Improved Cryopreservation Using Droplet-vitrification and Histological Changes Associated with Cryopreservation of Madder (Rubia akane Nakai)

An efficient protocol for cryopreservation of madder hairy root cultures has been developed using droplet-vitrification. In previous study, combining loading solution C4 (35% PVS3) and vitrification solution B5 (80% PVS3) was the most effective method. In this study, we tried three types of vitrification solution, B5, A3 (90% PVS2, on ice), and A5 (70% PVS2, on ice). Combining loading solution C4 and vitrification solution A5 (on ice) showed the best regeneration rate in this study. Histological changes of the cells within the hairy root of madder were also observed in different steps. The cells from the hairy roots of the control treatment were full and intact with different size of vacuoles and obvious cell nucleus having a dark nucleolus. After the stage of preparing for cryopreservation (after preculturing, loading, followed by dehydration solution AS or B5), intercellular spaces had become distinct, and within cells, the cytoplasms had become denser and week plasmolyses had appeared. The cell plasmolyses were much more apparent and we measured the degree of plasmolysis by calculating, the area of cell/the area of cytoplasm. The value of plasmolysis degree was the highest in the combination of preculture, loading solution C4, and dehydration solution A5, 1.97. Because the highest regeneration rates appeared in the treatment of AS for 20 min, we could assume that the optimal degree of plasmolysis for cryopreservation might be around 1.97. The changes in cell structure during cryopreservation might be a useful basis for the development of a proper long-term preservation method for madder germplasms.