Transgenic barley: A prospective tool for biotechnology and agriculture

    Transgenic barley: A prospective tool for biotechnology and agriculture ¨ Katar´ına Mr´ızov´a, Edita Holaskov´a, M. Tufan Oz, Eva Jiskrov´a, Ivo Fr´ebort, Petr Galuszka PII: DOI: Reference:

S0734-9750(13)00167-5 doi: 10.1016/j.biotechadv.2013.09.011 JBA 6742

To appear in:

Biotechnology Advances

Received date: Revised date: Accepted date:

8 June 2013 20 September 2013 24 September 2013

¨ M. Tufan, Jiskrov´ Please cite this article as: Mr´ızov´ a Katar´ına, Holaskov´ a Edita, Oz a Eva, Fr´ebort Ivo, Galuszka Petr, Transgenic barley: A prospective tool for biotechnology and agriculture, Biotechnology Advances (2013), doi: 10.1016/j.biotechadv.2013.09.011

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ACCEPTED MANUSCRIPT Transgenic barley: A prospective tool for biotechnology and agriculture Katarína Mrízová, Edita Holasková, M. Tufan Öz, Eva Jiskrová, Ivo Frébort and Petr Galuszka*

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Department of Molecular Biology, Centre of the Region Haná for Biotechnological and Agricultural Research, Palacký University, Šlechtitelů 11, 783 71 Olomouc, Czech Republic

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*Corresponding author. Tel.: +420 585 634 923; fax: +420 585 634 936.

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E-mail addresses: [email protected] (K. Mrízová), [email protected] (E. Holasková), [email protected] (M.T. Öz), [email protected] (E. Jiskrová),

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[email protected] (I. Frébort), [email protected] (P. Galuszka).

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Barley (Hordeum vulgare L.) is one of the founder crops of agriculture, and today it is the fourth most important cereal grain worldwide. Barley is used as malt in brewing and distilling industry,

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as an additive for animal feed, and as a component of various food and bread for human

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consumption. Progress in stable genetic transformation of barley ensures a potential for improvement of its agronomic performance or use of barley in various biotechnological and

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industrial applications. Recently, barley grain has been successfully used in molecular farming as a promising bioreactor adapted for production of human therapeutic proteins or animal vaccines. In addition to development of reliable transformation technologies, an extensive amount of various barley genetic resources and tools such as sequence data, microarrays, genetic maps, and databases have been generated. Current status on barley transformation technologies including gene transfer techniques, targets, and progeny stabilization, recent trials for improvement of agricultural traits and performance of barley, especially in relation to increased biotic and abiotic stress tolerance, and potential use of barley grain as a protein production platform have been reviewed in this study. Overall, barley represents a promising tool for both agricultural and biotechnological transgenic approaches, and is considered an ancient but rediscovered crop as a model industrial platform for molecular farming.

Key words: Barley; transgenesis; stress tolerance; pathogen resistance; molecular pharming; yield improvement. Abbreviations: AMP, antimicrobial peptides; AMY, amylase; HOR3-1, hordein D; GFP, green

ACCEPTED MANUSCRIPT fluorescent protein; GM, genetically modified; GP, Golden Promise; GUS, β-glucuronidase;

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PMF, plant molecular farming; T-DNA, transfer DNA.

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1. Introduction to barley as an important crop for biotechnology and agriculture

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The monocotyledonous family of Poaceae includes a great number of agriculturally important species such as maize (Zea mays L.), wheat (Triticum aestivum L.), and rice (Oryza sativa L.).

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Domesticated barley (Hordeum vulgare L.) is a member of this family, and is the fourth most important cereal grain with a worldwide production exceeding 130 million metric tons annually

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(FAO statistics; http://faostat.fao.org). Wide genetic variation of barley has produced cultivars particularly tolerant to cold, salinity, drought, and alkaline soil. Hence, it is possible to cultivate barley in diverse environments ranging from the area around the Arctic Circle to artificially

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irrigated fields in Sub-Saharan Africa. Besides human consumption, barley is used as malt in

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brewing and distilling industry and as an additive for animal feeding. For the past few years, barley grains have been successfully used as an important bioreactor adapted for the production

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of therapeutic proteins. Progress in genetic transformation of cereal crops ensures further improvement of their agronomic performance and use of these fast-growing species in various biotechnological and industrial applications. Barley has been considered as a model species for the agriculturally important Triticeae tribe of Poaceae family due to recent improvements in transformation efficiency, reduction of time required for preparation of stable transgenic lines, availability of extensive resources of barley germplasm and mutants as well as diploid feature and relatively low complexity of its genome. Knowledge of the full genome sequence is essential for understanding natural genetic variation and development of modern strategies for breeding programs. Recently, novel high-throughput sequencing technologies have been exploited to unravel the structure of barley genome (Mayer et al., 2011; Mayer et al., 2012). A systematic synteny analysis with model species from the Poaceae family with already annotated genomes (rice, maize, sorghum and Brachypodium) confirmed the existence of over 30.000 barley genes, and majority of these genes was located to specific chromosomal loci of the barley genome. It is assumed that full annotation of the barley genome will appear soon, as more than 90% of predicted gene structures are currently available

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public

databases

(http://webblast.ipk-gatersleben.de/barley/index.php).

Since

2003,

Affymetrix Inc. provides a GeneChip Barley Genome Array containing more than 22.000 unique probes designed on the basis of EST libraries (Close et al., 2004). The technology so far allowed

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over fifty whole-transcriptome comparative studies focused on malting properties, pest and disease control, abiotic stress tolerance, nutritional characteristics, and reproductive development

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(http://www.plexdb.org). Besides the quantification of gene expression, the advent of high-

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throughput RNA sequencing (RNA-seq) enables studies on structural variation of RNA population, role of alternative splicing and other post-transcriptional modifications in various

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physiological responses, and exploration of yet uncharacterized transcriptionally active regions of the barley genome. Alignment of deep RNA sequence data to predicted barley genes indicated,

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with high-confidence, that more than 70% of the transcripts might be regulated by alternative splicing (Mayer et al., 2012). Using RNA-seq approach, novel molecular markers for genotyping of barley and rye cultivars were determined and fixed for future breeding programs (Close et al.,

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2009; Haseneyer et al., 2011). Recently, high impact-differences in transcript levels during the

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early-stages of leaf senescence were detected in transgenic wheat plants with a down-regulated transcription factor modulating nutrient remobilization (Cantu et al., 2011). This study clearly

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showed that evaluation of large amount of sequencing data from different tissues can reveal very small but important variances induced by genetic manipulation of a single gene. Considering the progress in barley transformation and accessibility of methods for whole genome and transcriptome sequencing, a targeted genetic manipulation of barley and other cereal species becomes essentially vital for achievement of considerable increase in yield and tolerance to abiotic and biotic factors in the near future.

2. Methods for barley transformation Manipulation of plant genomes is achieved by various techniques of transgenic plant technology that aim stable expression and transmission of the introduced gene to progeny. The introduced gene can confer resistance to diseases and herbicides, provide tolerance to abiotic stresses, improve the yield and quality, or compel plants to produce pharmaceuticals or industrial chemicals. Efficiency of a transformation protocol depends on several factors including (1) the presence of a reproducible and highly efficient gene transfer technique, (2) the choice of explant

ACCEPTED MANUSCRIPT type which can easily regenerate, (3) the presence of a reliable regeneration method for the plant species, into which the novel gene is desired to be introduced, and (4) the availability of an effective screening and selection method for the recovery of transformants. Selection of promoter

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sequence is no less important for proper transgene expression in transgenic plants. The promoter sequences tested in transgenic barley and barley promoters evaluated in other plant species are

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summarized in Table 1. These promoters will be discussed in more detail further in the text.

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Additionally, expression patterns of tissue-specific or inducible promoters from barley genome are displayed in Fig. 1. Temporal and spatial expression patterns of the endogenous genes driven

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by these promoters provide valuable information for selection of the promoter to be used in development of transgenic barley with the intended transgene expression.

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The method of choice for DNA delivery into dicotyledonous plants is indirect transformation mediated by Agrobacterium tumefaciens. The cereals were not originally considered as being amenable to the Agrobacterium-mediated transformation, as they are not infected by

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Agrobacterium spp. in nature. However, recent advances led to efficient DNA delivery into

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wheat and barley using this method. Another most commonly employed method for DNA delivery into plants is particle bombardment. Genetic transformation of cereals and

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monocotyledonous plants using routine methods is challenging due to difficulties in DNA delivery as well as regeneration of the target plant species. Development and optimization of novel methods for DNA delivery into cereal genome have raised the possibilities for improvement of critical agricultural traits such as grain yield, tolerance to environmental stresses, and pathogen resistance in cereals, which gain more importance as the demand for food increases because of population increase. Although the recent progress in transformation efficiency to obtain stable progeny for the model barley cultivar Golden Promise is admirable, serious obstacles still remain for elite barley varieties or other genera of the Triticeae tribe such as rye, which are strongly recalcitrant to transformation and in vitro regeneration.

2.1 Biolistic transformation Biolistic transformation, also called the microprojectile bombardment, was developed in the late 1980s, originally as an alternative method for transformation of monocotyledonous plants, which were considered recalcitrant to Agrobacterium infection (Klein et al., 1987; Sanford et al., 1987).

ACCEPTED MANUSCRIPT It was shown for the first time in onion epidermal cells that accelerated tungsten microparticles are able to deliver DNA into the plant cells with integration of the DNA into the genome (Klein et al., 1987). However, only a transient expression could be achieved as the introduced gene was

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expressed only in the vegetative cells directly hit by the microparticles. Thus, reproductive or regenerative cells or tissues were investigated as possible targets for stable transformation using

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biolistics. In barley, different cells or tissues such as suspension cells, immature embryos (Kartha

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et al., 1989; Mendel et al., 1989), endosperm (Knudsen and Muller, 1991), callus tissue, anthers, microspore-derived embryos (Jahne et al., 1994; Wan and Lemaux, 1994; Leckband and Lörz,

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1998; Carlson et al., 2001), shoot meristematic cells (Zhang et al., 1999), and leaves (Shirasu et al., 1999) have been used as biolistic targets. Although particle bombardment was considered

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theoretically tissue and genotype independent, major success was achieved when immature embryos were used as targets (Kartha et al., 1989; Wan and Lemaux, 1994; McElroy et al., 1997; Cho et al., 1998; Harwood et al., 2000; Travella et al., 2005; Ritala et al., 2008). Thus today,

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using biolistic-mediated techniques.

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barley immature embryo is regarded as the most reliable and efficient tissue for transformation

Since the introduction of microprojectile bombardment, several protocols for highly efficient

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introduction of foreign genes into plant genomes and generation of stable transformants have been optimized. Progress in transformation efficiency has been achieved when tungsten particles were replaced by gold microprojectiles, which have less detrimental effects on living cells (Knudsen and Muller, 1991). Additionally, different devices and driving forces such as explosive charge of gun powder and high-pressure helium were employed for the acceleration of microprojectiles, resulting in higher transformation efficiency (Kartha et al., 1989; Mendel et al., 1989; Ritala et al., 1993; Jahne et al., 1994; McElroy et al., 1997; Cho et al., 1998; Leckband and Lörz, 1998; Shirasu et al., 1999; Zhang et al., 1999; Harwood et al., 2000; Carlson et al., 2001; Travella et al., 2005; Ritala et al., 2008). The critical step in stable transformation of barley is the regeneration of transgenic plants from target tissues. It was found that complementation of bombardment media with osmolytes such as sorbitol or mannitol led to higher rate of stable transformants (Kikkert et al., 2004). Furthermore, addition of growth hormones such as 6benzylaminopurine (BAP) or 2,4-dichlorophenoxyacetic acid (2,4-D) and cooper to the regeneration media significantly improved the regeneration of transgenic plants (Cho et al., 1998; Harwood et al., 2000).

ACCEPTED MANUSCRIPT The major disadvantage of biolistic-mediated transformation is the possible integration of multiple copies of the transgene which can lead to silencing or altered expression of the gene of interest or genetic instability over the generations (Travella et al., 2005). Another disadvantage of

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biolistics is the tissue and cell damage that can be caused by microprojectiles. Nevertheless, the majority of commercially available transgenic crops were developed by the particle

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bombardment. Due to the progressive increase of Agrobacterium-mediated transformation

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efficiency in vast range of plant species, the particle bombardment method, today, is primarily employed in transient expression studies or inoculation of plants with viral pathogens (Godinez-

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Hernandez et al., 2001; Ribas et al., 2005).

