Inheritance and Stability of 5-Methyltryptophan Resistance in Datura innoxia Selected In vitro

The resistance to 5-methyltryptophan (5MT) in Datura innoxiaplants regenerated from selected cultures was maintained forover 10 years and was inherited in progeny as if controlledby a single, nuclear, dominant gene. The resistance was causedby a feedback altered anthranilate synthase leading to highfree tryptophan levels. (Received August 18, 1995; Accepted February 5, 1996)     

Comparison of tropane alkaloid spectra between Datura innoxia grown in Egypt and Bulgaria

The alkaloid spectra of Datura innoxia plants grown in Egypt and Bulgaria were investigated by GC-MS. Thirty-eight alkaloids were detected in the roots, leaves and fruits of the plants. Five new alkaloids for D. innoxia are reported. Alkaloid spectra of Egyptian and Bulgarian plants differ significantly in respect to their alkaloid composition and main alkaloids accumulated in the plant organs.     

In vitro evaluation of Datura innoxia (thorn-apple) for potential antibacterial activity

Various parts of Datura innoxia were examined for potential antibacterial activity by preparing their crude aqueous and organic extracts against Gram-negative bacteria (Escherichia coli and Salmonella typhi) and Gram-positive bacteria (Bacillus cereus, Bacillus subtilis and Staphylococcus aureus). The results of agar well diffusion assay indicated that the pattern of inhibition depends largely upon the plant part, solvent used for extraction and the organism tested. Extracts prepared from leaves were shown to have better efficacy than stem and root extracts. Organic extracts provided potent antibacterial activity as compared to aqueous extracts. Among all the extracts, methanolic extract was found most active against almost all the bacterial species tested. Gram-positive bacteria were found most sensitive as compared to Gram-negative bacteria. Staphylococcus aureus was signifi cantly inhibited by almost all the extracts even at very low MIC followed by other Gram-positives. For Escherichia coli (a Gram-negative bacterium), the end point was not reached for ethyl acetate extract while it was very high for other extracts. The study promises an interesting future for designing a potentially active antibacterial agent from Datura innoxia.     

Influence of feeding precursors on tropane alkaloid production during an abiotic stress in Datura innoxia transformed roots

The effects of feeding tropane alkaloid precursors in transformed root culture of Datura innoxia Mill. were studied during a stress treatment. The permeabilizing effect of Tween 20 on tropane alkaloid production by hairy root cultures was studied in flasks with different feeding of precursors (L-ornithine, L-arginine, L-phenylalanine, DL-β-phenyllactic acid, and tropinone). It has been shown that the addition of various precursors alone (0.5 m mol l ^-1) was ineffective in stimulating hyoscyamine production. In contrast, a short treatment with Tween 20, combined with L-phenylalanine feeding, amplified the level of hyoscyamine released into the medium compared with the Tween treatment alone. Thus, the total hyoscyamine content per flask was increased (+ 40%) compared with the control. When DL-β-phenyllactic acid (0.5 m mol l ^-1) was used, this last effect became more pronounced (+ 60%). These results show that permeabilization with Tween modulates tropane alkaloid accumulation by a release of alkaloids into the medium and a restoration of hyoscyamine root content. The simultaneous feeding of DL-β-phenyllactic acid and tropinone during the Tween treatment gave a similar effect to that obtained with DL-β-phenyllactic acid and Tween, suggesting that the synthesis of the tropate moiety determines the flux at the level of the esterification of tropine. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro propagation of the alkaloid producing plant Datura insignis Barb. Rodr.

A method is presented for the in vitro propagation of Datura insignis Barb. Rodr. Nodal explants were cultured on Murashige and Skoog medium supplemented either with BA alone or in combination with 2,4-D or IAA. Shoot multiplication and elongation were obtained in various growth regulator concentrations. Best results were obtained in a medium with 1.0 mgl^-1 of BA. Individual shoots were excised and transfered to growth regulator-free medium for rooting. Additional multiplication was obtained by single-node culture using explants from these in vitro rooted shoots. Rooted plantlets were successfully grown in soil.     

