REGENERATION OF PLANTS FROM CULTURED GUARD-CELL PROTOPLASTS OF NICOTIANA-GLAUCA (GRAHAM)

Experiments were performed to optimize conditions for culturing guard cell protoplasts of Nicotiana glauca and to determine whether cultured guard cell protoplasts were totipotent. Guard cell protoplasts were i solated from adaxial epidermal tissue of leaves of Nicotiana glauca gr own under fluorescent light (800-900 mu mol m(-2) s(-1) of photons of photosynthetically active radiation). To increase the probability that guard cells were of uniform osmotic potential at the time leaves were harvested, leaves were collected in darkness, just before the beginni ng of each light period. Protoplasts were cultured in liquid media sim ilar to those used for culturing mesophyll cell protoplasts of Nicotia na tabacum but of modified pH and KCl, CaCl2, sucrose and glycine conc entrations. Concentrations of growth regulators were 0.3 mg 1(-1) alph a-napthaleneacetic acid (NAA) and 0.075 mg 1(-1) of 6-benzylaminopurin e (BAP). Protoplasts were incubated in darkness at 25 degrees C in 8-w ell microchamber slides at a density of 1.25 x 10(5) cells ml(-1). Cel l divisions began within 72-96 h of initiation of cultures. After 96 h , cell survival averaged 57% of the initial number of cells (n, 20; ra nge, 36-84%). After 8-10 weeks of culture, cell colonies were transfer red to a callus initiation medium containing agar and incubated under continuous white fluorescent light (25 mu mol m(-2) s(-1) of photons o f photosynthetically active radiation) for another 8-10 weeks. Green c allus tissue was then transferred to a commercial callus growth medium and incubated under similar conditions. After 8-10 weeks of further g rowth, callus was transferred to a commercial shoot differentiation me dium and incubated similarly. Multiple shoots appeared within 2 weeks. Shoots were transferred to a root differentiation medium and cultured under the conditions described above. When roots were sufficiently de veloped (6-8 weeks), plants were transplanted to soil and grown in a g rowth chamber. We conclude that guard cell protoplasts can be made to survive in culture at high percentages and that cultured guard cell pr otoplasts of Nicotiana glauca are totipotent.