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2.2 Agrobacterium-mediated transformation

Transformation of plants via Agrobacterium is known for more than 30 years (Herrera-Estrella et

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al., 1983; Gasser and Fraley, 1989). Dicotyledonous plants are natural hosts of Agrobacterium

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tumefaciens, and the bacterial infection causes crown gall disease. It was revealed that this bacterium is capable of transferring a particular segment (T-DNA) of tumor-inducing (Ti)

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plasmid to the nucleus of a plant cell where the T-DNA is randomly integrated into the genome (Binns and Thomashow, 1988). Hence, Agrobacterium became an essential tool for genetic engineering of dicotyledonous plants. Despite the general assumption that monocotyledonous plants are recalcitrant to Agrobacterium infection, first transgenic cereals were obtained by this method in the mid-1990s (Chan et al., 1993; Smith and Hood, 1995) after the identification of supervirulent strains of Agrobacterium. In 1997, Tingay and her colleagues reported, for the first time, successful transformation of barley plants using A. tumefaciens AGL1, after co-cultivation of wounded immature embryos with the bacterial culture. More than 50 independent transgenic barley lines were obtained, and stable expression of the selectable marker gene was confirmed in subsequent generations (Tingay et al., 1997). In succeeding years, several protocols for barley transformation were developed and further optimized (Bartlett et al., 2008; Hensel et al., 2008; Hensel et al., 2009). It was reported that various factors or parameters influence the success in development of transformed plants. These factors include the selected plant genotype and explant, choice of bacterial strain and vector, co-cultivation medium used and duration of the cocultivation, compositions of callus induction and regeneration media, as well as the tissue and

ACCEPTED MANUSCRIPT cell damage that can result from bacterial infection or handling during transformation (Cheng et al., 2004; Hensel et al., 2007; Holme et al., 2008; Sood et al., 2011). It was found that addition of phenolic compounds like acetosyringone to the co-cultivation medium increased the efficiency of

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bacterial infection (Cheng et al., 2004; Kumlehn et al., 2006; Hensel et al., 2007). Recently, it was reported that the presence of surfactants such as Tween-20 or Pluronic acid F 68 in the

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infection medium resulted in higher transformation efficiency, since these surfactants reduce the

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surface tension and facilitate bacterial access to the explants (Yadav et al., 2013b). Transformation efficiency can also be improved by treatment of the explants with dessication or

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high temperature, which enhance the competency of plant cells for T-DNA uptake (Cheng et al., 2003; Yadav et al., 2013b).

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The most reliable and promising target material for barley transformation is the immature embryo because of its high regenerative capacity. However, selection of transformants and plant regeneration are rather time-consuming steps when immature embryos are used. The whole

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process of transgenic barley development, from inoculation of immature embryos to the harvest

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of transgenic seeds takes about 10 months. Although the homozygous transgenic plants can be identified in young T1 generation, the time required for establishment of stable homozygous lines

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can be extended to at least two years. Additionally, somaclonal variations can be observed in transformations using immature embryos. Thus, novel tissues or cells such as ovules and pollens have been used as alternative targets for Agrobacterium-mediated transformation of barley (Holme et al., 2006; Kumlehn et al., 2006). The main advantage of ovule as a target explant is the comparably less time required for the development of transgenic plants. It was reported that cocultivation of cultured ovules with Agrobacterium immediately after pollination led to successful transformation events at early stages of embryo development. These innovations resulted in generation of high quality transgenic plants by direct regeneration without callus stages within 2 or 3 months (Holme et al., 2006; 2008). On the other hand, plant regeneration from transformed androgenetic pollen cultures requires calli formation, and the whole procedure takes approximately 3 months. However, the selection of transformed plants, though segregation analysis is necessary, is easy and quick (Kumlehn et al., 2006). Transformation of haploid cell cultures followed by genome doubling provides great opportunity for development of homozygous plants bearing the target gene in a short period of time.

ACCEPTED MANUSCRIPT Besides selection of a proper explant, the choice of a suitable barley cultivar is no less important for a successful transformation. Golden Promise (GP) is the most reliable cultivar, extremely amenable to Agrobacterium infection. Bartlett and her colleagues (2008) achieved a

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transformation efficiency close to 25 % when they employed immature embryos of GP cultivar, Agrobacterium strain AGL1, and optimal cultivation and regeneration conditions (Bartlett et al.,

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2008). Very recently transformation protocols for an Indian cultivar of barley were optimized for

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both Agrobacterium-mediated transformation and particle bombardment (Yadav et al., 2013a; b). On the other hand, other barley germplasms can generate callus tissue with a high frequency, but

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show very weak regeneration competence (for a review see Vyroubalová et al., 2011). For example, the transformation efficiencies of Australian cultivars Schooner, Sloop and Chebec

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were lower than 1 %, mainly because of low regenerative capacity (Murray et al., 2004). Not only the proper cultivar but also the target plant material influences the transformation success. Kumlehn et al. (2006) examined the regeneration ability of transgenic pollen culture of barley

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cultivar Igri. Overall, they obtained transgenic plants with an efficiency of 2.2 transformants per

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pollens from one spike, which approachs to reported efficiencies of embryo transformation (Kumlehn et al., 2006). In contrast, ovule cultures seem to be more germplasm independent. The

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embryogenic potential and regeneration capacity of non-transformed ovules of four barley cultivars (Femina, Salome, Corniche, and Alexis) did not show any significant differences compared to GP. However, the regeneration frequency of these cultivars after Agrobacteriummediated transformation changed dramatically with efficiencies two or three-times lower than that of GP (Holme et al., 2008). Crossing emasculated flowers with transgenic GP pollen renders a possible solution for transformation of elite barley cultivars. Transgenic calli prepared from embryonic axes of Swedish elite cultivar by this approach regenerated, in 60 % of the cases, healthy plants bearing the transgene (Nalawade et al., 2012). Overall, Agrobacterium-mediated transformation compared to the biolistic method has many advantages including a preferential integration of the transgene into transcriptionally active regions of chromosomes, low copy number insertion, no requirements for sophisticated instruments, and high transformation efficiency (Czernilofsky et al., 1986; Koncz et al., 1989). Despite some limitations, Agrobacterium-mediated transformation remains the most favorable and prospective method for barley transformation.

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2.3 Alternative methods of barley transformation

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Although Agrobacterium- and biolistic-mediated transformations are the most widely employed methods for introduction of target genes into barley plants, several direct DNA delivery methods

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have also been investigated. Typical plant material for direct transformation is protoplast derived

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from various tissues such as endosperm, aleurone, roots, and leaves. It was reported that transformation of barley protoplasts by polyethylene glycol (PEG) treatment led to transient

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expression of the target gene (Lee et al., 1989; Diaz, 1994; Jenes et al., 1994; Zhang et al., 1995). Fertile transgenic barley plants were obtained after PEG-mediated transformation of protoplasts derived from primary calli (Kihara et al., 1998), cell suspension cultures (Funatsuki et al., 1995)

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and scutellum (Nobre et al., 2000). Protoplast transformation by PEG-mediated DNA transfer is more convenient for transient expression studies, especially for analysis of promoter activity.

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Another direct DNA transfer method used was electroporation, which displayed high efficiency

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in transformation of protoplasts derived from roots and leaves but was less effective in transformation of protoplasts derived from endosperm (Diaz, 1994; Diaz et al., 1995).

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Several attempts to transform barley plants have also been made using microinjection, which is usually preferred for transformation of animal cells. Barley microspores as well as zygote protoplasts were investigated as potential targets for microinjection (Bolik and Koop, 1991; Holm et al., 2000). Little success was achieved after microinjection of a DNA cassette into the protoplasts of zygotes. The viability of protoplasts after microinjection was approximately 60 %, and the transgene integration into host genome was extremely low. Of the total number of six hundred protoplasts used, only 2 lines expressed the marker gene, suggesting that this method is not suitable for possible applications in barley transformation unless major optimization is achieved (Holm et al., 2000). Virus-induced gene silencing (VIGS) is a promising method for functional gene analysis in reverse genetics. VIGS is based on incorporation of target gene segment into viral genome and subsequent use of the modified virus for plant inoculation. The virus is replicated in host cells by production of double-stranded RNA, which is recognized and degraded by plant defensive ribonuclease enzymes into small RNA fragments. Then a single RNA strand is incorporated into RNA-induced silencing complex (RISC) and used for degradation of target RNA transcripts

ACCEPTED MANUSCRIPT based on sequence similarity (Baulcombe, 1996). Potential use of brome mosaic virus as well as barley streak mosaic virus (BSMV) for VIGS in barley, rice and maize was investigated. Target plant genes were successfully silenced using BSMV; however, the silencing signal was localized

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and did not spread to the whole plant, thus, only transient silencing was achieved (Holzberg et al., 2002; Ding et al., 2006; Oikawa et al., 2007). VIGS was used for silencing of the genes involved

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in pathogen resistance (Bruun-Rasmussen et al., 2007) as well as for identification of pathogen

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resistance-related loci under Blumeria graminis attack (Yuan et al., 2011). An analogous strategy termed host-induced gene silencing (HIGS) provides new opportunities to study loss-of-function

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phenotypes of candidate fungal genes. In this system, in planta expression of silencing-inducing RNA molecules can trigger suppression of corresponding fungal genes during colonization.

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Recently, Nowara et al. (2010) have successfully used this tool to study function of selected genes in barley powdery mildew fungus Blumeria graminis. HIGS has also been employed for validation of certain B. graminis candidate genes for their contribution to barley infection (Pliego

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et al., 2013).

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2.4 Gene targeting and removal of selection markers Maize transposons Activator/Dissociation (Ac/Ds) based system was adopted for barley as a prospective tool for identification and determination of gene function by targeted knock-out (Koprek et al., 2000; 2001; McElroy et al., 1997) or activation tagging (Ayliffe et al., 2007). Ac/Ds system comprises of autonomous Ac element that catalyzes transposition of both Ac and non-autonomous Ds elements (Kunze and Starlinger, 1989). Ds element contains usually a selectable resistance gene surrounded by inverted repeats, which transposes at frequencies sufficient for large-scale mutagenesis (Koprek et al., 2000; Ayliffe et al., 2007). The preference of Ac/Ds to transpose locally enables to place Ds element close to the gene of interest and cease or enhance, if additional ubiquitous promoter is part of the Ds element, their expression. The tagged gene is subsequently localized by flanking sequence rescue using high-efficiency thermal asymmetric interlaced or inverse PCR. The Ac/Ds system was recently used for tagging several genes on QTLs affecting malting quality and seed germination, thus became a valuable resource for deeper investigation of their role in these processes (Singh et al., 2012).

ACCEPTED MANUSCRIPT Novel strategies for a precise plant genetic manipulation based on chimeric nucleases with several zinc-finger domains (Townsend et al. 2009) or motifs from transcription activator-like effectors (TALEN; Bogdanove and Voytas 2011) were recently developed. These nucleases are

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designed to recognize unique sequences and subsequently after transformation into a plant cell create double strand breaks, which are repaired by endogenous cell machinery to leave mutation

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or selection marker insertion in required locus – usually gene of interest. The first targeted gene

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knock-out on cereal crop was performed on Zea mays plants in 2009 exploiting pairs of zincfinger nucleases (Shukla et al. 2009, Townsend et al. 2009). Nevertheless, due to easier handling

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and lower cost, TALEN technology has grown in importance and in last couple of years was successfully used on rice and Brachypodium (Li et al., 2012; Shan et al., 2013). Very recently,

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capacity of TALEN to introduce double strand breaks in specific loci of barley genome was proved (Wendt et al., 2013). Several independent plants with short deletions in the promoter of a barley phytase were regenerated after Agrobacterium mediated transformation of immature

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embryos with construct bearing specifically tailored TALE motifs recognizing short sequence in

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this promotor adjacent to FokI nuclease. The study clearly showed that TALEN technology can be used for production of a high amount of barley transformants with loss-of-function deletions at

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pre-selected gene regions in near future.

