In vitro propagation and production of cardiotonic glycosides in shoot cultures of Digitalis purpurea L. by elicitation and precursor feeding

Abstract

Digitalis purpurea L. (Scrophulariaceae; Foxglove) is a source of cardiotonic glycosides such as digitoxin and digoxin which are commercially applied in the treatment to strengthen cardiac diffusion and to regulate heart rhythm. This investigation deals with in vitro propagation and elicited production of cardiotonic glycosides digitoxin and digoxin in shoot cultures of D. purpurea L. In vitro germinated seedlings were used as a primary source of explants. Multiple shoot formation was achieved for three explant types (nodal, internodal, and leaf) cultured on Murashige and Skoog (MS) medium with several treatments of cytokinins (6-benzyladenine-BA; kinetin-Kin; and thidiazuron-TDZ) and auxins (indole-3-acetic acid-IAA; α-naphthaleneacetic acid-NAA; and 2,4-dichlorophenoxy acetic acid-2,4-D). Maximum multiple shoots (12.7 ± 0.6) were produced from nodal explants on MS + 7.5 μM BA. Shoots were rooted in vitro on MS containing 15 μM IAA. Rooted plantlets were successfully acclimatized. To further maintain the multiple shoot induction, mother tissue was cut into four equal parts and repeatedly sub-cultured on fresh shoot induction liquid medium after each harvest. On adaptation of this strategy, an average of 18 shoots per explant could be produced. This strategy was applied for the production of biomass and glycosides digitoxin and digoxin in shoot cultures on MS medium supplemented with 7.5 μM BA and several treatments with plant growth regulators, incubation period, abiotic (salicylic acid, mannitol, sorbitol, PEG-6000, NaCl, and KCl), biotic (Aspergillus niger, Helminthosporium sp., Alternaria sp., chitin, and yeast extract) elicitors, and precursors (progesterone, cholesterol, and squalene). The treatment of KCl, mycelial mass of Helminthosporium sp., and progesterone were highly effective for the production of cardenolides. In the presence of progesterone (200 to 300 mg/l), digitoxin and digoxin accumulation was enhanced by 9.1- and 11.9-folds respectively.

Enhanced Biosynthesis of Withanolides by Elicitation and Precursor Feeding in Cell Suspension Culture of Withania somnifera (L.) Dunal in Shake-Flask Culture and Bioreactor

“…There were numerous reports on precursor feeding strategies in plant cell/organ cultures to improve secondary metabolites production whereas the growth characteristics were considerably reduced upon precursor treatment depending upon the plant species tested. For example, inclusion of cholesterol and squalene in the culture medium confirmed extensive synthesis of digitoxin and digoxin in Digitalis purpurea culture, whereas their addition reduced the biomass accumulation [47]. Addition of mevalonic acid increased production of artemisinin in cell suspension culture of A. annua and its supplementation in the medium reduced the growth properties [38].…”

The influence of methyl jasmonate, cholesterol and l‐arginine on solasodine production in hairy root culture of Solanum mammosum

“…This could be supported by Patil et al. who noted that the decrease in biomass growth and the raise in the production of digitoxin and digoxin as a result of feeding the precursors in the shoot cultures of Digitalis purpurea .…”

“…On the other hand, adding l-arginine on day 20 of the culture (the late exponential growth phase and the early exponential phase for solasodine production) has possibly allowed this precursor to involve fully in the secondary metabolism, producing more solasodine. This could be supported by Patil et al [43] who noted that the decrease in biomass growth and the raise in the production of digitoxin and digoxin as a result of feeding the precursors in the shoot cultures of Digitalis purpurea.…”

Enhanced in vitro production of diosgenin in shoot cultures of Dioscorea deltoidea by elicitation and precursor feeding

“…Sivanandhan et al [27] reported that squalene and cholesterol reduced biomass accumulation in Withania somnifera suspension cultures. In another report, it was found that biomass production was decreased due to the addition of squalene and cholesterol in shoot cultures of Digitalis purpurea [12].…”