Journal of Experimental Botany, Vol. 63, No. 5, pp.

1791–1798, 2012
doi:10.1093/jxb/err380 Advance Access publication 3 December, 2011

GM CROPS-REVIEW PAPER

Advances and remaining challenges in the transformation of

barley and wheat
Wendy A. Harwood*
Department of Crop Genetics, John Innes Centre, Norwich Research Park, Norwich, NR4 7UH, UK
* To whom correspondence should be addressed. E-mail: wendy.harwood@jic.ac.uk

Received 5 August 2011; Revised 28 October 2011; Accepted 1 November 2011

Abstract
Highly efficient and cost-effective transformation technologies are essential for studying gene function in the major
cereal crops, wheat and barley. Demand for efficient transformation systems to allow over-expression, or RNAi-
mediated silencing of target genes, is greatly increasing. This is due to technology advances, such as rapid genome
sequencing, enhancing the rate of gene discovery and thus leading to a large number of genes requiring functional
analysis through transformation pipelines. Barley can be transformed at very high efficiency but the methods are
genotype-dependent. Wheat is more difficult to transform, however, recent advances are also allowing the
development of high-throughput transformation systems in wheat. For many gene function studies, barley can be
used as a model for wheat due to its highly efficient transformation rates and smaller, less complex genome. An
ideal transformation system needs to be extremely efficient, simple to perform, inexpensive, genotype-independent,
and give the required expression of the transgene. Considerable progress has been made in enhancing
transformation efficiencies, controlling transgene expression and in understanding and manipulating transgene
insertion. However, a number of challenges still remain, one of the key ones being the development of genotype-
independent transformation systems for wheat and barley.

Key words: Barley transformation, genotype dependence, transgene expression, transgene insertion, wheat transformation.

Introduction
Efficient, high-throughput, and cost-effective transforma-
tion technology is vital in order to allow the functional
analysis of genes of potential value in crop improvement
programmes. This application of crop transformation may
not lead to the development of a commercial GM crop but
rather will provide a valuable contribution in the process
leading to the development of improved conventional crops.
However, the direct use of GM technology in wheat and
barley may also have future applications in improving the
ability of these crops to withstand environmental challenges,
in increasing yield, and enhancing nutritional quality.
The two most common approaches utilizing stable trans-
formation technology for the analysis of gene function are
either to over-express the gene of interest or to silence it
using RNA interference (RNAi)-based silencing. RNAi-
based gene silencing technology provides highly specific
gene silencing that has been used extensively to investigate
ª The Author [2011]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
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gene function. However, alternative transient loss of func-
tion assay systems such as viral induced gene silencing
(VIGS) can also be used. VIGS has the advantage of being
more rapid than stable transformation and can be particu-
larly useful for plants that are very difficult to transform.
However, stable (RNAi) gene-silencing systems have a num-
ber of advantages including the ability to obtain a range of
levels of silencing, the ability to control tissue specificity,
and the fact that the silencing is heritable. In this review, the
focus is on stable transformation systems.
Barley is the fourth most important cereal crop in terms of
production. As well as being an important crop in its own
right, it is used as a diploid model for the more complex
hexaploid wheat. The first report describing a large number of
fertile transgenic barley plants was in 1994 (Wan and Lemaux,
1994). In this report, immature embryos were transformed
using microprojectile bombardment. Agrobacterium-mediated
1792 | Harwood

