The amino acid proline has been proven to play an important role in plants exposed to environmental stress (cold, salt and freezing). The interaction of exogenous proline with both NaCl and chilling temperature (4°C) on the growth of embryogenic cell lines of Larch (Larix X leptoeuropeae), Sitka spruce (Picea sitchensis (Bong.) Carr.) and Oak (Quercus robur L.) was investigated. Increasing the exogenous proline concentration increased the growth rate of the cells, both with chilling and salt stress. Freezing tolerance, quantified by K+ leakage, was increased by exogenously added proline. Plasmids expressing P5CS (A'-pyrroline-5-carboxylate synthetase), the rate limiting enzyme in cellular proline production, were introduced separately into Agrobacterium tumefaciens LBA4404. pBI-P5CS contained proline-overexpressing Vigna aconitifolia P5CS cDNA. pBI-P5CSF129A was a feedback-insensitive variant of this. Both vectors contain the NPTII and GUS coding regions that were used for selection of transgenic plants on kanamycin and as a reporter of transformation, respectively. Embryogenic calli of Larch, Sitka spruce and Oak were transformed with these plasmids, a number of transgenic lines isolated, and these examined for proline production, cold, salt and frost tolerance.