Compact callus cluster suspension cultures of Catharanthus roseus with enhanced indole alkaloid biosynthesis

Summary Compact callus clusters showing a certain level of cellular or tissue differentiation were established from Catharanthus roseus stem and leaf explants in a modified MS liquid induction medium supplemented with 5.37 μM α-naphthaleneacetic acid and 4.65 μM kinetin. In the induction medium most leaf explants developed into friable half-closed hollow callus clusters, whereas in the same medium containing 2,4-dichlorophenoxyacetic acid instead of α-naphthaleneacetic acid, most leaf explants were induced to form dispersed cell suspension cultures. Characteristics of these different types of suspension cultures were compared, and the results showed that the compact callus clusters could synthesize indole alkaloids 1.9- and 2.4-fold higher than the half-closed hollow callus clusters and dispersed cell cultures, respectively. The degree of compaction expressed by the ratio of fresh weight to dry weight of these suspension cultures was correlated to indole alkaloid production. Our studies also postulated that the level of cellular/tissue differentiation might be responsible for these different alkaloid synthesis capabilities. Sucrose regime affected some properties (the size, degree of compaction, differentiation level) of the compact callus cluster cultures and therefore influenced alkaloid production.     

Effects of Pargyline on Mitochondrial Amine Oxidase Activity, Indoleacetic Acid Synthesis and Growth of Crown-Gall Tumor Callus

Vinca rosea L. crown-gall tumor callus tissue cultures treated with N-benzyl-N methyl propargylamine (pargyline) exhibited a decrease in the level of endogenous indoleacetic acid from 0.42 μg/mg of protein to less than 0.30 μg/mg of protein. A simultaneous decrease in the specific activity of mitochondrial amine oxidase from 3000 units to less than 250 units at 1.0 μM, 0.01 mM, 0.1 mM and 1.0 mM pargyline, suggested a relationship between amine oxidase function and indoleacetic acid synthesis. Tryptamine incorporation into indoleacetic acid was also decreased at these concentrations. Pargyline inhibited tumor callus growth significantly (based on fresh weight measurements) at the highest concentration, 1.0mM. These data support the hypothesis of a coordinate metabolic system linking mitochondrial amine oxidase activity and indole acetic acid synthesis. Inhibitory action of pargyline on the enzyme is reflected in reduced indoleacetic acid levels and, ultimately, in reduced callus growth rates.     

Selection of high nicotine-producing cell lines of tobacco callus by single-cell cloning

Single-cell clones of Nicotiana tabacum callus showed wide variation in the production of nicotine. An efficient screening of numerous clones was made possible by the adoption of the ‘cell squash method’ devised for estimating the approximate alkaloid content of a small piece of callus. From these clones, several cell lines with higher yield of nicotine (1.0–3.4% of dry wt) have been isolated by repeated clonal selection.     

Regeneration of garlic callus as affected by clonal variation, plant growth regulators and culture conditions over time

 A long-term regeneration system for garlic (Allium sativum L.) clones of diverse origin was developed. Callus was initiated on a modified Gamborg's B-5 medium supplemented with 4.5 μM 2,4-D and maintained on the same basal medium with 4.7 μM picloram+0.49 μM 2iP. Regeneration potential of callus after 5, 12 and 16 months on maintenance medium was measured using several plant growth regulator treatments. The 1.4 μM picloram+13.3 μM BA treatment stimulated the highest rate of shoot production. Regeneration rate decreased as callus age increased, but healthy plantlets from callus cultures up to 16-months-old were produced for all clones. Regeneration of long-term garlic callus cultures could be useful for clonal propagation and transformation. Received: 24 September 1998 / Revision received: 27 January 1999 / Accepted: 26 February 1999     

Effects of medium components and light on callus induction, growth, and frond regeneration in Lemna gibba (Duckweed)