transformation of barley followed in 1997 (Tingay et al., remains one of the key challenges for both wheat and
1997) again using immature embryo explants. Following barley. In this review, each of these features of trans-
this first report of Agrobacterium-mediated transformation formation systems is considered in turn. The aim is not to
in barley, the technology was quickly adopted as the method provide an exhaustive review but to focus on some recent
of choice. A comparison of biolistic and Agrobacterium- advances leading to improved barley and wheat systems and
mediated transformation methods in barley was made by to identify the challenges still remaining.
Travella et al. (2005). It was found that Agrobacterium gave
higher transformation efficiencies as well as giving trans-
genic plants with lower transgene copy numbers and more Improving transformation efficiency
stable transgene expression. A recent report of routine and
There are three areas to consider when attempting to im-
highly efficient Agrobacterium-mediated barley transforma-
prove transformation efficiency, firstly, improving regenera-
tion describes a high-throughput method with an average
tion from the chosen target tissue, secondly, increasing the
transformation efficiency of 25% for the spring genotype
number of transformation events and, thirdly, improving
Golden Promise. (Bartlett et al., 2008).
the ability to select transformation events.
Wheat is considered to be the world’s most important
The first key requirement for a successful transformation
crop but it has lagged behind the other major cereal crops
in terms of development of efficient transformation meth- system is a highly regenerable target tissue. In barley, imma-
ture embryos have been the target tissue of choice (Fig. 1A)
ods. The first successful wheat transformation used micro-
although alternative targets such as microspores (Shim et al.,
projectile bombardment of embryogenic callus tissue (Vasil
2009) and ovules (Holme et al., 2008) have been used. In
et al., 1992). Agrobacterium-mediated transformation tech-
wheat, immature embryos have again been the most widely
niques were first reported in 1997 (Cheng et al., 1997).
used target, although alternatives such as inflorescence tissue
However, despite these first successful reports, the efficiency
(Amoah et al., 2001) and callus derived from mature embryos
of wheat transformation remained low and microprojectile
have been used (Wang et al., 2009) (Fig. 1B). A range of
bombardment remained the method of choice as it generally
gave higher efficiencies than Agrobacterium. Microprojectile culture media components have been evaluated, or the levels
of key nutrients manipulated, to enhance regeneration in
bombardment and Agrobacterium-mediated transformation
different culture systems. For example, Thidiazuron (TDZ)
were compared in wheat and, after large-scale experiments,
has been shown to act as a powerful growth regulator in
higher transformation efficiencies were achieved with Agro-
cereals (Schulze, 2007) with the potential to enhance regen-
bacterium (Hu et al., 2003). In addition, the quality of the
eration. Picloram has also been shown to be beneficial in
transformation events was higher as they had lower trans-
some cereal cultures giving better regeneration than 2,4-
gene copy numbers, thus agreeing with the findings in
dichlorophenoxyacetic acid (2,4-D) (Barro et al., 1999).
barley (Travella et al., 2005). High quality transformation
events are usually considered to be single copy insertions of Lipoic acid, an antioxidant, has also been shown to be
effective at increasing the number of responding embryo-
the gene of interest without additional backbone sequences
genic calluses in wheat transformation experiments (Dan
or other rearrangements and with stable expression of the
et al., 2009). However, simple modifications to culture
transgene over generations. Hu et al. (2003) used immature
media components have also had a very significant effect in
embryos as explants for transformation and, indeed,
enhancing regeneration. Bartlett et al. (2008) developed
throughout the history of the development of transformation
a high-throughput transformation system for barley and,
methodology, immature embryos have been the preferred
during the optimization of the system, changes were made
explant for both wheat and barley. Agrobacterium-mediated
transformation is now able to yield efficiencies up to 30% in to the level of copper in the culture media. It was found that
the addition of extra copper led to the regeneration of
wheat (Risacher et al., 2009). However, Agrobacterium-
double the number of shoots from each immature embryo
mediated transformation is limited to specific wheat geno-
and this, in turn, increased transformation efficiency.
types whereas biolistics methods are applicable to a much
wider range (Sparks and Jones, 2009).
Although high transformation efficiencies are essential to
minimize the effort required to produce sufficient numbers
of independent transgenic plants for analysis, there are
many other desirable features of transformation systems.
Simplicity and low cost is desirable to allow maximum
access to the technology. The ability to obtain the required
level and pattern of expression of the transgene is also
important and, in some cases, it is necessary to control, or
at least to determine and understand, the site of transgene
insertion. High-throughput transformation systems are
usually developed for a single responsive genotype and are Fig. 1. (A) Regeneration from immature embryos of barley cultivar
not transferrable to alternative genotypes. An ideal trans- Golden Promise. (B) Regeneration from mature-embryo-derived
formation system would be genotype independent but this callus of wheat cultivar Bobwhite.

