Cryopreservation of oncidium golden anniversary’s protocorm-like bodies using encapsulation-dehydration method.

Oncidium Golden Anniversary is an orchid hybrid that has high economic value in flower market worldwide. However, this new orchid hybrid is difficult to maintain and cultivate conventionally thus increase the risk of permanent loss. Therefore, cryopreservation technique has been developed for long-term conservation of the orchids. This study was aimed to establish the proliferation of protocorm-like bodies (PLBs) and to develop encapsulation-dehydration method for cryopreservation of Oncidium Golden Anniversary hybrid orchid. The best media for rapid proliferation of PLBs was in half-strength semi-solid MS media supplemented with 20 g/L sucrose and 1 mg/L BAP using either whole PLBs, half-moon PLBs and transverse thin cell layer (tTCL) PLB as an explant. The PLBs produced were used as starting material in encapsulation-dehydration method and analysed by 2,3,5- triphenyltetrazolium chloride (TTC) spectrophotometry test and survivability percentage. Some important parameters have been assessed were PLB size, preculture condition, sodium alginate media, calcium chloride solution, dehydration duration, and the addition of antioxidants. The maximum survivability with 93.3% was obtained when 3-4mm PLB size was precultured on half-strength MS media supplemented with 0.3M sucrose and 0.1μM melatonin for 1 day, encapsulated with 3% sodium alginate contained 0.4M sucrose, polymerized in 100mM calcium chloride solution consisting of 0.056M sucrose, dehydrated using silica gel for 4 hours before stored for 1 hour in liquid nitrogen. The cryopreserved PLBs were thawing rapidly at 40±2°C for 90 seconds and cultured on half-strength MS regrowth media without antioxidants. In order to reveal the damages produced by cryopreservation treatments, biochemical, microscopic and molecular analyses were determined in both cryopreserved and non-cryopreserved PLBs comparing with untreated PLBs. Biochemical analyses showed the fluctuation of antioxidant enzyme activities (CAT, APX, POX) in PLBs at various cryopreservation stages explained the inconsistent outcomes and low survivability post-cryopreservation. Meanwhile, reduction of chlorophyll, carotenoid and porphyrin contents resulted in a reduction of photosynthesis process in the cells. Histological analysis indicated that the main factors affecting the viability of PLBs were a degree of plasmolysis in PLBs due to dehydration and rupture of cells after freezing. Scanning electron microscopy analysis revealed that cryopreserved and non-cryopreserved PLBs shrinking in size while ultrastructural studies of optimized encapsulation-dehydration method show cryopreserved PLBs cells were suffered from damages that resulted in no growth of cells after freezing. Finally, DAMD and ISSR analyses confirmed the occurrence of 7.4% and 7.32% polymorphism, respectively in the cryopreserved PLBs. This research indicates that some further study need to be done to improve regrowth of cryopreserved PLBs.