Evaluation of viable regions for successful cryopreservation of orchid protocorms

In Orchidaceae, the protocorm, which forms after seed germination, serves as a crucial genetic resource for plant preservation and breeding. To establish a long-term conservation method for the protocorm, in this study, we developed cryopreservation methods. Protocorms of Bletilla striata, a terrestrial orchid species, were precultured for 3 days in a medium containing 0.3 M sucrose and then cryopreserved using vitrification. Although viable cell staining indicated the presence of many surviving cells in the protocorms, the regrowth rate was extremely low. We categorized the stained regions based on the presence or absence of staining in the shoot apical meristem (SAM). The staining rate in the SAM region was low after cryopreservation, which was considered the cause of the low regrowth rate. To enable cryopreservation of the protocorms, the V-Cryo-plate method was adopted, and the treatment times for the solutions used in dehydration were investigated. Treatment times of 45 min and 2 h for the loading and vitrification solutions were found to be appropriate, respectively. Furthermore, an investigation of the effect of adding plant growth regulators to the preculture medium showed that adding 0.1 µM 1-naphthalene acetic acid resulted in a high SAM survival rate of 88% and regrowth rate of 87%, comparable to that observed with survival cell staining. Therefore, survival in the SAM region is important for regrowth in cryopreserved protocorms. Confirmation of survival regions is an important indicator for bridging the gap between the survival and regrowth rates.