Despite the improvements made by adding or changing the
concentration of various media components, these changes
only improve regeneration in certain genotypes and in certain
culture systems. The regeneration of green plants in tissue
culture is known to be under genetic control and a number of
studies have identified qualitative trait loci (QTL) associated
with the regeneration response. In barley, for example,
Bregitzer and Campbell (2001) described the identification of
QTL for in vitro plant regeneration. Recently, Tyagi et al.
(2010) have identified a number of genes located within QTL
peaks associated with green plant regeneration in barley.
These genes included a ferredoxin-nitrite reductase (NIR)
gene. In rice, a NIR gene has been shown to be involved in
plant regeneration (Nishimura et al., 2005) and high NIR
activity was linked to high regeneration ability when a range
of varieties were examined. Other genes potentially involved
in regeneration response include hormone biosynthesis genes
and genes involved in hormonal response. Ethylene is known
to influence plant regeneration in barley and Jha et al. (2007)
showed that regeneration could be influenced by manipulat-
ing ethylene. For example, addition of 1-aminocyclopropane
1-carboxylic acid (ACC), an ethylene precursor, increased
regeneration in the genotype Morex but did not further
improve regeneration in Golden Promise. Studies such as
these indicate that, in order to improve regeneration efficiency
in a range of genotypes, it is first necessary to understand
the genes involved in the regeneration response and their
expression patterns and then to adapt or tailor the strategies
to improve regeneration to the required genotype.
The second approach to improving transformation effi-
ciencies is to increase the number of transformation events.
Recent studies have almost exclusively concentrated on
using Agrobacterium as the DNA delivery system and
looked at ways of improving the number of transformation
events. In order to do this, target tissues have been subjected
to a range of treatments, for example, vacuum infiltration
and sonication were used to improve transformation in
barley (Shrawat et al., 2007). In rice and maize, it has been
reported that treatment of immature embryos by centrifuga-
tion and heat improves transformation efficiency (Hiei et al.,
2006). The presence of additional virulence genes during
Agrobacterium-mediated transformation of durum wheat
was found to be necessary for successful transformation
(Wu et al., 2008). The addition of certain virulence genes has
also shown beneficial effects in rice transformation (Vain
et al., 2004). Recent studies in Arabidopsis have shown that
the over-expression of some histone genes increases suscep-
tibility to Agrobacterium and thus increases transformation
efficiency (Tenea et al., 2009). It is thought that the histone
proteins have a role in protecting the DNA introduced to
the plant cells. Other plant genes are also known to be
involved in Agrobacterium-mediated transformation and
are reviewed by Gelvin (2010). It is likely that, as our
understanding of the role of different plant genes in the
transformation process increases, there will be new opportu-
nities to exploit this knowledge to increase the frequency of
Agrobacterium-mediated transformation events in wheat and
barley.
Barley and wheat transformation | 1793
After regeneration efficiency from the target tissue and
the number of transformation events has been optimized, it
is still necessary to have an efficient system to identify the
resulting transformation events. Efficient, cost-effective
transformation systems must be able to select transgenic
plants and avoid non-transgenic ‘escape’ plants coming
through the system, as this leads to time-consuming
additional analysis. In a number of cereals, including wheat
and barley, the hygromycin resistance gene provides an
effective selection system that rarely allows escape plants to
survive (Harwood et al., 2009). However, a number of other
selectable marker genes, including the bar gene, conferring
resistance to the glufosinate group of herbicides, are also
effective (Harwood and Smedley, 2009; Wu et al., 2008).
Some selectable marker genes can provide a metabolic
advantage to the transformed cells and this is referred to
as positive selection, for example, the phosphomannose
isomerize gene (pmi). The pmi gene has been used to select
transformed tissues in many species including wheat
(Gadaleta et al., 2006) and has been promoted as being
more desirable than herbicide or antibiotic resistance genes
from a biosafety/regulatory point of view. Where trans-
formation is used simply as a research tool to determine
gene function, the presence of a selectable marker is usually
not a problem. In addition, ‘clean-gene’ technology, which
allows the use of the selectable marker gene of choice, and
then allows the selectable marker to be segregated away
from the gene of interest in the progeny, yielding plants
containing only the gene of interest, is readily available
(Thole et al., 2007). Marker gene elimination has been
demonstrated in barley (Matthews et al., 2001) and in wheat
(Permingeat et al., 2003) and thus efficient selection of
transgenic plants is not now an important limitation.
Controlling transgene expression
The ability to obtain the required level and pattern of
transgene expression is another key requirement for any
transformation system. The first consideration for controll-
ing transgene expression is the choice of promoter. This area
has been recently reviewed by Hensel et al. (2011) where the
