Short-term cold storage of encapsulated somatic embryos and retrieval of plantlets in grey orchid (Vanda tessellata (Roxb.) Hook. ex G.Don)

Abstract

Vanda tessellata (Roxb.) Hook. ex G.Don (grey orchid) is a horticultural and medicinally potent, and conservation prioritized orchid species. The present study investigates asymbiotic seed germination, callus induction, somatic embryogenesis, encapsulation of somatic embryos (SEs), and retrieval of plantlets of V. tessellata from the artificial seeds after short-term cold storage at − 4 °C for 12 months. Seeds cultured on full strength Murashige and Skoog’s (MS) medium containing 2.0 mg L−1 6-benzylaminopurine (BAP) had the highest frequency of asymbiotic seed germination (94%) as compared with half- and one-fourth strength MS media supplemented with various concentrations and combinations of 6-furfurylaminopurine (kinetin) and BAP. The protocorm like bodies (PLBs) gained dedifferentiation and proliferated into embryogenic calli on MS medium containing 2.0 mg L−1 BAP and 0.5 mg L−1 indole-3 acetic acid (IAA). Somatic embryos (SEs) were differentiated from the callus when cultured on MS medium in combination with 1.0 mg L−1 BAP and 0.5 mg L−1 IAA. The morpho-anatomical elucidations authenticated somatic embryogenesis and various developmental stages of SEs from embryogenic calli. Short-term cold storage of SEs was carried out by encapsulating with 2% sodium alginate and 100 mM calcium chloride and stored under dark at − 4 °C for 12 months to conserve this valuable germplasm. The stored artificial seeds were germinated (91%) on MS medium incorporated with additives and 0.5 mg L−1 each of BAP, kinetin, and IAA. Complete retrieval of plantlets was achieved in 8 week with formation of shoots (4.2 cm average length) and roots (2.5 cm). The well developed plantlets were acclimatized in the greenhouse with 90% survival rate. Vanda tessellata (grey orchid) is a horticultural and medicinally potent, and conservation prioritized orchid species. A reproducible protocol has been successfully established for asymbiotic seed germination, callus induction, somatic embryogenesis, encapsulation of somatic embryos, and retrieval of plantlets of V. tessellata from the artificial seeds after short-term cold storage at − 4 °C for 12 months.