Selection and stabilization of transgenic plants are, in majority of the cases, accompanied with perpetual production of selectable marker protein, whose gene is in close linkage with the gene of interest at the integration site. In some cases the presence of selectable markers, which are usually of bacterial origin, is no longer required in stable transgenic plants. Several techniques to remove the selectable marker, such as strategies employing transposon system or site-specific recombination, were developed and successfully employed in model plants. Both these strategies are based on excision of the region coding for selectable marker from the integration locus using flanking sites. Short and firmly defined flanking sequences are recognized by transposase or recombinase, and the gene coding for selection marker is excised and subsequently relocated or destroyed (Dale and Ow, 1991; Cotsaftis et al., 2002). The recombinase gene is introduced to the genome by crossing with an independently prepared transgenic line, while the transposition usually makes use of naturally occurring transposon elements. A similar principle was employed in a strategy of marker elimination by homologous recombination (Iamtham and Day, 2000). Another possibility is co-transformation by two unlinked sequences for marker and gene of

ACCEPTED MANUSCRIPT interest, where the genes are integrated at different regions, and selection marker can later be segregated out (Coronado et al., 2005; Matthews et al., 2001). Recently, the technique was used to compare different approaches for co-transformation (Kapusi et al., 2013). The transformation

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with a single Agrobacterium strain bearing two binary vectors was shown to be the most effective, resulting in approximately 1% efficiency. Conventional selectable marker linked to a

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negative selection system – a suicidal gene e.g. bacterial cytosine deaminase – which converts

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non-toxic pro-drug to cellular toxin after application to the putative transgenic plants, can significantly speed up the selection of marker-free segregants (Goedeke et al., 2007; Koprek et

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al., 2000).

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3. Transgenic barley with improved agricultural traits

Transgenic cereal crops with increased stress tolerance or yield are already available on the

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market for more than fifteen years. The most widely spread transgenics are maize varieties

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producing Bacillus thuringiensis endotoxin (Bt) against corn rootworm and other coleopteran pests (Devos et al., 2013). In 2012, Bt-maize was planted on almost 70% of whole corn acreage

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in U.S.A. Its cultivation is spread through whole American continent and also in few European countries, where it is still the only genetically modified (GM) crop distributed among farmers. Other examples of GM cereals commercially used in agriculture are drought stress tolerant maize expressing Bacillus subtilis cold shock protein B (Beazley et al., 2012) and maize expressing a synthetic gene coding a thermostable alpha-amylase derived from Thermococcales for enhanced bioethanol production (Wolt and Karaman, 2007). There are also rice and wheat GM varieties with herbicide tolerance and maize with increased lysine content, which have been approved for open field planting. These GM varieties are not in commercial production yet. Despite the big progress in generation of transgenic barley plants, none of the approaches for increasing agricultural performance of barley is close to commercialization so far.

3.1 Transgenic barley with increased tolerance to abiotic stress One of the most important problems, agriculture is facing today, is the adverse effect of environmental stresses, such as high salinity, drought and low temperature, on plant development

ACCEPTED MANUSCRIPT and seed production. Demand for drought and salt tolerant crops has increased with the worldwide climate change during the last few decades (Reynolds and Ortiz, 2010). Plants respond to environmental stresses through various physiological and biochemical

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processes. Much effort has been made for identification of stress-protective or adaptation-related

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genes which are activated during abiotic stress (Bray et al., 2000; Shinozaki and YamaguchiShinozaki, 2000). Overexpression of these genes can help plants tolerate abiotic stress. It was

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shown that activities of many stress-responsive genes were regulated by transcription factors, which are hence attractive targets for applications in transgenic studies. A promising example is

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the family of drought-responsive element binding proteins (DREBs) / C-repeat binding factors (CBFs). These transcription factors were used for increasing stress tolerance in many crops

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including Brassica junceae (Cong et al., 2008), rice (Oh et al., 2007), soybean (Chen et al., 2007) and some grasses (Zhao et al., 2007). Recently, two DREB/CBFs from wheat were used for modulation of stress tolerance in barley and wheat (Morran et al., 2011). Transcription factor

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genes, DREB2 and DREB3 were overexpressed under the control of constitutive duplicated

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CaMV35S promoter as well as drought-inducible ZmRAB17 promoter from maize. Transgenic plants constitutively expressing DREB genes showed increased tolerance to water deficiency.

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However, growth was retarded in these transgenic plants which reached only 70% of the size of control plants at flowering stage with lowered grain yield. The growth retardation was eliminated by using the promoter of RAB17. The ZmRAB17 expression is strongly up-regulated during drought stress and its promoter shows only basal activity at normal conditions. Barley plants overexpressing DREB genes driven by ZmRAB17 promoter were more tolerant to drought stress without undesirable effects on plant growth and development. Barley plants constitutively expressing DREBs also showed increased frost tolerance. Recently, barley lines with wheat DREB3 gene under the control of two cold-inducible promoters were generated (Kovalchuk et al., in press). Combination of the gene with rice WRKY71 promoter – naturally driving expression of a transcription factor inducible by stress hormone abscisic acid (Xie et al., 2006) – resulted in a significantly improved frost tolerance when 3-week-old seedlings were exposed to short term freezing stress. Increased stress tolerance is probably a result of DREB/CBF-mediated upregulation of whole gene battery with protective effect on cell membrane and integrity (Morran et al., 2011).

ACCEPTED MANUSCRIPT Another group of examples is the transcription factors belonging to the family of proteins characterized by the presence of highly conserved NAC (NAM, ATAF1/2 and CUC2) domain on N-terminus. NAC transcription factors were successfully used for modulation of stress tolerance

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in rice, where overexpression of stress-responsive SNAC1 gene led to enhanced drought and salt stress tolerance (Hu et al., 2006). A similar approach was used in wheat by overexpressing

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TaNAC69-1 gene (Xue et al., 2011) under the control of two barley dehydrin gene promoters, one

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with a constitutive pattern of expression - HvDHN8s and a second one inducible by drought HvDHN4s (Fig. 1; Xiao and Xue, 2001). TaNAC69-1 transgenic lines displayed up-regulated

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expression of a number of stress-induced genes, higher biomass accumulation under water deficiency, as well as improved water-use efficiency at early vegetative stages (Xue et al., 2011).

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Constitutive or inducible overexpression approach was used in various plant species (Arabidopsis, tomato, apple) for evaluation of the effect of transcription factor OsMYB4 from rice on abiotic stress tolerance (Park et al., 2010). Overall, variation of stress tolerance in transgenic

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plants suggested that the activity of OsMYB4 is most probably dependent on the host genomic

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background (Mattana et al., 2005; Vannini et al., 2007; Pasquali et al., 2008). In a recent study, OsMYB4 transcription factor was overexpressed in barley under the control of cold inducible

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AtCOR15a promoter. Transgenic barley plants expressing OsMYB4 showed increased frost tolerance and germination vigor under low temperature conditions with a minor effect on plant growth (Soltész et al., 2012).

A lot of effort has also been put into modulation of response to oxidative and salinity stress in cereal crops. Hordeum vulgare is considered one of the most salt tolerant crops and has several halophytic relatives such as sea barley grass (Hordeum marinum), but mechanisms providing the resistance have not yet been explored (Munns et al., 2006). Only few transgenic approaches increasing salinity tolerance in barley were tested so far. Recently, a gene coding for a putative protein kinase (AtCIPK16) from SOS (salt overly sensitive) family was identified in Arabidopsis genome, as the one being involved in the process of Na+ exclusion (Roy et al., 2013). Signaling pathway mediated via SOS kinases regulates the activity of the Na+/H+ antiporters (Qiu et al., 2002). Transgenic barley plants overexpressing AtCIPK16 exhibited an increase in salinity tolerance, which was expressed as 20–45% higher maintenance in biomass under exposure to long-term strong salinity stress (30 days, 300 mM NaCl) contrary to control plants (Roy et al., 2013).

ACCEPTED MANUSCRIPT It is well known that aluminum (Al) inhibits root growth, leading to decreased water and nutrient uptake in plants (Kochian, 1995; Ma, 2007). A potential candidate for the modulation of Al tolerance is wheat ALMT1 gene, which encodes a membrane-bound protein responsible for Al-

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activated malate efflux (Ryan et al., 1997; Zhang, 2001). It was previously shown in tobacco cells that the overexpression of ALMT1 led to an increased Al stress tolerance (Sasaki et al., 2004).

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Thus, the transgenic barley plants overexpressing ALMT1 gene were generated and tested for Al

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tolerance. When cultivated in a contaminated hydroponic culture, the transgenic plants showed robust root growth. In contrast, growth of the control plants was inhibited under the same

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conditions, and some deformations of root apices were observed (Delhaize et al., 2004). In addition, when transgenic barley plants constitutively overexpressing wheat ALMT1 gene were

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grown on acid soil, they showed significantly improved root growth, shoot biomass, and grain yield (Delhaize et al., 2009). Recently, transgenic barley plants overexpressing barley ALMT1 gene, the most similar to wheat ALMT1, were generated and characterized (Gruber et al., 2011).

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The HvALMT1 protein functions in transport of organic acids and does not need to be activated

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by Al treatment (Gruber et al., 2010) contrary to other ALMT transporters including the above mentioned wheat counterpart. Stable HvALMT1 overexpression resulted in reduced growth with

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severe leaf spotting leading to necrosis. Additionally, transgenic plants with the highest HvALMT1 expression were unable to set seeds. On the other hand, malate and succinate efflux was significantly increased by approximately 20- and 15-fold, respectively, leading to enhanced Al resistance in contaminated growth solutions. However, a practical exploitation of the gene seems to be limited due to continuous imbalance in organic acid homeostasis (Gruber et al., 2011). One of the major determinants of Al resistance in barley plants is the gene coding for Alactivated citrate transporter1 (AACT1; Furukawa et al., 2007). Primary function of AACT1 is to release citrate to the xylem for efficient iron translocation. Detailed study of Al-resistant cultivars revealed the presence of a specific 1kb insertion in 5´UTR region of the HvAACT1 gene. It was revealed, when transformed into Al-sensitive barley cultivar, that the insertion is responsible for enhanced expression of HvAACT1 in the root tips. Subsequent secretion of citrate into rhizosphere led to an increased Al tolerance in HvAACT1 transformants, as well as better adaptation in acid soils (Fujii et al., 2012). Excess of metallic ions such as Al is also associated with increased formation of reactive oxygen species (ROS). It was previously found that excess of Al induces directly or indirectly the

ACCEPTED MANUSCRIPT expression of many genes involved in response to oxidative stress (Richards et al., 1998). Thioredoxin (TRX) is an effective antioxidant protein that is induced by ROS during oxidative stress (Broin and Rey, 2003; Laloi et al., 2004). Transgenic barley plants overexpressing TRX

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gene from the grass Phalaris coerulescencs were developed to evaluate the response of barley to AlCl3 stress. Inhibitory effect of Al3+ salt on the root growth was observed in both transgenic as

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well as control plants. However, the roots of transgenic plants showed higher ability to elongate

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and lower degree of protein oxidation in comparison with the control plants. In addition, activity of some important antioxidative enzymes (catalase, glutathione peroxidase, glutathione reductase,

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and ascorbate peroxidase) was significantly increased (Li et al., 2010). The importance of TRX in improvement of resistance against mineral stress was confirmed in another study where the role

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of TRX in elimination of selenite ions was investigated (Kim et al., 2003). Previously developed transgenic barley line overexpressing the gene for TRX from wheat in starchy endosperm (Cho et al., 1999a) was treated with sodium selenite. Approximately 65% of homozygous transgenic

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grains had germinated in the presence of 2 mM sodium selenite in contrary to only 10%

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germination rate of controls with null allele. Moreover, 10-fold difference in the root and shoot length of transgenic compared to control seedlings was observed after imbibition in 1 mM

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selenite (Kim et al., 2003). These results suggested that barley plants overexpressing TRX have high tolerance to selenite- and aluminum-mediated inhibition of plant growth, and thus can find applications in the phytoremediation of polluted soil.

3.2 Pathogen resistant transgenic barley plants Stem rust, powdery mildew, and head blight, caused by pathogenic fungi of the order Pucciniales, Erysiphales and Hypocreales, respectively, are the most serious and widespread diseases of cereal crops worldwide. Every year, these fungal diseases are the reason for huge yield losses in agriculture. The biggest problem is the resilience of the causative pathogens to fungicides which results in increased usage and cost of chemicals in agriculture. Considering the potential risk of over-usage of agrochemicals on environment and human health, new approaches of plant protection are desirable. Many studies focusing on barley-powdery mildew interaction were conducted to identify the genes involved in the process (Hein et al., 2005; Eichmann et al., 2006; Shen et al., 2007).

ACCEPTED MANUSCRIPT Modified expression of some of these genes can contribute to enhanced resistance of barley to pathogens (Christensen et al., 2004; Proels et al., 2010). An example is the transmembrane protein MLO, which acts as a negative regulator of plant defense response. It was shown that

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some natural or induced mutation of MLO gene leads to resistance to Blumeria graminis f. sp. hordei, causative agent of powdery mildew in barley (Jørgensen, 1992). The absence of

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functional MLO protein results in a lowered accessibility of the pathogen spores to plant cells

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(Consonni et al., 2006). So far, transgenic barley with modified level of MLO protein has not yet been generated, however preliminary results obtained for wheat with transiently silenced MLO

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expression indicate that powdery mildew resistance can be achieved in barley by silencing or knock-down approaches (Varallyay et al., 2012).

