Production of chrysanthemic acid and pyrethrins by tissue cultures ofChrysanthemum cinerariaefolium

Tissue cultures ofChrysanthemum cinerariaefolium were established, and then used to study the production of pyrethrin insecticides, and their precursor chrysanthemic acid. Callus cultures and root-differentiated cultures did not contain pyrethrins whereas shoot differentiated callus was found to produce the pyrethrins. Chrysanthemic acid was isolated by extraction from callus cultures, and feeding¹⁴C-labelled chrysanthemic acid to a cell suspension ofC. cinerariaefolium established that the acid accumulates largely as a glucoside ester.Abbreviations MS Murashige and Skoog (1962) medium - 2,4-D 2,4-dichlorophenoxyacetic acid - 1AA Indoleacetic acid - BAP 6-Benzylaminopurine - GC-MS Gas chromatography - mass spectrometry     

Berberine synthesis by callus and cell suspension cultures of Coscinium fenestratum

Callus and cell suspension cultures of Coscinium fenestratum were established from sterile petiole segments on Murashige & Skoog (MS) medium, supplemented with 2,4-dichlorophenoxy acetic acid (2,4-D) and benzyl amino purine (BAP). The cells in the culture produced berberine as the major compound. NAA stimulated the product synthesis over 2,4-D. Presence of light inhibited the growth and enhanced the berberine synthesis.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxy acetic acid - HPLC high pressure liquid chromatography - IAA indole-3-acetic acid - NAA -naphthaleneacetic acid - TLC thin layer chromatography     

Biosynthesis of ribosome-inactivating proteins from callus and cell suspension cultures of Mirabilis expansa (Ruiz &Pavon)

 Callus and cell suspension cultures from the little known Andean crop Mirabilis expansa were developed and maintained on Murashige and Skoog medium supplemented with 2,4-dichlorophenoxyacetic acid (1 mg/l) and kinetin (0.1 mg/l). Callus and cell suspension cultures were screened with antibodies raised against ME1 (27.5 kDa) and ME2 (27 kDa), two ribosome-inactivating proteins (RIPs) previously found in roots of M. expansa. A 29-kDa protein found in callus and cell suspensions reacted strongly with ME1 antibodies. The 29-kDa protein, named MEC, was purified to homogeneity by ammonium sulfate precipitation and cation exchange perfusion chromatography. Amino acid N-terminal sequencing revealed close homology between MEC and ME1. The MEC amino acid sequence examined was highly conserved among RIPs from widely different sources. This new RIP was immunolocalized to the cell walls of callus and cell suspension cultures. Received: 20 August 1999 / Revision received: 10 December 1999 / Accepted: 21 December 1999     

Effects of Chemical and Physical Conditions on Growth of Camptotheca acuminata Cell Cultures

Callus was induced from Camptotheca acuminata, which produces an antitumor alkaloid, carnptothecin. Using the Murashige and Skoogs’ medium as the basal, cultural conditions were examined for C. acuminata suspension cultures. As a result, a medium, containing 0.1 mg/liter 2,4-D, 3 mg/liter kinetin and 0.05 mg/liter GA₃, was established as a medium that gave the best cell growth in suspension cultures. In addition, conditioning of medium and addition of 0.115 mm l-Trp and l-Phe to medium promoted remarkably growth of cell suspensions.     

Steroidal alkaloids in tissue cultures and regenerated plants of Solanum dulcamara

Callus and cell suspension cultures were established with shoots of the soladulcidine variety of the bittersweet Solanum dulcamara L. Plantlets were regenerated from undifferentiated callus. From mixotrophic callus as well as mixotrophic suspension cultures soladulicidine, solasodine and the corresponding neutral spirostanes tigogenin and diosgenin were isolated and identified by thin layer chromatography and mass spectrometry. Total alkaloid concentrations were about 0.2 mg/g dry weight (callus) and 0.1 mg/g dry weight (green suspension cultures). In the heterotrophic cell line only the neutral sapogenins could be detected. Alkaloid accumulation in callus of Solanum dulcamara could be enhanced by the induction of organogenesis. The shoots of the regenerated plants from the mixotrophic callus contained soladulcidine (1.6 mg/g dry weight) and tigogenin. Thus, in concentration and composition the regenerated plants equalled the source plant.Abbreviations 2.4-D 2.4-dichlorophenoxyacetic acid - NAA naphthylacetic acid - HPLC high performance liquid chromatography - TLC thin layer chromatography     

