Hyoscyamus Muticus Research Articles

Growth and the Uptake of Sucrose and Mineral Ions by Transformed Root Cultures ofDatura stramonium, Datura Candida×aurea, Datura wrightii, Hyoscyamus muticusandAtropa belladonna

Growth and the uptake of sucrose, anions (Cl(-), NO3(-), PO4(3-), and SO4(2-)), and cations (K(+), Na(+), Mg(2+), Ca(2+), and NH4(+)) by transformed roots of Datura stramonium, Datura wrightii. Datura candida x Datura aurea hybrid, Hyoscyamus muticus, and Atropa belladonna were examined during batch culture over 28 days in modified 14 litre stirred tank reactors containing Gamborg's B5 salts medium. All cultures completely removed NH4(+) and PO4(3-) from the medium, and Mg(2+) was totally removed by the Datura cultures. The other ions were consumed at different rates depending on the culture but were never totally removed from the medium. Amounts for each ion consumed per gram increase in biomass are presented and compared between the different cultures. More NO3(-) than any other ion was consumed by each of the cultures. Concomitant with the uptake of sucrose was a slow release of glucose into the medium. Roots were found to contain only low levels of free sugars. Total hyoscyamine production ranged from 115 mg to 633 mg per reactor.

Selection for Hyoscyamine and Cinnamoyl Putrescine Overproduction in Cell and Root Cultures of Hyoscyamus muticus

Hairy root cultures of Hyoscyamus muticus have been shown to produce stable levels of tropane alkaloids comparable to those found in whole plants. In contrast, cell cultures of this and other solanaceous species produce only trace amounts of alkaloids but can be used for selection of metabolic variants. We have taken advantage of both systems and the ability to convert between them in vitro in an effort to select for increased production of the tropane alkaloid hyoscyamine. Hairy roots were converted into cell suspensions by addition of 1 mg/L 2,4-dichlorophenoxyacetic acid to Murashige-Skoog medium (T. Murashige and F. Skoog [1962] Physiol Plant 15: 473-497) and screened for resistance to the amino acid analog p-fluorophenylalanine (PFP). Cells that could grow in media containing 400 [mu]M PFP were selected and cloned from single cells. The resistant cells accumulated high levels of cinnamoyl putrescines, which share the same biosynthetic precursors as hyoscyamine. Hairy root cultures were regenerated from both PFP-sensitive and PFP-resistant cells by removing 2,4-dichlorophenoxyacetic acid from the medium. Resistance to PFP continued to be expressed in regenerated roots. Higher levels of hyoscyamine were found in hairy roots regenerated from PFP-resistant cells than were found in controls. We suggest that the precursors overproduced by the PFP-resistant cells can be diverted into the hyoscyamine pathway upon the regeneration of root cultures.

Efficient plant regeneration from hairy root-derived protoplasts of Hyoscyamus muticus

Successful plant regeneration was achieved for the first time from hairy root-derived protoplasts of Hyoscyamus muticus. High yields (7 × 10(6) / g fresh weight) of protoplasts were isolated directly from the transformed roots of Hyoscyamus muticus using an enzyme mixture comprising 1 % macerozyme and 2 % cellulase in an osmoticum consisting of 0.2 M CaCl2 and 0.6 M mannitol. Protoplasts were first cultured in liquid NT/PRO I medium and further on semi-solid NT/PRO II agar medium. The procedure permits highly efficient formation of colonies. The plating efficiency varied from 1-9 %. The small individual colonies regenerated easily into shoots and roots at frequencies of 18 % and 70 %, respectively. The time required for the development of small plantlets from protoplasts was 8-11 weeks. The regenerated plants contained rolB from Ri-T-DNA and exhibited an altered phenotype compared to the control plants.