Nucleotide pools in suspension-cultured cells of Datura innoxia

A general picture of the metabolic events which govern to growth behaviour of a batch culture of suspended dedifferentiated cells of Datura innoxia is obtained by following both the uptake and accumulation of the medium phosphate and sucrose by the cells, and the synthesis of RNA, protein and starch. The results are compared with the changes in the nucleotide pools described in the preceding paper. The sequence of formation and the regulatory dependencies of cellular pools of phosphate, sucrose, nucleotides and RNA in the production of proteins, starch and cell mass, and in the control of proliferation and cell growth are discussed. Furthermore, the importance of the maintenance pools for metabolic survival during starvation is emphasized.     

Tropane alkaloids of Datura innoxia from Morocco

Fifty three alkaloids were identified in the organs (roots, stems, leaves, flowers, and seeds) of Datura innoxia by GC/MS. Seventeen of them are reported for the first time for this species and one nor-derivative, 3-phenylacetoxynortropane (28), for the genus Datura. Furthermore, four new tropane esters were tentatively identified as 3-acetoxy-6,7-epoxytropane (acetylscopine) (10), 3-acetoxy-6-propionyloxy-7-hydroxytropane (15), 6,7-dehydro-3-phenylacetoxytropane (25), and 3-(2'-phenylpropionyloxy)-6,7-epoxynortropane (dihydroaponorscopolamine) (37) on the basis of their mass spectral data. Hyoscyamine (44) and scopolamine (48) figure as main alkaloids in the roots and aerial parts, respectively.     

Shoot Differentiation in Callus Cultures of Datura innoxia

Datura innoxia Mill. callus cultures formed shoots in 2–4 weeks on media containing; a) gibberellic acid, b) indoleacetic acid, c) low concentrations of naphthylacetic acid, d) low concentrations of 2,4-dichlorophenoxyacetic acid, e) benzylaminopurine, f) no growth substance. Benzylaminopurine promoted shoot differentiation. Gibberellic acid inhibited shoot formation weakly, but inhibited proper leaf blade formation. Root differentiation was rare. The callus cultures of Datura innoxia grew rapidly (100-fold in 4 weeks) on a slightly modified Murashige and Skoog medium (0.5 mg/l thiamin · HCl, pH 5.5, no glycine) in light at 30°C. Callus grew well on any single one of the growth substances NAA (10^?5M), 2,4-D (10^?6M) or BAP (3 × 10^?6M). Growth was less and more erratic on GA or IAA. The callus cultures did not grow significantly better when BAP was combined with one of the auxins or with GA.     

Embryogenesis in suspension cultures of Datura innoxia Mill.

In vitro plant regeneration via embryogenesis was obtained in suspension cultures of Datura innoxia Mill. Embryogenesis was induced in suspension cultures raised from callus of androgenetic origin, using LS liquid medium supplemented with 0.22 mg/l 2,4-D. The total number of embryos formed was variable over time in culture. Embryos differentiated and matured in the liquid medium itself as also evidenced by histological observation. Embryos germinated to form plantlets on semisolid MS medium without growth substances. The regenerated plants had haploid number of chromosomes.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - Kn Kinetin - LS Linsmaier and Skoog (1965) - MS Murashige and Skoog (1962)     

Growth patterns and alkaloid accumulation in hairy root and untransformed root cultures of Datura stramonium

The aim of this work was to study the possible relationship between alkaloid production and growth measured as: biomass increase and cellular division frequency, in Datura stramonium in vitro root cultures (hairy root and normal cultures). A comparison of growth values on a fresh and dry weight basis showed that there were differences between transformed and non-transformed lines. The differential growth between lines occurred due to a real biomass increase and not because of water accumulation. On the other hand, the rate of cell division showed a similar pattern for all lines studied. Therefore, the differences in growth are not due to different cell division rates, nor to the presence of larger meristems, but to the development and growth of lateral roots and the presence of active intercalary meristematic zones in each line. The maximum alkaloid production occurred when the cultures were not growing. This suggests an inverse relationship. Finally, the data support a specific model of growth at the level of cell division in root cultures which has not been described before in the literature. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro organogenesis and plant formation in cucumber