Summary Basal media, plant growth regulator type and concentration, sucrose, and light were examined for their effects on duckweed (Lemna gibba) frond proliferation, callus induction and growth, and frond regeneration. Murashige and Skoog medium proved best for callus induction and growth, while Schenk and Hildebrandt medium proved best for frond proliferation. The ability of auxin to induce callus was associated with the relative strength of the four auxins tested, with 20 or 50 μM 2,4-dichlorophenoxyacetic acid giving the highest frequency (10%) of fronds producing callus. Auxin combinations did not improve callus induction frequency. Auxin in combination with other plant growth regulators was needed for long-term callus growth; the two superior plant growth regulator combinations were 10 μM naphthaleneacetic acid, 10 μM gibberellic acid, and 2 μM benzyladenine with either 1 or 20 μM 2,4-dichlorophenoxyacetic acid. Three percent sucrose was best for callus induction and growth. Callus induction and growth required light. Callus that proliferated from each frond’s meristematic zone contained a mixture of dedifferentiated and somewhat organized cell masses. Continual callus selection was required to produce mostly dedifferentiated, slow-growing callus cell lines. Frond regeneration occurred on Schenk and Hildebrandt medium without plant growth regulators but was promoted by 1 μM benzyladenine. Callus maintained its ability to regenerate fronds for at least 10 mo. Regenerated fronds showed a slower growth rate than normal fronds and a low percentage of abnormal morphologies that reverted to normal after one or two subcultures.     

Sensitivity of Cultured Tissue of Arabidopsis thaliana Races to Sulfanilamide

Callus of Arabidopsis thaliana (L.) Heynh. was examined for the kinetics of sulfanilamide inhibition of growth. Two geographic races of this species were compared for relative sulfanilamide sensitivity (as assessed by dry weight increase on drug-containing medium) and rate of drug uptake. It was found that low concentrations of sulfanilamide in the culture medium (～ 100 μM) inhibited callus growth and that low concentrations of p-aminobenzoate (20 μM) competitively antagonized the sulfanilamide effect. Racial variation for sulfanilamide sensitivity was demonstrated. A partially drug-resistant race had greater sulfanilamide uptake (per gram fresh weight) than a sensitive race. suggesting that relative drug-sensitivity was not determined by the capacity to exclude sulfanilamide.     

5‐Aminolevulinic acid promotes callus growth and paclitaxel production in light‐grown Taxus cuspidata suspension cultures

Cultured plant cells generally produce low levels of secondary metabolites, and elicitors of secondary metabolites usually inhibit callus growth. The aim of this study was to determine the effect of 5‐aminolevulinic acid (ALA), a chlorophyll precursor that promotes plant growth, on callus induction from leaves of Taxus cuspidata, and on callus growth on solid medium. ALA at 0.76, 7.6, and 76 μM had similar effects on callus induction and growth, while ALA at 760 μM had negative effects. Next, the effects of ALA concentrations on callus growth and paclitaxel production in suspension cultures in the dark were evaluated. The results showed that 0.76 and 7.6 μM ALA stimulated growth and paclitaxel production, while 76 μM ALA had negative effects. ALA is thought to promote cellular activity under light conditions. Therefore, the effects of light intensity on callus growth and paclitaxel production in the presence of ALA were evaluated. Our results showed that the best conditions for callus growth and paclitaxel production were 7.6 μM ALA under photosynthetically active radiation of 12 μmol photons m^?2 s^?1. Callus growth and paclitaxel production were inhibited under stronger light (24 μmol photons m^?2 s^?1). Together, these results show that ALA promoted callus growth and the production of paclitaxel by light‐grown cultured T. cuspidata cells.     

Effects of ethylene on somatic embryogenesis and galanthamine content in <Emphasis Type="Italic">Leucojum aestivum</Emphasis> L. cultures

The influence of ethylene on in vitro morphogenesis of Leucojum aestivum and galanthamine accumulation was studied. Calli were cultivated on Murashige and Skoog (MS) medium supplemented with 25 μM 4-amino-3,5,6-trichloropicolinic acid (picloram) and 0.5 μM benzyladenine (BA). During incubation under these conditions, callus cultures produced ethylene (9.5 nL/g fresh weight: F.W.) whereas no ethylene was found in somatic embryos cultivated on medium supplemented with 0.5 μM α-naphthalene acetic acid (NAA) and 5 μM zeatin. Application of the precursor of ethylene 1-aminocyclopropane-1-carboxylic acid (ACC) increased ethylene production in both cultures, and decreased callus growth by a factor of 1.2, whereas callus growth was enhanced by a factor of 1.1 in the presence of an inhibitor of ethylene silver nitrate (AgNO₃) or by a factor of 1.2 with an absorbent potassium permanganate (KMnO₄). ACC enhanced the induction of somatic embryos and the development of globular embryos. Removal of ethylene by KMnO₄ during somatic embryogenesis led to the development of plants with greater length. Silver thiosulphate (STS) induced galanthamine production in callus cultures (0.1% dry weight), whereas ACC induced galanthamine production in somatic embryo cultures (2% dry weight).     

