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Protoplasts were isolated from leaves of Pisum sativum L. cv. Sparkle aseptically with pectinase and cellulase and grown in a synthetic medium. Cell wall regeneration was observed microscopically and quantitatively controlled by the change of glucose concentrations and by the use of proteolytic enzyme. The protoplasts increased in size, elongated and regenerated cell walls in the sixth day of culture and gave rise to two cells, which divided repeatedly to form large colonies in 2 weeks. The phenomenon of "budding" was seen in 2 to 3 days of culture. In relation to WLS (wall like structure) formation, protoplast batches showing high frequencies of bud formation also had high WLS formation rates. With the increase of glucose concentrations, the number of protoplast showing bud formation increased but decreased above the value of 0.2 molal glucose. The budding phenomenon was remarkably inhibited in the medium with proteolytic enzyme. When protoplasts were stained with aceto-carmine, freshly isolated protoplasts showed 91% of mononuclear, 8% of binuclear, and 0.7% of polynuclear forms. After 9 days in culture, percentages of bi- and polynuclear forms increased 25.6% and 8.1%, respectively and that of mononuclear forms decreased correspondingly. This phenomenon was due to mitosis, not fusion.
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