A Simple and Reproducible Protocol for Callus Induction, Plant Regeneration and Cryopreservation of Grammatophyllum specinocum BL.

Grammatophyllum specinocum BL. is native to Thailand and it 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, plant regeneration, and cryopreservation of Grammatophyllum specinocum BL. The orchid seeds successfully germinated on ½ Murashige and Skoog (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 for promoting plantlet regeneration. Protocorms of G. specinocum were successfully cryopreserved using encapsulation-dehydration method with the following protocol: 24-h pretreatment of alginate-encapsulated protocorms with 0.5 M sucrose followed by 8-h dehydration. RAPD analysis was carried out to detect genetic stability of cryopreserved protocorms and genetic differences were not detected between non-cryopreserved and cryopreserved protocorms.