authors provide a list of promoters shown to be effective in
the cereals. A number of promoters give good constitutive
expression throughout the plant, for example, the maize
ubiquitin promoter that is widely used in both wheat and
barley. A comparison of gus gene activity in barley under
different promoters is described by Himmelbach et al. (2007)
who found the highest expression levels with the maize
ubiquitin promoter. A range of tissue-specific promoters
have also been shown to be effective in wheat and barley.
Seed-specific promoters were examined in both wheat and
barley (Furtado et al., 2009) and while some showed similar
activity in both species, one pericarp-specific promoter was
only active in barley. This highlights the need for care when
choosing an appropriate promoter to control transgene
expression. The range of available promoters has now been
extended to include those induced by biotic or abiotic stress.
For example, a promoter from a member of the barley
1794 | Harwood
Germin-like GER4 gene cluster has been shown to give high
levels of pathogen-induced expression (Himmelbach et al.,
2010).
In addition to the choice of promoter, the inclusion of
untranslated regulatory elements and of signal peptides also
affects the level and pattern of transgene expression. Signal
peptides can direct proteins to particular subcellular com-
partments and have been utilized extensively to direct
transgenic proteins to appropriate locations within the cell.
The signal peptides used in transgenic wheat and barley are
listed in the review by Hensel et al. (2011).
Another available tool for the manipulation of transgene
expression is to use intron-mediated enhancement. The in-
clusion of an expression-enhancing intron within the trans-
gene coding sequence has been shown to give higher levels
of expression in both wheat and barley. In wheat, the fourth
intron from the maize T3/T7-like DNA-dependent RNA
polymerase (RpoT) gene, enhanced expression when included
at position +165 within the luciferase transgene (Bourdon
et al., 2004). The same intron-containing luciferase transgene,
also gave enhanced expression in barley plants compared with
controls containing a luciferase gene without an additional
intron. However, inclusion of an alternative intron, the first
intron from the Arabidopsis polyubiquitin 10 gene (UBQ10)
gave even greater enhancement of expression in barley with
an almost 3-fold increase over the control plants (Bartlett
et al., 2009). Therefore intron-mediated enhancement offers
an additional tool to optimize the expression of transgenes.
Where gene-silencing (RNAi) constructs are used as
opposed to over-expression constructs, the level of gene
silencing will be affected by choices made in the design of
the construct. Choice of the size and section of the gene to
include in the ‘hairpin’ construct may affect the specificity
and level of silencing (Helliwell and Waterhouse, 2005).
This is in addition to the effect of the promoter and any
other regulatory elements on the activity of the silencing
construct.
Fig. 2. FISH carried out using a biotin or digoxigenin-labelled probe made to fragments of the integrated plasmid, containing the bar
gene, together with a labelled marker probe pTa71, used to identify barley chromosome 5. In the ideogram of chromosome 5H, the
transgene insertion sites are shown by red spots and the pTa71 marker in blue. Individual transgenic line names are given above the
corresponding chromosome pictures and alongside their position on the chromosome ideogram.
Understanding and controlling transgene insertion
Knowledge of the exact genomic location of an inserted
transgene is important for risk assessment, traceability, and
to increase understanding of the transformation process.
Transgene insertion sites have been examined using a variety
of methods. Fluorescence in situ hybridization (FISH) has
been used to provide an efficient method of physically
mapping transgenes in barley (Harwood et al., 2004). When
this method was used to map 23 transgene insertion sites,
the pattern of insertion appeared to be non-random (Salvo-
Garrido et al., 2004) with evidence of clustering of inde-
pendent transgene insertions and insertion in gene-rich
areas of the genome. Figure 2 shows the analysis of trans-
gene insertion using FISH in barley and the distribution of
insertion sites along chromosome 5H. Transgenes can also
be genetically mapped in relation to known genetic markers
(Salvo-Garrido et al., 2004) to provide further evidence of
the location of the transgene insertion.
Despite the availability of physical and genetic methods
to gain information on the site of transgene insertion,
molecular analysis of transgene insertion has yielded the
greatest volume of information. Analysis of the junction
between the inserted transgene and the host plant DNA can
yield valuable information that can inform safety assess-
ment of the particular transformation event. For example, it
can detect unexpected rearrangements at the transgene
insertion site and determine whether the insertion has
disrupted known endogenous genes or regulatory sequen-
ces. It can also show the presence of plasmid backbone
sequences that have been inserted along with the T-DNA.
Wu et al. (2006) showed that, in wheat, approximately two-
thirds of lines contained some vector backbone DNA.
A variety of PCR-based methods are available to isolate
transgene junction sequences (Cullen et al., 2011). The
region of plant DNA adjacent to the transgene is also
referred to as the transgene flanking region. Isolation of
 Barley and wheat transformation | 1795

