Effects of light conditions on growth and defense compound contents of Datura inoxia and D. stramonium.

We examined the effects of light conditions on plant growth and production of defense compounds in the toxic species Datura inoxia and D. stramonium. Specifically, we investigated morphological and physiological traits, including the contents of nitrogen-based tropane alkaloids (atropine and scopolamine) as defense compounds, under three light conditions: 100%, 80%, and 50% of full sunlight. Both species showed similar morphological and physiological responses to exposure to different intensities of light. Although the total plant mass decreased under lower light conditions, the total leaf area per plant increased. The reason being that the leaf mass per plant did not decrease, while the leaf mass per unit area decreased. Leaf nitrogen and chlorophyll concentrations and the chlorophyll/nitrogen ratio increased under lower light conditions, whereas the chlorophyll a/b ratio decreased. These morphological and physiological changes may be seen as ways to increase light acquisition under low light conditions. Leaf atropine and scopolamine concentrations did not differ among the three light conditions for both species. In conclusion, both Datura species underwent morphological and physiological changes under low light conditions, enabling them to use carbon and nitrogen to increase light acquisition while maintaining their chemical defense capability.

Datura innoxia plants hydroponically-inoculated with Agrobacterium rhizogenes display an enhanced growth and alkaloid metabolism.

BACKGROUND: The production of secondary metabolites through the culture of entire plants is of great interest. Soilless culture, such as hydroponics, enables the control of plant growth and metabolism. Specific environmental conditions must be developed to maximize the productivity of medicinal plants used as efficient natural bioreactors. METHODS: The nutrient solution of newly established hydroponic cultures ofDatura innoxia Mill. were inoculated with Agrobacterium rhizogenes (A.r.) wild strains (TR7, TR107, 11325 or 15834). Growth and the alkaloid contents of roots and aerial parts were analyzed. Axenic cultures were also performed with modified TR7 strains containing the egfp or gus reporter gene. In vitro isolated root cultures enabled the phenological and molecular demonstration of gene transfer. RESULTS: A.r.TR 7 led to a greater improvement in plant secondary metabolism and growth. Positive expression of the reporter genes occurred. Isolation and subculture of some of the roots of these plants showed a hairy root phenotype; molecular tests proved the transfer of bacterial genes into the roots isolated from the plants. CONCLUSIONS: Hyoscyamine and scopolamine productivity is enhanced after A.r. inoculation in the nutrient solution of hydroponic plants. Transformation events occur in the original roots of the plants. This leads to chimeric plants with a part of their roots harboring a hairy root phenotype. Such semi-composite plants could be used for successful specialized metabolite bioproduction in greenhouses.

Establishment, Culture, and Scale-up of Brugmansia candida Hairy Roots for the Production of Tropane Alkaloids.

Brugmansia candida (syn. Datura candida) is a South American native plant that produces tropane alkaloids. Hyoscyamine, 6ß-hydroxyhyoscyamine (anisodamine), and scopolamine are the most important ones due to their anticholinergic activity. These bioactive compounds have been historically and widely applied in medicine and their demand is continuous. Their chemical synthesis is costly and complex, and thereby, these alkaloids are industrially produced from natural producer plants. The production of these secondary metabolites by plant in vitro cultures such as hairy roots presents certain advantages over the natural source and chemical synthesis. It is well known that hairy roots produced by Agrobacterium rhizogenes infection are fast-growing cultures, genetically stable and able to grow in hormone-free media. Additionally, recent progress achieved in the scaling up of hairy root cultures makes this technology an attractive tool for industrial processes. This chapter is focused on the methods for the induction and establishment of B. candida hairy roots. In addition, the scaling up of hairy root cultures in bioreactors and tropane alkaloid analysis is discussed.

Phylogenetic correlations among chemical and physical plant defenses change with ontogeny.

