Plantlet regeneration of Paris polyphylla Sm. via thin cell layer culture and enhancement of steroidal saponins in mini-rhizome cultures using elicitors

Abstract

An efficient regeneration protocol for the medicinal plant, Paris polyphylla Sm. was developed through the formation of mini-rhizomes (MRs) using transverse thin cell layer (tTCL) culture technique. MRs were induced from tTCL explants derived from the basal and middle stem portions while apical portion failed to show any kind of response. Highest response percentage (86.6 %) of MRs formation with a maximum fresh weight (1.05 ± 0.08 g) was achieved from basal sections cultured on ½ MS medium supplemented with 0.5 mg/l 6-benzylaminopurine (BAP). MRs transferred to plant growth regulator free medium gave rise to shoot buds that eventually regenerated into plantlets and were successfully acclimatized with a survival percentage of more than 95 % under greenhouse conditions. Quantification through reverse-phase HPLC showed 1.41-fold higher content of total steroidal saponins in MRs cultured on medium supplemented with 0.5 mg/l BAP as compared to the field-grown rhizome. Elicitation of MRs liquid culture with chitosan, salicyclic acid (SA) and yeast extract enhanced the production of steroidal saponins but resulted in reduced growth rate. Highest total steroidal saponins content (87.66 ± 1.66 mg/g DW) was achieved in cultures treated with SA at 50 mg/l after 30 days of elicitation which is 3.6 times higher than the in vivo rhizome. The developed protocol would facilitate the conservation of this valuable medicinal plant and could be used as a ready stock to meet the demands of the pharmaceutical industry for steroidal saponins productions.