The induction of cell division in plant suspension cultures.

Techniques are described whereby a culture medium can be conditioned by separation from a dense cell suspension either by a sinter or by a dialysis membrane. The enhanced growth promoting activity of the conditioned, as compared with new medium, is revealed by its removal from contact with the dense suspension and inoculation with stationary phase cells at or below the minimum effective density needed for cell growth. To obtain a conditioned medium of high activity, it is necessary to use an appropriate volume ratio of culture medium to conditioning cell suspension and to limit the conditioning period. Conditioning of the culture medium reduces, by comparison with new medium, the minimum effective cell density from approximately 12,000 cells down to 500-1,000 cells per ml. There thus exists a population dependent requirement which is not met by the conditioned medium as it is now prepared. Experiments indicate that this requirement may be met by volatile factors (possibly carbon dioxide) released by high cell density suspensions and which are lost in the transfer of conditioned medium to the test flasks. The retention of the activity of conditioned medium in various situations was studied as a preliminary to work on the chemical basis of conditioning. Results point to the release of amino acids and gibberellin as accounting for a part of the conditioning effect. Another factor is the rise in medium pH from 5.2 to 6.4. Conditioning is not considered to involve reduction of supra-optimal concentrations of medium constituents. An improved new synthetic medium, including these growth factors, lowers the minimum inoculum density from 12,000 to 1,000-2,000 cells per ml. Suggestion are made as to how this medium might be further improved, leading to the ability to initiate cell divisions in cells present at very low densities in a medium of known composition.