SEQUENTIAL HORMONE REQUIREMENT FOR GROWTH AND ORGANOGENESIS OF PETUNIA-HYBRIDA PROTOPLASTS-DERIVED CALLI

A procedure to regenerate buds or roots from little calli derived from Petunia hybrida leaf protoplasts has been developed. It comprises three culture periods on simple, wholly defined media. Protoplasts were first plated at high density for 7 days to induce initial divisions. Microcolonies were then plated at low density and grown iittle calli for 10-30 days, either in Petri dishes or in large volume cultures. During this step, auxin and cytokinin concentrations, and interactions between these hormones, influenced markedly the plating efficiency of microcolonies and the capacity of calli to sustain transfer to low osmotic strength media. All calli regenerated buds within 20 days after transfer onto a cytokinin-rich solid medium, in which osmoticum, sugar and cytokinin concentrations have been optimized. Auxin requirement depended on auxin and cytokinin concentrations supplied during the previous growth step. When the calli were transferred onto an auxin-rich solid medium, roots formed mostly from those which had previously grown in the presence of large cytokinin concentrations. These sequential hormone effects on growth and regeneration make this system suitable for further analysis of the physiological determination of competence and for a molecular approach of hormone action on organogenesis. © 1990.