Role of calcium and differentiation in enhanced sesquiterpene elicitation from calcium alginate-immobilized plant tissue

Calcium alginate immobilization of Hyoscyamus muticus plant tissue culture is shown to enhance the rapid formation of sesquiterpenes in response to exposure to extracts of the fungal pathogen Rhizoctonia solani. The studies were carried out with suspension-cultured cells and roots at low tissue loadings to demonstrate that the enhancement is not due to either the degree of differentiation or the presence of metabolic gradients. The fungally elicited response of root tissue is shown to be much less sensitive to both the physical and chemical environment than free cell suspensions. By examining the effects of the immobilization components (Ca 2+, alginate), and immobilization in barium alginate gels, it is concluded that the enhanced sesquiterpene production in immobilized tissue is mediated through calcium.

Cloning and Bacterial Expression of a Sesquiterpene Cyclase from Hyoscyamus muticus and Its Molecular Comparison to Related Terpene Cyclases

Genomic and cDNA clones for vetispiradiene synthase, a sesquiterpene cyclase found in Hyoscyamus muticus, were isolated using a combination of reverse transcription-polymerase chain reactions and conventional cloning procedures. RNA blot hybridization demonstrated an induction of mRNA consistent with the induction of cyclase enzyme activity in elicitor-treated cells, DNA blot hybridization indicated a gene family of 6 to 8 members, and bacterial expression of 3 cDNA clones indicated that each coded for a vetispiradiene synthase enzyme activity catalyzing the synthesis of a single reaction product. Intron-exon organization of the vetispiradiene synthase gene was identical with that previously described for 5-epi-aristolochene synthase (tobacco sesquiterpene cyclase) and casbene synthase (castor bean diterpene cyclase), and the vetispiradiene synthase amino acid sequence was 77% identical with and 81% similar to the tobacco sesquiterpene cyclase. Regions of the vetispiradiene synthase sequence centered around amino acids 60, 100, and 370 were conspicuously different relative to the tobacco sesquiterpene cyclase. The sequence similarity between the tobacco and H. muticus enzymes is suggested to be reflective of the conservation of several partial reactions common to both enzymes, and the differences may be reflective of a partial reaction unique to each enzyme.

Virulence of different Agrobacterium strains on hairy root formation of Hyoscyamus muticus

Hyoscyamus muticus accession was evaluated for its response to inoculation with Agrobacterium rhizogenes strains LBA9402, A4, 15834, and Agrobacterium tumefaciens strain C58 CI pRT GUS104. The Agrobacterium strain used for the transformation has a significant influence on the phenotype of the clone as well as on the growth rate and hyoscyamine production of these root culture clones. The most virulent strains were C58 CI pRT GUS104 and LBA9402. More roots were obtained on LSO medium than on B50 medium. Acetosyringone addition and the time from wounding affected root formation. The alkaloid content was highest in clones C58 and A4 (≈90mg·l(-1)). There are great differences between individual hairy root clones, and hence they are not as uniform as has often been speculated. The Agrobacterium strain used for the transformation has a great influence in this respect.

Effect of nitrogen and sucrose on the primary and secondary metabolism of transformed root cultures of Hyoscyamus muticus

Effect of nitrogen and sucrose on the primary and secondary metabolism of transformed root cultures of Hyoscyamus muticus

Use of Binding Measurements To Predict Elicitor Dosage Requirements for Secondary Metabolite Production from Root Cultures

AbstractThe strength of binding of a fungal elicitor derived from Rhizoctonia solani to Agrobacterium‐transformed root cultures of Hyoscyamus muticus is assessed. Two techniques are used to estimate the equilibrium binding constant: (1) a dose response, which involves the measurement of sesquiterpene formation in the presence of different amounts of elicitor, and (2) elicitor‐contacting experiments in which the root tissue is exposed to the elicitor for a short period of time and then the unbound elicitor concentration is determined by a bioassay. The equilibrium binding constants estimated by these two techniques were on the same order of magnitude. The value predicted from the dose response technique was 6.44 (mmol of glucose equiv/L of medium)−1, while that obtained from elicitor contact was 1.89 (mmol of glucose equiv/L of medium)−1. The higher binding constant measured by dose response may result from feedback inhibition of secondary metabolite formation at higher elicitor dosages. The magnitude of the binding'constant indicates that elicitor dosage is dependent upon both the amount of tissue in the reactor and the amount of medium present to dilute the added elicitor. Since proposed reactor configurations for plant root culture vary from 200 to 2000 g of tissue per liter of medium, elicitor dosage requirements will vary significantly depending upon the reactor operational conditions. This influence of elicitor binding on reactor design and operation is discussed.

