Selected potential encapsulation-dehydration parameters on Dendrobium Bobby Messina protocorm-like bodies using TTC analysis

Protocorm-like bodies (PLBs) of Dendrobium Bobby Messina were cryopreserved using an encapsulation-dehydration technique and analysed for survival using the 2,3,5-triphenyltetrazolium chloride (TTC) spectrophotometry analysis. PLBs with the size range of 1-2 and 3-4 mm were selected from four-week old cultures and pretreated with (0.0, 0.2, 0.4, 0.6, 0.8, 1.0, 1,2 M sucrose) and/or (0.0, 0.2, 0.4, 0.6, 0.8, 1.0, 1,2 M sorbitol) supplemented with half strength semi-solid MS media at 24°C for (0- 5 days) under 16 hours photoperiod. Precultured PLBs were encapsulated in 3 % sodium alginate and 0.1M calcium chloride, both supplemented with half strength liquid MS media and 0.4M sucrose. The beads were osmoprotected in 0.75M sucrose supplemented with half strength liquid MS media on an orbital shaker (110 rpm) at 24°C for 24 hours under 16 hours photoperiod. Osmoprotected beads were dehydrated for (0-10 hours) hours in a parafilm-sealed culture jar containing 50g of oven-sterilized silica gel followed by rapid freezing in liquid nitrogen. After thawing in a 40 °C water bath for 90-120 seconds, the cryopreserved beads were cultured on growth recovery media containing half strength semi-solid MS media supplemented with 2 % sucrose. Survival of the cryopreserved PLBs was assessed with the TTC spectrophotometric test. The highest survival of cryopreserved 3-4mm PLBs was obtained through the use of pretreatmen