Morphogenesis and secondary metabolites production in the medicinal plant castilleja tenuiflora benth. Under nitrogen deficiency and starvation stress in a temporary immersion system

Castilleja tenuiflora Benth, is a medicinal plant that synthesizes secondary metabolites such as phenylethanoid glycosides (PhGs), flavonoids, lignans, and iridoid glycosides with pharmacological properties. Nutrient deficiency stress in in vitro culturing of plants has been shown to be a good biotechnological strategy to stimulate secondary metabolism. Our aim was to evaluate morphogenesis (growth and development) and the production of secondary metabolites in C. tenuiflora shoots in response to stress by nitrogen (N) deficiency and starvation in temporary immersion bioreactors. Shoots were grown in B5 liquid medium with 25.74 mM (control), 1.32, 0.66 (deficiency) and 0 mM (starvation) N concentration. In all treatments, matter and shoot multiplication rate (SMR) decreased over 40%, chlorophylls content decreased, roots length increased, and leaves showed chlorosis respect to control. Under 0.66 mM N deficiency, the enzyme phenylalanine ammonia lyase (PAL) was more active (twice more than control) and total phenolic compounds were enhanced. At day 21: maximum concentration of PhGs (verbascoside and isoverbascoside) and iridoids (aucubin) was observed in 0.66, 1.32 and 0 mM N treatments (283 ± 0.5, 90 ± 0.3 and 12.5 ± 0.1 mg g⁻¹ DM, respectively); these concentrations were 175, 143 and 34% greater than respective controls on the same day. Additionally, presence of anthocyanins was evident in stems of all plants developed under N deficiency and starvation. Despite the fact that, the matter, SMR and chlorophylls decreased because N stress deficiency, the production of secondary metabolites was enhanced, demonstrating that in vitro culture in temporary immersion bioreactors under suitable nutrients deficit such as N is an appropriate strategy to stimulate secondary metabolism in C. tenuiflora.