LOCALIZATION OF TARGET-CELLS AND IMPROVEMENT OF AGROBACTERIUM-MEDIATED TRANSFORMATION EFFICIENCY BY DIRECT ACETOSYRINGONE PRETREATMENT OF CARROT ROOT DISKS

Localization of target cells for Agrobacterium-mediated transformation in the carrot root disc model has been achieved after inoculation with a disarmed A. tumefaciens strain harbouring a GUS-intron construct. The first GUS positive cells could be detected on both sides of the discs 48 h after inoculation. The transformed cells were always more numerous on the apical side, mainly localized in the intrafascicular cambium and in the immature phloem strands. The kinetics of free endogenous IAA levels on both sides after wounding have been determined, indicating that rapid IAA accumulation on the apical side was not simply due to polar migration from the basal side. Attempts to optimize transformation efficiency were made by pretreating the discs with various concentrations of acetosyringone (AS) and/or naphthalene acetic acid (NAA). Surprisingly, while 25 muM AS applied to bacteria prior to the inoculations was ineffective, the same AS concentration applied as a pretreatment to the discs strongly increased the number of transformed cells in the target tissues and decreased the lag time for the appearance of the first GUS positive cells. NAA pretreatment on the basal side enhanced the AS effect. AS pretreatment was found both to advance the reentry of competent cells with a potential for cell division into the S phase of the cell cycle and to stimulate bacterial attachment to the cell walls. The relationship between transformation efficiency and DNA synthesis in the host cells is discussed. AS treatment of plant tissues prior to inoculation is proposed as a means of increasing the transformation rates.