Theory predicts patterns of defense across taxa based on notions of tradeoffs and synergism among defensive traits when plants and herbivores coevolve. Because the expression of characters changes ontogenetically, the evolution of plant strategies may be best understood by considering multiple traits along a trajectory of plant development. Here we addressed the ontogenetic expression of chemical and physical defenses in 12 Datura species, and tested for macroevolutionary correlations between defensive traits using phylogenetic analyses. We used liquid chromatography coupled to mass spectrometry to identify the toxic tropane alkaloids of Datura, and also estimated leaf trichome density. We report three major patterns. First, we found different ontogenetic trajectories of alkaloids and leaf trichomes, with alkaloids increasing in concentration at the reproductive stage, whereas trichomes were much more variable across species. Second, the dominant alkaloids and leaf trichomes showed correlated evolution, with positive and negative associations. Third, the correlations between defensive traits changed across ontogeny, with significant relationships only occurring during the juvenile phase. The patterns in expression of defensive traits in the genus Datura are suggestive of adaptation to complex selective environments varying in space and time.

Abiotic induction affects the costs and benefits of inducible herbivore defenses in Datura wrightii.

We evaluated the costs and benefits of continuous high-level expression of defenses relative to naturally-induced defenses in field-grown Datura wrightii in the presence and absence of herbivores. We induced D. wrightii plants with monthly applications of the plant hormone methyl jasmonate (MeJA) and assessed levels of inducible proteinase inhibitors (Pins). MeJA application increased Pin production by 124 %, whereas the increase in Pins due to herbivory was more modest (36 %). Pin induction was costly and significantly reduced plant fitness compared to unmanipulated plants both in the presence and absence of herbivores. Although MeJA-treated plants exposed to herbivory suffered significantly less herbivore damage than unmanipulated plants exposed to herbivory, this was not accompanied by a corresponding fitness benefit. In contrast to glasshouse studies in which protected plants never expressed Pins, Pin induction occurred in field-grown plants not treated with MeJA and completely protected from herbivory. Subsequent experiments confirmed that putative herbivore defenses can be induced abiotically in D. wrightii as: 1) Pin levels did not differ significantly between field-grown plants protected from herbivory and plants exposed to chronic herbivory over the full season; and 2) plants exposed to ambient UV-B light in the absence of herbivory expressed low levels of Pins after two wk of exposure, whereas plants protected from UV-B remained uninduced. The costs of induced responses may be relatively easily determined under field conditions, but there may be many inducing agents in the field, and the benefits of induction may be difficult to associate with any single inducing agent.

Ontogeny and season constrain the production of herbivore-inducible plant volatiles in the field.

Herbivores may induce plants to produce an array of volatile organic compounds (herbivore-induced plant volatiles, or HIPVs) after damage, and some natural enemies of herbivores are attracted by those HIPVs. The production of HIPVs by the undomesticated species Datura wrightii was quantified in response to damage by its natural community of herbivores or the plant hormone methyl jasmonate (MeJA) over plant's 6-month growing season. Patterns of HIPV production were compared to the seasonal abundance of D. wrightii's two most abundant herbivores, the chrysomelid beetle Lema daturaphila and the mirid bug Tupiocoris notatus, and their shared generalist predator, the lygaeid bug Geocoris pallens. HIPV production was especially high in the spring, when plants were growing vegetatively, but HIPV production declined after plants began to flower and produce fruit, and these volatiles no longer were inducible by September. The composition of the HIPV blends also changed seasonally. HIPV production and composition were partially restored by "rejuvenating" plants back to the vegetative growth stage independently of season by cutting them back and allowing them to resprout and regrow vegetatively. HIPV production of D. wrightii in the field is limited to the earlier ontogenetic stages of growth, despite the fact that both herbivores and their shared natural enemy inhabited plants throughout the full season. The adaptive value of HIPV production in D. wrightii may be constrained by plant ontogeny to the vegetative stages of plant growth.

The dark side of the mycorrhiza.

Plant association with arbuscular mycorrhizal (AM) fungi is usually regarded as mutualistic. However, this positive effect could disappear if the benefit of the fungal-plant association changes with colonization density. In order to test the conditionality of this interaction, we evaluated plant performance and tolerance to defoliation across five levels of commercial AM fungal inoculum concentrations. Additionally, we evaluated if plant performance and tolerance were similarly affected by a whole soil community collected under a native congener. Along the gradient of inoculation, plant performance exhibited a peak at intermediate inoculum concentration, indicating the presence of an optimum level of AM fungal concentration that maximized AM fungal benefit. Root colonization by fungal hyphae increased linearly across the experimental inoculation gradient. Paralleling root colonization, plant tolerance to defoliation decreased linearly along the inoculum gradient. Plant performance was similar under the whole soil and commercial treatments. Our results show a negative correlation between tolerance to defoliation and AM fungal inoculum concentration, indicating that AM fungi colonization could constrain the evolution of plant tolerance to herbivory.

