mechanisms and control of genetic recombination in plants
Biotechnology relies to a large extent on our ability to introduce foreign
genes into cells. A major problem with present day technology is the non-predictability
of the integration of such transgenes. DNA introduced into plant cells
mostly integrates at random, i.e. at non-predetermined positions of the
genome. The biological process ultimately responsible for random integration
is known as illegitimate recombination. DNA integrated at random frequently
contains multiple copies and often copies are scrambled. Multiple copies
also often induce gene silencing and hence instability in the expression
of the introduced genes. In addition, the DNA integrates at loci of unknown
stability and capacity for expression and randomly integrated copies may
induce unpredictable and undesirable mutations in the host genome. Gene
targeting is based on homologous recombination and thus allows the integration
of DNA at predetermined positions and therefore allows precision manipulation
of genes. To make gene targeting commonly available as a tool for plant
biotechnology, illegitimate and homologous recombination pathways need
to be studied so as to allow the development of reliable and efficient
gene targeting methods.
aims to identify and analyse the control and molecular mechanisms of genetic
recombination in plant cells. An understanding of these processes is expected
to provide the means to:
the chromosomal location and structure of transgenic loci;
2) control the level of recombination;
3) understand the way in which plants sense chromosomal integrity and
Approach and methodology
Genetic and biochemical methods were used to identify key parameters or
key genes that determine the efficiencies of homologous and non-homologous
recombination in plant cells. Factors stimulating the homologous pathway
were over-expressed and the expression of factors promoting non-homologous
recombination was suppressed. The effects on gene targeting were then
studied in model systems.
Main findings and outcome
Homologous and non-homologous recombination pathways govern the fate of
DNA after transformation. To improve gene targeting, either the non-homologous
pathway needs to be suppressed or the homologous pathways must be stimulated.
Several avenues were pursued to stimulate the homologous route. Mutants
were generated which exhibit elevated levels of homologous recombination
and these mutants are currently being analysed. DNA damage repair and
homologous recombination share part of their pathways. Therefore mutants
sensitive to DNA damaging agents were isolated. One of the genes isolated
this way contributes to the maintenance of chromosomes and has also been
shown to be important for efficient homologous recombination in plants.
Over-expression of a bacterial recombination protein has been shown to
stimulate homologous recombination in plants. Despite this, the protein
does not increase the frequencies of gene targeting when Agrobacterium
is used for transformation, presumably because this recombination protein
cannot access its substrate when the Agrobacterium DNA delivery
system is used. In line with this observation, once recombination is initiated,
however, this protein improves the precision of the recombination reaction.
To elucidate the non-homologous pathway of recombination, various plant
genes involved are being analysed. To facilitate this study, a yeast system
was developed which allows the analysis of the T-DNA transfer process
in yeast. This system makes the power of yeast genetics and its repertoire
of recombination genes available to plants. A key discovery was that the
non-homologous pathway apparently is suppressed in tissues with high rates
of homologous recombination. This finding allows a rational approach to
the suppression of non-homologous recombination in the cells routinely
used for plant transformation.
Although our understanding of the general biology of recombination in
plants is constantly improving, we still lack the knowledge for precision
engineering of plants' genes. A further investment in basic mechanisms
of recombination will be necessary to develop gene targeting as a tool
for the genetic manipulation of plants.
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SMC-like protein is required for efficient homologous recombination
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October 1997 September 2000
Université Blaise Pascal
Max-Planck-Institut für Züchtungsforschung
University of Ghent (BE)
Project website address
B. Hohn, J. Paszkowski
Friedrich Miescher Institute
Leiden University (NL)
Wageningen Agricultural University (NL)
Institute for Plant Genetic and Crop Plant Research
Institute of Plant Breeding