1 Barley Breeding

Barley (Hordeum vulgare L.) is one of the first crops to be domesticated and on the other hand one of the most genetically diverse cereal species [1]. The cultivation history of the barley was started with the first seeding about 10,000 years ago by the farmers in Near East [1, 2]. In Europe, barley is nowadays the second most important cereal crop. It is mainly used for animal feed and beverage production. Although human diet is not the primary use, barley offers several health benefits and is still an important source of calories in Northern and Eastern Europe as well as in other parts of the world such as North Africa, Middle East and Asia.

Over decades, breeders have utilized many strategies to introduce novel heritable mutations into plant genomes in order to develop new improved varieties. The use of numerous physical, chemical and biological mutagens such as gamma rays [3], X-rays [4], ethyl methanesulfonate [5], sodium azide [6], Agrobacterium tumefaciens [7] and transposon-based molecular markers [8] has facilitated the rapid extension of genetic diversity throughout the last century. However, these approaches have significant limitations, including the non-specific character of the produced mutations, the huge number of unnecessary mutations and the occasional undesired deletions, duplications or rearrangements of large genomic fragments [9]. Methods of haploid production are also an important tool in barley breeding, being time-saving and providing genetically fixed breeding lines.

Current breeding goals for barley depend on local conditions and vary a lot. In order to meet the increasing demand for livestock feed, starch, and a range of alcoholic (such as beer, whisky, and ethanol) and non-alcoholic (such as barley tea, barley coffee, and malt drink) beverages, barley breeding must focus on developing high-yielding and stress-tolerant varieties that can thrive even in challenging climatic conditions. Global changes such as the predicted increase in human population and diet, set some common goals both for breeding and agricultural crop production [10]. Thus, breeding for disease resistance, high yielding and stability are crucial components worldwide to ensure food security and satisfy the increasing demand. Together with the accelerating global changes, barley breeding has been challenged to speed up the process and multiplex a variety of traits in new varieties.

2 Genome Editing Advancements in Barley

Among gene editing technologies there are three major classes of synthetic endonucleases applied in plants: (a) zinc finger nucleases (ZFNs) are endonucleases linked to a multi-zinc-finger DNA-binding domain [11]; (b) transcription activator-like effector nucleases (TALENs) are composed of multiple transcription-factor-like DNA-binding domains linked to endonuclease domain [12]; (c) the widely used CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) system is composed of single guide RNAs (sgRNAs) enabling targeting multiple genes simultaneously and the Cas nuclease [13, 14]. First, TALEN-mediated genetic modifications in barley were induced in embryogenic pollen and leaf epidermis of winter barley variety ‘Igri’ [15, 16]. At the same time, Lawrenson and colleagues demonstrated for the first time the use of CRISPR/Cas9 technology in immature embryos of barley variety ‘Golden Promise’ to generate stable and inheritable mutations [17]. Biolistic transformation method for inducing CRISPR/Cas9-mediated InDels in ‘Golden Promise’ was presented only couple of years later [18].

Barley genome assembly was completed on the North American barley variety ‘Morex’ in 2017 [19]. Soon thereafter several protocols optimizing CRISPR/Cas protocols for barley were published [20,21,22,23]. The first Cas nuclease utilised in site-directed mutagenesis of barley was SpCas9 originated from Streptococcus pyogenes. Recently, the use of two versions of high efficiency endonuclease LbCas12a from Lachnospiraceae bacterium coupled with CRISPR was reported in barley [24].

Most CRISPR/Cas-mediated approaches focus on “negative effects” and generation of null alleles or loss-of-function alleles by targeting coding regions, while many agronomically important traits are associated with gain-of-function alleles. New strategies employed in other plants target non-coding promoter regions. Li and co-workers [25] engineered allelic variation by editing tomato KLUH promoter around a single-nucleotide polymorphism (SNP) located in a conserved putative cis-regulatory element [25]. Among twenty-one mutant alleles with various insertions and deletions, five mutant alleles showed a consistent increase in fruit weight. Moreover, promoter editing has proven useful in altering plant architecture in tomato [26], in developing resistance against Xanthomonas in rice [27, 28] and citrus [29], and in engineering drought tolerance in maize [30, 31]. Editing of non-coding cis-regulatory elements (CRE) offers considerable potential for crop improvement via fine-tuning of gene expression that cannot be achieved by simple knockout mutations. However, its widespread application is still hampered by the lack of precise knowledge about functional motifs in CRE [32]. Recent advancement CRISPR-Combo enables genome editing (targeted mutagenesis or base editing) and gene activation in plants simultaneously [33].

