Mannitol and sorbitol concentration optimization for effective Epipactis flava Seidenf. in vitro slow growth storage

Epipactis flava Seidenf., a Thai rheophytic orchid, is endangered due to habitat destruction and climate change. In vitro conservation serves as an effective technique for the ex situ preservation of orchid diversity. This study investigated the in vitro storage of E. flava seedlings under slow growth conditions using Murashige and Skoog (MS) medium supplemented with either sorbitol or mannitol individually at concentrations of 1.0%, 2.0%, 3.0%, and 4.0% (w/v) for 24 wk without subculture. The storage medium added with 3.0% (w/v) sucrose was used as the control. After 24 wk of storage, the storage medium containing 2.0% mannitol provided the highest survival rate (97.6%) while the highest leaf formation (70.8%) was achieved with 3.0% sucrose. The addition of 1.0 to 2.0% sorbitol, 1.0% mannitol, and 3.0 to 4.0% mannitol showed the highest shoot bud formation (100%). At the end of 24 wk, the plantlets were transferred to fresh medium for an 8-wk growth recovery. The plantlets derived from the storage medium with 3.0% mannitol exhibited the highest survival rate (73.5%). Conversely, the highest number of shoot buds (9.9 shoot buds per plantlet) was found on plantlets derived from storage medium with 1.0% sorbitol while the highest number of shoots (4.8 shoots per plantlet) was achieved in plantlets derived from 1.0% mannitol. Consequently, these results suggested that the storage medium with 3.0% mannitol is optimal for conserving plantlets for up to 24 wk, and all plantlets could successfully produce shoot buds and shoots during in vitro recovery. Hence, this strategy has potential in the conservation and utilization of this endangered species.