Plant Molecular Biology 52: 247–258, 2003.
© 2003 Kluwer Academic Publishers. Printed in the Netherlands.
Transgene integration, organization and interaction in plants
, Richard M. Twyman
, Rita Abranches
, Eva Stoger
Rexagen Corporation, Genome Centre, Norwich Bio-Incubator, Colney Lane, Norwich, NR4 7UH, United King-
Department of Biology, University of York, Heslington, York, YO10 5DD, United Kingdom.
Department of Cell and Developmental Biology, John Innes Centre, Norwich Research Park, Colney Lane, Nor-
wich, NR4 7UH, United Kingdom.
Biologie VII, RWTH Aachen, Worringerweg 1, 52074 Aachen, Germany.
Fraunhofer Institute for Molecular Biology and Applied Ecology, Auf dem Aberg 1, D-57392 Schmallenberg,
Author for correspondence: email@example.com
Received 27 November 2001; accepted 24 January 2003
Key words: cereal transformation, clean DNA, ﬂuorescent in situ hybridization (FISH), transgene integration,
It has been appreciated for many years that the structure of a transgene locus can have a major inﬂuence on the level
and stability of transgene expression. Until recently, however, it has been common practice to discard plant lines
with poor or unstable expression levels in favor of those with practical uses. In the last few years, an increasing
number of experiments have been carried out with the primary aim of characterizing transgene loci and studying
the fundamental links between locus structure and expression. Cereals have been at the forefront of this research
because molecular, genetic and cytogenetic analysis can be carried out in parallel to examine transgene loci in
detail. This review discusses what is known about the structure and organization of transgene loci in cereals, both
at the molecular and cytogenetic levels. In the latter case, important links are beginning to be revealed between
higher order locus organization, nuclear architecture, chromatin structure and transgene expression.
The production of transgenic plants is achieved us-
ing two alternative strategies. One exploits the natural
ability of Agrobacterium tumefaciens to transfer DNA
from a resident plasmid into the plant genome (Kado,
1998). The other is a group of unrelated techniques
collectively referred to as ‘direct DNA transfer’, which
includes methods such as microinjection (Crossway
et al., 1986), transformation of protoplasts mediated
by polyethylene glycol or calcium phosphate (Datta
et al., 1990; Jongsma et al., 1987), particle bom-
bardment (Christou, 1992), electroporation (Fromm
et al., 1994) and transformation using silicon carbide
whiskers (Frame et al., 1994). These and other trans-
formation techniques have been reviewed by Twyman
et al. (2002). The only similarity among the di-
rect transfer techniques is that external physical or
chemical factors mediate DNA delivery into the cell.
The integration mechanism of the foreign DNA is
poorly understood, both in the case of Agrobacterium-
mediated transformation and direct DNA transfer. The
position of integration and the structure of the trans-
gene locus can vary considerably among independent
transformants, and each of these factors may have a
profound effect on the level and stability of trans-
gene expression. Of particular interest is the role of
transgene organization in silencing phenomena, and