A simple and reproducible protocol for plant regeneration and cryopreservation of Grammatophyllum specinocum BL.

Abstract

Grammatophyllum specinocum BL. is native to Thailand and is at risk of extinction due to human-caused environmental changes and commercial exploitation. Cryopreservation is an efficient approach for conservation of plant germplasms. The aim of this study was to establish a simple and reproducible protocol for callus induction and plant regeneration, and cryopreservation of Grammatophyllum specinocum BL. The orchid seeds successfully germinated on halfstrength Murashige and Skoog (½ MS) medium. Amendment of 0.5 mg/l 6-benzylaminopurine (6-BA) into MS medium was suitable for callus induction whilst a combination of 1 mg/l 6-BA and 2 mg/l naphthaleneacetic acid (NAA) was effective in promoting plantlet regeneration. Protocorms of G. specinocum were successfully cryopreserved using encapsulation-dehydration method with the following protocol: 24-h preculture of protocorms with 0.75 M sucrose solution in the dark followed by encapsulation of the precultured protocorms in Ca-alginate beads, 24-h pretreatment of encapsulated beads with 0.5 M sucrose solution in the dark, and 8-h dehydration before plunging into liquid nitrogen. Random amplified polymorphic DNA (RAPD) analysis was carried out to detect genetic stability of cryopreserved protocorms and genetic differences not detected between noncryopreserved and cryopreserved protocorms.

Keywords

• cryopreservation
• plant regeneration
• orchid
• RAPD