Induction of somatic embryogenesis and genetic transformation of ohio buckeye (Aesculus glabra willd.)

Summary Mature embryo axes of the Ohio buckeye were germinated on a medium containing 1 mg gibberellic acid (GA) per 1. Three wk following germination, stem, petiole, and leaf blade tissues were excised and placed on media containing either 1 mg (4.5 μM) 2,4-dichlorophenoxy acetic acid (2,4-D) per 1, 1 mg (4.7 μM) kinetin per 1, 1 mg of both 2,4-D (4.5 μM) and kinetin (4.7 μM per 1, or 2 mg of both 2,4-D (9.1 μM) and kinetin (9.3 μM) per 1. Embryogenic tissue was formed only from stem segments after 2–3 mo. of culture on media containing both 2,4-D and kinetin. Embryogenic tissue could be either maintained on solid medium for proliferation of embryogenic callus or placed in liquid medium for proliferation of embryogenic suspension cultures. For transformation of suspension cultures, tissues were inoculated with Agrobacterium EHA105 containing the binary plasmid Vec035, briefly sonicated, and cultured in the presence of 100 μM acetosyringone for 2 d. To eliminate Agrobacterium, tissues were washed and placed in liquid proliferation medium containing either 500 mg Cefotaxime per 1 or 400 mg TimentinŖ per 1. Selection on 20 mg hygromycin per 1 was initiated 2 wk after inoculation, and after an additional 10 wk, hygromycin-resistant tissue was isolated and separately cultured. Although some hygromycinresistant clones were recovered with no sonication treatment, four to five times more clones were obtained following sonication. Putative transformed clones were confirmed to be transgenic via both histochemical β-glucuronidase (GUS) assay and southern hybridization analyses. Development of transgenic embryos occurred on a growth regulator-free medium containing 3% sucrose. After 2 mo. of embryo development, the embryos were transferred to fresh medium for germination.     

Isopropyl m-Chlorocarbanilate and Its Hydroxylated Metabolites: Their Effects on Cell Suspensions and Cell Divison in Soybean and Carrot

Hydroxylated metabolites of isopropyl m-chlorocarbanilate (chlorpropham) are found in intact soybean plants (Glycine max Merr.). The metabolites are isopropyl 2-hydroxy-5-chloro-carbanilate (2OH) and isopropyl 3-chloro-4-hydroxycarbanilate (4OH). The phytotoxicity of these metabolites and chlorpropham was tested in cell suspensions and roots of intact soybean seedlings and cell cultures of carrot (Daucus carota L.). The growth of soybean cell suspensions was inhibited with 50 μM chlorpropham. Ten μM chlorpropham usually slowed initial growth of the cultures while 5 μM and 0.1 μM chlorpropham had no effect. The 2OH and 4OH metabolites had no significant effect on dry weight over the same concentration range. Some metabolism of chlorpropham, 2OH and 4OH occurred during 6 and 48 h of incubation with soybean cells. The results are interpreted to mean that all three analogs penetrated into the cells, were metabolized, and some of the metabolites excreted back into the medium. Mitotic index studies of intact 3-day-old soybean roots showed that 2OH inhibited mitosis to a greater extent than chlorpropham, whereas 4OH produced only a slight and insignificant reduction compared to controls. Chlorpropham, 2OH and 4OH (at 50 μM) all reduced the growth of wild carrot cultures grown in the presence or absence of 2,4-D. Therefore, hydroxylation of chlorpropham at the 2′ or 4′ positions of the 5′ chlorinated benzene ring is not sufficient to render the compound nonphytotoxic in all plant systems.     