Fertile somatic hybrid between sexually incompatible Hyoscyamus muticus and Hyoscyamus albus

Somatic hybrid plants have been regenerated from fused protoplasts of a chlorophyll deficient mutant of H. muticus (2n=28) with wild type protoplasts of H. albus (2n=68). The inability of protoplasts of H. albus to regenerate was utilized in complementation with achlorophyllous, but regenerating, protoplasts of H. muticus for the selection of green somatic hybrid colonies and plants. The somatic hybrid plants showed intermediate morphological characters, and possessed 82-120 chromosomes, with a modal number of 96 which is also the amphidiploid complement of the two species. The isozyme patterns indicated the presence and expression of genes from both parents. The hybrid plants produced 33-78% viable pollen and set viable seeds upon selfing and backcrossing in a directional manner.

An unusual root tip formation in hairy root culture of Hyoscyamus muticus

Hairy root cultures of Hyoscyamus muticus were established using Agrobacterium rhizogenes ATCC 15834. In one out of 8 clones established, an unusual root tip formation was observed after transfer of cultures from half-strength Murashige and Skoog (1962) to White's medium (1939). This phenomenon was associated with the production of a fine brownish cell suspension culture. Hairy root development resumed after transfer of the root tips from White to half-strength Murashige and Skoog medium. After plating the isolated brownish cells on hormone-free half-strength Murashige and Skoog or White solid medium, callus proliferation was observed, and then redifferentiation of hairy roots occurred. The polymerase chain reaction analysis of the H. muticus hairy root (clone Z2) revealed that only the tl region of the T-DNA was integrated. The growth and the production of five tropane alkaloids by this clone were examined.

Somatic Embryogenesis and Plant Regeneration from Tissue Cultures of Hyoscyamus muticus L.

Somatic embryogenesis and regeneration of plantlets was achieved In callus cultures derived from cotyledonary leaf pieces of Hyoscyamus muticus L on MS medium enriched with 2 mg/l 2,4–0 and 0.5 mg/l BAP. For embryogenesis and organogenesis varying concentrations of NAA with or without BAP were added In the medium. Organogenesis was also achieved when callus was transferred to the hormone free medium.

Reversed-phase liquid chromatographic isolation of lubimin and solavetivone from Hyoscyamus muticus “hairy” root cultures

A high-performance liquid chromatographic reversed-phase procedure was developed to estimate the concentration of sesquiterpene phytoalexins secreted into the media by Agrobacterium transformed root cultures of Hyoscyamus muticus. An isolation procedure based on solid-phase extraction (C 18 Sep-pak) results in very rapid analysis and gives full recovery, compared to sequential chloroform extraction. A simple isocratic procedure permits quantification of sesquiterpene production by these root cultures in response to exposure to fungal elicitors. Calibration curves for lubimin and solavetivone content were obtained by fractionating sufficient material to provide a measurable weight. The identity of these compounds was confirmed by TLC, UV, GC-MS, and NMR. This experimental system is being used to examine the regulation of sesquiterpene biosynthesis, and to develop large-scale processing techniques for the production of secondary metabolises from plant roots grown in bioreactor systems.

Enhanced recovery of solavetivone from Agrobacterium transformed root cultures of Hyoscyamus muticus using integrated product extraction

The integrated recovery of solavetivone from fungus elicited "hairy root" cultures of Hyoscyamus muticus is examined using volatile organic solvents and solid-phase adsorbents in an external loop extraction configuration. Hexane and pentane are shown to be toxic when added directly to the culture; however, growth of roots is not inhibited when cultures are exposed to media saturated with these hydrocarbons. Solid-phase neutral adsorbents, XAD-7 and XAD-16, display higher capacity and better solavetivone partitioning capability than the hydrocarbons; however, their selectivity for the sesquiterpene solavetivone is poor in comparison with hexane. In both cases, the integration of product recovery through extraction resulted in a doubling of product formation by alleviating feedback repression. Implications of these results to the recovery of secondary metabolites from plant root cultures are discussed. .