transgene flanking sequences is far more straightforward in been demonstrated in soybean (Li et al., 2010). Although
transgenic lines generated using Agrobacterium-mediated technical challenges still remain with the technologies for
techniques compared with biolistic-derived lines, as the achieving targeted transgene insertion, these technologies
T-DNA borders provide a starting point for the various should allow the goal of targeted transgene insertion in
DNA walking techniques. T-DNAs may insert within genes wheat and barley to be achieved.
or within their regulatory sequences, thus providing a source
of mutations that can be utilized for the analysis of gene
Genotype dependence in wheat and barley
function. Large T-DNA insertion mutant populations have
transformation
been generated in rice and Arabidopsis (Sallaud et al., 2004),
and these have been extensively utilized in functional One key remaining challenge for most crop transformation
genomics studies. There is no such resource available in systems is to overcome genotype dependence. In barley, the
wheat or barley where the large genome size and the fact most successful, high-throughput transformation systems
that the majority of the genome is made up of repetitive use the spring variety Golden Promise (Bartlett et al., 2008).
elements, mean that generating enough T-DNA insertions to This genotype has excellent regeneration from immature
target a useful number of genes would be an enormous task. embryo target tissues as well as good susceptibility to
It is well known that if a population of independent Agrobacterium infection. An assessment of the transforma-
transgenic lines is developed, each containing a single copy tion efficiency for Golden Promise compared with three UK
of the same transgene, then a range of different transgene malting barley varieties showed that, whereas Golden
expression levels will be observed (Bartlett et al., 2009). This Promise gave transformation efficiencies up to 35%, the
is explained by ‘position effects’, a term used to describe the other three varieties failed to yield any transformation
variation caused by the particular genomic environment of events (WA Harwood, unpublished data). Figure 3A–D
the transgene. Many attempts have been made to target shows examples of the appearance of callus, following
transgenes to specific locations within the plant genome, in transformation, from all four varieties. Callus resistant to
order to remove this source of variation and to deal with the selective agent hygromycin is only seen from Golden
the regulatory concerns that arise due to the inability to
control the exact site of transgene insertion. In 2009, the
first report of the use of zinc-finger nucleases to target
a transgene to a specific locus in a crop plant emerged
(Shukla et al., 2009). In this example, a herbicide tolerance
gene was targeted to a specific location within the maize
genome. Zinc fingers can be used to make specific small
insertions or deletions at target sites, to generate specific
sequence changes using a homologous recombination-based
approach, or to target a transgene to a particular genomic
location (Porteus, 2009). Following the successful demon-
stration of this technology in maize, it should offer a method
for targeted insertion in wheat and barley.
There is an increasing need to introduce multiple trans-
genic traits into crop plants. The grouping of a range of
separately transformed transgenes into a cultivar of choice
can be complicated and time-consuming if a number of
independently segregating traits are involved. A solution
would either be to introduce all the transgenes at once or to
target new transgenes to an existing transgenic locus.
Available methods for the simultaneous introduction of
multiple transgenes to plants are reviewed by Dafny-Yelin
and Tzfira (2007). Naqvi et al. (2010) review successes in
metabolic engineering in plants by the introduction of mul-
tiple genes either using Agrobacterium or direct DNA
transfer methods. For example, corn modified to have
enhanced levels of three separate vitamins by the modifica-
tion of three different metabolic pathways was described by
Naqvi et al. (2009). However, the option to add additional
transgenes to an existing transgenic locus is attractive and Fig. 3. Transformation of (A) Optic, (B) Oxbridge, (C) Tipple, and
more flexible. Ow (2011) reviews the available options for (D) Golden Promise showing the development of transformed
