The study focused on finding a suitable cryoprotectant (CPA) and an optimum freezing protocol for the cryopreservation of the endosymbiotic dinoflagellates (Symbiodinium, clade G) of Junceella fragilis wherein the success of experiments is crucial to both scientific and ecology studies. A two-step freezing technique was developed. The viability of the thawed dinoflagellates was assayed using the adenosine triphosphate (ATP) bioassay for the first time and was further confirmed through the culturing of dinoflagellates in vitro. The results suggested that 30 min was the most suitable holding time for the dinoflagellates, and the samples produced highest viability when suspended at 5 cm from the surface of LN2. Results also showed that 1 M methanol with 0.4 M sucrose was the most effective CPA, yielding the highest viability (56.93%). Although cell densities of both cryopreserved and control group suffered an initial decline of culture, the cell densities were maintained throughout the remaining duration. In the present study, the cryopreservation of clade G endosymbiont algae was studied for the first time and the method described here could be applied for future studies on symbiotic algae cryopreservation.

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Cryopreservation has been extensively used in various mega‐industries and has recently been applied in genetic banking for conservation purposes. Compared with conventional cell preservation methods, cryopreservation can maintain the viability of cryopreserved cells for an indefinite time at a relatively lower cost and lesser manpower, with lower probabilities of contamination and genetic changes. This study presents the crucial role of sugar, a cryoprotectant supplement in cryopreservation. Sugar molecules typically interact with the lipid bilayer during the freezing phase to maintain plasma membrane integrity when cells undergo dehydration. When combined with other permeable cryoprotectants such as glucose with methanol and trehalose with dimethyl sulphoxide, sugar prolongs and enhances cellular post‐thaw viability. Moreover, terrestrial and marine organisms have benefited from the inclusion of sugar in the cryopreservation protocol. For wide range of cells such as the sperm, oocytes, embryos and larvae of marine vertebrates and invertebrates, as well as marine algae, cryopreservation with sugar produced positive results compared with cryopreservation without sugar. Not all sugar is beneficial, and the type and concentration of sugar should be applied according to the species. Moreover, the freezing method may also affect the function of sugar. Nevertheless, understanding the role of sugar in cryobiology and conducting a preliminary trial of sugar for cryopreserving cells would benefit future research on cryopreservation.

Quantification by real-time RT-PCR requires a stable internal reference known as a housekeeping gene (HKG) for normalising the mRNA levels of target genes. The present study identified and validated stably expressed HKGs in post-thaw Symbiodinium clade G. Six potential HKGs, namely, pcna, gapdh, 18S rRNA, hsp90, rbcl, and ps1, were analysed using three different algorithms, namely, GeNorm, NormFinder, and BestKeeper. The GeNorm algorithm ranked the candidate genes as follows in the order of decreasing stability: pcna and gapdh > ps1 > 18S rRNA > hsp90 > rbcl. Results obtained using the NormFinder algorithm also showed that pcna was the most stable HKG and ps1 was the second most stable HKG. We found that the candidate HKGs examined in this study showed variable stability with respect to the three algorithms. These results indicated that both pcna and ps1 were suitable for normalising target gene expression determined by performing real-time RT-PCR in cryopreservation studies on Symbiodinium clade G. The results of the present study would help future studies to elucidate the effect of cryopreservation on gene expression in dinoflagellates.