Biochemical analyses of Sabin Blue PLBs during cryopreservation by vitrification.

Sabin Blue is an important orchid hybrid that has been grown extensively as cut flower, potted plant and is also popular for its deep purplish blue flowers.  The most efficient long term conservation method of this hybrid is through cryopreservation. Cryopreservation involving the vitrification method consists of explants exposure to highly concentrated cryoprotective solution followed by freezing rapidly in liquid nitrogen. However, these treatments involved highly concentrated cryoprotectant that could incur toxicity to the explants. Hence, cryopreservation protocol requires biochemical analyses in understanding the damages or injuries occurred during cryopreservation treatments. In this study, biochemical analyses revealed a general reduction in chlorophyll, carotenoid and porphyrin content to 0.40 µg/g F W (thawing stage), 31.50 µg/g F W unloading stage and 2230.41 µg/g F W (thawing stage), respectively in comparison to the control treatments. In addition, increased level in proline content were obtained at different cryopreservation stages with highest level (5.42 µmole/g F W) recorded at the PVS2 dehydration stage. Fluctuated outcomes were obtained in catalase (CAT), ascorbate peroxidase (APX) and peroxidase (POX) enzyme activities in PLBs exposed to different cryopreservation stages. Lowest values recorded for CAT enzyme activity were obtained at the dehydration stage (3.94 U/g). Lowest POX enzyme activities were obtained at the dehydration (122.36 U/g) and growth recovery (106.40 U/g) stages. Additionally, lowest APX enzyme activities values were recorded at the thawing (7.47 U/g) and unloading (7.28 U/g) stages. These have contributed to low regeneration of  Sabin Blue protocorm like bodies (PLBs) following cryopreservation. Hence, in the future experimental design, exogenous antioxidant could be included in the cryopreservation procedures to improve the existing protocol.