Effects of Cryoprotectants in Combination on the Survival of Frozen Sugarcane Cells

Abstract

Cryoprotection of suspension cultures of sugarcane cells (Saccharum sp.) during freezing to various temperatures was tested using glucose, dimethylsulfoxide, and ethylene glycol at various concentrations, alone and in combinations. Viability of the thawed cells was assessed by triphenyl-tetrazolium chloride reduction, cell growth, and microscopic examination. Enhanced cryoprotection-as much as a doubling in viability value-was achieved by employing glucose and dimethylsulfoxide in mixtures, as compared with the lesser cryoprotective effect of either compound alone, at 1.9 molar total concentration in all cases; the mixture was most effective at a concentration of about 1.9 molar, with a molar ratio of the two components of about 1:3, respectively. Much of the increase in viability value arose from a decrease in toxic effect that came about through mixing the cryoprotective agents. Binary mixtures containing ethylene glycol and either glucose or dimethylsulfoxide were less effective and more toxic than comparable glucose-dimethylsulfoxide mixtures. Use of the optimized latter mixture allowed freezing of these tropical cells to-23 C with little decrease in survival, or to-40 C, still with the capability for delayed growth. Many benefits, experimental and practical, are to be expected from the viable, low temperature frozen storage of plant tissues (1, 14, 26). In attempting to attain this goal, investigators have examined and attempted to understand or imitate the yearly adaptation undergone by temperate zone plants during their natural freeze-hardening process (8, 9). During freeze-hardening, a large number of physical and chemical changes take place within the plant. These include tissue dehydration and the degradation and resynthesis of whole classes of chemical compounds of both low and high mol wt, whose cryoprotective roles are poorly understood (8, 15). In studying the protection of cultured plant cells against freezing damage in vitro, investigators have added chemical agents, singly or sometimes in arbitrary appearing combinations (4, 7, 12, 13, 17, 22). A drawback in the use of cryoprotective agents is a toxic effect on the treated cells, in itself a severe deterrent to their survival. In an attempt to rationalize, systematize, and comprehend the roles of such substances in freezing protection, we initiated an exploration of the effects of single additives at various concentrations, and of combinations of them to maximize the cryoprotective effect while, if possible, minimizing chemical toxicity. A better understanding of the effects of cryoprotective substances on the complex cellular reorientations involved in survival of cell freezing, and also of the origins of freezing damage itself and of natural freeze protection, could be derived from such information. Cultures of sugarcane were used as the test material. As with many other vegetatively propagated crops (and in contrast to those plant species whose pure lines are best propagated through seeds) the seeds of the multiple polyploidic sugarcane cannot be relied upon for a straight line display of characteristics (16, 21). Finding a viable freezing procedure for maintaining identified clonal stocks would fill an immediate horticultural need. Investigation was initiated with Glu,' DMSO, and ethylene glycol, used earlier as a cryoprotective combination (4). The compounds are all low in mol wt but quite different in their chemical character and rate of cellular penetration. These substances , alone and in combination, were studied as cryoprotectants for cane cells grown in liquid suspension culture. While both positive and negative reports indicating synergistic ("complemen-tary") protective effects from the combining of cryoprotectants have appeared in the literature for various plant and animal tissues (2-4, 7, 9, 11-13, 17, 22), the results in this paper support the positive findings. They offer quantitative evidence of more than additive cryoprotection when sugarcane cells are treated with a combination of cryoprotective compounds. MATERIALS AND METHODS The cell cultures used were Saccharum cv. H50-7209 (trispecific hybrid) obtained from Dr. P. H. Moore, USDA, Honolulu. All suspensions were subcultured weekly and maintained on a rotary shaker (150 rpm) at 28 C. The medium for growing cane cells consisted of a modified Murashige-Skoog formula containing 2,4-D (3 mg/liter) and 10lo coconut water (6). Actively growing cells were usually harvested for freezing experiments 6 to 9 days after inoculation. The cell suspension was concentrated to a convenient cell density by decanting supernatant medium, after allowing the heterogeneous mixture of cell clump sizes to settle for 10 min. Aliquots (1-2 ml) from the gently stirred suspension were then distributed into graduated tubes in a routine randomized manner and chilled on ice. An equal volume of cold cryoprotective solution was added progressively in 0.3-ml increments , with stirring, over a period of 15 to 35 min. By the method of Sugawara and Sakai (22), modified, the sample suspensions were immersed in a-10 C bath for 2 min, then nucleated by chilling each sample with an external wedge of dry ice and placed in successive baths as follows:-10 C,-15 C,-23 C,-40 C, and-196 C, 4 min at each temperature, to the final temperatures indicated in individual experiments. After 4 min at the final temperature, samples were thawed by swirling in a 40 C bath just to the point of thawing, then diluted and centrifuged, read for packed cell volume, and washed, all at close to 0 C. The dilution and washing steps were carried out with a simplified medium containing only major inorganic salts and 3% sucrose, added stepwise to make 10 ml. Cell viability (survival) was determined ' Abbreviations: TTC: triphenyltetrazolium chloride; Glu: glucose; DMSO: dimethylsulfoxide. 598