Dimethyl sulfoxide can initiate cell divisions of arrested callus protoplasts by promoting cortical microtubule assembly.

A serious problem in the technology of plant cell culture is that isolated protoplasts from many species are reluctant to divide. We have succeeded in inducing consecutive divisions in a "naturally" arrested system-i.e., protoplasts from a hibiscus cell line, which do not divide under standard conditions-and in an artificially arrested system-i.e., colchicine-inhibited callus protoplasts of Nicotiana glutinosa, which do readily divide in the absence of colchicine. In both cases, the reinstallation of a net of cortical microtubules, which had been affected either by colchicine or by the protoplast isolation procedure, resulted in continuous divisions of the formerly arrested protoplasts. Several compounds known to support microtubule assembly in vitro were tested for their ability to promote microtubule assembly in vivo. Best results were obtained by addition of dimethyl sulfoxide to the culture medium. Unlimited amounts of callus could be produced with the dimethyl sulfoxide method from protoplasts which never developed a single callus in control experiments.