Enhanced protoplast division by media ultrafiltration

Leaf protoplasts of lucerne (alfalfa- Medicago sativa L.) and tobacco ( Nicotiana tabacum L.) were cultured in standard liquid culture medium and in medium that had been passed through a 10-kDa cut-off ultrafilter. The proportion of lucerne cells that divided was increased by 50–400% in ultrafiltered medium over that in standard medium. The effect was seen in six independent experiments performed over a period of 9 months. The inhibitory effect was detected in each of four separate batches of glucose that were examined from the same manufacturer. Ultrafiltration of medium used to culture tobacco protoplasts gave a 10% increase in the proportion of cells that divided. High molecular weight inhibitors of protoplast division were detected as contaminants in a number of components of the lucerne protoplast culture medium, including glucose, minor sugars and sugar alocohols, coconut water and casein hydrosylate. Gel filtration showed that the major inhibitory contaminant in glucose had a molecular weight greater than 200 000.