Tropane alkaloids GC/MS analysis and low dose elicitors' effects on hyoscyamine biosynthetic pathway in hairy roots of Algerian Datura species.

Plant secondary metabolites play a major role in plant adaptation to stress. Species belonging to Solanaceae family and Datura genus produce tropane alkaloids: D. stramonium, D. tatula and D. innoxia. These species are cultivated for their hyoscyamine (HS) content, whence the interest of this study to induce transformed roots of these species with strain A4 of Agrobacterium rhizogenes. Hairy roots (HRs) of Datura were established at high frequency by infecting vitroplants. All HRs (343 independent lines) were next employed to study the production of HS and growth. A screening of HRs alkaloid content by GC/MS is performed; it reveals, for the first time, the production of 13 alkaloids by the selected root lines. The selection of high productive line offers an interesting option to enhance the productivity. As HS is the dominant compound, the lines of Datura species were selected for their characteristics for biomass and HS production. The elicitors salicylic acid (SA) and acetyl salicylic acid (ASA) were also used to increase HS production. The results showed that the optimal concentration of the two elicitors (AS and ASA) was 0.1 mM. The highest HS content (17.94 ± 0.14 mg g-1 D.W.) obtained in HRs of D. tatula treated with 0.1 mM of acetyl salicylic acid.

The Desirability Optimization Methodology; a Tool to Predict Two Antagonist Responses in Biotechnological Systems: Case of Biomass Growth and Hyoscyamine Content in Elicited Datura starmonium Hairy Roots.

BACKGROUND: The use of the desirability function approach combined with the response surface methodology (RSM), also called Desirability Optimization Methodology (DOM), has been successfully applied to solve medical, chemical, and technological questions. It is particularly efficient for the determination of the optimal conditions in natural or industrial processes involving different factors leading to the antagonist responses. OBJECTIVES: Surprisingly, DOM has never been applied to the research programs devoted to the study of plant responses to the complex environmental changes, and thus to biotechnological questions. MATERIALS AND METHODS: In this article, DOM is used to study the response of Datura stramonium hairy roots (HRs), obtained by genetic transformation with Agrobacterium rhizogenes A4 strain, subjected to the jasmonate treatments. RESULTS: Antagonist effects on the growth and tropane alkaloid biosynthesis are confirmed. With a limited number of experimental conditions, it is shown that 0.06 mM jasmonic acid (JA) applied for 24 h leads to an optimal compromise. Hyoscyamine levels increase by up to 290% after 24 h and this treatment does not significantly inhibit biomass growth. CONCLUSIONS: It is thus demonstrated that the use of DOM can efficiently - with a minimized number of replicates - leads to the optimization of the biotechnological processes.

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.