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Recently, importance of BAX inhibitor 1 (BI-1), a conserved cell death-regulator protein, in plant-pathogen interaction was investigated in barley. Transgenic barley plants overexpressing HvBI-1 gene were more susceptible to B. graminis f. sp. hordei (Babaeizad et al., 2009).

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Increased level of BI-1 reduced the frequency of hypersensitive cell death reactions and

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consequently suppressed general defense responses in the infected plants. In contrast, young seedlings of barley overexpressing HvBI-1 were more resistant to Fusarium graminearum, the

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agent causing head blight or root rot diseases (Babaeizad et al., 2009). In a subsequent study, it was shown that down-regulation of HvBI-1 by transient gene silencing restricted the susceptibility to B. graminis f. sp. hordei infection. Reduced amount of BI-1 in transgenic barley cells enhanced the resistance to fungal hyphae penetration (Eichmann et al., 2010). In addition, a study on mutualistic symbiosis of fungal root endophyte Piriformospora indica and barley revealed that HvBI-1 influences P. indica development in barley. Invasive growth of P. indica was significantly reduced in transgenic barley overexpressing HvBI-1 compared to wild type plants. P. indica probably interacts with the host cell death machinery to make successful invasion. This indication was further supported by the finding that P. indica occupies mainly dead or dying root cells (Deshmukh et al., 2006). Mutualistic symbiosis of P. indica and barley has several benefits for barley plant including enhanced root resistance to Fusarium culmorum, systematic resistance to B. graminis, protection from abiotic stress caused by high salt concentrations, and higher yield (Waller et al., 2005). Interaction between barley and fungal pathogens is also influenced by ROP (also called RAC) proteins, which belong to the Ras superfamily of small G-proteins. Their role in the resistance to

ACCEPTED MANUSCRIPT B. graminis was initially investigated in barley epidermis cells derived from leaf segments (Schultheiss et al., 2002; 2003). Transient knock-down of HvRACB led to a lowered susceptibility to B. graminis after inoculation (Schultheiss et al., 2002). Additionally, mutations

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in GTPase domain of three barley ROP proteins HvRACB, HvRAC3 and HvROP6, resulting in continuous activation, led to an increased accessibility of B. graminis into barley epidermis cells

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(Schultheiss et al., 2003). A stable transgenic line constitutively overexpressing activated

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HvRACB mutant was generated and studied (Schultheiss et al., 2005). Besides the higher susceptibility to B. graminis, transformants showed some shoot and root deformation, such as

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delayed shoot development and stunted roots, as well as enhanced water loss under stress conditions. The function of other ROP proteins in susceptibility to powdery mildew was

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confirmed in subsequent studies (Pathuri et al., 2008; Hoefle et al., 2011). Some effort was also made for development of barley resistant to stem rust fungus Puccinia graminis f. sp. tritici. Resistance bearing locus RPG1 was introduced into barley breeding

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program a long time ago. Owing to the progress of molecular biology techniques, RPG1 gene

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was finally cloned by a map-based approach, and identified as a putative receptor kinase (Brueggeman et al., 2002). The P. graminis susceptible barley cultivar Golden Promise, lacking

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the RPG1 locus, was transformed with RPG1 gene cloned from resistant cultivar Morex. Transgenic barley plants showed high resistance to stem rust after pathogen inoculation (Horvath et al., 2003).

Another approach to improve disease resistance in barley employs antimicrobial peptides, which are involved in defensive reactions of plants. A promising example is a proline-rich antifungal peptide Metchnikowin. It was previously shown that synthetic Metchnikowin inhibits the growth of pathogenic fungi at very low concentrations (Schäfer and Kogel, 2009). The transgenic barley plants expressing the same peptide displayed enhanced resistance to powdery mildew, Fusarium head blight, and root rot (Rahnamaeian et al., 2009; Rahnamaeian and Vilcinskas, 2012). In another approach, detoxification of fungal toxins was tested as a strategy against F. graminearum which releases deoxynivalenol, a mycotoxin from the group of eukaryotic proteosyntesis inhibitors,

during

infection.

Transgenic

barley

overexpressing

3-OH

trichothecene

acetyltransferase enzyme deactivating deoxynivalenol showed significant reduction in disease symptoms when tested in greenhouse conditions. However, the results were not approved in a field trial, as no reduction in deoxyvalenol concentration was observed (Manoharan et al., 2006).

ACCEPTED MANUSCRIPT Authors attributed the discrepancy to possible effects of somaclonal variation detected in the transgenic lines or the fact that other fungi causing similar symptoms interfered with plants in the field experiment.

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Cultivated barley varieties are often infected by viruses, among which the barley yellow dwarf

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virus (BaYDV) is the most widely distributed viral disease of cereals. Standard transgenic approaches based on anti-viral RNA silencing or coat protein overproduction were successfully

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tested in oat and barley (McGrath et al., 1997; Wang et al., 2000). In another study, Hv-eIF4E gene coding for eukaryotic translation initiation factor 4E was identified to play an important role

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during the infection by barley yellow mosaic virus (BaYMV). Several non-silent single nucleotide polymorphisms in Hv-eIF4E sequence were determined in different barley accessions.

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Stable transformation of a resistant barley genotype with the genomic fragment or a full-length cDNA of Hv-eIF4E derived from susceptible cultivars induced susceptibility to BaYMV. Obtained results illustrate that mutations in a basic component of translation machinery complex

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can thus form a mechanism for virus resistance (Stein et al., 2005).

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In summary, plant defensive reactions to pathogen attack are extremely complex, and many genes and proteins are involved in response mechanisms. Thus, better understanding of these

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mechanisms and intensive studies are necessary to achieve and develop new barley cultivars with targeted resistance and preserved developmental and morphological properties.

3.3 Transgenic barley plants with improved quality and yield Increasing quality and yield of barley plants has become one of the major goals in barley molecular breeding in the last few years. Most of the studies have focused on altering the grain constituents such as starch, proteins, lipids, cell walls components, and mineral content. Starch is composed of amylose (glucose units joined by α-1,4 glycosidic linkages) and amylopectin (glucose units joined by α-1,4 and α-1,6 glycosidic linkages), which vary in their degree of polymerization. Starch from majority of barley cultivars contains about 20-30% of amylose and 70-80% of amylopectin. There is a big effort to produce starch with enhanced amylose to amylopectin ratio. It was previously found that proportion of amylose in starch has direct positive correlation with content of resistant starch (RS) (Regina et al., 2006; Li et al., 2008a; Shrestha et al., 2010). RS is a portion of dietary starch that is resistant to enzymatic hydrolysis, and thus it is

ACCEPTED MANUSCRIPT fermented in the large intestine by anaerobic gut bacteria instead of the degradation in stomach and small intestine. RS is associated with many promoting effects on human health; hence, increasing RS content is desirable. Silencing of starch branching enzymes, which are necessary

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mainly for amylopectin synthesis, could enhance the amylose content. Using this approach, the amount of amylose-enriched starch was successfully increased in wheat (Sestili et al., 2010) and

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rice (Wei et al., 2010a; 2010b). Recently, transgenic barley plants with silenced starch branching

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enzymes producing amylose-only starch were also produced (Carciofi et al., 2012a). In these plants, amylose and amylopectin contents were 99.1% and 0.9%, respectively. Unfortunately,

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such transgenic barley seeds showed a wrinkled phenotype with enlarged endosperm cavities. Additionally, the total yield of T2 generation of transgenic plants declined by approximately 22%

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in comparison to control plants (Carciofi et al., 2012a). The role of a barley protein called limit dextrinase inhibitor (LDI) in starch metabolism was investigated in transgenic barley plants designed to down-regulate LDI using RNA antisense technology (Stahl et al., 2004). As a result,

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enhanced limit dextrinase (α-dextrin 6-glucanohydrolase) activity led to an increase in starch

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degradation as well as reduced amylose to amylopectin ratio. In another study on starch content via modulation of its metabolism, transgenic barley callus was generated by overexpression of

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granular bound starch synthase Ia (Carciofi et al., 2012b). Callus cells produced significantly increased content of amylose, up to 4% of total dry weight. Another important approach for the improvement of barley targets β-glucans, which are the main components of cereal cell wall. The β-glucans are hydrolyzed by β-glucanase, which is prone to irreversible degradation at temperatures above 55°C. Increased depolymerisation of β-glucans, which is desirable for malting and brewing processes and the feed industry could be achieved by use of a thermotolerant fungal endo-1,4-β-glucanase (fEBG) or hybrid bacterial EBG (hEBG) derived from Bacillus spp. Two barley cultivars (Kymppi and Golden Promise) were transformed with fEBG or hEBG gene under the control of α-amylase promoter. Transgenic barley plants produced β-glucanases in scutelum and aleurone tissues of germinating grains, and the enzymes remained active after 2 hours of incubation at 65°C (Jensen et al., 1996; Nuutila et al., 1999). It was shown that the transgene was transmitted through four consecutive generations with stable expression (Jensen et al., 1998). The malt from these transgenic barley plants was used as a supplement to broiler chicken diet in a succeeding study (von Wettstein et al., 2000). Broiler chicken diet is predominantly based on corn grain, since barley grains is a low-energy feed.

ACCEPTED MANUSCRIPT Chicken lacks the enzyme for utilization of β-glucans, major component of barley grains. Supplement of bacterial β-glucanase to barley diet provided efficient depolymerisation of βglucans and increased the nutritive value of barley-based diet for poultry and improved growth in

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chicken (Rickes et al., 1962). Similar positive effects were observed when the malt from transgenic barley grains producing hEBG was added to the chicken diet. Chicken fed with the

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transgenic barley malt showed an equal weight gain and feed efficiency compared to chicken fed

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with corn only. This indicated the use of transgenic barley grains producing hEBG as an alternative for corn grains, which are more expensive and required in larger amounts for human

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consumption (von Wettstein et al., 2000). Moreover, transgenic barley plants overexpressing hEBG under the control of endosperm-specific barley hordein D gene promoter were generated.

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The hEBG was linked to hordein D signal peptide that delivered the protein into storage vacuoles. These transgenic plants produced 40 times more hEBG than the transgenic plants overexpressing hEBG driven by α-amylase promoter (Horvath et al., 2000). Some effort has also

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been made in the modulation of β-glucan biosynthesis, which is mediated in part by cellulose

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synthase-like (CslF) gene family. Recently, transgenic barley plants overexpressing barley CslF6 gene under the control of an endosperm-specific oat globulin promoter were prepared. Plants

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produced up to 80% more β-glucans in grains compared to the wild type plants (Burton et al., 2011). Improvement of β-glucan content in cereal grains has benefits for human health as well, since β-glucans can reduce the risk of non-insulin dependent diabetes, obesity and colorectal cancer and also reduce serum cholesterol level (Dikeman and Fahey, 2006; Truswell, 2002). Several studies have focused on improvement of mineral content as well as content of essential amino acids in barley. Micronutrient malnutrition affects over billions of people worldwide where deficiency of zinc, iron and iodine are the most frequently observed (Palmgren et al., 2008; White and Broadley, 2009). Zinc is an essential nutrient influencing various physiological and metabolic processes. Transgenic barley plants overexpressing Arabidopsis zinc transporter gene AtZIP1 under the control of ubiquitin promoter were generated to improve zinc uptake. Despite the finding that transgenic barley grains were smaller than control ones, total zinc and iron content was 2 times higher. Additionally, contents of magnesium and calcium increased about 0.5 times (Ramesh et al., 2004). An approach focused on the improvement of mineral content of plants uses overexpression of genes coding for phytases, which dephosphorylate phytic acid and liberate orthophosphate. Phytic acid is the major form of phosphorus storage compound in cereal

ACCEPTED MANUSCRIPT seeds but unfortunately, monogastric animals like pigs and poultry are unable to utilize it, since they lack phytase in their gastrointestinal tract (Steen et al., 1998). Hence, increasing phytase activity that would lead to high phosphate bioavailability in barley seeds is desirable.Recently,

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barley plants with increased phytase activity were generated by cisgenesis, which implies transformation with endogenous genetic material (Holme et al., 2012). Authors transformed the

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complete copy of barley endogenous phytase gene, including the promoter, introns, and the

