Cryostorage of Cloned Amino Acid Analog-Resistant Carrot and Tobacco Suspension Cultures

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Abstract
Five clones were isolated from 5 different amino acid analog-resistant D. carota L. cv. Sativa and N. tabacum L. cv. Xanthi cell lines. The individual clones were similar in their resistance to DL-5-methyltryptophan, S-(2-aminoethyl)-L-cysteine, or azetidine-2-carboxylic acid and in their corresponding free amino acid levels. The cell suspensions were stored using a controlled freezing rate at -196.degree. C with concentrations up to 40% of the 4 cryoprotectants: manitol, proline, dimethylsulfoxide, glycerol and combinations of dimethylsulfoxide and glycerol. No less than 55% post-thaw viability, determined by phenosafranin dye exclusion, was obtained after storage using a cryoprotectant mixture of 10% glycerol and 10% dimethylsulfoxide. Growth of the cryostored cells could be obtained consistently only by using feeder plate methodology with this combination of cryoprotectants. Post-thaw viability and percentage of cells demonstrating growth, as estimated by growth kinetics, were similar. This indicates that little selection occurred during the freezing and recovery process. The amino acid analog-resistant traits were unaltered following cryostorage. Suspension cultures of D. innoxia Mill. were frozen similarly with maximum post-thaw viability of 38%, but subsequent growth was not obtained. Protoplasts of D. innoxia, tobacco and carrot were also cryostored using a mixture of 10% dimethylsulfoxide and 10% glycerol as cryoprotectants. Viabilities of no less than 40% were obtained, only the carrot protoplasts regenerated cell walls and underwent cell division.