Silver nanoparticles and plant growth regulators improved micropropagation of Anoectochilus roxburghii (Wall.) Lindl. via protocorm-like bodies

A high-frequency micropropagation protocol for Anoectochilus roxburghii (Wall.) Lindl. was developed via protocorm regeneration, induction and proliferation of protocorm-like bodies (PLBs), shoot regeneration, in vitro rooting, and acclimatization. Silver nanoparticles (AgNPs) were used for surface disinfection of floral-buds to regenerate protocorm which was divided into node, intercalary meristem, and internode to induce PLBs under the influence of plant growth regulators - PGRs (auxin and cytokinin), and silver nanoparticles (AgNPs). In addition, the effect of organic supplements (coconut water, potato starch and peptone) on PLB proliferation and shoot regeneration, and the influence of AgNPs on in vitro rooting, acclimatization, and kinsenoside accumulation were also evaluated. After 30 days, flower-bud explants regenerated protocorms a rate of 83.00% and 3.45 protocorms/explant on Schenk & Hildebrandt (SH) medium supplemented with 1.0 mg/L 6-Benzylaminopurine (BAP) or Thidiazuron (TDZ) only after 60 days. PLBs were induced predominantly at the node–intercalary meristem junction from protocorms cultured on SH medium containing BAP, α-naphthaleneacetic acid (NAA), and 1.5 mg/L AgNPs. The highest PLB proliferation (50.33 PLBs/explant) and shoot regeneration (35 shoots/PLB cluster) were achieved on SH medium supplemented with 20% coconut water, 1.0 mg/L BAP, 0.5 mg/L NAA, and 1.5 mg/L AgNPs after 90 days. Plantlet growth (plantlet height, leaf number and width, root number and length, fresh/dry weight, and SPAD index – chlorophyll content) was maximal on SH medium with 2.0 mg/L AgNPs. Plantlets from media containing 2.0 g/L AgNPs were acclimatized in a peat moss: perlite (1:1) substrate and reached 85% survival after 12 months in the greenhouse. Kinsenoside contents increased with cultivation time (3, 6, 9, 12 months) peaking at 2.591% in the whole plant after 12 months. This study establishes an efficient micropropagation protocol for A. roxburghii via protocorm and PLB regeneration.