The above-mentioned findings have opened the great potential of rapid characterisation of gene function in barley, followed by advancement in precision and increased speed in breeding. New breeding techniques (NBTs) now enable switching on/off target genes in barley or convert allelic variants into more advantageous alleles without genetic linkage drag. These approaches could support traditional breeding by overcoming the limits of random mutagenesis and at the same time without developing transgenic plants. However, there are several bottlenecks in the successful utilisation of CRISPR/Cas system in barley such as transformation efficiency and the risk for off-targets. Transformation efficiency is one of the obstacles hindering the use of CRISPR/Cas in the production of new barley varieties. The risk of off-target mutations can be minimized with the help of large number of bioinformatic and computational tools developed to date, which facilitate gRNA site selection and evaluation of the probability for off-target events [34].

Thus, genome editing with the CRISPR/Cas system has been presented to be applicable in barley and a suitable technology for precision breeding. So far, the examples of using genome editing in barley have mainly focused on the validation of mutagenesis protocols and gene function, however, many genes and traits applicable in breeding are yet to be tested and verified.

3 Using Genome Editing to Target Disease Resistance, Yield and Stability

The discovery of the CRISPR/Cas system about ten years ago has brought an extensive precision to the portfolio of site-specific mutagens and a wide range of inducible modifications, which have many putative applications in breeding. Efficient, easy to use and highly target-specific single-guide RNAs (sgRNA) enable crop breeders to boost specifically either yield, biomass, abiotic/biotic stress tolerance, disease, pest resistance or any other trait [35,36,37]. Recent experiments combining customisable endonucleases and doubled haploid technology facilitate and accelerate the induction of multiple homozygous and inheritable mutations even further [38]. There are also several recent reviews available about the potential of using CRISPR/Cas genome editing in barley [39,40,41,42,43,44,45]. Here we shall highlight the potential of genome editing in barley breeding with the focus on breeding for high yielding, disease resistant and stable varieties.

Overall plant immunity and genome stabilization could be one of the targets to induce disease resistant barley varieties. There are a few examples in barley, where disease resistance has been tackled with the aid of CRISPR/Cas technology. The cosmopolitan fungal pathogen Fusarium graminearum causes fusarium head blight (FHB), which not only reduces crop yield but also accumulates mycotoxins in barley grains. 2-oxoglutarate Fe(II)-dependent oxygenase (2OGO) has been identified as a susceptibility factor and plant immunity suppressor in Arabidopsis. Barley orthologue Hv2OGO was shown to complement the CRISPR/Cas9-induced knock out mutation in Arabidopsis and may have a similar role in controlling resistance to FHB in barley [46]. In addition, seven MORC proteins in barley, paralogs of Microrchidia (MORC) protein family, were shown to be involved in plant immunity. CRISPR/Cas-induced double knockout mutants of HvMORC1 and HvMORC6a showed increased disease resistance to fungal pathogens Blumeria graminis and Fusarium graminearum [47, 48]. A large number of mildew locus o (mlo) mutants have been found or generated in various barley varieties, which exhibit strong resistance to powdery mildew fungus Blumeria graminis f. sp. hordei. Recently, CRISPR/Cas9-mediated reverse genetics approach was employed to elucidate the molecular function of MLO [49].

Wheat dwarf virus (WDV) is an economically important, insect-transmitted DNA virus, which infects also barley, causing severe yield losses. Direct antiviral utilization of the CRISPR/Ca9 system was presented in barley by establishing WDV resistance via targeting sgRNA sequences against viral genome [50]. Eukaryotic virus translation initiation factor E (eIF4E) is a plant cellular translation initiation factor and an essential target in potyvirus infection. Barley plants with modified HveIF4E were generated, but viral resistance is yet to be tested [51].

Soil-borne bymoviruses barley yellow mosaic virus (BaYMV) and barley mild mosaic virus (BaMMV) infect young winter barley seedlings in autumn and can cause yield loss up to 50%. PROTEIN DISULFIDE ISOMERASE LIKE 5–1 (PDIL5-1) from ancient landraces and wild relatives of barley confers resistance to all known strains of these viruses. Novel genome-edited PDIL5-1 alleles were shown also to be resistant to BaMMV, without any adverse effects on growth or yield [52].

