Nutritional requirements of protoplast-derived, haploid tobacco cells grown at low cell densities in liquid medium

Preliminary attempts to define a completely synthetic medium able to support divisions of haploid tobacco mesophyll protoplasts at low initial densities have failed. High protoplast concentrations together with large amounts of naphtaleneacetic acid in the medium (3 mg l(-1) NAA) were required for maximal induction of protoplast division. However, cell suspensions derived from haploid protoplasts after four days of preculture at high initial cell densities could be diluted to densities as low as 1-4 cells ml(-1), provided the concentration of NAA in the medium was lowered to below 0.3 mg l(-1). The optimal NAA supply for low cell density growth was affected by the nature of the nitrogen source.A simple minimal medium which supports the growth of these haploid cells with a plating efficiency of 30-40%, independent of the cell density in the range of 1-4 to 3·10(4) cells ml(-1), has been established. In this medium inositol was the only vitamin stringently required for growth.Growth of cells at low densities was also possible in a medium initially containing 3 mg l(-1) NAA, provided it was conditioned by the growth of protoplasts at high densities. Preliminary experiments with [(14)C]NAA showed that the amount of free NAA remaining in the medium after preincubation at high densities was drastically reduced. Simultaneously, NAA conjugates accumulated in the medium. The implications of these results are discussed.