Inhibition of Deoxyribonuclease Activity in the Medium Surrounding Plant Protoplasts

After 1 hour, exogenous deoxyribonucleic acid was degraded within a culture medium at 25 C (pH 6) containing protoplasts of Daucus carota L. var. sativa. Low temperature incubation (1 C) or the addition of 45 millimolar sodium citrate to the medium eliminated DNase activity for at least 4.5 hours. This DNase activity was not reduced at pH 7 or 9, nor by addition of 200 millimolar adenosine 5′-triphosphate. Techniques were developed to ensure high protoplast plating efficiencies and high regenerative capabilities after low temperature treatment and the addition of sodium citrate to the medium. Results indicated citrate concentrations to 45 mM and 1 C temperatures revealed little or no effect on protoplast regeneration capacities. Protoplast viability was 90 to 95% at the time of plating as determined by phenosafranin staining and an estimated 50 to 60% of these undergo cell division in the solid agar medium.