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

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


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:

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

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.

Efficient, high-throughput, and cost-effective transforma- gene function. However, alternative transient loss of func-
tion technology is vital in order to allow the functional tion assay systems such as viral induced gene silencing
analysis of genes of potential value in crop improvement (VIGS) can also be used. VIGS has the advantage of being
programmes. This application of crop transformation may more rapid than stable transformation and can be particu-
not lead to the development of a commercial GM crop but larly useful for plants that are very difficult to transform.
rather will provide a valuable contribution in the process However, stable (RNAi) gene-silencing systems have a num-
leading to the development of improved conventional crops. ber of advantages including the ability to obtain a range of
However, the direct use of GM technology in wheat and levels of silencing, the ability to control tissue specificity,
barley may also have future applications in improving the and the fact that the silencing is heritable. In this review, the
ability of these crops to withstand environmental challenges, focus is on stable transformation systems.
in increasing yield, and enhancing nutritional quality. Barley is the fourth most important cereal crop in terms of
The two most common approaches utilizing stable trans- production. As well as being an important crop in its own
formation technology for the analysis of gene function are right, it is used as a diploid model for the more complex
either to over-express the gene of interest or to silence it hexaploid wheat. The first report describing a large number of
using RNA interference (RNAi)-based silencing. RNAi- fertile transgenic barley plants was in 1994 (Wan and Lemaux,
based gene silencing technology provides highly specific 1994). In this report, immature embryos were transformed
gene silencing that has been used extensively to investigate using microprojectile bombardment. Agrobacterium-mediated
ª The Author [2011]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
For Permissions, please e-mail:
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.
Barley and wheat transformation | 1793