In vitro organogenesis was achieved from callus derived from hypocotyl explants of Cucumis sativus L. cv. Poinsett 76. Calli were induced from hypocotyl explants excised from 7-d-old seedlings grown on Murashige and Skoog (MS) medium containing 87.64 μM sucrose, 0.8 % agar, 3.62 μM 2,4-dichlorophenoxy acetic acid and 2.22 μM 6-benzyladenine (BA). Regeneration of adventitious buds from callus (25 shoots explant⁻¹) was achieved on MS medium supplemented with 8.88 μM BA, 2.5 μM zeatin and 10 % coconut water after two subcultures in the same medium at 30-d interval. Gibberellic acid (1.75 μM) favoured shoot elongation and indole 3-butyric acid (7.36 μM) induced rooting. Rooted plants were hardened and successfully established in soil.     

Promotive effect of polyvinylpolypyrrolidone on pollen embryogenesis in Datura innoxia

Pollen embryos of Datura innoxia Mill are produced in larger numbers from anthers on agar-gelled medium containing polyvinylpolypyrrolidone than on control. The best response is observed with 0.5% polyvinylpolypyrrolidone. The effect is possibly due to adsorption of substances (phenolics) emanating from cultured anthers and inhibiting the development of pollen grains into embryos.     

Biosynthesis of C6-C3 acids in Datura innoxia

Datura innoxia plants were fed via the roots with cinnamic acid-[2¹⁴C], (±)-phenyllactic (2-hydroxy-3-phenylpropanoic) acid-[2¹⁴C] and phenylalanine-[2-¹⁴C]. In each case apohyoscine, hyoscine, hyoscyamine and littorine were isolated from the aerial parts, and hyoscine, hyoscyamine and littorine from the roots. Cinnamic acid was not incorporated into the acid moieties of the alkaloids. Phenyllactic acid served as a better precursor than phenylalanine for tropic acid (hyoscine and hyoscyamine) and atropic acid (apohyoscine). Phenylalanine served as an effective precursor for the phenyllactic acid moiety of Littorine.     

In vitro organogenesis using multipotent cells

The establishment of efficient methods for promoting stem cell differentiation into target cells is important not only in regenerative medicine, but also in drug discovery. In addition to embryonic stem (ES) cells and various somatic stem cells, such as mesenchymal stem cells derived from bone marrow, adipose tissue, and umbilical cord blood, a novel dedifferentiation technology that allows the generation of induced pluripotent stem (iPS) cells has been recently developed. Although an increasing number of stem cell populations are being described, there remains a lack of protocols for driving the differentiation of these cells. Regeneration of organs from stem cells in vitro requires precise blueprints for each differentiation step. To date, studies using various model organisms, such as zebrafish, Xenopus laevis , and gene-targeted mice, have uncovered several factors that are critical for the development of organs. We have been using X. laevis , the African clawed frog, which has developmental patterns similar to those seen in humans. Moreover, Xenopus embryos are excellent research tools for the development of differentiation protocols, since they are available in high numbers and are sufficiently large and robust for culturing after simple microsurgery. In addition, Xenopus eggs are fertilized externally, and all stages of the embryo are easily accessible, making it relatively easy to study the functions of individual gene products during organogenesis using microinjection into embryonic cells. In the present review, we provide examples of methods for in vitro organ formation that use undifferentiated Xenopus cells. We also describe the application of amphibian differentiation protocols to mammalian stem cells, so as to facilitate the development of efficient methodologies for in vitro differentiation.     

Enhancement of pollen embryo formation in Datura innoxia by charcoal

In recent years liquid medium has been shown to be better than agar-gelled medium for production of haploids by anther culture. However, on addition of charcoal to agar medium the anther response in Datura innoxia Mill, increases dramatically and is better than in liquid medium. For anthers with pollen at the premitotic stage, the best result was observed with 1% charcoal in Difco agar and 1.5% in Normal agar. The effect is possibly due to adsorption of substances inhibitory to androgenesis and emanating from anthers, as well as to substances present in the nutrient medium and agar.     