In vitro cultures of Solanum malacoxylon Sendt.: nutritional requirements and sterol production

Callus cultures were established from hypocotyl, root and leaf explants of Solanum malacoxylon. The growth rate of calli was evaluated on Murashige and Skoog medium as well as Gamborg's B5 medium. Sterols were isolated from calli grown on both media however the B5 proved to be more effective in promoting the production of these metabolites. No calcitriol was found in the cultures. Feeding experiments with vitamin D₃ were scarcely effective in promoting the production of vitamin D₃ hydroxylated metabolites.Abbreviations GC/MS gas-liquid chromatographic-mass spectrometric analysis - CHCL₃ chloroform - GLC gas liquid chromatography - PCV packed cell volume - TLC thin layer chromatography     

Somatic embryogenesis and organogenesis in Encephalartos dyerianus and E. natalensis

Callus cultures were initiated from zygotic embryos of Encephalartos dyerianus and E. natalensis. Callus of both species were transferred onto a modified B5 medium containing different combinations of 2,4-dichlorophenoxyacetic acid and kinetin. Somatic embryogenesis and shoot organogenesis occurred in both species. The embryos were dicotyledonary. To date none of the embryos have matured.Abbreviations ABA abscisic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

Production of higher levels of trigonelline by cell cultures of Trigonella foenum-graecum than by the differentiated plant

Callus cultures of Trigonella foenum-graecum contained 3 to 4 times more trigonelline than the seeds of this plant and 12 to 13 times more than the roots and shoots. Even higher levels of this alkaloid were produced by suspension cultures. This high productivity was maintained during successive subculturing of calli and cell suspensions for eight months. Thus, trigonelline is to be added to the group of the few metabolites whose synthesis in cell cultures exceeds its production in the differentiated plants. Media that had supported the growth of suspension cultures contained one third or more of the total alkaloid, whereas media of callus cultures contained about one tenth of this substance. Trigonelline accumulated in callus and suspension cultures with aging. Raising the level of nicotinic acid in the nutrient medium resulted in some increase of trigonelline production by the culture.Abbreviations 2.4 D 2.4-dichlorophenoxyacetic acid - IAA indoleacetic acid - IPA indolepropionic acid - NAA -naphthaleneacetic acid - GA Gibberellic acid - K kinetin     

Induction and development of somatic embryos from cell suspension cultures of soybean

Glycine max (L.) Merr. (soybean) andGlycine soja Sieb. and Zucc. cell suspension cultures were grown and used as inoculum sources for growing callus on agar-solidified nutrient media. Concentrations and chemical forms of the growth regulators in liquid and solidified media were altered in an attempt to achieve in vitro plant regeneration. Numerous embryoids, particularly ofG. soja, were produced on basal nutrient media supplemented with 100 ppm casein hydrolysate, 0.1 μM abscisic acid, 2.25 μM 2,4-dichlorophenoxyacetic acid, and 15 μM adenine or 0.46 μM kinetin. Often the roots of the embryoids elongated. This was enhanced in the presence of an inhibitor of gibberellin synthesis (1 to 20 μM Amo 1618). Callus recovered from aG. soja suspension culture produced one shoot structure when grown on a solid medium containing 0.2 μM Amo 1618 and 80 μM glutathione. The shoot structure consisted of two distinct buds, one producing two leaves. The shoot did not develop into a plant. Although regeneration of soybean plants was not achieved, these observations suggest that it may be achievable. The investigations reported in this paper (no. 81-3-100) were performed in connection with a project of the Kentucky Agriculture Experimental Station and the paper is published with the approval of the Director.     

