Isolation of mesophyll protoplasts of Nicotiana rustica and their regeneration into plants flowering in vitro

Protoplasts were isolated from leaves of in vitro grown plants of Nicotiana rustica L. by the one step enzymatic method. With 3% cetlulase in 0.5 M mannitol at 25°C and pH 4.6, within 10-12 h about half the total cells were transformed into protoplasts. The enzyme activity had two pH maxima, one at pH 3.5 and the other at pH 6.5, indicating the presence of isoenzymes. A time-course study at different temperatures indicated that ai 30°C the protoplasts' liberation was quicker but it resulted in their subsequent bursting. The protoplasts were cultured in liquid as well as on agar-jelled medium of Ohyama and Nitsch (1972) supplemented with 2.4-D and benzylaminopurine, each at I mg/1. and 14% sucrose. Regular divisions could be seen only on agar-jelted medium. Colonies on transfer to Murashige and Skoog (1962) medium, containing IAA and kinetin, differentiated into plantlets. These plants, within 2 months, flowered in vitro and set seed of which about 20% germinated