<Emphasis Type="Italic">Cupressus</Emphasis> callus and cell suspension cultures: Effect of seiridins on their growth and sensitivity

Summary Cupressus macrocarpa and C. arizonica were examined for callus and cell culture production in vitro. Both species produced callus on agar-solidified MSCY medium supplemented with vitamins, antioxidants, 0.14 μM kinetin (KIN), and 10 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Suspension cultures of both species were established in liquid MSCY medium. Seiridin (SE) and iso-seiridin (ISE), two phytotoxic butenolides produced by Seiridium cardinale, S. cupressi, and S. unicorne, the causal agents of many canker diseases of cypress, were tested on callus or cell suspension cultures. In the medium without other plant growth regulators (PGR), SE promoted cell proliferation of cypress better than ISE, for callus initiation, callus maintenance, and cell suspension cultures. The growth rates of cypress callus tissues and suspension cultures of both cypress species on media containing 50–150 μM SE or ISE were measured. At concentrations of 50 μM and higher, growth rates increased exponentially with the SE concentration. A comparison with KIN and 2,4-D indicated that 50 μM SE promoted growth of callus tissues and cell suspension cultures more than 100 μM ISE. SE can also interact with, or counteract, KIN and 2,4-D. It was demonstrated that SE could replace KIN in the medium for C. arizonica. SE could be involved in cell enlargement and proliferation processes. The less susceptible cypress species (C. arizonica) had a high content of terpenoids than that of the more susceptible species (C. macrocarpa). SE could be a useful tool as a phytohormonal-like regulator to manipulate physiological changes at the cellular level and as an elicitor of sensitivity or tolerance of cypress germplasm to the phytotoxin.     

Somatic embryogenesis and plant regeneration from floral explants of cacao (Theobroma cacao L.) using thidiazuron

Summary A procedure for the regeneration of cacao (Theobroma cacao) plants from staminode explants via somatic embryogenesis was developed. Rapidly growing calli were induced by culturing staminode explants on a DKW salts-based primary callus growth (PCG) medium supplemented with 20 g glucose per L, 9 μM 2,4-D, and thidiazuron (TDZ) at various concentrations. Calli were subcultured onto a WPM salts-based secondary callus growth medium supplemented with 20 g glucose per L, 9 μM 2,4-D, and 1.4 nM kinetin. Somatic embryos were formed from embryogenic calli following transfer to a hormone-free DKW salts-based embryo development medium containing sucrose. The concentration of TDZ used in PCG medium significantly affected the rate of callus growth, the frequency of embryogenesis, and the number of somatic embryos produced from each responsive explant. A TDZ concentration of 22.7 nM was found to be the optimal concentration for effective induction of somatic embryos from various cacao genotypes. Using this procedure, we recovered somatic embryos from all 19 tested cacao genotypes, representing three major genetic group types. However, among these genotypes, a wide range of variation was observed in both the frequency of embryogenesis, which ranged from 1 to 100%, and the average number of somatic embryos produced from each responsive explant, which ranged from 2 to 46. Two types of somatic embryos were identified on the basis of their visual appearance and growth behavior. A large number of cacao plants have been regenerated from somatic embryos and established in soil in a greenhouse. Plants showed morphological and growth characteristics similar to those of seed-derived plants. The described procedure may allow for the practical use of somatic embryogenesis for clonal propagation of elite cacao clones and other applications that require the production of a large number of plants from limited source materials.     

Production of pyrroloquinazoline alkaloid from leaf and petiole-derived callus cultures of <Emphasis Type="Italic">Adhatoda zeylanica</Emphasis>