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terminator using Agrobacterium-mediated transformation, while the selection cassette was segregated out. Finally two homozygous cisgenic lines, which had up to 3 times higher phytase

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activity, were stabilized (Holme et al., 2012). Concurrently, transgenic barley plants with fungal phytase gene under the control of α-amylase promoter were prepared in our laboratory, and

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transgenic barley lines showed significantly increased phytase activity in several subsequent homozygous generations. These plants are being cultivated under second round of low-scale field experiment (Fig. 2), and feeding trials with transgenic seeds on monogastric animals are currently progress

(Ohnoutková

et

al.,

2010;

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in

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http://bch.cbd.int/database/record.shtml?documentid=104335). Another study was focused on improvement of lysine content in barley. Lysine is an essential

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amino acid that has vital functions in mammalian metabolism (Rodgers et al., 2005; Kim et al., 2006). Synthesis of lysine in plants is limited by a feedback inhibition of the key enzyme dihydrodipicolinate synthase (DHDPS) (Azevedo et al., 1997). This regulation is missing in bacteria. Hence, transformation of bacterial DHDPS gene into plants could lead to improved lysine content. Transgenic barley plants overexpressing bacterial DHDPS gene driven by ubiquitin promoter were generated successfully. The lysine content in transgenic barley plants was approximately 20-30% higher compared to that in control plants. Moreover, the level of threonine was also increased. This observation was explained by the fact that lysine and threonine are both synthesized by the aspartate biosynthetic pathway (Ohnoutková et al., 2012). Several studies were conducted on regulation of cytokinin homeostasis and its direct effect on grain yield and size in barley. Cytokinins are important plant hormones, which influence plant morphology and diverse physiological processes such as senescence, sink activity and stress tolerance (Mok and Mok, 2001; Werner et al., 2001; Balibrea-Lara et al., 2004). Previously, it was shown that mutation of cytokinin dehydrogenase (CKX) gene led to a higher plant productivity in rice (Ashikari et al., 2005). Recently RNA-interference technology was

ACCEPTED MANUSCRIPT implicated to generate transgenic barley plants with silenced HvCKX1 gene (Zalewski et al., 2010). The total grain yield of transgenic barley increased by up to 20% compared to the plants transformed with empty vector. Cytokinins were found to regulate sink strength via activation of

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cell wall invertases and hexose transporters (Balibrea-Lara et al., 2004). Accelerated mobilization and flow of sucrose induced by enhanced cytokinin content in aleurone regulatory layer, where

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HvCKX1 is mainly expressed, could result in a higher starch accumulation in the endosperm, and

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thus enhanced grain filling (Zalabák et al., 2013). Some effort has been made to develop early flowering barley plants using Arabidopsis cryptochrome gene AtCRY2, which is responsible for

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the control of flowering time in Arabidopsis. Two barley cultivars (El-Dwaser and El-Taif) were transformed with AtCRY2 gene under the control of its native promoter using particle

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bombardment. Transgenic barley plants expressing AtCRY2 gene flowered about 15 days earlier during long day conditions (16 h light / 8 h dark) and about 25 days earlier during short-day conditions (8 h light / 16 h dark), compared to the control plants. It was proposed that early

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flowering transgenic barley might enable multiple cultivations within a single season (El-Assal et

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al., 2011).

In future, research will be focused on transgenic approaches to improve the technical quality of

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barley grain for economically profitable production of biofuel (Dunwell, 2009), or to alter grain composition for improvement of nutrient quality of animal feed.

4. Barley as a tool in plant molecular farming Although expression of stable and functional proteins remains a bottleneck for basic and applied research, certain plant-based heterologous expression systems are well established and considered as promising and competitive. Plant-based expression also called plant molecular farming (PMF) is a recent approach in biotechnology, where plant cells or tissues are used as biofactories for large scale production of recombinant proteins, secondary metabolites or chemicals. Plants address advantages of other biological systems but lack their pitfalls (Basaran and RodriguezCerezo, 2008). The main advantage of PMF is the possibility of large scale commercial production of high-value compounds within the growing plant tissues for lower costs (Fischer et al., 2004; Ma et al., 2003). Additionally, use of plants for commercial production avoids the ethical case deliberation of animal expression systems. Plants as production platforms are considered safe since there is a low to no risk of product contamination by animal or human

ACCEPTED MANUSCRIPT viruses and pathogens (Daniell et al., 2001; Fischer et al., 2004). Moreover, plants are able to perform complex eukaryotic post-translational modifications like glycosylation or SS-bond formation required for production of functional proteins structurally similar to their native

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counterparts (Ma et al., 2003; Ramessar et al., 2008).

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Industry is faced with various effective plant-based expression strategies ranging from plant cell suspensions to field-grown transgenic crops including cereals (Twyman et al., 2003). There are

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currently four different cereals (maize, rice, wheat, and barley) whose grains are commonly used as a vehicle for large scale production of peptides or proteins (Ramessar et al., 2008). The use of

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cereal seeds for PMF possesses critical benefits as grains provide biochemically inert and stable environment which allows long term storage of recombinant proteins or peptides at ambient

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temperature without loss of product quality, and enable simplified downstream processing (Stoger et al., 2002; Xu et al., 2012). After homozygous transgene fixation, it is also possible to grow the seeds on a field, and thus, increase the amount of recombinant product logarithmically.

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However, these benefits must be weighed against unique challenges arised by strict regulatory

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demands. Field-grown cereals represent an open-air bioreactor, and their cultivation must meet expected requirements of absolute isolation from food supply to avoid the risk of food

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contamination (Elbehri, 2005). One of these cereal host expression systems, barley, will be discussed in detail.

4.1 Barley grain as a biomanufacturing platform The use of barley grain as a tool for molecular farming has emerged as a favorable strategy, as it is a plant storage organ whose natural properties enable efficient, robust, and scalable heterologous production of desired compounds. From the chemical point of view, this end-use organ is comprised of four major constituents namely carbohydrates, proteins, minerals, and phytochemicals. Carbohydrates represent about 80% of the mature barley grain (MacGregor and Fincher, 1993). Starch, the product of photosynthetic carbon fixation, comprises about 50-70% of barley grain (Henry, 1988). It is presented in the form of starch granules mainly in the endosperm. Proteins are the second most abundant group of barley seed components. Their presence is within the range of 8-15% on a dry-weight basis, and they are mainly accumulated in endosperm. Barley seed proteins can be classified according their functions as structural, storage,

ACCEPTED MANUSCRIPT or metabolic and protective proteins (Fig. 3). Another possible classification is based on extraction properties of proteins, an approach established by Osborne (1895). According this classification, there are four different protein fractions in barley grains, namely albumins (4% of

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total soluble protein, TSP), globulins (18% of TSP), prolamins (37% of TSP), and glutelins (37% of TSP) (Zimolka, 2006; Gubatz and Shewry, 2010). For the purposes of PMF, knowledge on

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strong, seed-preferable expression of transgenes (Fig. 1).

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types of barley seed proteins is essential, as their promoters might be useful tools for driving

Besides efficient machinery that enables correct folding of the heterologous protein, developing

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barley grains possess various types of protease inhibitors. Some of the examples for protease inhibitors include Bowman-Birk type trypsin inhibitor (BBBI), α-amylase/subtilisin inhibitor

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(BASI), chymotrypsin inhibitor 2 (CI-2), and different types of serpins, as well as the proteins extracted by chloroform and methanol, so called CM proteins (Fig. 3). Besides their functions in defense against pathogens, some of these may protect barley grain cells against endogenous

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proteases. High content of protease inhibitors in barley grain together with a low content of water

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during dormancy allow long-term storage of heterologous proteins of interest at ambient temperature (Eskelin et al., 2009; Patel et al., 2000). Therefore, it is possible to decouple the

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process of protein production from the process of subsequent downstream processing. Extraction and purification of the heterologous products are largely assisted by the fact that barley grain has relatively low content of secondary metabolites, is free of endotoxins, and has a simple protein profile. Moreover, barley holds certain agronomical advantages such as availability of powerful methods for harvest, transport and storage of barley grains. Last but not least, domesticated diploid barley is a self-pollinating species. Thus, outcrossing with other non-transgenic plants is extremely rare (Ritala et al., 2002). Additionally, barley holds the GRAS (generally regarded as safe) status from the U.S. Food and Drug Administration (FDA). Taken together, barley grains provide unique features that address many of the drawbacks of other expression systems.

4.2 Seed-specific promoters suitable for molecular farming For the purpose of PMF, achievement of high levels of recombinant products in desired plant tissues is crucial. Transgene expression and target production can be increased by optimization of various parameters like the rate of transcription, transcript stability, and efficiency of translation

ACCEPTED MANUSCRIPT (optimization of codon usage) as well as stable accumulation of desired product (Stoger et al., 2002; Streatfield, 2007). Different seed-specific promoters suitable for molecular farming in barley grains are summarized in this section (Table 1); as these promoters hold the key to match

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the requirements for high protein accumulation. The use of promoters able to drive tissue-specific expression (Fig. 1) possesses several benefits over exploiting their ubiquitous counterparts.

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Proteins recombinantly produced in all parts of plant body may have negative pleiotropic effects

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on the vegetative growth, and thus influence yield. With the use of strong grain-specific promoters, it is possible to achieve higher accumulation levels of proteins in seeds. For example,

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maize constitutive ubiquitin-1 (ZmUBI-1) promoter was compared with rice endosperm-specific glutelin B-1 (OsGLUB-1) promoter in regard to product accumulation in barley T1 grains.

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Average accumulation amounts of recombinant products under the control of ZmUBI-1 promoter were 3- to 50-fold lower than that under the control of OsGLUB-1 promoter (Eskelin et al., 2009).

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Barley grain presents various tissues suitable for expression and deposition of heterologous

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products. The largest group of promoters widely used in PMF is endosperm-specific. Most of these promoters are derived from seed storage protein genes of barley or other cereals. One of the

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most commonly used endosperm-specific promoters is rice OsGLUB-1 promoter (Patel et al., 2000; Kamenarova et al., 2007; Eskelin et al., 2009). It has been used to express and stably store the cell wall degrading enzyme xylanase in developing barley grains (Patel et al., 2000). Xylanase improves the efficiency of feed grain conversion in monogastric animals. Expression of the fungal chimeric xylanase gene was driven under the control of OsGLUB-1 or barley hordein B-1 (HOR2-4) promoters. Although hordeins in ripe grain form about 50% of the total endosperm protein, the activity of recombinant xylanase was at least 2-fold higher in OsGLUB-1 than that in HOR2-4 transgenic lines. The rice GLUB-1 promoter has also been used in another comparative study on expression of full-length gene coding for collagen α-1 chain (COL1A1) and its more stable 45-kDa fragment in barley seeds. In one of the homozygous doubled haploid lines, accumulation levels of 45-kDa COL1A1 fragment reached 0.07% of total extractable protein. Whereas, accumulation of this collagen fragment driven by barley germination-specific aleurone α-amylase (α-AMY) promoter reached only 0.028% of total extractable protein in T0 lines (Eskelin et al., 2009). On the other hand, promoter of α-AMY gene represents a good candidate when aleurone-specific expression is desired, as indicated by expression data (Fig. 1). In other

ACCEPTED MANUSCRIPT studies, OsGLUB-1 promoter was successfully used for production of human lactoferrin in barley grains (Kamenarova et al., 2007; Tanasienko et al., 2011). Another strong endosperm-specific promoter suitable for PMF is barley endogenous hordein D

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(HOR3-1) promoter. This promoter is 3- to 5-times more active than barley hordein B and C

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promoters according to transient GUS expression assays (Sörensen et al., 1996). The possibility to employ HOR3-1 promoter to direct endosperm-specific expression of the gene coding for

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structural E2 protein of the CSFV (classical swine fever virus) was patented by Nelsen-Salz et al. (2003). The immunogenic E2 protein serves as a vaccine against the mammalian CSFV. Barley

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HOR3-1 promoter is one of the promoters used by ORF Genetics Ltd. (Iceland) for commercial production of biologically active recombinant human growth factors with a yield analogous to

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prokaryotic expression systems. Recently, Erlendsson et al. (2010) published a report describing HOR3-1 promoter-driven production of codon-optimized human FLT3 ligand. In the highest producing line, the estimated level of recombinant product reached up to 60 mg kg-1 of seeds.