Modeling and optimization of hairy root growth in fed-batch process.

This article proposes a feeding strategy based on a kinetic model to enhance hairy roots growth. A new approach for modeling hairy root growth is used, considering that there is no nutrient limitation thanks to an appropriate feeding, and the intracellular pools are supposed to be always saturated. Thus, the model describes the specific growth rate from extracellular concentration of the major nutrients and nutrient uptakes depend on biomass growth. An optimized feeding strategy was determined thanks to the model to maintain the major nutrient levels at their optimum assuming optimal initial concentrations. The optimal feed rate is computed in open loop using kinetic model prediction or in closed loop using conductivity measurements to estimate biomass growth. Datura innoxia was chosen as the model culture system. Shake flask cultures were used to calibrate the model. Finally, cultures in bioreactor were performed to validate the model and the control laws.

In vitro organogenesis and alkaloid accumulation in Datura innoxia.

The kinetics of tropane alkaloids accumulation in different organs such as roots, leaves, stems, flowers and seeds of Datura innoxia was investigated by GC-MS. Twenty-six tropane alkaloids were detected. The ester derivatives of tropine (3alpha-tigloyloxytropine and 3-tigloyloxy-6-hydroxytropine) are the major compounds. Undifferentiated callus were established from the stem explants of Datura innoxia using Murashige and Skoog (MS) medium supplied with 6-benzylaminopurine (BA, 1 mg l(-10) and indole-3-acetic acid (IAA, 0.5 mg l(-1)) in combination for 6 weeks. Callus differentiation was initiated by subculture onto solid MS medium, free from hormones, for more than 10 months. Initially, shoots were formed after four weeks from subculture. Further subculturing in basal MS medium without growth regulators initiated the rooting of a shooty callus after 6 weeks. Investigation of the alkaloid content of the unorganized and organized callus revealed that callus (either green or brown) yielded only trace amounts of alkaloids. On the other hand, re-differentiated shoots contained mainly scopolamine while re-differentiated roots biosynthesized hyoscyamine as the main alkaloid.

Constitutive and jasmonate-inducible traits of Datura wrightii.

Plants in the family Solanaceae possess numerous traits that are induced from damage from herbivores. Many of these also can be induced by exposing plants to the plant hormone jasmonic acid or its volatile ester methyl jasmonate. Datura wrightii (Solanaceae) is dimorphic for leaf trichome morphology in most southern California populations. Trichome phenotype is governed by a single gene, and the glandular trichome condition is dominant and under developmental control. This study addressed two major objectives. The first was to determine if mature plants with glandular or nonglandular trichomes responded differentially to methyl jasmonate. The second objective was to determine if exposure of seedlings to methyl jasmonate during the period of trichome differentiation altered either the phenotype or the density of trichomes that mature plants expressed. Methyl jasmonate induced from 200 to 800 microg/ml of proteinase inhibitor activity and increased the activity of polyphenol oxidase by more than threefold depending on the experiment. These increases did not differ significantly between plants expressing glandular or nonglandular trichomes. Methyl jasmonate exposure did not increase the activity of peroxidase or the concentration of scopolamine or hyoscyamine, the two major alkaloids of Datura. Exposure to methyl jasmonate during trichome differentiation did not affect either the final trichome phenotype or the density of either type of trichome, but did increase the production of acylsugars in glandular trichomes by 44%. Because trichome phenotype was not inducible, and because both trichome phenotypes showed similar increases in proteinase inhibitors and polyphenol oxidase activity, the methyl-jasmonate-inducible responses of D. wrightii are independent of trichome phenotype in D. wrightii.

Xanthan gum: an economical substitute for agar in plant tissue culture media.

Xanthan gum, a microbial desiccation-resistant polysaccharide prepared commercially by aerobic submerged fermentation from Xanthomonas campestris, has been successfully used as a solidifying agent for plant tissue culture media. Its suitability as a substitute to agar was demonstrated for in vitro seed germination, caulogenesis and rhizogenesis of Albizzia lebbeck, androgenesis in anther cultures of Datura innoxia, and somatic embryogenesis in callus cultures of Calliandra tweedii. Culture media used for eliciting these morphogenic responses were gelled with either 1% xanthan gum or 0.9% agar. Xanthan gum, like agar, supported all these responses.