using recombinase-mediated approaches for gene stacking. callus on plates containing hygromycin as the selective agent.
Recombinase-mediated methods for the insertion of addi- (E, F) Callus development without transformation: (E) Golden
tional transgenes at a previous transgene insertion site have Promise, (F) Maythorpe.
1796 | Harwood
Promise and from one immature embryo of the variety
Tipple. However, the Tipple callus failed to regenerate
plants. This demonstrates the challenge of trying to trans-
form a particular commercial genotype. Golden Promise is
a gamma-ray induced mutant of the cultivar Maythorpe
(Forster, 2001). Thus it is possible that a mutation present
in Golden Promise confers the high transformability.
However, when Maythorpe was transformed alongside
Golden Promise it was possible to achieve transformation
efficiencies of 25% in Maythorpe, demonstrating that both
varieties were highly transformable (WA Harwood, un-
published data). Figure 3E and F show the similarity of the
regenerable callus generated from Maythorpe compared
with callus from Golden Promise. This clearly demonstrates
that transformability in barley is under genetic control.
There are examples throughout the literature of particu-
lar wheat and barley genotypes being amenable to trans-
formation. In wheat, more varieties can be transformed
using biolistic techniques than using Agrobacterium. Sparks
and Jones (2009) describe a biolistics protocol that has
allowed the transformation of 35 wheat genotypes while
only a few genotypes can be transformed using Agro-
bacterium. This is probably because, although a number of
genotypes have the ability to regenerate green plants from
immature embryos, only a few of them are also susceptible
to Agrobacterium. There has been one report of a method
for the transformation of barley that is considered to be
genotype-independent. This involves the infection of in
vitro-cultured ovules with Agrobacterium (Holme et al.,
2008). However, isolation of the ovule target tissue is
a skilled procedure and not suitable for a high-throughput
transformation system.
High-throughput transformation systems in wheat and
barley must use a target tissue that is easily isolated and
highly regenerable. For both wheat and barley the most
suitable target is immature embryos. Agrobacterium is cur-
rently the preferred DNA delivery system due to the ad-
vantages offered in terms of low copy number and stability
of transgene expression. As both the regeneration of green
plants from immature embryo target tissues and susceptibil-
ity to Agrobacterium are under genetic control, an under-
standing of the genes involved is probably a prerequisite for
developing strategies to overcome genotype dependence.
Conclusions
Transformation efficiencies in both wheat and barley
continue to increase and this is allowing the demand for an
evaluation of gene function using transgenic tools to be met.
However, the pace of gene discovery is also increasing, with
the availability of more sequenced crop genomes and
improved genomics tools, meaning that even more genes
will need to go through a transformation pipeline to allow
the study of gene function. A range of tools are available to
help achieve the level and specific pattern of transgene
expression required, but there are still gaps in the range of
promoters available for use in wheat and barley that need to
be addressed. Transgene integration has been widely studied
and it is now possible to target transgenes precisely to
particular genomic locations. However, further advances in
this technology are required before it can be used routinely
in wheat and barley. A key remaining challenge is the
genotype dependence of most wheat and barley transforma-
tion systems and this continues to restrict the application of
the technology. It is, however, likely that, by understanding
and manipulating plant genes important in either the plant
regeneration process or in susceptibility to Agrobacterium, it
will be possible to address issues of genotype dependence
and to improve transformation efficiencies further.
Acknowledgements
Support by grant in aid to the John Innes Centre from the
UK Biotechnology and Biological Sciences Research Coun-
cil is gratefully acknowledged. Haroldo Salvo-Garrido is
gratefully acknowledged for contributing Fig. 2 and Eva
Medvecka for Fig. 1. Penny Sparrow is thanked for critical
reading of the manuscript.
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