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The HOR3-1 promoter was also used for expression of an engineered termostable endo-1,4-β-

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glucanase in barley endosperm tissues (Horvath et al., 2000). Accumulation of heat stable product in homozygous T2 seeds reached, on average, 5.4% of the extractable proteins. For high

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level expression of the recombinant enzyme, it was fundamental to optimize the codon and drive synthesis as a precursor with hordein D signal peptide. Fusion of heterologous protein with a hordein leader sequence enhances expression. A patented report describes the use of barley HOR3-1 promoter for production of natural sweetener thaumatin from African perennial herb Thaumatococcus daniellii. Authors prepared constructs, where GC-optimized thaumatin sequence was fused with hordein D or thaumatin signal sequences on N- or C-terminus (Stahl et al., 2009a). Additionally, α-AMY promoter was used for production of thaumatin with α-amylase inhibitor signal peptide (Rogers, 1997). In another study, barley HOR3-1 promoter was used to control the expression of a sequence coding for a hybrid protein comprised of codon-optimized human homeobox B4 protein and carbohydrate binding module from Thermotogota maritima. Inventors of this patented report described yet another strategic tool to maximize production of the chimeric protein in barley. Their approach was based on suppression of abundant endogenous barley storage proteins, as there is always competition for limited amount of different resources such as amino acids and translational machinery (Orvar, 2005).

ACCEPTED MANUSCRIPT Successful production of an edible vaccine in barley endosperm for porcine against F4-positive enterotoxigenic Escherichia coli (ETEC) was reported. In this study, immunogenic fimbral adhesin (FAEG), with adhesion to F4, was produced in endosperm and shown to be

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heterogeneously glycosylated and immunologically active. Effects of 3 different barley endosperm-specific promoters, namely HOR2-4, β-amylase (β-AMY), and trypsin inhibitor (TI)

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were evaluated. The TI promoter was determined as the most active in endosperm tissue using

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GUS expression. Hence, it was employed for high level accumulation of recombinant FAEG, which constituted up to 1% of grain TSP (Joensuu et al., 2006). The findings of this study are in

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accordance with previous expression studies, as TI gene seems to be strongly expressed in endosperm tissue, and expression driven by TI promoter does not show any leakage in other

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tissues (Fig. 1).

The barley γ-hordothionin promoter has also been used for the purpose of molecular farming in barley kernels. This promoter was employed to drive expression of codon-optimized human

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serum albumin gene that was produced as a fusion protein with γ-hordothionin signal peptide

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(Stahl et al., 2009b). To complete the list on most widely used promoters for molecular farming in barley grains, it is important to point out the promoter derived from a gene coding for wheat

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endosperm-specific high-molecular-weight glutenin Bx17 (HMW Bx17). The promoter was used to target expression of an anti-glycophorin single-chain antibody fused to an epitope of the HIV, which might be used as a reagent for detection of the virus in blood, into the barley endosperm. Using barley endosperm-specific expression strategy, high-level expression of the fusion antibody (150 μg g-1 grain) was achieved (Schünmann et al., 2002). An overview of the promoters used so far for PMF in the barley grain is presented in Table 1 and Fig. 4. Some other promoters have also been proven to direct strong, seed-specific expression of transgene, and hence could be powerful tools for PMF. Among these, barley endogenous aleurone-specific LTP2 (lipid transfer protein 2; Opsahl-Sorteberg et al., 2004) promoter was used to direct expression of Zea mays CKX1 (cytokinin oxidase/cytokinin dehydrogenase) gene. The strength and organ-specificity of the promoter were analyzed in homozygous barley plants. According to CKX activity assay (Frébort et al., 2002), no detectable change in the activity of the enzyme was observed in roots or leaves of transgenic plants. However, the CKX activity in crude protein extract from transgenic grains was significantly increased compared to that in the extract from the control plants. Additionally, transgenic plants were similar in height and root biomass to

ACCEPTED MANUSCRIPT non-transgenic ones (Fig. 5A). However, significant decrease in the number and size of grains was observed in transgenic plants. Most of the grains were not filled (Fig. 5B). The results were in accordance with the fact that the ZmCKX1 gene belongs to a family of strong morphogenes

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(Ye et al., 2008) deviating hormonal homeostasis in the plant body, and any leakage of the promoter out of the targeted tissue would lead to significant morphological changes (Holásková

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4.3. Products in cereal-based molecular farming

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et al., unpublished results).

Proteins and peptides are produced in plants for therapeutic purposes, industrial or agricultural applications, or for basic research. Earlier studies used tobacco and potato as model plants for

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production of antigens, proteins, and pharmaceuticals (Sijmons et al., 1990; Arakawa et al., 1997). However, recent improvements in transformation systems led to employment of cereals

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which enable higher product yield and require less processing. Cereal seeds display properties

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like dormancy, seedling vigor, nutrient storage, and strength against environmental constraints (Boothe et al., 2010), which are considered advantageous towards storage of expressed protein or

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pharmaceutical.

Among cereals, maize, rice, wheat, and barley have several advantages such as established technologies to generate transgenic plants, easy optimization of traits through breeding, and possibility of processing the seed into an edible form. Vaccine antigens, pharmaceuticals, and recombinant proteins produced in expression systems employing various plants (Daniell et al., 2009; Xu et al., 2012) including cereals (Ramessar et al., 2008) have been reviewed in comprehensive studies. Additionally, companies using different plant species and approaches for commercial production of various proteins have been summarized (Spok, 2007). Historically, the first industrial proteins commercially produced in cereal seeds include recombinant egg white avidin and bacterial β-glucuronidase (Hood et al., 1997; Hood et al., 1999). Avidin was the first plant-made protein that was marketed; and it is still on the market as a reagent (Sigma-Aldrich Co., A8706) produced in maize for research purposes. Considering the recent advances in biosensors based on immunochemical detection and quantitation relying on avidin-biotin interaction, it is apparent that recombinant avidin from maize keeps a huge potential as a commercial product.

ACCEPTED MANUSCRIPT Maize seeds have been used for production of recombinant human proteins and bacterial antigens as reagents for research or medicine. A full length mammalian α1 chain of collagen type I (Zhang et al., 2009) and heat-labile toxin B subunit (LTB) from enterotoxigenic E. coli (Chikwamba et

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al., 2002; Tacket et al., 2004) were produced in maize grains. Immunogenicity of LTB in human after oral administration demonstrated the feasibility of using edible transgenic plants to deliver

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protective antigens as novel oral vaccines (Tacket et al., 2004). On the other hand, vaccines and

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pharmaceuticals produced in food crops, specifically maize, raised substantial public concern. After the crisis in 2002, when the U.S. originated biotech company ProdiGene Inc. faced fines for

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violating Plant Protection Act (Fox, 2003), U.S. government has been trying to create incentives for biopharming companies to use non-food crops (e.g. tobacco). This has led to stronger

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regulation and reduced number of biotech companies working in the field for the production of drugs and chemicals in food and feed crops. To manage the safety and environmental impacts of plant-originated pharmaceuticals, the U.S. Department of Agriculture (USDA) and European

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Food Safety Authority (EFSA) have issued guidance for field testing of plants intended for

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industrial and pharmaceutical use (USDA-APHIS, 2008; EFSA, 2009).The public concern and the governmental regulations additionally stimulated the search for self-pollinating, biologically

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and environmentally safe hosts for industrial production of proteins and pharmaceuticals. Human serum albumin (HSA) was one of the first pharmaceutical proteins expressed in plants (Sijmons et al., 1990). Recently, expression of HSA in rice endosperm for large-scale production has been demonstrated (He et al. 2011). It was shown that the level of recombinant HSA reached 10% of TSP of the rice grain, and large-scale production yielded a productivity rate of 2.75 g kg-1 rice. As a self-pollinating crop plant, rice has been extensively used for production of pharmaceuticals, vaccine antigens, and industrial proteins (Ramessar et al. 2008). Recombinant human lactoferrin and lysozyme, which have been produced in rice (Huang et al., 2008; Yang et al., 2003), are available on the market (Sigma-Aldrich Co., L4040 and L1667) as research grade chemicals. These proteins have also been expressed in barley (Kamenarova et al., 2007; Stahl et al., 2002). Although gene integration and protein expression have been demonstrated using Southern and Western blotting, respectively, commercial adaptability in a large-scale production and expression levels as percentage of soluble proteins of barley were not indicated in these studies (Kamenarova et al., 2007; Stahl et al., 2002). On the other hand, recombinant lactoferrin produced in rice is currently under study as a novel, orally administered, preventive treatment for

ACCEPTED MANUSCRIPT antibiotic-associated diarrhoea (AAD). Phase II clinical trials on the formulation VEN100™ (Ventria Bioscience) containing lactoferrin as the main ingredient has been completed. It was reported that the Phase II clinical study showed a reduction in the risk of AAD by approximately

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50% with no observed adverse effects when VEN100 was administered in conjunction with

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antibiotics in a long-term care setting (http://ventria.com/).

Various proteins, vaccine antigens and pharmaceuticals have also been produced in grains of

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wheat and barley for the purpose of PMF (Table 2). Nowadays the main commercial focus of PMF in the Triticeae cereals concerns with the expression of various proteins in the barley

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endosperm (ORF Genetics Ltd, Iceland and Ventria Bioscience, CO). Besides human lactoferrin and lysozyme, a considerable number of other recombinant proteins have been expressed with

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immense potentials for large-scale production. Barley is a self-pollinating species, which is considered an advantage over other cereals. It displays low risk of uncontrolled gene flow and is considered biologically and environmentally safe. The pharmaceuticals produced in barley

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include human serum albumin, antithrombin III, α1-antitrypsin, lactoferrin, and lysozyme

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(Kamenarova et al., 2007; Stahl et al., 2002), mammalian collagen (Eskelin et al., 2009; Ritala et al., 2008), human growth factor FLT3 ligand (Erlendsson et al., 2010), barley lipoxygenase 2

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(Sharma et al., 2006), and thaumatin (Stahl et al., 2009a). In one of the earliest studies, Stahl et al. (2002) employed various human proteins to reveal the patterns and sites of transgene integrations into barley genome. Though the study solely aimed characterization of the integrations, transgenes were successfully integrated into barley genome under the control of various seed-specific promoters from barley (Fig. 1). Mammalian collagen that has various uses in medicine and food industry has been expressed in barley suspension cells (Ritala et al., 2008) and endosperm tissues (Eskelin et al., 2009). One of the biologically active human growth factors expressed in endosperm tissue of barley seeds, in a recent study, was human FLT3 ligand (Erlendsson et al., 2010). Additionally, the purification of the FLT3 ligand using sequential chromatography on immobilized metal ion affinity resin and cation exchange resin near to homogeneity was demonstrated. Barley endosperm was proposed as a viable platform for the bioproduction of human-derived growth factors since the yield of active FLT3 obtained from barley seed extracts was comparable to that from a bacterial expression system (Erlendsson et al., 2010). The barley-made human growth factor FLT3 ligand is currently on the market and priced

ACCEPTED MANUSCRIPT 5 to 10 times lower compared to counterparts obtained frombacteria or human cell culture lines, respectively (http://www.orfgenetics.com). Small antimicrobial peptides (AMPs), also called peptide antibiotics, are active against a broad

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range of pathogenic organisms attacking plants or animals (Thevissen et al., 2007). They present

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a new-generation of biocidal agents for plant protection as well as medical applications. Several studies reported expression of various synthetic, fungal or animal AMPs in plants including

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tobacco, rice, potato, and others (Arce et al., 1999; Coca et al., 2006; Imamura et al., 2010; Nadal et al., 2012). The AMPs include defensins, thionins, magainins, cathelicidins, non-specific lipid

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transfer proteins, and many others. Heterogeneous expression of AMPs in transgenic plants provided enhanced resistance against fungal and bacterial pathogens (Montesinos, 2007). For

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instance, cecropins, which constitute a family of AMPs, are key components of the immune response in insects. It was demonstrated that transgenic rice plants expressing the cecropin A gene from the giant silk moth Hyalophora cecropia showed improved resistance to Magnaporthe

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grisea, the causal agent of the rice blast disease (Coca et al., 2006). Various AMPs of insects

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(Langen et al., 2006; Osusky et al., 2000), frog (Yevtushenko and Misra, 2007) or human (Aerts et al., 2007) have been used to render the transformed plants more resistant to fungal pathogens.

