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Much has been written about the enormous potential of genetic modification
for improving the agronomic properties and nutritional value of crops and
for enabling the production of improved raw materials and novel products in
plants. However, coordinate manipulation of multiple genes or traits in
plants is still difficult to achieve, and this hurdle limits the speed of
technological advance, August 2005 by Claire Halpin.

In 2004, only 9% of commercially-grown GM crops contained ?stacked? or
?pyramided? traits developed by introducing two or more novel genes.
However, at the research level, a variety of conventional and more novel
methods for multi-gene manipulation are currently being developed or tested,
presenting the hopeful prospect of soon overcoming one of the major
challenges currently facing plant biotechnology.

Conventional strategies

Probably the most obvious way that two or more transgenes can be introduced
into one plant is to add them sequentially by either (a) crossing a plant
containing one transgene with individuals harboring another transgene, or
(b) by retransforming a transgenic plant with additional transgenes. Many
examples can be drawn to illustrate the success of these strategies1. For
example, sequential crossing has been used to pyramid different pest or
disease-resistance genes into crops, to produce novel proteins such as
secretory IgA antibodies in plants, to introduce new biochemical pathways
for biodegradable plastic biosynthesis, or to effect complex manipulations
to existing pathways for lignin biosynthesis or fruit ripening.
Retransformation has also been used in species as diverse as Arabidopsis and
trees to successfully introduce or manipulate biochemical pathways
controlling flower color or salinity tolerance, and pathways involved in
producing starch, fatty acids, and lignin. However, iterative procedures
such as these are severely limited by the fact that the introduced
transgenes can segregate in subsequent generations, as they are not linked,
but sited at different, random loci in the plant?s genome. Significant time,
effort, and cost will be associated with maintaining the larger progeny
populations that will be needed to identify plants that retain all
transgenes. Nevertheless, retransformation can have considerable advantages
for species in which segregation is not a problem, because vegetative
propagation methods are usually employed. However using vegetative
propagation means that different selectable marker genes used during rounds
of retransformation cannot be eliminated by out-crossing and will accumulate
in the plants, potentially creating a significant hurdle for regulatory
approval and public acceptance. Recently, several systems that enable the
removal of selectable marker genes2 or that allow for transformation without
the use of selectable markers have been developed that may help to overcome
this limitation.

Perhaps the most promising approach taken to date to introduce multiple
transgenes into plants has been the strategy of co-transformation. Multiple
transgenes on different T-DNAs (harbored together or separately within one
or several Agrobacterium strains) or multiple DNA fragments3 (if using
biolistics) are inoculated or bombarded together onto plant tissues. The
technique is quick, works with a variety of species, and has the major
advantage that transgenes tend to co-integrate at the same chromosomal
position and will therefore be inherited together in most progeny.
Co-transformation has already been used commercially to produce maize with
stacked herbicide tolerance and insect resistance traits. It was also used
to engineer ?Golden rice?, increase isoflavone levels in soybean, produce
rice resistant to multiple insects, concurrently improve several wood
quality traits in trees, and to allow Arabidopsis to produce a biodegradable
plastic co-polymer. Some reports suggest that co-transformed transgenes tend
to integrate as high copy number repeats that might promote transgene
silencing or complicate the regulatory approval process. Not enough work has
yet been done to determine whether this will be a real problem when working
with large numbers of transgenes, but finding transgenics with stable
expression and relatively simple integration patterns is not a problem when
small numbers of transgenes are involved. Techniques for resolving loci with
complex integration patterns, via recombination, have been tested in wheat
and may offer a future solution to this problem. Similarly, more work is
needed to develop methods that would promote coordinate expression of
multiple co-transformed genes. Existing literature, though sparse, suggests
that as the number of co-transformed genes increases, so does the difficulty
in identifying plants expressing all transgenes at effective levels.

Another strategy that has been used with great success to stack traits in
commercial GM crops has been to physically link two or more genes by
positioning them contiguously on DNA that will transfer as a single entity
into a plant (i.e., on a single T-DNA for Agrobacterium-mediated
transformation). At a research level, as many as four or five genes enabling
biodegradable plastic production have been linked within one T-DNA and
introduced into Arabidopsis or oilseed rape. The practical problems
associated with assembling large multi-gene cassettes are being addressed by
the development of several new vector systems in which individual gene
cassettes constructed in unidirectional shuttle vectors can be easily
transferred to special plant transformation vectors that have been
engineered to contain an array of rare endonuclease cleavage sites matching
those of the shuttle vectors. Large numbers of genes might also be
transferred by assembling several T-DNA cassettes, each of moderate size
(harboring just two or three linked genes), and by co-transforming these
different T-DNAs. However even physically linked genes are not necessarily
going to be coordinately expressed at similar levels. In addition, factors
such as the number of transgenic loci, the number of insertions at a given
locus, and the stability of each locus can influence transgene expression.
Repeated sequences within transgenes, either in coding or regulatory
regions, can also lead to variability in expression by activating gene
silencing. Current opinion is divided on whether flanking linked genes with
matrix-associated regions can improve coordinated expression and/or reduce
transgene silencing.

