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 wit h a disarmed A. tumefaciens strain harbouring a GUS-intron construct. The first GUS positive cells could be detected on both sides of the di scs 48 h after inoculation. The transformed cells were always more num erous on the apical side, mainly localized in the intrafascicular camb ium and in the immature phloem strands. The kinetics of free endogenou s IAA levels on both sides after wounding have been determined, indica ting that rapid IAA accumulation on the apical side was not simply due to polar migration from the basal side. Attempts to optimize transfor mation efficiency were made by pretreating the discs with various conc entrations of acetosyringone (AS) and/or naphthalene acetic acid (NAA) . Surprisingly, while 25 muM AS applied to bacteria prior to the inocu lations was ineffective, the same AS concentration applied as a pretre atment to the discs strongly increased the number of transformed cells in the target tissues and decreased the lag time for the appearance o f the first GUS positive cells. NAA pretreatment on the basal side enh anced the AS effect. AS pretreatment was found both to advance the ree ntry of competent cells with a potential for cell division into the S phase of the cell cycle and to stimulate bacterial attachment to the c ell walls. The relationship between transformation efficiency and DNA synthesis in the host cells is discussed. AS treatment of plant tissue s prior to inoculation is proposed as a means of increasing the transf ormation rates.