Occurrence of an Inhibitor of Tissue-Type Plasminogen Activator in Seeds and in Vitro Cultures of Erythrina caffra Thunb

The level of an inhibitor of tissue-type plasminogen activator (t-PA) increased slowly during the early developmental stage of seeds of Erythrina caffra Thunb. Thereafter, the inhibitor increased exponentially until the seeds reached maturity. At maturity, the t-PA inhibitor levels in the cotyledons were 38 times higher than the levels at the onset of seed development. The t-PA inhibitor accumulated at a faster rate than the storage proteins, which reached a concentration 15 times higher than the protein concentration at the onset of seed development. During the imbibition and germination process, the t-PA inhibitor decreased gradually. The inhibitor kept on decreasing during the growth of the seedlings until the 10th day after imbibition, when it leveled off at 4.1% of that of the initial inhibitor concentration. The inhibitor remained at this level until the cotyledons were shed at day 22. The total protein in the cotyledons decreased at a slower rate than the inhibitor and reached a minimum concentration at day 20 of 3.6% of the initial protein concentration in the cotyledons. Callus cultures of root, shoot, leaf, and cotyledonary tissue was established and maintained on Murashige-Skoog medium supplemented with 3% sucrose, 10 micromolar benzyladenine, and 5 micromolar 2,4-dichlorophenoxyacetic acid. A shoot cell suspension culture was established on Murashige-Skoog medium supplemented with 3% sucrose, 1 micromolar benzyladenine, and 0.5 micromolar 2,4-dichlorophenoxyacetic acid (pH 5.7) and shaken at 60 revolutions per minute. The level of t-PA inhibitor in root, shoot, leaf, and cotyledonary callus was substantially lower than in the corresponding intact tissue. The t-PA inhibitor levels in the linear growth phase was higher than in the lag or stationary growth phases of the cell suspension culture. A hydrolysate of the cell walls of tomato and E. caffra Thunb, as well as polyamines and organic acids, did not increase the concentration of t-PA inhibitor in suspension cultures or intact leaf tissue of E. caffra. The t-PA inhibitor levels of suspension cultures were increased by Na₂SO₄ but not by I-cysteine in the nutrient medium.     

In vitro culture ofBellevalia romana (L.) Rchb. I. Plant regeneration through adventitious shoots and somatic embryos

Summary Callus induction, adventitious shoot and root formation, and somatic embryogenesis were investigated in root, cotyledon and mesocotyl cultures ofBellevalia romana (L.) Rchb. grown on a synthetic nutrient medium containing different plant hormones. The combination of naphtaleneacetic acid plus benzylaminopurine was very effective in causing callus growth and plant regeneration from mesocotyl explants. On the contrary 2,4-dichlorophenoxyacetic acid caused suppression of shoot bud development in the same type of callus. Both cotyledon and root derived calli showed a low growth rate and did not regenerate shoots but only roots. Differentiation of somatic embryos which eventually developed into plantlets was promoted by 2,4-dichlorophenoxyacetic acid in suspension cultures. The results are discussed in relation to studies on nuclear behaviour during different morphogenetic pathways.     

Miconazole: An effective antifungal agent for plant tissue culture

Miconazole at concentrations between 5 and 20 mgl^-1 inhibited hyphal growth and sporulation in a wide range of fungi commonly associated with plants. These fungi included 4 Oomycetes, 11 Deuteromycetes, 4 Ascomycetes and 4 common airborne contaminants of plant tissue culture. The phytotoxicity of 20 mgl^-1 miconazole was also tested against a wide range of in vitro plant cultures. Shoot cultures from 15 species showed either no response or a slight growth reduction in the presence of miconazole. Although the growth of shoot cultures in 2 other species was significantly reduced by miconazole, a positive growth rate was maintained. Callus and hairy root cultures from 5 species were more sensitive to miconazole than shoot cultures, although they also retained a positive growth rate in the presence of the antifungal agent. Reconstruction experiments demonstrated the effectiveness miconazole had for the rescue of plant material from in vitro cultures contaminated from fungi.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BA 6-benzylaminopurine - EBI ergosterol biosynthetic inhibitor - IAA indole-3-acetic acid - MZ miconazole - NAA -naphthaleneacetic acid     

Antifeedant effects of in vitro culture extracts of the neem tree,Azadirachta indica against the desert locust (Schistocerca gregaria (Forskål))

Callus and micropropagated shoots were initiated from leaf explants of the neem tree, Azadirachta indica A. Juss. A variety of whole plant and in vitro cell cultures from neem seedlings of Ghanian origin were tested for insect antifeedant compounds using the desert locust (Schistocerca gregaria (Forskål)). Feeding suppression occurred when whole extracts of seed, leaf, callus, suspension and shoot cultures were tested in no-choice feeding bioassays. Controls of sucrose, carrot callus and the plant growth medium showed no feeding deterrence. Azadirachtin, the main known antifeedant in neem seed kernels, was quantified from a seed extract by HPLC but was not detected in any of the other extracts. Antifeedancy was determined during batch growth of a suspension culture which had been in culture for 5 months; results indicated that antifeedants were still being formed and that levels increased after maximum biomass was attained.     