Yield stability is the genotype’s ability to produce consistently high yield in diverse environments. Breeding for high yield and stability is a complex process that requires the consideration of various factors such as genetics, environment and management practices. Breeding for yield requires the selection of high-yielding genotypes with desirable agronomic traits such as plant height [53, 54], physiological maturity [55], disease and pest resistance [56], and lodging resistance [57]. Previous studies on CRISPR/Cas editing technology in barley targeted HvPM19 multi-copy genes (PM19-1 and PM19-3), associated with grain dormancy [17]. Lawrenson and colleagues transformed the two PM19 genes independently into variety ‘Golden Promise’. Genome-editing of the cytokinin oxidase/dehydrogenases (HvCKX1 and HvCKX3) in barley, which are regulating endogenous cytokinin metabolism, has shown their importance in regulating root length, tillering and yield [20, 58]. Galli and colleagues described transformation of HvMORC1 and HvMORC6a CRISPR/SpCas9 constructs to regulate transposable elements to increase biotic stress resistance and agronomic traits in barley [48,64,]. Cellulose synthase-like gene superfamily (HvCslF3, HvCslF6, HvCslF9 and HvCslH1) genes are related to low grain (1,3; 1,4)-β-glucan content in barley, which is a preferred trait in brewing and distillation processes [59, 60]. D-hordein gene (HvHor3) has also been targeted to change the D-hordein composition and other grain phenotypic features [61, 62]. Gene-editing of the caffeic acid O-methyltransferase 1 (COMT1) for use in lignocellulosic biomass and lignin biosynthesis has also been utilized [63,64,65].

The terms “phenotypic stability and yield stability” are often used to refer to phenotypic variations of the genotypes. Moreover, according to Becker and Leon a stable genotype is one that performs consistently despite significant statistical differences in environmental variables [66]. Stability of quality traits to produce superior varieties in cereals is very important for breeders because genotype ranks affect selection efficiency for genotypes that perform well under different environmental stress factors such as drought [67], salinity [68], diseases [69] to produce superior varieties in cereals. Thus, several studies have reported that the yield stability is correlated with the biomass [70], photosynthetic capacity [71], flowering time [72]. Newly developed targeting and genome editing technologies provide an opportunity to manipulate specific genomic sequences for improved yield stability. Recently, most of the genome editing studies in barley focus on grain quality, by targeting phytase activity [73], flavoenzyme activity [20], high amylose content [74], D-hordein content [62], grain size and composition [75], lignocellulosic content in secondary cell walls [63] and vitamin E biosynthesis in grains [76].

4 Challenges in Barley Genome Editing

Although, the CRISPR/Cas is currently actively studied in many research centres and breeding companies, there are still a number of limitations in the application of the protocol. The limiting factors are: small number of suitable barley genotypes, low transformation efficiency, possible off-targets in other parts of the genome exhibiting high sequence similarity, availability of mutable cut sites in the target sequence, and biallelic mutations due to inefficient cutting of the genomic DNA [20, 77, 78].

Callus regeneration is the final step in the Agrobacterium-mediated transformation and CRISPR/Cas genome editing protocols used mainly for barley. However, the varietal dependency in the efficiency of barley callus regeneration was observed already in 1980-ies both for winter and spring barley [79]. Barley variety ‘Golden Promise’ has since then been used as the standard for callus regeneration and transformation. Thus, genotypic restrictions on plant regeneration have hindered the implementation of transformation and genome editing tools on most barley varieties for over four decades.

Barley transformation protocols have been optimized, updated and improved over time. Several enhanced protocols have been published, mainly for Agrobacterium-mediated transformation of immature embryos, which provide average transformation efficiencies of 25% in the background of ‘Golden Promise’ [80,81,82,83]. For instance dicamba in the callus induction and maintenance media was generally superior to 2,4-D in promoting transformation and addition of CuSO4 resulted in formation of more green plants [84]. The addition of L-cysteine as an antioxidant was reported to hinder the browning of embryos and boost the efficiency of transformation [85]. Albinism, which can appear among regenerated barley, is caused by the inability of proplastids to transform into chloroplasts. Pre-treatment with mannitol may help to reduce albino barley plants [86]. Recently, anther culture-based system was shown to enable effective creation of transgenic plants not only from ‘Golden Promise’ but also from four other Australian commercial barley varieties [87].

