Development of Cryopreservation Procedure Using Vitrification Method of Cymbidium aloifolium (L.) Sw., Medicinal Orchid

Attachai Trunjaruen, Pitakpong Maneerattanarungroj and Worasitikulya Taratima

Background and Objective: Cymbidium aloifolium is a multipurpose orchid that is used as an ornamental and medicinal plant. This study determined the most effective protocol for protocorm vitrification. Materials and Methods: Protocorms size 2-3 mm were precultured using liquid ND medium containing 0, 0.5 and 1.0 M sucrose for 24 hrs before incubated in loading solution for 20 min and moved to PVS2 then dehydrated for 0, 1, 2, 3, 4 and 5 hrs at 0°C. Treated protocorms were plunged into LN immediately after treatment with PVS2 and kept for 24 hrs. After thawing, cryopreserved protocorms were recovered in unloading solution for 20 min before culture on solid ND medium. Suitable conditions for protocorm cryopreservation by vitrification were identified to estimate genetic stability using the RAPD technique and Genomic Template Stability (GTS) calculation. Results: Results demonstrated the interaction between sucrose concentration and PVS2 duration with both factors affecting the survival of C. aloifolium protocorms after regrowth on ND medium for 12 weeks. The results reveal that treatment of preculture in 0.5 M sucrose and 2 hrs of PVS2 gave significantly the highest survival percentage (22%) and regeneration percentage (13%). Both survival and regeneration percentages decreased when sucrose concentration and PVS2 exposure period were increased. Results showed differences in RAPD banding patterns between cryopreserved and non-cryopreserved plantlets. GTS value at 79.02% indicated a genetic alteration in cryopreserved plantlets but the variation in morphology was not observed. Conclusion: These finding useful for further development of protocols of C. aloifolium and other Cymbidium orchids.

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