Novel strategies

Several alternative novel strategies to express multiple genes from a single
promoter have been developed and may present the best prospects for ensuring
coordinated expression of introduced genes in plants.

Plastid transformation holds tremendous promise as a way of easily
expressing polycistronic constructs (indeed entire bacterial operons) in
plants4. Extremely high levels of protein production can be achieved.
However, relatively few crop plants can yet be transformed in this way, and
the system will never be suitable for proteins requiring certain
post-translational modifications or targeting to other sub-cellular
locations.

Internal ribosome entry sites (IRESs) are viral sequences that can directly
recruit ribosomes to internal positions within mRNAs and initiate
translation in a cap-independent manner. IRESs have been used in gene
therapy research in animal systems and a few reports describe their use in
polycistronic constructs in plants. Limitations of the strategy are that,
although cistrons expressed in this way are coordinately regulated, they are
expressed at different levels due to the relative inefficiency of internal
initiation and IRES-mediated expression can be lower than normal
cap-dependent expression.

The expression of multiple proteins can also be coordinated by expressing
them initially in a single open reading frame as a polyprotein that could
subsequently be processed into component polypeptides. Several research
groups have tested the value of connecting protein sequences via short
linker sequences that are substrates for plant proteinases. However this
approach depends on temporal and spatial coincidence between the expression
of the polyprotein and the endogenous plant proteinase that will process it,
and this limits the applications for which this strategy will be useful.
Several groups have constructed chimeric polyproteins in which a processing
protease (the potyvirus NIa protease) is encoded within the polyprotein
itself. By separating coding sequences by the protease?s heptapeptide
cleavage sequence, discrete polypeptide products can be processed
post-translationally. Polyprotein cleavage is efficient but expression
levels can be low, and targeting of component proteins to different
subcellular compartments is complicated if polyprotein translocation occurs
more rapidly than polyprotein cleavage. An alternative polyprotein strategy
separates component polypeptides by the 20 amino acid 2A peptide of certain
picornaviruses. This novel peptide mediates an intra-ribosomal ?skip? during
translation so that a peptide bond is not formed at the end of the 2A
sequence, yet translation continues. Thus 2A peptides can effect
co-translational polyprotein ?cleavage? allowing unimpeded subsequent
targeting of individual polypeptides to different sub-cellular
compartments5. Chimeric polyproteins incorporating 2A have been widely
tested in eukaryotic systems including mammalian, human, insect, yeast, and
fungal cells, and several reports now describe their successful use in
plants.

Perspectives

An increasing diversity of conventional and novel techniques are being used
to stack genes and/or traits in transgenic plants and some have already been
used in commercial production. All methods have individual advantages and
limitations and further refining and supplementation of this ?toolkit? is
still necessary in order to provide more durable and cleaner transgenic
technologies for the future. Advancing the technologies for multiple gene
manipulation in plants is a major challenge facing 21st century plant
biotechnology. Meeting this challenge is essential if the full opportunities
for GM crops to effect revolutionary change in agriculture, industry,
nutrition, and even medicine are to be realized.

Acknowledgements

The author?s research is supported by the UK Biotechnology and Biological
Sciences Research Council (BBSRC).

References

1. Halpin C. (2005) Gene stacking in transgenic plants ? the challenge for
21st century plant biotechnology. Plant Biotech. J. 3, 141-155
2. Hare PD & Chua NH. (2002) Excision of selectable marker genes from
transgenic plants. Nat. Biotechnol. 20, 575-580
3. Altpeter F et al. (2005) Particle bombardment and the genetic enhancement
of crops: myths and realities. Mol. Breeding 15, 305-327
4. Bock R & Khan S. (2004) Taming plastids for a green future. Trends
Biotechnol. 22, 311-318
5. El Amrani A et al. (2004) Coordinate expression and independent
subcellular targeting of multiple proteins from a single transgene. Plant
Physiol.135, 16-24

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