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THE PLANT CELL, Vol 8, Issue 9 1589-1599, Copyright © 1996 by American Society of Plant Biologists
Approaching the Lower Limits of Transgene Variability
L. Mlynarova, LCP. Keizer, W. J. Stiekema and J. P. Nap
Department of Molecular Biology, Centre for Plant Breeding and Reproduction Research (CPRO-DLO), P.O. Box 16, NL-6700 AA Wageningen, The Netherlands
The inclusion of chicken lysozyme matrix-associated regions (MARs) in T-DNA
has been demonstrated to reduce the variation in [beta]-glucuronidase (GUS)
gene expression among first-generation transformed plants. The residual
variation observed between transgenic plant lines with MARs at the T-DNA
borders was investigated. By definition, any phenotypic variance between or
within genetically identical plants is caused by random or environmental
variation. This variation therefore sets a lower limit to the variation in
GUS activities. The variance of GUS activity in offspring plant populations
of genetically identical individuals was used as an estimate of
environmental variation. For transgenic plants with MARs at the T-DNA
borders, the variation between independent transformants could not be
distinguished from the environmental variation. The variation could be
attributed mainly to the variation in the GUS activity measurement.
Therefore, the MAR element approached the maximal possible reduction of
transgene variability given current technology and sample sizes. The role
of MARs in offspring plants was evaluated by comparing such populations of
transgenic plants for the magnitude of and variation in GUS activity.
Pairwise comparisons showed that the presence of MARs reduced variation in
offspring generations in the same manner as demonstrated for primary
transformants. The populations carrying a doubled cauliflower mosaic virus
35S promoter-GUS gene tended to be more variable than the Lhca3.St.1
promoter-GUS gene-carrying populations. This tendency indicated an
intrinsic susceptibility of the doubled cauliflower mosaic virus 35S
promoter to variation. Homozygous plants were approximately twice as active
as the corresponding hemizygous plants and tended to be more variable than
the hemizygous plants. We hypothesized that the magnitude of environmental
variations is related to a higher susceptibility to transgene silencing.
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