Hordeum vulgare was one of the very first crops used in cereal improvement programs based on different induced mutation strategies. In 1930, Stadler studied the mutagenic effects of different types of radiation on maize and barley, describing chlorophyll-deficient and virescent phenotypes in seedlings [3]. In 1938, Nilsson-Ehle and Gustafsson tested X-rays and UV light on the barley cultivar (cv.) ‘Gull’ and isolated several mutants, which were named albina, xantha, alboviridis, viridis, tigrina, striata and maculata, categorizing them by their carotenoid and chlorophyll contents and distribution within the leaf blade [4]. The characteristics of several mutated lines were recognized as being very valuable for potential use in agriculture, since they exhibited alterations in grain yield, straw stiffness, straw length and tillering capacity, as well as changes in spike firmness, kernel maturation and pigmentation [5]. Later on, two varieties of barley ‘Trebi’ and ‘Moister’ were exposed to the radiation generated by the first aerial atomic explosion at Bikini atoll in 1946 [6]. Meanwhile, Gustafsson and Mackey applied mustard gas to barley to observe the effect of chemical mutagenesis [7], whereas Ehrenberg and collaborators tested various mutagenic compounds on barley and evaluated their impact on chlorophyll accumulation [8]. After these pioneering experiments, a broad range of chemical and physical mutagens were tested systematically. During this phase, alkylating agents able to generate G/C to A/T transitions in DNA, such as EMS (Ethyl Methane Sulfonate), ENU (N-nitroso-N-ethylurea), MNU (N-nitroso-N-methylurea), DES (diEthyl Sulfate) and sodium azide (NaN₃), were widely used for the mutagenesis of barley. The first chemically induced barley variety, ‘Luther’, was released in the US in 1966. ‘Luther’ was obtained by exposing the variety ‘Alpine’ to DES. In 1965, in Czechoslovakia, the variety ‘Diamant’ was obtained after gamma-ray irradiation. This new variety was ~ 15 cm shorter than the parental ‘Valticky’ and displayed an increase in grain yield amounting to about 12% [9]. At around the same time, in the UK, ‘Golden Promise’ was registered. This semi-dwarf cultivar originated from exposure of the salt-sensitive variety ‘Maythorpe’ to gamma rays [10]. The generation of ‘Golden Promise’ represented an important step towards the development of tissue culture and barley transformation techniques (see below).

Genetic studies of barley have not been restricted to breeding programs. The plant has also been used as a model species to dissect the molecular mechanisms that underlie plant development and physiology and, for a large part of the 20th century, it served as a major experimental system for the investigation of chloroplast biogenesis and photosynthesis. In particular, several studies during the 1970s characterized different aspects of plastid structure and development, such as plastid growth, replication and differentiation. Dark-grown barley seedlings were used to determine the protochlorophyll content and structure of the etioplasts. Exposure to different light conditions allowed chloroplast development to be characterised from both structural and biochemical points of view [11,12,13,14].

The organization of chloroplast membranes was analysed in chloroplast preparations solubilised with digitonin and fractionated by electrophoresis, proving the existence of distinct sets of membranes [15]. The functionality and structural organization of thylakoids were also studied in barley mutants altered in chlorophyll biosynthesis [16] and revealed the impact of such changes on thylakoid membrane organization. For instance, the chlorina-f2 mutant, which is impaired in chlorophyll b accumulation, led to the discovery of light-harvesting chlorophyll-binding proteins [17,18,19,20]. Chlorina-f2 was also used to assess the impact of protein-chlorophyll complexes on the ultrastructure of thylakoid membranes, shedding light on the organisation of the photosynthetic apparatus [17,21]. In addition, the tigrina-d mutant [22], originally suggested to be involved in the early steps of tetrapyrrole biosynthesis prior to ALA formation, was recently identified as the barley orthologue [23] of the FLU gene of Arabidopsis thaliana, a nuclear-encoded, plastid-localized protein that plays a key role in the negative-feedback control of chlorophyll biosynthesis, with an essential role during the dark-to-light switch [24]. Moreover, the barley xantha mutants helped to elucidate key steps in chlorophyll biosynthesis [25]: xantha-l was shown to code for a mutated form of Mg-protoporphyrin IX monomethyl ester cyclase, while xantha-f, -g, and -h carry genetic lesions at three distinct loci encoding the three Mg-chelatase subunits [26,27].

From a physiological point of view, Smith et al. [28] documented changes in chloroplast activity during de-etiolation of barley seedlings by measuring the Hill reaction in relation to chlorophyll accumulation. The correlation between plastid ultrastructure, chlorophyll synthesis and development of photosynthetic activity was also evaluated by measuring O₂ evolution [29].

Besides the characterization of the photosynthetic apparatus, barley played an important part in the dissection of the chloroplast’s gene expression machinery. Indeed, evidence for a fully nuclear-encoded transcriptional activity in plastids, later named the Nuclear-Encoded RNA Polymerase (NEP; [30]), was first reported in barley, based on analysis of the albostrians mutant. In particular, the synthesis of RNA was reported in the white sectors of albostrians leaves, which harbor plastids that are devoid of ribosomes. These data provided initial evidence for the existence of a nuclear-encoded and plastid-localized RNA polymerase [31]. In addition, ribosome-free plastids of albostrians were helpful in distinguishing between the set of plastid genes preferentially transcribed by NEP, such as rRNA, rpo and rps15, and the set transcribed by the Plastid-Encoded RNA polymerase (PEP), which is enriched in photosynthesis-related genes such as psbA, rbcL, atpI-H [31,32]. Furthermore, the barley albostrians mutant was essential to the initial detection of communication between organellar and nuclear genomes. By analyzing albostrians, which is characterized by reduced amounts and/or activities of nucleus-encoded chloroplast proteins including the small subunit of ribulose-1,5-bisphosphate carboxylase⁄oxygenase (Rubisco), ferredoxin NADP+ reductase, and enzymes of the Calvin cycle, Börner provided the first evidence for plastid signals that control nuclear gene expression, leading to the discovery of chloroplast-to-nucleus retrograde communication [33,34,35].

4. Influences