ANAEROBIC STRESS IN MEDICAGO-SATIVA CELL-SUSPENSIONS

The metabolic responses of an alfalfa (Medicago sativa clone RL34) cell suspension culture to anoxia stress were determined over a period of 72 h. Sealing a 6-day-old suspension in a test tube caused complete depletion of oxygen within 35 min and caused significant loss of viability. For example, the ability to form somatic embryos was reduced at 8 h; mitochondrial respiration and retention of electrolytes were reduced at 24 h. The pH of the suspension rose during anoxia from pH 5.2 to over 6.0 at 72 h. Ethanol accumulated in the medium to about 9.5 mM at 72 h, as a consequence of increased alcohol dehydrogenase activity. The nitrogenous components also changed. The protein content of the cells declined dramatically after 24 h with some accumulation in the extracellular medium. At the same time, the ammonia content of the medium increased moderately. Free amino acids, primarily arginine and tyrosine, accumulated 4 to 5 fold in the medium, whereas lysine accumulated over 10 fold in the cells. Proteolysis and the accumulation of these basic amino acids is apparently the reason why acidosis is not observed in this culture system during anoxia.