Insights into the phytochemical accumulation, antioxidant potential and genetic stability in the in vitro regenerants of Pholidota articulata Lindl., an endangered orchid of medicinal importance

The present study enumerates an efficient and genetically stable mass multiplication protocol through in vitro culture technology with enhanced production of secondary metabolites for an endangered orchid Pholidota articulata Lindl. Higher frequency plantlet regeneration of P. articulata was accomplished in Gamborg B 5 medium supplemented with chitosan as compared to the other conventional plant growth hormones used. Chitosan-treated seeds showed the highest germination (97.06%) and the developing plantlets cultured in medium supplemented with 4 mg/l of chitosan showed the best rooting (37.33/ plantlet), shooting (8.67/ plantlet), and high acclimatizing ability (83.33%). Mass scale propagation of Pholidota articulata was obtained in B 5 medium supplemented with chitosan using axillary buds as secondary explants from the in vitro – raised plantlets. These micropropagated plantlets were assessed for clonal fidelity using inter-specific sequence repeats (ISSR) and start codon targeted polymorphism marker (SCoT) and were found to be genetically uniform wherein 91.8% of monomorphism was recorded. A comparative analysis of secondary metabolites and antioxidant potential among the in vivo and in vitro plants revealed a higher accumulation capacity in chitosan-treated plantlets. An interrelationship between phytochemicals’ accumulation and chitosan concentration in the medium was deduced using the matrix plot which showed that 4 mg/l of chitosan was optimum for the accumulation of phytochemicals. Also, a correlation between the accumulated phytochemicals and the antioxidant activity in P. articulata could be shown using the Pearson correlation and Principal Component Analysis. The comprehensive in vitro regeneration protocol developed in this study can be of enormous importance for both ex-situ conservation and harvest of various bioactive compounds from P. articulata thereby reducing the pressure on its natural populations.