Bacterial contamination in cryopreservation of coconut zygotic embryos

This experiment was carried out from the end of 2015 as an international cooperative project among the Rural Development Administration (RDA), the Philippine Coconut Authority (PCA) and Bioversity International by Korea's RDA fund for sustainable use of coconut genetic resources and long-term safety preservation. For the implementation of strategic plan for conservation of coconut genetic resources, preparation of microbial-free clean embryos is necessary for embryo culture, material transfer and cryobanking. From the first round of cryopreservation experiments, bacterial contamination of in vitro embryos was one of the main problems to be solved. Hence, it is recommended to develop a system to get clean in vitro embryos and to control the contamination during the course of experiments. The application of nutrient agar medium before preculture was efficient for the detection of bacterial contamination. The 16S ribosomal RNA gene was used for the identification of bacterial strains. Among the contaminants, 20 strains were classified in 15 species of bacteria. There were two strains closely related to Sphingomonas echinoides ATCC 14820T and Bacillus safensis FO-36bT and four strains highly related to Sphingomonas panni C52T. Most of the bacteria identified were considered as non-pathogenic. Endogenously-borne contamination from coconut embryos poses the highest risk during the course of the experiments. Some of the bacteria did not initially appear in nutrient agar upon receipt of embryos but could break out, spread, and contaminate clean embryos both during preculture and post-culture stages. A combination of antibiotics, cefotaxime and rifampicin was applied to reduce the bacterial growth during the germination of embryos. Surface sterilization of embryos assisted with vacuum was successful to decrease the bacterial contamination. In preculture-desiccation procedure, 90% germination and 35% regeneration was achieved in cryopreserved 'Malayan Yellow Dwarf' embryos which were progressively precultured with sucrose 17.5% (1 day), -35% (1 day), and -50% (1 day) followed by silica gel desiccation for 17 h.