Assessment of wool waste and hair waste as soil amendment and nutrient source.

A field and two container experiments were conducted to assess uncomposted wool and hair wastes as a nutrient source for crops and to evaluate their potential to improve soil biological and chemical properties. Overall, addition of wool or hair waste to soil increased yields of basil (Ocimum basilicum L. 'Trakia'), thorn apple (Datura innoxia Mill. 'Inka'), peppermint (Mentha x piperita L. 'Black Mitchum'), and garden sage (Salvia officinalis L. 'Desislava'), increased NH(4)-N and NO(3)-N in soil, increased total N (and protein) content in plant tissue, stimulated soil microbial biomass, and decreased mycorrhizae colonization of plant roots of thorn apple but not in basil. Wool and hair waste additions to soil altered slightly the content and composition of plant secondary metabolites (essential oils or alkaloids); however, overall the constituents remained within the "typical" range for the respective crops. Scanning electron microscopy (SEM) and energy dispersive X-ray (EDX) analysis demonstrated that wool and hair wastes decompose slowly under field or greenhouse conditions, and act as a slow release S, N, P, and K fertilizer. These results, along with the measured concentrations of NO(3)-N in soil at harvest, suggest that the addition of wool or hair waste of only 3.3 g kg(-1) of soil may support two to five harvests or crops under greenhouse conditions and two to four field seasons in field production systems, and would improve soil biological and chemical characteristics. Further research is needed to optimize the rate of application of these waste materials to the nutrient requirements of specific crops to avoid nitrate leaching into the ground water. In addition, the effect of wool and hair waste on other environmental end points should also be further investigated before specific recommendations for growers are provided.

Comparative study of responses in four Datura species to a zinc stress.

The effects of zinc toxicity on the growth and the photosynthetic activities of four Datura species (Datura metel, Datura innoxia, Datura sanguinea, Datura tatula) were studied using various ZnSO4 concentrations (0, 1, 2.5 and 5 mM) added in the Coic Lessaint solution. Growth, photosynthesis, chlorophyll fluorescence and chlorophyll concentration were measured after 20 days of zinc stress. These parameters were severely reduced by this heavy metal. The zinc excess involves the stomate closing, the increase of CO2 concentration in the leaves, the inhibition of certain enzyme of the Calvin cycle, a degradation of photosystem and the chlorophyll decomposition. These phenomena allow the decrease of the net photosynthesis to be partially explained. These key parameters to assess photosynthetic performance allow the plants to be classified according to their resistance to zinc. Compared with the three other species, D. innoxia showed a very strong capacity to protect itself against toxic zinc concentrations; a large amount of ZnSO4 (5 mM) was required to inhibit 43% of the photosynthesis.

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.

Length of time in tissue culture can affect the selected glyphosate resistance mechanism.

Usually, stepwise selection of plant suspension cultures with gradually increasing concentrations of the herbicide glyphosate results in the amplification of the target enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS; EC 2.5.1.19) gene that leads to resistance by increasing EPSPS mRNA and enzyme activity. We show that glyphosate selection with newly initiated suspension cultures can produce resistant lines with resistance mechanisms other than gene amplification and that usually as the cultures age gene amplification becomes the predominant mechanism. Gene amplification did not occur in 3 lines selected from 5-month-old Datura innoxia Mill. cultures but did occur in all 10 lines selected after 52 months. Selection with Nicotiana tabacum L. (tobacco) less than 5 months old produced 2 lines out of 24 with no EPSPS amplification while all 17 lines selected from older cultures contained amplified genes. Lines selected from the oldest culture (35 years) also exhibited amplification of several different genes, indicating the expression of different EPSPS genes or an enhanced gene amplification incidence. None of the 15 lines selected from 2 different 5-month-old Daucus carota L. (carrot) lines exhibited amplification while amplification led to the resistance of all 7 lines selected from one of the original carrot lines (DHL) after 3 years. However, the other line (Car4) was exceptional and produced only non-amplified lines (9 of 9) after 8 years in culture. These results show that plant tissue cultures change with time in culture and that several different new mechanisms can result in glyphosate resistance.

