosynthesis induced by pravastatin is not overcome by zeatin or a cytokinin-like compound, whereas the inhibition of alkaloid accumulation can be partially overcome. The use of pravastatin shows that the availability of mevalonate for primary and secondary metabolism is differently regulated in Catharanthus roseus cells.     

Influence of auxins on alkaloid accumulation by a transgenic cell line of Catharanthus roseus

The transgenic cell line of Catharanthus roseus (L.) G. Don S10 was used to study the effect of the presence of the synthetic auxins naphthalene acetic acid and 2,4-dichlorophenoxyeacetic acid in the culture medium on the accumulation of terpenoid indole alkaloids. Line S10 carries a recombinant, constitutively over-expressed version of the endogenous strictosidine synthase gene. The experiments were carried out using a two-stage culture system, consisting of a growth phase of 7 to 10 days and a production phase of 14 or 30 days. The hormonal composition was a crucial factor when formulating both the growth and the production media. It was determined that the presence of naphthalene acetic acid during the production phase led to lower levels of alkaloid accumulation. The presence of 2,4-dichlorophenoxyacetic acid in the growth medium reduced culture aggregation and repressed secondary metabolism. Cultures grown in medium containing 2,4-dichlorophenoxyacetic acid showed reduced capacity to supply biosynthetic precursors, which resulted in low levels of accumulation of terpenoid indole alkaloids. Cultures grown in 2,4-D-containing medium showed reduced capacity to supply biosynthetic precursors and higher rates of catabolic activity, which resulted in low levels of TIA accumulation. The expression of the gus and strictosidine synthase transgenes, measured at the enzyme level, was similarly high under all conditions tested. This revised version was published online in June 2006 with corrections to the Cover Date.     

Influence of auxins on somatic embryogenesis and alkaloid accumulation in Leucojum aestivum callus

In vitro cultures of Leucojum aestivum are considered as an alternative for the production of galanthamine, which is used for the symptomatic treatment of Alzheimer’s disease. We studied the effects of auxins 2,4-dichlorophenoxyacetic acid (2,4-D), 4-amino-3,5,6-trichloropicolinic acid (picloram), 3,6-dichloro-o-anisic acid (dicamba) at concentrations of 25 and 50 µM on the induction of embryogenic callus and its capacity to induce somatic embryogenesis and alkaloid accumulation. The embryogenic response of the explants was from 30% for 25 µM of dicamba to 100% for picloram (for both 25 and 50 µM). 2,4-D (50 µM) stimulated greater callus proliferation and somatic embryo induction as compared to the other auxins. Polyethylene glycol (PEG) stimulated somatic embryo maturation. Callus grown on media containing 50 µM of auxins produced fewer phenolic compounds as compared with callus grown on media containing 25 µM of auxins. GC-MS analyses showed seven alkaloids in the in vivo bulbs and two to four in callus culture. Galanthamine was detected in callus cultivated with 2,4-D (25, 50 µM), picloram (25 µM), and dicamba (50 µM). Other alkaloids, trisphaeridine, tazettine, and 11-hydroxyvittatine were accumulated only in callus growing on medium with picloram (50 µM).     

Somatic embryogenesis and plantlet regeneration in the soybean Glycine max

A tissue culture procedure for the regeneration of somatic embryos and plantlets from somatic cells of the soybean Glycine max is described. Bean pods of soybean cv. TGM119 were immersed in liquid nitrogen for 20 minutes. Young embryos were excised from the immature seeds and cultured to form calli. Calli grown from the young embryos were incubated in liquid culture for two weeks. The liquid suspension culture was filtered to obtain single cells. The soybean cells were cultured for one month in a liquid medium in hanging drop cultures for development into proembryoids. The proembryoids were maintained on a solid growth medium for 40 days. The resultant callus tissue was transferred into MS media containing selected combinations and concentrations of 2,4-Dichlorophenoxyacetic acid, Naphthaleneacetic acid, Kinetin, Benzyladenine and Indoleacetic acid. In the presence of Benzyladenine (0.2 mg/l) and Indoleacetic acid (0.01 mg/l), globular and heart shaped somatic embryos were formed on the surface of the calli. Calli containing somatic embryos were transferred into liquid medium and incubated under low light conditions. After six months further incubation, more than 1,000 plantlets and a large number of somatic embryoids at various developmental stages were obtained per flask.Abbreviations KT kinetin - CM coconut milk - BA benzyladenine - NAA napthalene acetic acid - IAA indole acetic acid - 2,4-D 2,4 dichlorophenoxy acetic acid - MS Murashige and Skoog medium     

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     

Participation of an active transport system in berberine-secreting cultured cells of Thalictrum minus

The release of the benzylisoquinoline alkaloid berberine from cultured cells of Thalictrum minus into the medium proved to be temperature-dependent and was suppressed by such inhibitors of the plasma membrane-bound ATPase as vanadate and diethylstilbestrol. These results indicate that berberine is secreted through an energy-requiring process located in the plasma membrane of berberine-producing T. minus cells. This is the first finding that a secondary metabolite of plant cell culture is secreted by an active transport system.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - DES diethylstilbestrol - DMSO dimethyl sulfoxide - LS Linsmaier and Skoog (1965) - NAA 1-naphthaleneacetic acid     

Release and crystallization of berberine in the liquid medium of Thalictrum minus cell suspension cultures

Cell suspension cultures of Thalictrum minus L. var. hypoleucum Miq. were found to produce a large amount of berberine (400–800 mg/l) when 5–10 M 6-benzyladenine was added to Linsmaier and Skoog's medium containing 100 M 1-naphthaleneacetic acid. Most of the berberine produced was released continuously from the cells into the liquid medium, and an excess of berberine crystallized as its nitrate in the medium. When the cells were cultured in a modified LS medium containing 20 mM KNO₃ and 40 mM NH₄Cl in place of 20.6 mM NH₄NO₃ as nitrogen source, most of the alkaloid crystallized to form berberine chloride instead of nitrate. Minor alkaloids, thalifendine and magnoflorine, were also isolated from the medium and identified.Abbreviations LS Linsmaier and Skoog (1965) - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - BA 6-benzyladenine