MORPHOGENESIS AND SECONDARY METABOLITES PRODUCTION IN THE MEDICINAL PLANT Castilleja tenuiflora Benth. UNDER NITROGEN DEFICIENCY AND STARVATION STRESS IN A TEMPORARY IMMERSION SYSTEM

Abstract

G. Inei-Shizukawa, H. A. Velasco-Bedrán, G. F. Gutiérrez-López and H. Hernández-Sánchez Abstract 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. Keywords: abiotic stress, Aucubin, nitrogen starvation, phenylethanoid glycosides, RITA® bioreactor. ResumenCastilleja tenuiflora Benth., es una planta medicinal que sintetiza metabolitos secundarios como feniletanoides glicosilados (PhGs), flavonoides, lignanos, e iridoides; que presentan actividad farmacológica. El estrés por deficiencia de nutrientes en el cultivo de plantas in vitro ha mostrado ser una estrategia biotecnológica apropiada para estimular su metabolismo secundario. En este trabajo se evaluaron la morfogénesis (crecimiento y desarrollo) y la producción de metabolitos secundarios en brotes de C. tenuiflora en biorreactores de inmersión temporal expuestos a estrés por deficiencia y ausencia de nitrógeno (N). El estrés se aplicó a un cultivo de los brotes cultivados en medio líquido B5 con 25.74 mM (control), 1.32, 0.66 (deficiencia) y 0 mM (ausencia) de N. En comparación con el control, todos los tratamientos mostraron una disminución de más del 40% en la biomasa y elíndice de multiplicación de brotes, el contenido de clorofila disminuyó, la longitud de las raíces aumentó y las hojas mostraron clorosis. En defi...

Trichoderma Asperellum, an inoculant for production of steviol glycoside in Stevia Rebaudiana bertoni plants micropropagated in a temporary immersion bioreactor

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Production of secondary metabolites using tissue culture-based biotechnological applications

“…As examples of recent studies using bioreactors, the followings can be given: phenolic acids, flavonoids and dibenzocyclooctadiene lignans in Schisandra chinensis ( Szopa et al., 2018 ; Szopa et al., 2019 ); verbascoside, baicalin, wogonoside, luteolin, luteolin-7-glucoside in Scutellaria alpine ( Grzegorczyk-Karolak et al., 2017 ); flavonoids in Gynura procumbens ( Pramita et al., 2018 ); isoflavonoids in Pueraria tuberosa ( Kanthaliya et al., 2019 ); essential oils, p-cymene, geranyl acetate, δ-cadinene, shyobuone, methyl everninate, alloaromadendrene, ledene oxide (II) in Ledum palustre ( Jesionek et al., 2018 ); phenylethanoid glycosides (verbascoside,isoverbascoside) and aucubin in Castilleja tenuiflora ( Cortes-Morales et al., 2018 ); thapsigargin in Thapsia garganica ( Lopez et al., 2018 ); rosmarinic acid and phenolics in Salvia nemorosa ( Heydari et al., 2020 ); flavonoids (Quercetin, Kaempferide, Epicatechin gallate, quercetin-3-o-glucose, Kaempferol-3-rutinoside) in Orostachys cartilaginous ( Hao et al., 2020 ); antifungal saponins SC-2 and SC-3 in Solanum chrysotrichum ( Salazar-Magallón and Huerta de la Peña, 2020 ); six phenolic acids [rosmarinic acid, methyl rosmarinate cafeic acid hexoside, cafeic acid, salvianolic acid F (I) and salvianolic acid F (II)] and four phenylethanoids (verbascoside, leucosceptoside, isoverbascoside and martynoside) in Salvia viridis ( Grzegorczyk-Karolak et al., 2022 ); and some phenolic acids, flavonoids (diosmin, catechin, rutin, and myricetin), a stilbenoid (resver- atrol) and phenylethanoid glycosides (acteoside and echinacoside) in Scrophularia striata ( Ahmadi-Sakha et al., 2022 ).…”

Callus induction and phytochemical profiling of Yucca carnerosana (Trel.) McKelvey obtained from in vitro cultures

“…Plant cell tissue and organ culture offer a useful biotechnological tool to enhance the production of biomass and secondary metabolites (Cisneros-Tórres et al, 2019;Cortes-Morales et al, 2018;Khan et al, 2019). This technique provides tools such as in vitro mass propagation, leading to greenhouse transfer, which has already been reported for Yucca spp.…”