Despite the improvements made by adding or changing the After regeneration efficiency from the target tissue and
concentration of various media components, these changes the number of transformation events has been optimized, it
only improve regeneration in certain genotypes and in certain is still necessary to have an efficient system to identify the
culture systems. The regeneration of green plants in tissue resulting transformation events. Efficient, cost-effective
culture is known to be under genetic control and a number of transformation systems must be able to select transgenic
studies have identified qualitative trait loci (QTL) associated plants and avoid non-transgenic ‘escape’ plants coming
with the regeneration response. In barley, for example, through the system, as this leads to time-consuming
Bregitzer and Campbell (2001) described the identification of additional analysis. In a number of cereals, including wheat
QTL for in vitro plant regeneration. Recently, Tyagi et al. and barley, the hygromycin resistance gene provides an
(2010) have identified a number of genes located within QTL effective selection system that rarely allows escape plants to
peaks associated with green plant regeneration in barley. survive (Harwood et al., 2009). However, a number of other
These genes included a ferredoxin-nitrite reductase (NIR) selectable marker genes, including the bar gene, conferring
gene. In rice, a NIR gene has been shown to be involved in resistance to the glufosinate group of herbicides, are also
plant regeneration (Nishimura et al., 2005) and high NIR effective (Harwood and Smedley, 2009; Wu et al., 2008).
activity was linked to high regeneration ability when a range Some selectable marker genes can provide a metabolic
of varieties were examined. Other genes potentially involved advantage to the transformed cells and this is referred to
in regeneration response include hormone biosynthesis genes as positive selection, for example, the phosphomannose
and genes involved in hormonal response. Ethylene is known isomerize gene (pmi). The pmi gene has been used to select
to influence plant regeneration in barley and Jha et al. (2007) transformed tissues in many species including wheat
showed that regeneration could be influenced by manipulat- (Gadaleta et al., 2006) and has been promoted as being
ing ethylene. For example, addition of 1-aminocyclopropane more desirable than herbicide or antibiotic resistance genes
1-carboxylic acid (ACC), an ethylene precursor, increased from a biosafety/regulatory point of view. Where trans-
regeneration in the genotype Morex but did not further formation is used simply as a research tool to determine
improve regeneration in Golden Promise. Studies such as gene function, the presence of a selectable marker is usually
these indicate that, in order to improve regeneration efficiency not a problem. In addition, ‘clean-gene’ technology, which
in a range of genotypes, it is first necessary to understand allows the use of the selectable marker gene of choice, and
the genes involved in the regeneration response and their then allows the selectable marker to be segregated away
expression patterns and then to adapt or tailor the strategies from the gene of interest in the progeny, yielding plants
to improve regeneration to the required genotype. containing only the gene of interest, is readily available
The second approach to improving transformation effi- (Thole et al., 2007). Marker gene elimination has been
ciencies is to increase the number of transformation events. demonstrated in barley (Matthews et al., 2001) and in wheat
Recent studies have almost exclusively concentrated on (Permingeat et al., 2003) and thus efficient selection of
using Agrobacterium as the DNA delivery system and transgenic plants is not now an important limitation.
looked at ways of improving the number of transformation
events. In order to do this, target tissues have been subjected
Controlling transgene expression
to a range of treatments, for example, vacuum infiltration
and sonication were used to improve transformation in The ability to obtain the required level and pattern of
barley (Shrawat et al., 2007). In rice and maize, it has been transgene expression is another key requirement for any
reported that treatment of immature embryos by centrifuga- transformation system. The first consideration for controll-
tion and heat improves transformation efficiency (Hiei et al., ing transgene expression is the choice of promoter. This area
2006). The presence of additional virulence genes during has been recently reviewed by Hensel et al. (2011) where the
Agrobacterium-mediated transformation of durum wheat authors provide a list of promoters shown to be effective in
was found to be necessary for successful transformation the cereals. A number of promoters give good constitutive
(Wu et al., 2008). The addition of certain virulence genes has expression throughout the plant, for example, the maize
also shown beneficial effects in rice transformation (Vain ubiquitin promoter that is widely used in both wheat and
et al., 2004). Recent studies in Arabidopsis have shown that barley. A comparison of gus gene activity in barley under
the over-expression of some histone genes increases suscep- different promoters is described by Himmelbach et al. (2007)
tibility to Agrobacterium and thus increases transformation who found the highest expression levels with the maize
efficiency (Tenea et al., 2009). It is thought that the histone ubiquitin promoter. A range of tissue-specific promoters
proteins have a role in protecting the DNA introduced to have also been shown to be effective in wheat and barley.
the plant cells. Other plant genes are also known to be Seed-specific promoters were examined in both wheat and
involved in Agrobacterium-mediated transformation and barley (Furtado et al., 2009) and while some showed similar
are reviewed by Gelvin (2010). It is likely that, as our activity in both species, one pericarp-specific promoter was
understanding of the role of different plant genes in the only active in barley. This highlights the need for care when
transformation process increases, there will be new opportu- choosing an appropriate promoter to control transgene
nities to exploit this knowledge to increase the frequency of expression. The range of available promoters has now been
Agrobacterium-mediated transformation events in wheat and extended to include those induced by biotic or abiotic stress.
barley. For example, a promoter from a member of the barley
1794 | Harwood

