Use of green fluorescent protein as a reporter of induced biochemical production in Hyoscyamus muticus root cultures

Agrobacterium-transformed root cultures of Hyoscyamus muticus synthesize antimicrobial compounds when treated with fungal elicitors, thereby providing a potential means of producing these biochemicals in alternative bioreactor systems. In this thesis, it is shown that defense gene induction via fungal elicitation is localized to contacted tissue whereas jasmonic acid, a potential systemic signal produced upon elicitation, stimulates induction in contacted and adjacent, non-contacted tissue. To determine treatment effects upon defense gene induction and localization, Hyoscyamus muticus hairy roots containing the green fluorescent protein reporter gene under the control of the vetispiradiene synthase promoter were generated via root transformation with Agrobacterium tumefaciens. Vetispiradiene synthase is a sesquiterpene cyclase enzyme responsible for the formation of sesquiterpene antimicrobials upon fungal elicitation. Roots constitutively expressing green fluorescent protein were also generated as a control for root transformation. Transgenic roots were exposed to localized treatments of agar-immobilized fungal elicitor, jasmonic acid, ethanol (co-solvent), nutrient medium, and combinations thereof. The induction of the sesquiterpene cyclase promoter in contacted and non-contacted regions was monitored acropetally and basipetally by the production of green fluorescence. Fungal elicitation resulted in promoter induction localized to treated regions only and stimulated preferential formation of the sesquiterpene lubimin. Localized treatment with jasmonic acid induced the promoter in contacted and adjacent, non-contacted regions and induced production of the sesquiterpene solavetivone. Low concentrations of ethanol induced the promoter in non-contacted regions but to a lesser extent than jasmonic acid, although no sesquiterpene formation was detected with ethanol treatment. Combinations of ethanol and fungal elicitor were also able to induce the sesquiterpene cyclase promoter in non-contacted regions. Results suggest that fungal elicitation may stimulate jasmonic acid production but not in sufficient amount to induce signal transport to non-contacted regions in the absence of ethanol. Regarding large-scale biochemical production, fungal elicitation of liquid-submerged systems would provide the comprehensive solid-liquid contacting necessary to induce the localized response observed with this treatment. Because jasmonic acid is capable of inducing product synthesis in non-contacted regions, a liquid-dispersed reactor system with limited solid-liquid contacting efficiency that achieves high root yield may be sufficiently induced by jasmonic acid treatment.