Summary Callus cultures of Adhatoda zeylanica Medicus were established from leaf and petiole explants. Accumulation of a bioactive pyrroloquinazoline alkaloid, vasicine, in callus cultures was detected and confirmed by thin layer chromatography, electron-ionization mass spectra, ¹³C NMR and high-pressure liquid chromatography analysis. The mass of vasicine obtained from leaf-derived callus cultures was found as 188 and this is comparable to that of the authentic sample. The retention time for leaf-derived extract was 10.065 and for the petiole-derived extract was 9.78 (authentic sample had 9.6 retention time) on high-performance liquid chromatography. The mass and NMR spectra were compared with the spectra obtained from the authentic sample of vasicine. Different growth regulators greatly influenced the growth of callus cultures. The accumulation of vasicine was more in leaf-derived callus grown on Murashige and Skoog (MS) medium with 2.3 μM kinetin, and 4.5 μM 2,4-dichlorophenoxyacetic acid. This is the first report on in vitro production of a pharmacologically important compound vasicine and its characterization by mass spectrometry and ¹³C NMR studies from callus cultures of Adhatoda zeylanica.     

Miscanthus×giganteus plant regeneration: effect of callus types,ages and culture methods on regeneration competence

The perennial rhizomatous grass, Miscanthus×giganteus is an ideal biomass crop due to its rapid vegetative growth and high biomass yield potential. As a naturally occurring sterile hybrid, M. ×giganteus must be propagated vegetatively by mechanically divided rhizomes or from micropropagated plantlets. Plant regeneration through somatic embryogenesis is a viable approach to achieve large‐scale production of plantlets in tissue culture. Effect of the callus types, ages and culture methods on the regeneration competence was studied to improve regeneration efficiency and shorten the period of tissue culture in M. ×giganteus. Shoot‐forming calli having a yellow or white compact callus with light‐green shoot‐like structures showed the highest regeneration frequency. Percentage of shoot‐forming callus induction from immature inflorescence explants was 41% on callus induction medium containing 13.6 μM 2,4‐d and 0.44 μM benzyladenine (BA). The use of a regeneration medium containing 1.3 μM NAA and 22 μM BA was effective at shortening the incubation period required for plantlet regeneration, with 69% of total regenerated plantlets obtained within 1 month of incubation on regeneration medium. Embryogenic‐like callus morphotype could maintain regeneration competency for up to 1 year as suspension cultures. Field grown regenerated plants showed normal phenotypic development with DNA content and plant heights comparable to rhizome propagated plants. Winter survival rates of the regenerated plants planted in 2006 and 2007 at the University of Illinois South Farm, Urbana‐Champaign, Illinois, were 78% and 56%, respectively.     

Indole alkaloid production by hairy root cultures of Catharanthus roseus

Hairy root cultures of Catharanthus roseus were established by infection with six different Agrobacterium rhizogenes strains. Two plant varieties were used and found to exhibit significantly different responses to infection. Forty-seven hairy root clones derived from normal plants and two derived from the flowerless variety were screened for their growth and indole alkaloid production. The growth rate and morphological appearance showed wide variations between the clones. The alkaloid spectra observed were qualitatively but not quantitatively very similar to that of the corresponding normal plant roots. No vindoline or deacetyltransferase activity could be detected in any of the cultures studied. O-acetylval-lesamine, an alkaloid which has not been previously observed in C. roseus was identified from extracts of hairy root clone No. 8. Two root clones were examined for their growth and alkaloid accumulation during a 26-day culture period. Alkaloid accumulation parallelled growth in both clones with ca. 2 mg ajmalicine and catharanthine per g dry weight being observed.Dedicated to Dr. Friedrich Constabel on the occasion of his 60th birthday     

Isolation and Identification of Tubaic Acid and β-Tubaic Acid from Derris Roots

A tobacco callus strain, OMT-53, was selected from many cultures as a desirable strain having high nicotine producing capacity. Several culture conditions were examined, aiming to get higher nicotine production with the callus strain, OMT-53. It was revealed that the nicotine production was remarkably enhanced when the callus tissues were cultured at a limited concentration of α-NAA in culture medium. The optimal concentrations of sucrose and nitrogen in the culture medium were 3 % and 840 mg N/L respectively. Some precursors in nicotine biosynthesis were examined, and only ornithine gave a slightly positive effect at 2x10^-4m concentration. Cultures at 25°C produced the highest yield for nicotine. Considerable amounts of nicotine (ca. 20% of total nicotine) were also recognized in the culture medium. Under the best culture condition mentioned above, nicotine production in tobacco callus tissues has been elevated to 2.14% on D.W, basis at 4 weeks’ culture. This value is near to that of the intact tobacco plants.     