Structural Characterization and Biological Evaluation of 18‐Nor‐ent‐labdane Diterpenoids from Grazielia gaudichaudeana

The phytochemical investigation of Grazielia gaudichaudeana aerial parts yielded 15 compounds, including diterpenes, triterpenes, sterols and flavonoids. With exception to ent‐kaurenoic acid diterpenes, the compounds isolated are being described for the first time in this species. Some unusual ¹H‐NMR chemical shifts of 18‐nor‐ent‐labdane ( 7 – 9 ) led us carry out a conformational analysis by theoretical calculations in order to support the experimental data. Moreover, due to the limitation of studies focused on pharmacological potential of Grazielia gaudichaudeana, the present study was carried out to investigate the antioxidant, antiproliferative, antiviral, antileishmanial and antimicrobial activities from the extract, fractions and isolated compounds obtained from this species. Ethyl acetate fraction showed significant activity in the antiproliferative assay, with GI₅₀ range of 3.9 to 27.2 μg mL^?1. Dichloromethane fraction, rich in diterpenoids, inhibited all human tumor cell lines tested, and the nor‐labdane 7 showed potent cytotoxic activity against glioma and ovary cancer cell lines.     

Plant regeneration through somatic embryogenesis in protoplast cultures of sandalwood (Santalum album L.)

Summary Protoplasts were isolated from embryogenic cell suspension cultures derived from proliferating shoot segments of a 20-year-old sandalwood tree (Santalum album Linn.). Under appropriate conditions, isolated protoplasts divided in liquid culture medium and produced embryogenic cell aggregates and globular embryos. Plating of cell aggregates on a fresh medium facilitated the differentiation of somatic emryos which further developed into plantlets.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ Gibberellic acid - IAA indoleacetic acid - IBA indolebutrytic acid - MES 2-(N-morpholino)ethane sulfonic acid - MS Murashige and Skoog's medium as modified in the text     

Production of a hepatoprotective cerebroside from suspension cultures of Lycium chinense

Suspension cultures derived from Lycium chinense Miller seedlings produced significant amounts of a hepatoprotective cerebroside. Callus was induced from the stem of aseptic seedlings of L. chinense and maintained on MS solid media supplemented with 1.0 ppm 2,4-D and 0.1 ppm kinetin. Suspension cultures were established, and the cells were grown in the same liquid media in the dark. Lyophilized cells were extracted with a combined reagent of chloroform and methanol (2:1, v/v). An aqueous suspension of the evaporated cell extract was partitioned with chloroform, and the chloroform layer was subjected to silicic acid column chromatography followed by semi-preparative reverse phase C8 high pressure liquid chromatography. The purified compound showed hepatoprotective activity comparable to that shown by silymarin, and the structure was identified as 1-O-(β-d-glucopyranosyl)-(2S,3R,4E,8Z)-2-N-2′-hydroxy-(palmitoyl)-4,8-sphingadiene on the basis of spectral data. The content of the compound in cultured cell was tenfold higher than that of the fruit of L. chinense. The biosynthesis of the compound in cultured cell systems appears to parallel cell growth. Received: 12 June 1998 / Revision received: 30 July 1998 / Accepted: 14 August 1998     

Growth of and quassin accumulation by cultures of Quassia amara

Callus and suspension cultures were established from Quassia amara, a member of the Simaroubaceae. Analysis of the tissue culture showed that quassin was present in both callus and suspension cultures. The effect of variation in auxin and cytokinins on both callus growth and the presence of quassin was examined. The suspension culture was grown in a 7 liter bioreactor when good yields of quassin were achieved.Abbreviations IAA indole-3-acetic acid - IBA indolebutyric acid - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - 6BA 6-benzyladenine - IpA N⁶ (-isopentenyl) adenine - IpAR N⁶ ( isopentenyl) adenine riboside - td doubling time     

