HIGH-FREQUENCY, HEAT TREATMENT-INDUCED INACTIVATION OF THE PHOSPHINOTHRICIN RESISTANCE GENE IN TRANSGENIC SINGLE CELL-SUSPENSION CULTURES OF MEDICAGO-SATIVA

WALTER C, BROER I, HILLEMANN D, Pühler A (1992)
MOLECULAR & GENERAL GENETICS 235(2-3): 189-196.

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Abstract
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.
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WALTER C, BROER I, HILLEMANN D, Pühler A. HIGH-FREQUENCY, HEAT TREATMENT-INDUCED INACTIVATION OF THE PHOSPHINOTHRICIN RESISTANCE GENE IN TRANSGENIC SINGLE CELL-SUSPENSION CULTURES OF MEDICAGO-SATIVA. MOLECULAR & GENERAL GENETICS. 1992;235(2-3):189-196.
WALTER, C., BROER, I., HILLEMANN, D., & Pühler, A. (1992). HIGH-FREQUENCY, HEAT TREATMENT-INDUCED INACTIVATION OF THE PHOSPHINOTHRICIN RESISTANCE GENE IN TRANSGENIC SINGLE CELL-SUSPENSION CULTURES OF MEDICAGO-SATIVA. MOLECULAR & GENERAL GENETICS, 235(2-3), 189-196.
WALTER, C., BROER, I., HILLEMANN, D., and Pühler, A. (1992). HIGH-FREQUENCY, HEAT TREATMENT-INDUCED INACTIVATION OF THE PHOSPHINOTHRICIN RESISTANCE GENE IN TRANSGENIC SINGLE CELL-SUSPENSION CULTURES OF MEDICAGO-SATIVA. MOLECULAR & GENERAL GENETICS 235, 189-196.
WALTER, C., et al., 1992. HIGH-FREQUENCY, HEAT TREATMENT-INDUCED INACTIVATION OF THE PHOSPHINOTHRICIN RESISTANCE GENE IN TRANSGENIC SINGLE CELL-SUSPENSION CULTURES OF MEDICAGO-SATIVA. MOLECULAR & GENERAL GENETICS, 235(2-3), p 189-196.
C. WALTER, et al., “HIGH-FREQUENCY, HEAT TREATMENT-INDUCED INACTIVATION OF THE PHOSPHINOTHRICIN RESISTANCE GENE IN TRANSGENIC SINGLE CELL-SUSPENSION CULTURES OF MEDICAGO-SATIVA”, MOLECULAR & GENERAL GENETICS, vol. 235, 1992, pp. 189-196.
WALTER, C., BROER, I., HILLEMANN, D., Pühler, A.: HIGH-FREQUENCY, HEAT TREATMENT-INDUCED INACTIVATION OF THE PHOSPHINOTHRICIN RESISTANCE GENE IN TRANSGENIC SINGLE CELL-SUSPENSION CULTURES OF MEDICAGO-SATIVA. MOLECULAR & GENERAL GENETICS. 235, 189-196 (1992).
WALTER, C, BROER, I, HILLEMANN, D, and Pühler, Alfred. “HIGH-FREQUENCY, HEAT TREATMENT-INDUCED INACTIVATION OF THE PHOSPHINOTHRICIN RESISTANCE GENE IN TRANSGENIC SINGLE CELL-SUSPENSION CULTURES OF MEDICAGO-SATIVA”. MOLECULAR & GENERAL GENETICS 235.2-3 (1992): 189-196.
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