Production of solasodine by hairy root,callus, and cell suspension cultures of Solanum aviculare Forst.

The production of the steroidal alkaloid solasodine, an alternative to diosgenin as a precursor for the commercial production of steroid drugs, was studied in hairy root, callus, and cell suspension cultures of Solanum aviculare Forst. through manipulation of culture medium. The individual and combined effects of medium components on the growth index and the production of solasodine were analyzed using factorial analysis of variance. Solasodine content was optimized to 6.2 mg g⁻¹in the hairy root, 1.4 mg g⁻¹callus, and 0.7 mg g⁻¹in cell suspension cultures (dry weight). An improved isocratic reversed phase high performance liquid chromatographic method provided selective determination of the solasodine content of these samples. Analysis of growth and solasodine content of hairy root cultures and callus cultures demonstrated that the production of solasodine was shown to be growth-dependent in hairy root cultures but not in callus cultures. This revised version was published online in June 2006 with corrections to the Cover Date.     

Response to NaCl of alfalfa plants regenerated from non-saline callus cultures

Salinity restricts crop productivity in many arid environments. Inadvertent selection for tolerance to osmotic stress may occur under cell or tissue culture conditions and could affect the performance of regenerated plants. The effect of NaCl on forage produced by alfalfa (Medicago sativa L.) plants regenerated from non-saline callus cultures was examined in this study. Plants of Regen-S, which was selected for improved callus growth and regeneration in non-saline cultures, had higher forage weight when grown on SHII medium at NaCl levels up to 100 mM compared to its parental cultivars, Saranac and DuPuits. Five additional original-regenerant plant pairs, each derived from non-saline callus cultures of different alfalfa plants, were evaluated in a solid (soil-like) substrate under saline and non-saline conditions. Weight of forage produced by rooted stem cuttings of regenerated plants was 33% higher at 50 mM NaCl compared to cuttings of explant donor plants. Self progenies from four of five regenerants had higher relative forage weight at 100 mM NaCl (percent of 0 NaCl treatment) than the original plants indicating increased NaCl tolerance.     

Variation in nicotine content of tobacco callus cultures

Plants ofNicotiana tabacum L. cv. Burley 21 which showed no difference in nicotine content were used to establish callus cultures. Cultures were initiated from different plants and from different leaves within each plant. The nicotine content of the calli was determined, and the results subjected to an analysis of variance. Differences between plants and differences within plants significantly affected the nicotine content of the cultures. The differences between plants were transmitted sexually and asexually, providing evidence that they are genetically determined. No such differences in nicotine content were found between the plants from which the cultures were established, indicating that nicotine production in vitro involves additional genes to those which are needed for nicotine production in the plant. The differences within plants were further investigated by establishing callus cultures from pith explants taken from different parts of the stem. Explants from apical pith tissue gave calli having far more nicotine and more roots than cultures derived from basal pith explants. This results may reflect the proximity of the apical pith explants to the site of auxin synthesis in the stem apex. Callus cultures derived from pith explants showed greater growth and nicotine production than those derived from leaf explants when the calli were induced on Murashige-Skoog medium containing -naphthalene acetic acid. This result is in conflict with the widely held belief that explants from different parts of the plant give cultures with similar yields of species-specific compounds.Abbreviations HN High nicotine - LN low nicotine     

Saponins in tissue culture of <Emphasis Type="Italic">Primula veris</Emphasis> L.

Roots of Primula veris L. contain considerable amounts of triterpene saponins, which are used in medicine as expectorants. P. veris is in many places an endangered plant, and its production in the field is laborious and a low yielding process. Plant tissue culture provides an alternative means for producing secondary metabolites. Shoot apex, callus, suspension, and root cultures of P. veris were developed for saponin production. In these cultures, the content of triterpene saponins, with focus on primula acid I, the most dominant saponin in Primula species, was determined and compared to that in soil-grown plants. The highest content of primula acid I was observed in root cultures, on average 29.5 mg/g dry weight. Some culture lines contained higher amounts of primula acid I (62.6 mg/g dry weight) than the roots of plants grown in soil.