A study of chlorophyll synthesis in Chlorella vulgaris (3) revealed striking differences in the growth patterns of this alga when cultured in nutrient solutions containing different amounts of magnesium. The differences in growth patterns were so variable and of such magnitude as to make difficult the finding of a suitable basis of comparison for physiological studies. Since Chlorella species are often the subject of such studies, a more exact knowledge of their growth habits seems desirable. The detailed studies of Chlorella made by PEARSALL and LoOSE (7) and MYERS (6) and numerous papers contributed by other workers have done much to advance the understanding of algal growth under various modifications of the culture conditions. This paper limits itself to comparisons of Chlorella growth at different magnesium concentrations. Data on certain aspects of this problem have been previously reported by SAIZEVA (9) and by VAN HILLE (5). Materials and methodsConical 500-ml. Pyrex flasks with sealed-in aeration tubes were autoclaved. Each flask was then filled with 350 ml. of sterile culture medium from a transfer flask in which as much as three liters of medium could be autoclaved in one batch (2). The full nutrient solution was prepared with reagent grade chemicals and contained the following components per liter of solution: KNO3, 10 m.mol.; Ca(NO3)2, 3 m.mol.; MgSO4, 2 m.mol.; KH2PO4, 2 m.mol.; ferric tartrate, 1 ml. of 0.5% solution; the micronutrients B, Mn, Zn, Cu, and Mo, in the amounts used by ARNON (1); glucose, 10 gm.; and Pyrex-redistilled water, to volume. The pH value of the autoclaved solution was 4 to 4.5. This solution was found to give consistently high culture densities, of the order of 200,000 cells per cu. mm. after a growth period of three weeks. In the media with lower magnesium contents, equivalent amounts of K2SO4 were substituted for the deficit in MgSO4. Glucose was supplied in the media in order to maintain a source of sugar for all cultures, whether or not they developed a chlorosis, and hence minimize the influence of starvation effects on cell growth.The magnesium concentrations used in these experiments were: 49 p.p.m. (full-magnesium), 2.8, 1.0, 0.5, and 0.2 p.p.m. Triplicate cultures of the first three concentrations were made; the latter two concentrations were run in

We describe a method for the possible cryopreservation of embryogenic callus of Piceaabies and Pinustaeda at −196 °C and the regeneration of somatic embryos from thawed cells of subcultured embryonal–suspensor masses. Piceaabies and Pinustaeda were frozen without cryoprotective agent, in the presence of dimethyl sulfoxide (10%), or in a mixture of polyethylene glycol, glucose, and dimethylsulfoxide (10, 8, and 10% w/v, respectively). Cell masses placed in plastic vials or aluminum envelopes were frozen at 1 °C/min to −30 °C and then immersed for 10 min in liquid nitrogen. Cells were thawed rapidly and placed on modified MS subculture medium. Six to seven somatic embryos per gram of fresh weight were regenerated from each piece of frozen cell mass as compared with 12–13 embryos per gram from unfrozen cells. Post-thaw cell growth was inhibited initially by up to 5 weeks. Inhibition was reversed after the third 10-day subculture. Results suggest that the long-term storage of embryogenic cell lines in liquid nitrogen may be feasible for tree improvement programs in circumstances where testing of progeny may take several years.

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 triphenyltetrazolium 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 -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 METHODSThe 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 gr...

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