Evolutionary ecology of Datura stramonium: equal plant fitness benefits of growth and resistance against herbivory.

This study evaluated how natural selection act upon two proposed alternatives of defence (growth and resistance) against natural enemies in a common garden experiment using genetic material (full-sibs) from three populations of the annual plant Datura stramonium. Genetic and phenotypic correlations were used to search for a negative association between both alternatives of defence. Finally, the presence/absence of natural enemies was manipulated to evaluate the selective value of growth as a response against herbivory. Results indicated the presence of genetic variation for growth and resistance (1--relative damage), whereas only population differentiation for resistance was detected. No correlation between growth and resistance was detected either at the phenotypic or the genetic level. Selection analysis revealed the presence of equal fitness benefits of growth and resistance among populations. The presence/absence of natural herbivores revealed that herbivory did not alter the pattern of selection on growth. The results indicate that both strategies of defence can evolve simultaneously within populations of D. stramonium.

Treatment of domestic wastewater by an hydroponic NFT system.

The objectives in this work were to investigate a conceptual layout for an inexpensive and simple system that would treat primary municipal wastewater to discharge standards. A commercial hydroponic system was adapted for this study and the wastewater was used to irrigate Datura innoxia plants. Influent and effluent samples were collected once a month for six months and analysed to determine the various parameters relating to the water quality. The legal discharge levels for total suspended, biochemical oxygen demand and chemical oxygen demand were reached with the plant system after 24 h of wastewater treatment. Total nitrogen and total phosphorus reduction were also obtained. NH4(+)-N was reduced by 93% with nitrification proving to be the predominant removal process. Significant nitrification occurred when the BOD5 level dropped 45 mg/l. Similar results were obtained for six months although the sewage composition varied widely. D. innoxia develops and uses the wastewater as the unique nutritive source.

Steroidal compounds from in vitro regenerated shoots of Datura metel.

Regeneration of adventitious shoots from young leaves of Datura metel is described. Shoot buds developed on MS medium with 2 mg/l and elongated on hormone-free solid basal medium. The microshoots failed to produce alkaloids, but a number of steroidal compounds were detected. The C(28) sterol 3beta,24xi-dihydroxy-ergosta-5,25-dienolide, regarded as the precursor of withanolides and related steroidal lactones, was identified. The withanolide 12-deoxywithastramonolide was also detected.

Study on the weed-crop competition for nutrients in maize.

Considering the effect of crop-weed competition the rate of weed growing, the competitiveness of the occurring weed species and the duration of competition are determining factors. Experiments were carried out on fields in order to collect data on the effect of early weed competition on maize, including the competition for nutrients and the possible rate of nutrient removal by weeds. From 7 sampling areas of the 9.2 ha field weeds and maize samples were collected 1 month after the sowing of maize. We determined the total numbers and the species numbers of weeds by plots. The removed plant species and maize were weighed then dried until the weight balance was reached. The samples were tested for N, P, K and Ca. Comparison was done with the weight and nutrient element content of maize plants taken from the treated, weed-free area. At the same time comparative analyses were made with the mass and nutrient contents of maize plants. There were 12 occurring weed species in this experiment. Based on the rate of weed cover the following species were dominant: Datum stramonium L., Cannabis sativa L., Amaranthus chlorostachis Willd., Chenopodium album L., Chenopodium hybridum L. Our experiments revealed that in the areas being likely to produce high weed populations and showing a considerable high nutrient removal by weeds, the competition between weed plants and maize occurs at an earlier stage of the vegetation period of maize than on fields with moderate weed populations. Weeds have utilised significant amount of nutrients which has been many fold of maize in case of unit area.

[Effect of space flight on ultrastructure in medicinal plant Datura innoxia Mill].

OBJECTIVE: To substantiate the effect of space environment on medicinal plants. METHOD: The seeds of Datura innoxia were set up in retrievable satellites. After returning to earth, the ultrastructural changes were observed by means of electron microscope. RESULTS: In cells of weightless group and hit group, some changes were found in chloroplast grana, starch grains and vesicles in chloroplasts. No obvious changes were observed in nuclei. CONCLUSION: Space environment has some effect on the ultrastructure in D. innoxia, and chloroplast is more sensitive to space environment than other organs.