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Cathelicidins and related AMPs are a family of small peptides found in many organisms. They are efficient effector molecules of mammalian innate immunity. A DNA fragment coding for the peptide LL-37, a derivative of human cathelicidin, was introduced into Chinese cabbage (Brassica rapa); and subsequently, improved resistance to bacterial and fungal pathogens was demonstrated in transgenic plants (Jung et al., 2012). It was proposed that AMPs expressed in specific tissues of barley might be used to improve plant protection against pathogens (Rahnamaeian et al., 2009). The broad spectrum activities of AMPs cover not only important plant pathogenic organisms but also clinically important human pathogens like Staphylococcus aureus, Pseudomonas aeruginosa and the human immunodeficiency virus (HIV). Besides improvement of plant defense, AMPs expressed in plants and produced in large-scale with high quality and low cost might be utilized in various medical applications. Therefore, AMPs such as cathelicidins, defensins, and magainins are considered critical targets for commercial production using PMF employing barley endosperm tissue as the production platform. Heterologous protein expression systems including PMF face critical obstacles that have to be resolved for commercial production (Jana and Deb, 2005; Steffensen and Pedersen, 2006; Desai

ACCEPTED MANUSCRIPT et al., 2010; Hopkins et al., 2012). Plant-based molecular farming and use of barley endosperm as the biofactory for commercial production pledge considerable promise to overcome certain obstacles. On the other hand, rapidly increasing need for recombinant proteins and

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pharmaceuticals in medicine and agriculture requires further improvement of plant expression technology as well as expression in barley endosperm, which in turn will help improve various

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applications in PMF and agriculture.

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5. Concluding remarks

Modern plant biotechnology allows researchers to overcome the limitations of classical breeding and meet the growing demand for food and nutritive quality. By overexpression of a single gene

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coding for a protein kinase regulating Na+ exclusion, barley could maintain 20 - 45% higher biomass under strong salinity stress (Roy et al., 2013). It is not usual to achieve such a significant

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improvement through traditional breeding. On the other hand, the results were obtained only

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under the controlled greenhouse conditions. Therefore it is essential to test generated transgenic lines under natural field conditions. As of the end of 2012, over one hundred low-scale field trials

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with transgenic barley have been performed in the U.S.A. (http://www.nbiap.vt.edu). More than fifty independent transgenic barley lines are currently under long-term (till 2017) field testing for evaluation

of

mainly

higher

drought

tolerance

or

nutrient

quality

in

Australia

(http://www.ogtr.gov.au). Contrary to other cereals, currently no transgenic barley variety is commercially available, excluding the lines used for molecular farming, which are in property of several biotech companies. In Europe, hitherto not more than 10 field trials with transgenic barley were accomplished (http://gmoinfo.jrc.ec.europa.eu/). Moreover, field trials for GM crops including barley run by academic, governmental, or private research institutions are often destroyed by activists in Europe (Kuntz, 2012). Persisting skepticism of the public to GM crops is one of the major limitations in testing and introduction of novel genetically modified varieties with improved agronomical and nutritional traits into the market. Thus, considerable effort needs to be invested in public education to drive reasonable judgment concerning GM food based on scientific facts but not on rumor and ignorance. Nevertheless, safety concerns including potential risks associated with uncontrolled release of transgenes in environment have to be continuously addressed and strictly monitored. One of the major concerns about transgenic crops is the use of

ACCEPTED MANUSCRIPT genetic materials from distant species, e.g. bacteria or human. The doubts are linked to ethical considerations and dispersal of new gene combinations in the environment. A promising solution to minimize potential risks of interspecies transgene mingling is cisgenesis or intragenesis

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concept, which implies the fact that a plant is transformed with genes and promoters originated from its own genome or closely related species with which the target plant is capable of sexual

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hybridization. An alien gene conferring resistance to selective agent is co-transformed on

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independent T-DNA segments and later segregated out of the pure cisgenic or intragenic line. First cisgenic barley lines have already been developed recently (Holme et al., 2012). In another

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approach, plant genes instead of bacterial ones are used for selection of transformed cells. One of these selection systems utilizes a plant gene encoding for phosphomannose-isomerase that

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converts mannose-6-phosphate to fructose-6-phosphate. Therefore only the transformed cells are capable of utilizing mannose as a carbon source. The system has already been tested on maize plants (Negrotto et al., 2000). Constitutive overexpression of an Arabidopsis endogenous gene

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coding for a member of the ABC transporter family allows transgenic plants to be selected on

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kanamycin concentration lethal for wild type plants (Mentewab and Stewart, 2005). Evaluation of these systems in transgenic approaches to develop plants with enhanced agronomical traits is thus

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of a high interest nowadays. Finally, it should be indicated that discussion concerning exemption of cisgenic plants from strict EU directive for GM crop handling has recently been started (EFSA, 2012).

Acknowledgements

The authors thank Dr. Ludmila Ohnoutková for sharing unpublished data and Kateřina Janošíková for technical assistance with preparation of figures. This work was supported by the OP RD&I grant ED0007/01/01 Centre of the Region Haná for Biotechnological and Agricultural Research and the grant KONTAKT II LH13062 from the Ministry of Education Youth and Sports, Czech Republic and IAA601370901 from the Grant Agency of the Academy of Science, Czech Republic. Petr Galuszka and Ivo Frébort acknowledge the support of the Operational Program

Education

CZ.1.07/2.3.00/20.0165).

for

Competitiveness

-

European

Social

Fund

(project

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ACCEPTED MANUSCRIPT

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Zalewski W, Galuszka P, Gasparis S, Orczyk W, Nadolska-Orczyk A. Silencing of the HvCKX1

productivity. J Exp Bot 2010;61:1839-51.

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gene decreases the cytokinin oxidase/dehydrogenase level in barley and leads to higher plant

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Zhang C, Baez J, Pappu KM, Glatz CE. Purification and characterization of a transgenic corn grain-derived recombinant collagen type I alpha 1. Biotechnol Progr 2009;25:1660-8. Zhang J, Tiwari VK, Golds TJ, Blackhall NW, Cocking EC, Mulligan BJ, et al. Parameters

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Cell Tiss Org 1995;41:125-38.

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influencing transient and stable transformation of barley (Hordeum vulgare L.) protoplasts. Plant

Zhang L, Castell-Miller C, Dahl S, Steffenson B, Kleinhofs A. Parallel expression profiling of

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barley-stem rust interactions. Funct Integr Genomics 2008;8:187-98. Zhang S, Cho MJ, Koprek T, Yun R, Bregitzer P, Lemaux PG. Genetic transformation of commercial cultivars of oat (Avena sativa L.) and barley (Hordeum vulgare L.) using in vitro shoot meristematic cultures derived from germinated seedlings. Plant Cell Rep 1999;18:959-66. Zhang WH. Malate-permeable channels and cation channels activated by aluminum in the apical cells of wheat roots. Plant Physiol 2001;125:1459-72. Zhang Y, Shewry PR, Jones H, Barcelo P, Lazzeri PA, Halford NG. Expression of antisense SnRK1 protein kinase sequence causes abnormal pollen development and male sterility in transgenic barley. Plant J 2001;28:431-41. Zhao JS, Ren W, Zhi DY, Wang L, Xia GM. Arabidopsis DREB1A/CBF3 bestowed transgenic tall fescue increased tolerance to drought stress. Plant Cell Rep 2007;26:1521-8. Zimolka J, 2006. Ječmen - formy a užitkové směry v České republice. Profi Press, Praha, 2006. Zou X, Seemann JR, Neuman D, Shen QJ. A WRKY gene from creosote bush encodes an activator of the abscisic acid signaling pathway. J Biol Chem 2004;279:55770-9.

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Figure 1. Expression profile of barley genes, of which promoters have already been used in

PT

transgenic plants, in different tissues and organs (A) and upon various stimuli (B). Data are generated via Genevestigator software (Hruz et al., 2008). Intensity signal for each probe in

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different organ and tissues was obtained from 806 independent Hv_22k Barley Genome 22k

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chips processed with RNA extracted from various cultivars of wild type barley. Conditions, upon which regulation of expression was studied, are described in more detail in following references: Caldo et al., 2006; 2Millett et al., 2009; 3Boddu et al., 2007; 4Zhang et al., 2008; 5Chen et al.,

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2010; 6Molitor et al., 2011; 7McGrann et al., 2009; 8Delp et al., 2009; 9Tommasini et al., 2008; Guo et al., 2009; 11Mangelsen et al., 2011; 12Walia et al., 2007; 13Greenup et al., 2011; 14Öz et

al., 2009;

Gardiner et al., 2010; §not published; *cultivar well adapted to drought stress;

drought sensitive cultivar.

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#

15

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10

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Figure 2. Experimental set up of small-scale GM barley field trial. One month old (A) and two months old (B) transgenic barley plants with increased production of

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Aspergilus niger phytase (T5 homozygous generation of α-AMY::phytase) grown on authors’s experimental field in Olomouc (2012 season) are displayed. The field was at least one hundred metres far from other cultivated barley plants, bordered by fence, protected with two metres wide barrier of wild type barley plants (Golden Promise) and marked appropriately.

Figure 3. Schematic summary of the basic groups of mature barley seed proteins. Classification is based on protein function. Proteins whose gene promoters have already been used for the purpose of molecular farming in barley grains are marked in boxes. A, aleurone; αAMY, α-amylase; BASI, barley α-amylase/subtilisin inhibitor; BP1, barley peroxidase 1; BPH, barley peroxidase homolog; BSSP, barley seed specific peroxidase 1; CI, chymotrypsin inhibitor; CM, chloroform methanol extracted proteins; EM, embryo; EN, starchy endosperm; LTP, lipid transfer proteins; ND, non-determined, SERPINS, serin protease inhibitors; TI, trypsin inhibitor. Adapted from Gubatz and Shewry, 2010; Hayes et al., 2003; Eggert et al., 2010.

ACCEPTED MANUSCRIPT Figure 4. Tested barley promoters and their spatial specificity. Specificity of tested barley promoters as well as promoters used in mature transgenic barley plants (A), grains (B) and seedlings (C) is shown. Arrows indicate the organs where the

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promoters are mainly expressed. AB stress inducible, promoters which are induced by abiotic and

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biotic stress stimuli. Abbreviations of promoters are given in Table 1.

Figure 5. Barley LTP2 promoter and its spatial specificity.

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(A) Phenotype of T2 generation of barley homozygous LTP2::ZmCKX1 transgenic plants (right) is compared with that of nontransgenic plants (left). Wild-type and transgenic plants are

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morphologically indistinguishable. (B) Changes in phenotype of the spikes of barley homozygous LTP2::ZmCKX1 T2 plants (right) and nontransgenic plants (left) are displayed. Transgenic plants

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produce significantly lower number of grains on a spike, and most of the grains are empty.

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UBI1

Ubiquitin-1

Barley Barley Barley

AAX95098.1 NP_001105409.1 S51061.1

Barley,wheat, oat

Al-Saady et al., 2004

ACS44709.1

GUS, GFP RNAi construct against HvCKX1 RNAi construct against HvRBOHF2 RNAi construct against HvWHY1 RNAi construct against HvBI

Barley Barley

Murray et al., 2004 Zalewski et al., 2010

NP_001148453.1

Barley

Proels et al., 2010

Barley

Melonek et al., 2010

Barley

Eichmann et al., 2010

Barley

GUS

Barley

Barley

GUS

Barley

Dunn et al., 1998 Molina et al., 1996 Brown et al., 2001

Barley A.thaliana Barley Barley Barley Barley Barley Barley A.tumefaciens

GUS Osmyb4 GUS GUS, GFP GUS GUS, GFP GUS, GFP GUS Metchnikowin

Barley Barley Barley Barley Barley Barley Barley Wheat Barley

Dal Bosco et al., 2003 Soltész et al., 2012 Robertson, 2003 Xiao and Xue, 2001 Himmelbach et al., 2010 Xiao and Xue, 2001 Zou et al., 2004 Freeman et al. 2011 Rahnamaeian et al., 2009

GUS

COR14b COR15a DHN1-2 DHN4s GER4a-f HVA1s HVA22 HSP17 MAS

Cold-regulated Cold-regulated Dehydrin 1-2 Dehydrin 4s Germin-like proteins Late embryogenesis abundant Abscisic acid-induced protein Heat shock protein Mannopine synthase

CE P

AC

Abiotic and biotic stress inducible BLT4.9 Low temperature responsible LTP4.3 (lipid transfer protein) BLT101.1 Low temperature responsible

ID number of gene driven

Tingay et al., 1997 Wan and Lemaux, 1994 Wan and Lemaux, 1994

CR

Whole viral genome

GUS GUS Bar

US

ScBV

Rice Maize Cauliflower Mosaic Virus Sugarcane Bacilliform Badnavirus Maize

MA N

Actin 1 Alcohol dehydrogenase 1 Whole CaMV genome

TE D

Constitutive ACT1 ADH1 CMV35S

IP

T

Table 1 List of promoters used for barley transformation and barley promoters used for transformation of other plants. Promoter Gene driven Origin Transgene Target plant Reference

