MEIOTIC STABILITY OF TRANSGENE EXPRESSION IS UNAFFECTED BY FLANKING MATRIX-ASSOCIATED REGIONS

DNA repeats are associated with gene instability and silencing phenome na in plants. Therefore, the presence of a direct repeat of matrix-ass ociated region (MAR) DNA, that considerably reduced position effects b etween independent transformants, may increase (epi)genetic instabilit y. To investigate the influence of such a repeat on the stability of t he expression of embedded transgenes, the meiotic stability of transge ne expression was assessed in eighteen homozygous 1-locus transgenic t obacco lines carrying the kanamycin resistance (NPTII) and the beta-gl ucuronidase (GUS) gene. Half of the lines carry a 3 kb direct repeat o f MAR DNA flanking the transgenes. Large progeny populations, totallin g over a million seedlings, were screened for kanamycin resistance wit h the help of a newly developed high-density seedling screen. Kanamyci n-sensitive seedlings were detected in selfed progeny at a frequency o f 0.5-5.9 x 10(-4). The frequency became as high as 2 x 10(-2) when em bryo development occur-red under heat and/or drought stress. In backcr ossed progeny only, a joint loss of NPTII and GUS gene expression was observed at an average frequency of 2.9 x 10(-5). In selfed and backcr ossed progeny we observed similar frequencies of reversion to kanamyci n sensitivity, indicating that epigenetic silencing mechanisms rather than MAR repeat-related homologous recombination underlie the reversal to kanamycin sensitivity. Different lines, hence different areas of t he tobacco genome, differed in their genetic stability. No significant differences in reversal frequencies were apparent between lines with or without the MAR elements. The use of the MAR repeat is, therefore, not compromised by any increased (epi)genetic instability.