Asymbiotic in vitro seed culture of Paphiopedilum fairrieanum, P. venustum and Dendrobium densiflorum on low-cost substrata

Asymbiotic germination of immature seeds of orchids represents a valuable method for large-scale seedling production, addressing both commercial demand and the conservation of rare and endangered species. In this study, we explored the efficacy of three low-cost substrates: Polyurethane foam (PUF), Coconut husk chips (CHC), and Tree Leaf litter (TLL), as substitutes for the expensive agar-gelled medium in the in vitro seedling culture of three commercially important orchid species, namely, Paphiopedilum fairrieanum (Lindl.) Stein., P. venustum (Wall. ex Sims) Pfitzer, both endangered Slipper orchids, and Dendrobium densiflorum Lindl. ex Wall. During a 120-day culture period, we recorded germination time, germination frequency, and the proportion of different growth stages on these substrates, comparing them to the agar-gelled medium. All substrates were fortified with half Murashige and Skoog medium, 0.5 mg/L α-Napthaleneacetic acid, 10% Coconut water, and 2% Sucrose. Subsequently, in vitro hardening of rooted seedlings was conducted using composites prepared from cost-effective materials, including charcoal, brick pieces, and coconut husk chips. Further, secondary hardening was done on the same composite that additionally contained garden soil, sand, and a top cover of moss. Among the tested substrates, agar-gelled medium exhibited the highest seed germination and seedling formation for Paphiopedilum, while it performed equally well as PUF for D. densiflorum. Conversely, the germination time for all species was one week shorter when using PUF. In comparison, CHC resulted in a lower germination frequency and prolonged germination time. Although protocorms (stage 3) of all species developed maximally on CHC, only D. densiflorum reached the rooted seedling stage. The performance of TLL was significantly inferior for all species, with none of them progressing to protocorm (stage 3). After primary hardening, ~80% of seedlings of P. fairrieanum and D. densiflorum, and ~70% of seedlings of P. venustum survived. Following 90 days of secondary hardening inside a 70% shade net house, about two-thirds, three-fourths, and half of the total transplants of P. fairrieanum, P. venustum, and D. densiflorum, respectively, thrived in the pots. Our findings suggest that PUF presents a cost-effective alternative for cultivating P. fairrieanum, P. venustum, and D. densiflorum. Additionally, CHC holds promise for low-cost production of their protocorms, which, upon multiplication on suitable media, could be developed into a large number of seedlings.