High frequency, heat treatment-induced inactivation of the phosphinothricin resistance gene in transgenic single cell suspension cultures of Medicago sativa.

One descendant of the Medicago sativa Ra-3 transformant T304 was analysed with respect to the somatic stability of the synthetic phosphinothricin-N-acetyltransferase (pat) gene which was used as a selective marker and was under the control of the 5'/3' expression signals of the cauliflower mosaic virus (CaMV) gene VI. In order to quantify gene instability, we developed a system for culturing and regenerating individual cells. Single cell suspension cultures derived from T304 and the ancestral non-transgenic M. sativa cultivar Ra-3, were established. The cells were regenerated into monoclonal calli. In transgenic calli, the phosphinothricin (Pt)-resistance phenotype was retained after more than 2 months of non-selective growth. In contrast, up to 12% of the suspension culture cells grown under nonselective conditions and at constant temperature (25 degrees C) lost the herbicide-resistance phenotype within 150 days. Surprisingly, a heat treatment (37 degrees C), lasting for 10 days, during the culture period resulted in an almost complete (95%) loss of the Pt resistance of the suspension culture cells. However, the frequency of cell division was identical in cultures grown under normal and heat treatment conditions. A biochemical test revealed that no phosphinothricin-N-acetyltransferase activity was present in heat treated, Pt-sensitive cells. The resistance level of the Pt-sensitive transgenic cells was equivalent to that of the wild-type cells. A PCR analysis confirmed the presence of the pat gene in heat treated, Pt-sensitive cells. From these results it is concluded that the Pt resistance gene was heat-inactivated at a high frequency in the M. sativa suspension cultures.