Micropropagation protocol of Nepenthes alata Blanco for the enhancement of secondary metabolite biosynthesis and in vitro bioactivity

Nepenthes alata Blanco is an ornamental carnivorous plant characterized by its unique pitcher morphology and its potential as a source of valuable bioactive compounds. The present study aimed to develop an efficient micropropagation protocol for large-scale in vitro propagation and to evaluate the phytochemical profile and biological activities of the mother plant and tissue culture-derived shootlets. Micropropagation optimal sterilization conditions were achieved using 0.2% mercuric chloride for 20 min. For shootlet induction, Murashige and Skoog (MS) medium supplemented with 1–3 mg/L benzylaminopurine (BAP) recorded the highest numbers of shootlet, while 3 mg/L isopentenyl adenine (2iP) produced highest shootlet length and leaf number. For root induction, half MS free of hormone contains 1 g/L activated charcoal (AC) or 2 mg/L naphthalene acetic acid (NAA) observed the highest rooting%. Successful acclimatization was achieved using peat moss alone or mixed 1:1 (v/v) with sand or perlite, resulting in good plantlet length and high leaf number. Analysis of the mother plant (NA) and seven tissue culture lines (NA1–NA7) including hormone-free (NA1), BAP-treated (NA2–NA4, 1–3 mg/L), and 2iP-treated (NA5–NA7, 1–3 mg/L) samples, showed that among the tissue culture lines, NA2 exhibited the highest total phenolic and condensed tannin contents. NA2 was also associated with enhanced antioxidant activity (DPPH and ABTS) and marked in vitro inhibitory activity against the evaluated inflammation-related enzymes, carbohydrate-hydrolyzing enzymes, and acetylcholinesterase. GC–MS analysis of the hexane-soluble fraction demonstrated variation in the non-polar metabolite profiles of the analyzed samples. The tissue culture lines generally exhibited lower relative abundances of triterpenoids and naphthoquinones and higher relative abundances of fatty acids. These results demonstrate that optimized micropropagation provides an efficient approach for the large-scale propagation of N. alata enhancing the production of bioactive metabolites.