Folate Metabolism in Datura innoxia (In Vivo and in Vitro Folylpolyglutamate Synthesis in Wild-Type and Methotrexate-Resistant Cells)

In vivo folylpolyglutamate pools of the wild-type (Px4) and methotrexate-resistant (MTX161) Datura innoxia cell lines were detected by incorporation of [14C]p-aminobenzoate into folates. The folylpolyglutamate derivatives were cleaved to p-aminobenzoylpolyglutamates and separated according to glutamyl chain length by high-performance liquid chromatography. Hexaglutamates were the predominant form in both Datura cell lines. The proportions of individual folylpolyglutamates were unaffected by culturing the cells in medium containing products of one-carbon metabolism such as glycine, adenine, thymidine, or methionine. Radiolabeling of the hexaglutamates was greatly reduced in the presence of 10-8 M methotrexate (MTX) in the Px4 cells but not in the MTX161 cells. Tetrahydrofolate, 5, 10-methylenetetrahydrofolate, and folinic acid were effective substrates for the folylpolyglutamate synthetase from Datura cells in vitro, whereas MTX and folate were poor substrates. In vivo, MTX can be slowly converted into its polyglutamate derivatives up to MTXGlu4 or MTXGlu5 in Datura cells in the longer term. Significantly lower levels of MTX polyglutamates in MTX161 cells were found compared with those of Px4 cells during prolonged (10 d) exposure to MTX. Although in vivo and in vitro folylpolyglutamate synthesis was found to be similar in both cell lines, about a 4-fold increase in specific activity of [gamma]-glutamyl hydrolase (GGH) was detected in the MTX161 cell line. The increase in GGH in the resistant cells suggested that breakdown of polyglutamylated forms of MTX may play a role in acquired MTX resistance.     

Growth and Plating of Cell Suspension Cultures of Datura innoxia

Suspension cultures of Datura innoxia Mill, were successfully grown on a modified Murashige and Skoog medium with 2,4–D, NAA or BAP as growth substances, provided the micronutrient levels were reduced to 1/10. Normal amounts of micronutrients were toxic. Attempts to identify the toxic elements did not succeed. Cultures grew exponentially on a shaker at 27°C in the light. Their doubling times varied from 1.1 days on 2,4–D (10^–6M) or NAA (10^?5M)+ 1 g/1 casein hydrolysate to 2.7 days on BAP (3 × 10^?7M) and 5.1 days on supraoptimal levels of 2,4-D (10^?5M). Cultures grew on NH₄⁺-N alone (from ammonium malate) or on NO₃^?-N alone. Dry weight yield was proportional to the amount of nitrate-N added (47 mg/mg N). Filtered suspension cultures containing single cells (plating cultures) could be grown in agar in petri dishes when NAA or 2,4-D were used as growth substances. Cells grew at densities above 500 units/ml in the agar. Most colonies grew from cell aggregates but division in single cells was observed. The highest plating efficiency was about 50% on 10^?6 M 2,4-D + 1 g/1 casein hydrolysate.     

Radiation-induced Organogenesis and Isoenzyme Patterns in Long-term Callus Cultures of Datura innoxia

Effect of gamma irradiation on growth, shoot organogenesis andenzyme activities and isoenzyme patterns of -amylase and peroxidaseduring differentiation in long-term calluses of Datura innoxiahave been investigated. Radiation in doses of 0.2 and 1.0 kRstimulated the shoot regeneration frequency as well as the numberof shoots per regenerating callus. The 0.2 kR dose could induceshoot organogenesis even in calluses incubated in the dark oncallusing medium, although with less frequency. Such cultures,however, showed profuse shoot regeneration when sub-culturedonto regeneration medium under light conditions. A higher radiationdose (5.0 kR) was lethal to both growth and shoot differentiation.Prior to shoot regeneration, -amylase and peroxidase specificactivities increased to four- to fivefold and 7–24-fold,respectively. While the amylase isoenzyme pattern remained unchanged,specific changes in the isoperoxidase pattern were observedduring shoot differentiation in callus cultures. The most significantchange was the appearance of fast-moving anodic bands priorto visible shoot differentiation. Thus, such isoperoxidasesprovide useful biochemical markers for shoot differentiation. Datura innoxia, shoot organogenesis, isoenzyme pattern, gamma-radiation, growth regulators