Z66529.1 Z66528.1 AB370200.1 Z25537.1 AJ512944.1 U01377.1 AF043087 EF409517.1 X93171.1 X78205.1 L19119.1 X64560.1 AB179739

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Raventos et al., 1995 Xiao and Xue, 2001 Horvath et al., 2003 Nagaraj et al., 2001 Um et al., 2007 Leckband and Lörz, 1998 Ouellett et al. 1998

X54325.1 AF333275.1 AF509747.1 X83233 AF109124.1 AH004641.1 AF031235.1 M93342.2

GUS, GFP

Barley Barley Barley Barley Barley Barley, wheat Barley, wheat, rye, rice, Brassica, alfalfa, cucumber, tomato, pepper Barley

WSI18j Root specific EXPB

Water stress-induced

Rice

Xiao and Xue, 2001

AF246702.1

Expansin B

Barley

GFP

A.thaliana, rice

AY351785

Dioxygenase

Barley

GUS

Tobacco

IDS3

Dioxygenase

Barley

GUS

NAS1

Nicotinamine synthase

Barley

GUS

Phosphate transporter Root abundant factor

Barley Barley

GUS, GFP GFP

Higuchi et al. 2001; Ito et al., 2007 Schünmann et al. 2004 Jung et al. 2007

AK253031

PHT1 RAF Leaf specific BTH7 Grain specific α-AMY

A.thaliana, tobacco A.thaliana, tobacco Rice A.thaliana

Kwasniewski and Szarejko 2006, Won et al. 2010 Kobayashi et al. 2003; Yoshihara et al. 2003 Kobayashi et al. 2007

IDS2

Leaf thionin

Barley

GUS

Tobacco

Holtorf et al. 1995

L36883.1

α-amylase

Barley

Collagen α-1 Thaumatin Antithrombin III, α-1-antitrypsin, serum albumin, lysozyme Thermotolerant (1,3-1,4)-βglucanase

Barley Barley Barley

Eskelin et al., 2009 Rogers, 1997 Stahl et al., 2002

Barley

Jensen et al., 1996

T

Cat GUS, GFP HvRPG1 GUS AtNDPK2 GUS, VST1 Luc

IP

Maize Rice Barley Barley Sweet potato Grape Wheat

CR

Pathogenesis related protein Responsive to ABA Stem rust resistance Fructan 6 fructosyltransferase Peroxidase Stilbene synthase Cold-specific

AC

CE P

TE D

MA N

US

PRMS RAB16Bj RPG1 6-SFT SWPA2 VST1 WCS120

D15051.1 AB024007.1

AF543197 DQ102384

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Barley Barley

EM

Early-maturing

Wheat

GL9HI GLO1 GLUB-1

Globulin 1 Glutenin B1

Wheat Oat Rice

High-molecular-weight glutenin Bx17

Wheat

HMWGLU1

High molecular weight glutenin subunit 1Dx5

Wheat

HOR1

Hordein C

Barley

T

IP

Nuutila et al., 1999

Barley, wheat

Z12961.1

GUS Thermotolarant βamylase Anthocyanin thermotolerant (1,3-1,4)-ßglucanase GFP

Barley Barley

Furtado et al., 2003; Furtado et al., 2009 Okada et al., 2000 Kihara et al., 2000

wheat Barley

Doshi et al., 2006 Jensen et al., 1996

AF155129.1 M62740.1

Barley

X52103.1

GUS GFP Xylanase Collagen α-1 Lactoferrin

Barley Barley Barley Barley Barley

Lysozyme fusion of antiglycophorin single chain and epitope of HIV sucrose nonfermenting-1related protein kinase GUS

Barley Barley

Furtado and Henry, 2005; Furtado et al., 2009 Kovalchuk et al., 2012 Vickers et al., 2006 Patel et al., 2000 Eskelin et al., 2009 Kamenarova et al., 2007; Tanasienko et al., 2011 Huang et al., 2006 Schünmann et al., 2002

Barley

Zhang et al., 2001

Barley

Entwistle et al., 1991; X60037.1 Muller and Knudsen, 1993;

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HMW Bx17

X05166.1

Matthews et al., 2001

CR

Dehydrin 12 (1,3-1,4)-β-glucanase isoenzyme

Barley

Lanahan et al., 1992 Caspers et al., 2001

Barley

US

DHN12 EII

β-AMY

Barley

MA N

Barley

Barley Barley

TE D

bifunctional αamylase/subtilisin inhibitor β-amylase

ASI

GUS Endo-β-1,4xylanase Endo-β-1,4glucanase α-amylase, αglucosidase GFP

AF414082

JF332038.1 AY795082.1 X54314.1

KC254854.1

Hordein D

Barley

Xylanase

Barley

GUS GFP GUS E2 of the CSFV virus FLT3 ligand Thermotolerant (1,3-1,4)-ßglucanase Thaumatin Chimeric human homeobox B4 GFP

Barley Barley Wheat Barley

IP

Barley

MA N

TE D

γ-hordothionin Isoamylase 1 Jekyll Lemma 1

Barley Barley Barley Barley

LEM2 LTP1 LTP2

Lemma 2 Lipid transfer protein 1 Lipid transfer protein 2

Barley Barley Barley

LTP6 MRP1 PR9a PR60 PR602 TI

Lipid transfer protein 6 Myb-related protein 1

Barley Maize Rice Wheat Rice Barley

GFP GUS GUS GUS GUS Fimbral adhesin

Trypsin inhibitor

AC

CE P

γ-HORTHIO ISO1 JEKYLL LEM1

GUS Lactoferrin Serum albumin GFP GFP GFP GFP GFP Anthocyanin GUS

Sörensen et al., 1996 Cho et al., 1999b; Patel et al., 2000 Sörensen et al., 1996 Furtado et al., 2009 Pistón et al., 2008 Nelsen-Salz et al., 2003

Barley Barley

Erlendsson et al., 2010 Horvath et al., 2000

Barley Barley

Stahl et al., 2009a Orvar et al., 2005

Barley

Cho et al., 2002; Furtado et al., 2009 Pistón et al., 2008 Stahl et al., 2002 Stahl et al., 2009b Sun et al., 2003 Radchuk et al., 2006 Skadsen et al., 2002 Somleva and Blechl, 2005 Abebe et al., 2006 Doshi et al., 2006 Kalla et al., 1994; OpsahlSorteberg et al., 2004 Federico et al., 2005 Barrero et al., 2009 Li et al., 2008b Kovalchuk et al., 2009 Li et al., 2008b Joensuu et al., 2006

US

HOR3-1

Hordein B

CR

HOR2-4

T

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Wheat Barley Barley Barley Barley Barley Wheat Barley Wheat Rice Barley Barley Barley Barley Barley Barley

809030

X84368.1

HVU22951 AF142588.1 AM261729.2 AF330255.1 AY684928 X60292 X69793.1 AY662492.1 AJ318519.1 EU264060.1 EU264062.1 EU264061.1 X65875.1

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Functional or clinical evaluation / Preventive indication / Usage

Reference / Company Kamenarova et al., 2007

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Table 2 Selected list of pharmaceutical and industrial proteins produced in barley and wheat. Product Plant used Tissue / Promoter Expression level Pharmaceutical proteins Human lactoferrin Barley Endosperm or NA whole plant/ Rice glutelin B1 or Maize Ubiquitin1 Barley

Endosperm/ Barley hordein D

Human serum albumin

Barley

Aleurone/ Barley α-amylase

NA

Human lysozyme

Wheat

> 3-40% TSP

Human lysozyme

Barley

NA

Human milk protein.

Huang et al., 2006

Human lysozyme

Barley

Endosperm/ Wheat HMW Glutenin 1Bx17 Endosperm/ Rice Glutelin B1 Aleurone/ Barley α-amylase

NA

Stahl et al., 2002

Human collagen type I α1 chain (COL1A1)

Barley

Suspension cells/ Maize Ubiquitin1

2-9 μg l-1

Human full length COL1A1 and 45 kDa COL1A1 fragment

Barley

Endosperm/ Rice Glutelin B1

13-45 mg kg-1 seed

Human growth factor FLT3 ligand

Barley

Endosperm/ Barley hordein D

Up to 60 mg kg-1 seed

PCR and nucleotide sequencing were used for determination of genome integration sites. Similar to COL1A1 purified from yeast based on Western blotting, pepsin resistance, and mass spectroscopy. Mass spectroscopy and analysis of amino acid composition revealed low level of hydroxylation at proline residues. Biological activity of FLT3 ligand was demonstrated in human acute myeloid leukemia cells.

US

CR

Human lactoferrin

An iron binding protein with antimicrobial activity. Southern and Western blot analyses for verification of transgene integration and expression. PCR and nucleotide sequencing were used for determination of genome integration sites. PCR and nucleotide sequencing were used for determination of genome integration sites. Human milk protein.

AC

CE P

TE D

MA N

NA

Stahl et al., 2002

Stahl et al., 2002

Huang et al., 2010

Ritala et al., 2008

Eskelin et al., 2009

Erlendsson et al., 2010

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Human α1-antitrypsin

Barley

Aleurone/ Barley α-amylase

NA

Single chain Fv antibody ScFvT84.66

Wheat

Whole plant/ Maize Ubiquitin1

NA

Single chain Fv antibodies against CD4 and CD28 Human antithrombin III

Wheat

50-180 μg g-1 seed NA

ISOkine, DERMOkine, various growth factors and cytokines

Barley

Whole plant/ Maize Ubiquitin1 Endosperm or aleurone/ Barley hordein D or Barley αamylase Endosperm/ Barley hordein D

Heat-stable phytase from Aspergilus niger

Wheat

Endo-xylanase from Bacillus subtilis

Wheat

Legumin A from pea

Wheat

ETEC Fimbrial adhesin FaeG F4 (K88)

Barley

Synthetic anti glycophorin ScFv-HIV epitope fusion

Barley

NA

Molecular farming of human proteins and growth factors. Various applications in medical research and diagnostics.

ORF Genetics Ltd.

Endosperm/ Wheat HMW Glutenin 1-D1 Whole plant/ Maize Ubiquitin1 Endosperm/ Wheat HMW Glutenin 1-D1 Endosperm/ Wheat LMW Glutenin G1D1 Endosperm/ Barley Trypsin Inhibitor Endosperm/ Wheat HMW Glutenin 1Bx17

NA

Molecular farming of second generation biofuels.

Harholt et al., 2010

NA

Grains with improved digestibility for non-ruminant animal feed. Grains with improved baking quality.

Brinch-Pedersen et al., 2000 Harholt et al., 2010

NA

Grains with altered protein composition.

Stoger et al., 2001

NA

Edible vaccine for pigs partially effective against ETEC-induced diarrhoea. Antibody. Usage as a diagnostic reagent for HIV.

Joensuu et al., 2006

US

CR

IP

T

Stahl et al., 2002

MA N

TE D

Wheat

AC

Industrial proteins and enzymes Ferulic acid esterase from Aspergilus niger

CE P

Barley

PCR and nucleotide sequencing were used for determination of genome integration sites. Antibody against carcinoembryonic antigen. Tumor-associated diagnostic reagent. Recombinant antibody fragments for ocular use. PCR and nucleotide sequencing were used for determination of genome integration sites.

NA

NA

Stoger et al., 2000

Brereton et al., 2007 Stahl et al., 2002

Schünmann et al., 2002

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Barley

Thaumatin from Thaumatococcus daniellii

Barley

TE D CE P AC

Grains containing thermostable 1,31,4-β-glucanase for better malting. Grains with altered oxygen availability. Plants with modified oxylipin status.

Horvath et al., 2000 Wilhelmson et al., 2007 Sharma et al. , 2006

Grains containing a natural sweetener for brewing industry.

Stahl et al., 2009a

T

Lipoxygenase 2 from barley

NA

IP

Barley

NA

NA

CR

Vitreoscilla haemoglobin

Endosperm/ Barley hordein D Whole plant/ Maize Ubiquitin1 Whole plant/ Cauliflower Mosaic Virus 35S Endosperm/ Barley hordein D

> 2 g kg-1 seed

US

Barley

MA N

Heat stable (1,3-1,4)-β-glucanase

AC

CE P

TE

D

MA

NU

SC R

IP

T

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Figure 1

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T

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TE

D

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Figure 2

CE P AC

Figure 3

TE

D

MA

NU

SC R

IP

T

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Figure 4

TE

D

MA

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SC R

IP

T

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Figure 5

CE P

TE

D

MA

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