Germin-like GER4 gene cluster has been shown to give high Understanding and controlling transgene insertion
levels of pathogen-induced expression (Himmelbach et al.,
2010). Knowledge of the exact genomic location of an inserted
In addition to the choice of promoter, the inclusion of transgene is important for risk assessment, traceability, and
untranslated regulatory elements and of signal peptides also to increase understanding of the transformation process.
affects the level and pattern of transgene expression. Signal Transgene insertion sites have been examined using a variety
peptides can direct proteins to particular subcellular com- of methods. Fluorescence in situ hybridization (FISH) has
partments and have been utilized extensively to direct been used to provide an efficient method of physically
transgenic proteins to appropriate locations within the cell. mapping transgenes in barley (Harwood et al., 2004). When
The signal peptides used in transgenic wheat and barley are this method was used to map 23 transgene insertion sites,
listed in the review by Hensel et al. (2011). the pattern of insertion appeared to be non-random (Salvo-
Another available tool for the manipulation of transgene Garrido et al., 2004) with evidence of clustering of inde-
expression is to use intron-mediated enhancement. The in- pendent transgene insertions and insertion in gene-rich
clusion of an expression-enhancing intron within the trans- areas of the genome. Figure 2 shows the analysis of trans-
gene coding sequence has been shown to give higher levels gene insertion using FISH in barley and the distribution of
of expression in both wheat and barley. In wheat, the fourth insertion sites along chromosome 5H. Transgenes can also
intron from the maize T3/T7-like DNA-dependent RNA be genetically mapped in relation to known genetic markers
polymerase (RpoT) gene, enhanced expression when included (Salvo-Garrido et al., 2004) to provide further evidence of
at position +165 within the luciferase transgene (Bourdon the location of the transgene insertion.
et al., 2004). The same intron-containing luciferase transgene, Despite the availability of physical and genetic methods
also gave enhanced expression in barley plants compared with to gain information on the site of transgene insertion,
controls containing a luciferase gene without an additional molecular analysis of transgene insertion has yielded the
intron. However, inclusion of an alternative intron, the first greatest volume of information. Analysis of the junction
intron from the Arabidopsis polyubiquitin 10 gene (UBQ10) between the inserted transgene and the host plant DNA can
gave even greater enhancement of expression in barley with yield valuable information that can inform safety assess-
an almost 3-fold increase over the control plants (Bartlett ment of the particular transformation event. For example, it
et al., 2009). Therefore intron-mediated enhancement offers can detect unexpected rearrangements at the transgene
an additional tool to optimize the expression of transgenes. insertion site and determine whether the insertion has
Where gene-silencing (RNAi) constructs are used as disrupted known endogenous genes or regulatory sequen-
opposed to over-expression constructs, the level of gene ces. It can also show the presence of plasmid backbone
silencing will be affected by choices made in the design of sequences that have been inserted along with the T-DNA.
the construct. Choice of the size and section of the gene to Wu et al. (2006) showed that, in wheat, approximately two-
include in the ‘hairpin’ construct may affect the specificity thirds of lines contained some vector backbone DNA.
and level of silencing (Helliwell and Waterhouse, 2005). A variety of PCR-based methods are available to isolate
This is in addition to the effect of the promoter and any transgene junction sequences (Cullen et al., 2011). The
other regulatory elements on the activity of the silencing region of plant DNA adjacent to the transgene is also
construct. referred to as the transgene flanking region. Isolation of

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.
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
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 be addressed. Transgene integration has been widely studied
Tipple. However, the Tipple callus failed to regenerate and it is now possible to target transgenes precisely to
plants. This demonstrates the challenge of trying to trans- particular genomic locations. However, further advances in
form a particular commercial genotype. Golden Promise is this technology are required before it can be used routinely
a gamma-ray induced mutant of the cultivar Maythorpe in wheat and barley. A key remaining challenge is the
(Forster, 2001). Thus it is possible that a mutation present genotype dependence of most wheat and barley transforma-
in Golden Promise confers the high transformability. tion systems and this continues to restrict the application of
However, when Maythorpe was transformed alongside the technology. It is, however, likely that, by understanding
Golden Promise it was possible to achieve transformation and manipulating plant genes important in either the plant
efficiencies of 25% in Maythorpe, demonstrating that both regeneration process or in susceptibility to Agrobacterium, it
varieties were highly transformable (WA Harwood, un- will be possible to address issues of genotype dependence
published data). Figure 3E and F show the similarity of the and to improve transformation efficiencies further.
regenerable callus generated from Maythorpe compared
with callus from Golden Promise. This clearly demonstrates
that transformability in barley is under genetic control. Acknowledgements
There are examples throughout the literature of particu-
lar wheat and barley genotypes being amenable to trans- Support by grant in aid to the John Innes Centre from the
formation. In wheat, more varieties can be transformed UK Biotechnology and Biological Sciences Research Coun-
using biolistic techniques than using Agrobacterium. Sparks cil is gratefully acknowledged. Haroldo Salvo-Garrido is
and Jones (2009) describe a biolistics protocol that has gratefully acknowledged for contributing Fig. 2 and Eva
allowed the transformation of 35 wheat genotypes while Medvecka for Fig. 1. Penny Sparrow is thanked for critical
only a few genotypes can be transformed using Agro- reading of the manuscript.
bacterium. This is probably because, although a number of
genotypes have the ability to regenerate green plants from
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