Studies of cotyledon protoplast cultures from B napus,B. campestris and B. oleracea. II: Callus formation and plant regeneration

Cotyledons from twelve cultivars of Brassica; B. napus (Westar, Eureka, Global, Pivot and Narc 82); B. campestris: (Arlo, Sonja, Bunyip and Wonk Bok) and B. oleracea (Phenomenal Early, Sugar Loaf and Earliball) were used for protoplast isolation and culture in a comparative study of cell colony and callus formation, and plant regeneration. The formation of cell colonies and callus from protoplast cultures were significantly influenced by the light conditions of seed germination. All twelve cultivars showed callus formation from protoplast cultures derived from cotyledons of seedlings grown in dark for 3 days followed by 1 day dim light (dark/dim light-grown). Callus was obtained in all five liquid media used: modified K8P(1), modified K8P(2), modified MS, modified B and modified NN. In contrast, only six cultivars exhibited callus formation from the protoplasts isolated from cotyledons of seedlings germinated under light conditions for 7 days (light-grown) and in only three media: modified K8P(1), modified MS, modified B.Callus, derived from protoplast cultures isolated from dark/dim light-grown cotyledons and grown on K3 or MS series solid media for about 1 month, could develop shoots when further transferred onto MS series regeneration media. All five cultivars of B. napus, three of the four cultivars of B. campestris (Arlo, Sonja and Bunyip) and one of the three cultivars of B. oleracea (Sugar Loaf) exhibited shoot regeneration from protoplast cultures within 2–3 months after protoplast isolation. The frequency of shoot regeneration ranged among 1–22.5%. A high degree of reproducibility was observed in cultivars Westar, Eureka, Global, Arlo, Bunyip and Sugar Loaf. In contrast, among the six cultivars that formed callus in protoplast culture derived from light-grown cotyledons, only three cultivars from B. napus (Westar, Eureka, Global) exhibited shoot regeneration 5.5 months after protoplast isolation. Regenerated shoots from cultivars Westar, Eureka and Bunyip and Sugar Loaf, which derived from protoplasts of dark/dim light germinated seedling and were induced to root on rooting media, survived in soil and grew to produce silique and set seeds.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - BA benzylaminopurine - EDTA ethylenediaminetetraacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - KT kinetin - GA₃ gibberellic acid - MS Murashige and Skoog medium - NAA -naphthaleneacetic acid - PAR photosynthetically active radiation     

A genetic analysis of cell culture traits in tomato

Summary Tomato genotypes superior in regenerating plants from protoplast and callus cultures were obtained by transferring regeneration capacity from Lycopersicon peruvianum into L. esculentum by classical breeding. The genetics of regeneration and callus growth have been studied in selfed and backcross progenies of a selected plant (MsK93) which has 25% L. peruvianum in its ancestry. Segregation data showed that the favourable cell culture traits of L. peruvianum are dominant. Regeneration capacity from established callus cultures was controlled by two dominant genes. Callus growth on primary expiants, callus growth of established cultures and shoot regeneration from explants had high heritabilities (0.47, 0.78, 0.87, respectively). Callus growth and regeneration capacity were not correlated within the populations studied.     

CYTOKININ-GIBBERELLIN REGULATION OF SHOOT DEVELOPMENT AND LEAF FORM IN TOBACCO PLANTLETS

Callus and plantlets derived from callus cultures of Nicotiana tabacum var. Wisc. #38 were grown on medium containing serial combinations of gibberellic acid (GA₃) and the cytokinin 6-(3-methyl-2-butenylamino)purine (2iP). Increasing levels of both growth substances resulted in the production of greater amounts of both callus and shoot tissue. More buds were induced when the cytokinin level was increased, and this effect was counteracted by raising the GA₃/2iP ratio. Furthermore, the size and form of the shoots depended on the GA₃/2iP ratio. High ratios resulted in tall, spindly plants with narrow leaves while low ratios resulted in short shoots with rounded leaves.