In addition to changing hormone and nutrient levels, techniques that modify the innate gene expression of plants could enhance the effectiveness of transformation. There are examples from maize, rice, sorghum and sugarcane (Saccharum officinarum) that overexpressing Baby boom (Bbm) and Wuschel2 (Wus2) genes produced high transformation frequencies in previously nontransformable lines [88]. BBM is a transcription factor among the superfamily of the APETALA 2/ETHYLENE RESPONSE FACTOR (AP2/ERF) DNA-binding domain, subfamily AP2 [89, 90]. Two TaBBM genes in wheat have been identified as orthologues for maize Bbm [91], however, barley counterparts are still to be uncovered. WUS is a bifunctional homeodomain transcription factor, which mainly acts as a repressor but can become also an activator [92, 93].

Another promising approach for increasing transformation efficiency, could be the expression of fusion protein combining wheat GROWTH-REGULATING FACTOR 4 (GRF4) and its cofactor GRF-INTERACTING FACTOR 1 (GIF1) [94]. The concept was proven to have the desired effect in wheat, rice, triticale as well as in the dicot crop citrus hybrid Carrizo citrange [94]. Studying the effect of GRF-GIF fusion protein in reducing varietal gap in barley transformation would be a compelling avenue for exploration.

Lately, there have been several reports addressing the transformation efficiency by aiming to dissect its genetic determinants in transformable genotypes. Three significant and seven suggestive Transformation Amenability (TFA) regions were identified in ‘Golden Promise’, which likely include necessary factors for Agrobacterium-mediated transformation in barley [7, 95]. Additionally, the transformation efficiency (TRA1) locus was identified in the barley mutant M1460 on chromosome 2H incorporating 225 gene sequences [96]. Thus, there are suggestions in the literature that certain genetic components could affect the amenability to Agrobacterium-mediated transformation. However, further research is needed before this data could be used to overcome recalcitrance.

5 Social and Legislative Aspects of Using Genome Editing in Barley Breeding

Transgenic and genome editing technologies have a number of challenges, including regulatory barriers, public acceptance and the time and cost of risk assessments needed prior to commercialization. Genome edited products do not align with the prevailing definitions of genetically modified organisms (GMOs) in the majority of legal frameworks [75, 97]. The United States Department of Agriculture (USDA) [98] and the Australian Government Office of the Gene Technology Regulator [99] have determined that CRISPR-edited crops without foreign DNA are exempt from regulation as genetically modified organisms with regard to the regulation and commercialization of such products that enables to support and usage of the genome edited crop production. Afterwards, the European Parliamentary Research Service has ruled the legislation process based on the societal acceptance to promote the safety of humans, other animals, the non-living environment, and safe agriculture based on current developments in international genome editing laws in any genome editing application organized with the CRISPR/Cas system [100]. Recent developments in England reveal a change in the law permitting the commercial use of the gene edited products [101]. Cambridge-based researchers support utilizing this technology to develop crop varieties that are resilient to climate change and anticipate that it will create new employment opportunities. By enabling the production of better-adapted crops in less time and facilitating prompt market access, this approach holds significant promise.

Consequently, genome editing enables to breed in less time and with greater precision, maximize crop genetic potential, generate germplasms that are more resistant to pests, biotic and abiotic challenges, and extend shelf life of plant products to reduce food waste. However, barley genome editing protocols still have a number of limitations. Acquiring knowledge about genome editing techniques and their applications is crucial for shaping regulatory frameworks that will impact the feasibility of utilizing novel barley varieties and support food security (Fig. 10.1).

Fig. 10.1
An illustration of the genome editing techniques and their advantages. The techniques are Z F Ns, C R I S P R or Cas, and T A L E Ns. The advantages are accelerated breeding, easier multiplexing of traits, and legislative challenges.

Implementing genome editing in barley breeding. NBTs such as ZFNs, TALENs and CRISPR/Cas system enable to accelerate breeding and multiplex different traits. Application of NBTs in breeding is yet accompanied by the challenges in legislative rules

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