Cryopreservation can be a stable, long-term method of germplasm conservation, but successful application can be challenging for tropical material. To optimize survival and re-growth from cryopreserved tissues derived from protocorm-like bodies (PLBs) of hybrid Cymbidium Twilight Moon ‘Day Light’, the effects of explant type (intact PLBs, half-PLBs, or PLB longitudinal thin cell layers) and various explant treatments were studied. Encapsulation in alginate beads was essential, and intact PLBs were best for cryopreservation, based on survival and ability to form neo-PLBs and/or percentage re-growth. Osmotic hydration of intact PLBs in 2% sucrose for 24 h increased neo-PLB formation and re-growth, with the best responses seen when PLBs were excised from alginate beads prior to re-growth after cryopreservation. Both non-transgenic and transgenic PLBs were amenable to cryopreservation for up to 1 year using these methods. This optimized protocol will improve the viability of hybrid Cymbidium germplasm after long-term cryopreservation.
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