Growth of plant root cultures in liquid- and gas-dispersed reactor environments.

The growth of Agrobacterium transformed "hairy root" cultures of Hyoscyamus muticus was examined in various liquid- and gas-dispersed bioreactor configurations. Reactor runs were replicated to provide statistical comparisons of nutrient availability on culture performance. Accumulated tissue mass in submerged air-sparged reactors was 31% of gyratory shake-flask controls. Experiments demonstrate that poor performance of sparged reactors is not due to bubble shear damage, carbon dioxide stripping, settling, or flotation of roots. Impaired oxygen transfer due to channeling and stagnation of the liquid phase are the apparent causes of poor growth. Roots grown on a medium-perfused inclined plane grew at 48% of gyratory controls. This demonstrates the ability of cultures to partially compensate for poor liquid distribution through vascular transport of nutrients. A reactor configuration in which the medium is sprayed over the roots and permitted to drain down through the root tissue was able to provide growth rates which are statistically indistinguishable (95% T-test) from gyratory shake-flask controls. In this type of spray/trickle-bed configuration, it is shown that distribution of the roots becomes a key factor in controlling the rate of growth. Implications of these results regarding design and scale-up of bioreactors to produce fine chemicals from root cultures are discussed.

A Soluble Auxin-Binding Protein from Hyoscyamus muticus Is a Glutathione S-Transferase

We have used the photoaffinity label azido-[3H]IAA (5-N3-[7-3H]indole-3-acetic acid), a biologically active analog of indole-3-acetic acid, to identify auxin-binding proteins (ABPs) in the soluble fraction of Hyoscyamus muticus. A 25-kD polypeptide previously described (H. Macdonald, A. M. Jones, P. J. King [1991] J Biol Chem 266: 7393-7399) has now been purified to homogeneity by conventional methods. Binding of azido-[3H]IAA to the purified protein was reduced by active auxins but not by inactive indoles. Partial amino acid sequences of the purified protein showed high homology to glutathione S-transferase (GST) from tobacco (ParB) and from maize (GT32). The conclusion that the 25-kD ABP is a GST is further supported by high GST activity in fractions highly enriched in the 25-kD polypeptide and recognition of the ABP by antibodies against GST from wheat and maize. Furthermore, purification of a protein from a soluble protein extract from H. muticus by affinity chromatography on glutathione-agarose also yielded a 25-kD polypeptide that was indistinguishable in its N-terminal amino acid sequence and biochemical characteristics from the protein purified by conventional methods. Possible functions of GST in auxin action are discussed.

Stimulation of solavetivone synthesis in free and immobilized cells of Hyoscyamus muticus by Rhizoctonia solani fungal components

Three elicitor fractions including spent culture medium, hydrolyzed mycelial pellet, and hydrolyzed mycelial biomass supernatant were tested for their ability to induce sesquiterpene formation in freely suspended and immobilized cells. Immobilization enhanced the effectiveness of the hydrolysate elicitors, but not that derived from spent culture medium. When effectiveness of the elicitors is compared based on product yield, spent culture medium is shown to be far more effective than hydrolysates as an elicitor.

Production of solavetivone by immobilized cells of Hyoscyamus muticus

The secretion of solavetivone, a phytoalexin, by the cells of Hyoscyamus muticus in response to fungal elicitation has been enhanced by gel entrapment in calcium alginate. The immobilized cells produced 53% higher product and exhibited sustained biosynthetic activity in repeated batch cycles when compared with suspended cells. Providing a non-elicited media exchange and a sufficient interval of time between repeated infection (elicitation) gave greater productivity. Apparently the cells need a period to recover from the infection.

Regeneration of intergeneric somatic hybrid plants between Atropa belladonna L. and Hyoscyamus muticus L.

In the present experiments, cell suspension derived protoplast of Atropa belladonna L. (2 n = 72) var acuminata and protoplasts of an achlorphyllous mutant cell line of Egyptian henbane, Hyoscyamus muticus (2 n = 28) were fused to obtain intergeneric somatic hybrid plants. The somatic hybrid plants possessed variable chromosome complements. Those plants with a chromosome number of around 100 and with both parental genomes distinguished on the basis of chromosome size were easier to establish in the glasshouse. Plants with multiple complements (chromosome number = 178) were grossly aberrant types and very difficult to maintain. The work involved complementation at the intergeneric level within the same tribe and evidence of chromosome elimination of Atropa chromosomes was obtained. The somatic hybrid plants developed with intermediate types of leaves and altered characteristics reflecting expression of both the genomes. In one of the somatic hybrid clones, SH-15, the pentamerous orientation of the whorls of sepals, petals and anthers in both the parents was altered to a hexamerous form. Further, the parents possess a bilocular ovary and this was altered to a trilocular form in this somatic hybrid. Plants that have responded better in their development in the glasshouse had a near tetraploid chromosome number of H. muticus and a near haploid chromosome number of A. belladonna. The presence of complementary bands of esterases and peroxidases as studied in the line SH-15, affirmed the hybrid nature.

Interpreting the role of phosphorus and growth rate in enhanced fungal induction of sesquiterpenes from Hyoscyamus muticus root cultures

The effect of thiamine limitation in combination with fungal elicitation on sesquiterpene (solavetivone) production was studied in Agrobacterium-transformed “hairy-root” cultures of Hyoscyamus muticus as a potential means of manipulating the growth rate independent of phosphorus availability. Limiting the initial supply of thiamine did not affect the growth of these cultures compared to growth at the control level of thiamine (0.01 g/l). There was also no enhancement in sesquiterpene production when thiamine supply was limited. Serial culturing in thiamine-free media suggests that these root cultures are not strictly auxotrophic for thiamine, in contrast to previously published results for untransformed root culture. The effect of phosphate limitation combined with elicitation on the production of solavetivone was examined at constant media volume to provide a constant elicitor concentration and to eliminate feedback-inhibition effects. Limiting the initial supply of phosphate to elicited cultures resulted in a twofold increase in solavetivone production as compared to the elicitation at control media phosphate levels (1.1mm). Because growth was attenuated, production per unit cell mass increased 11-fold compared to the control. The effect of phosphate limitation on solavetivone production at constant cell mass and elicitor per root mass was studied. Limiting the initial supply of phosphate to elicited cultures under these conditions did not result in enhanced production of solavetivone. The initially observed enhanced production of solavetivone at limiting initial phosphate concentrations is therefore due to factors other than the growth rate or phosphate involvement in secondary metabolism.

Genetic variation in meiotic behaviour and fertility in tetraploid Hyoscyamus muticus: correlation with diploid meiosis

Autotetraploidy was induced in 19 morphologically distinct genotypes of the medicinal solanaceous plant Hyoscyamus muticus (Egyptian henbane, 4x= 56). The multivalent association frequencies at metaphase I of meiosis ranged from 0.328 to 0.628 and were positively correlated with the chiasmate association frequency in the diploid progenitors. The frequent absence of chiasmata in one of the two chromosome arms in most of the tetraploids was attributed to interference between the arms and to pairing partner exchange in one of the chromosome arms instead of around the centromers. The bivalent frequency in the tetraploids was positively correlated with pollen fertility and seed set. The latter ranged from 7.5 to 68.5 per cent. The asymmetry of the karyotype was not reflected in the distribution of open bivalents in the diploid. The regression coefficients of metaphase I chromosome associations over fertility in the tetraploid suggested that, in the range covered, one multivalent less resulted in an increase of about 12 per cent in seed set. The regression of open bivalent frequency in the diploid progenitor over seed set in the tetraploid suggested that one open bivalent more in the diploid would result in almost 18 per cent seed set in the tetraploid. It is concluded that the selection of diploid progenitors with high frequencies of open bivalents should enhance the fertility of the artificial polyploids in H. muticus.