Understanding the mechanisms regulating the development of cereal seeds is essential for plant breeding and increasing yield. However, the analysis of cereal seeds is challenging owing to the minute size, the liquid character of some tissues, and the tight inter-tissue connections. Here, we demonstrate a detailed protocol for dissection of the embryo, endosperm, and seed maternal tissues at early, middle, and late stages of barley seed development. The protocol is based on a manual tissue dissection using fine-pointed tools and a binocular microscope, followed by ploidy analysis-based purity control. Seed maternal tissues and embryos are diploid, while the endosperm is triploid tissue. This allows the monitoring of sample purity using flow cytometry. Additional measurements revealed the high quality of RNA isolated from such samples and their usability for high-sensitivity analysis. In conclusion, this protocol describes how to practically dissect pure tissues from developing grains of cultivated barley and potentially also other cereals.
Cross-talk between exogenous salicylic acid (SA) and endogenous phytohormone pathways affects the antioxidant defense system and its response to salt stress. The study presented here investigated the effects of SA treatment before and during salt stress on the levels of endogenous plant growth regulators in three barley cultivars with different salinity tolerances: Hordeum vulgare L. cvs. Akhisar (sensitive), Erginel (moderate), and Kalaycı (tolerant). The cultivars' relative leaf water contents, growth parameters, proline contents, chlorophyll a/b ratios, and lipid peroxidation levels were measured, along with the activities of enzymes involved in detoxifying reactive oxygen species (ROS) including superoxide-dismutase, peroxidase, catalase, ascorbate-peroxidase, and glutathione-reductase. In addition, levels of several endogenous phytohormones (indole-3-acetic-acid, cytokinins, abscisic acid, jasmonic acid, and ethylene) were measured. Barley is known to be more salt tolerant than related plant species. Accordingly, none of the studied cultivars exhibited changes in membrane lipid peroxidation under salt stress. However, they responded differently to salt-stress with respect to their accumulation of phytohormones and antioxidant enzyme activity. The strongest and weakest increases in ABA and proline accumulation were observed in Kalaycı and Akhisar, respectively, suggesting that salt-stress was more effectively managed in Kalaycı. The effects of exogenous SA treatment depended on both the timing of the treatment and the cultivar to which it was applied. In general, however, where SA helped mitigate salt stress, it appeared to do so by increasing ROS scavenging capacity and antioxidant enzyme activity. SA treatment also induced changes in phytohormone levels, presumably as a consequence of SA-phytohormone salt-stress cross-talk.
- MeSH
- antioxidancia metabolismus MeSH
- biomasa MeSH
- časové faktory MeSH
- chlorofyl a metabolismus MeSH
- chlorofyl metabolismus MeSH
- ječmen (rod) účinky léků růst a vývoj fyziologie MeSH
- kyselina salicylová farmakologie MeSH
- látky reagující s kyselinou thiobarbiturovou metabolismus MeSH
- listy rostlin účinky léků fyziologie MeSH
- prolin metabolismus MeSH
- reaktivní formy kyslíku metabolismus MeSH
- regulátory růstu rostlin farmakologie MeSH
- solný stres účinky léků MeSH
- voda MeSH
- výhonky rostlin účinky léků růst a vývoj MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The production of doubled haploid (DH) barley plants through anther culture is a very useful yet simple in vitro technique. DH plants derive from divisions of haploid microspores that have undergone a developmental switch under the appropriate conditions. The successive divisions lead to the formation of an embryo or callus rather than the formation of mature pollen grains. Plants that regenerate from these embryos are often either haploid, in which case their chromosome set can be doubled by treatment with colchicine, or spontaneous double haploids. The efficiency of DH plant production is highly variable depending on the genotype of the source material. Despite this limitation, DH plants have been widely used in breeding and research programs. Compared to conventional approaches, breeding strategies that makes use of DH plants achieve a homozygous state, allowing transgene or mutation stabilization in the genome, within a considerably shorter time, thus accelerating workflow or reducing work volume.
- MeSH
- barvení a značení MeSH
- DNA rostlinná genetika MeSH
- haploidie MeSH
- ječmen (rod) růst a vývoj MeSH
- kultivační média MeSH
- květy růst a vývoj MeSH
- pyl růst a vývoj MeSH
- regenerace MeSH
- somatická embryogeneze rostlin MeSH
- sterilizace MeSH
- techniky tkáňových kultur metody MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The discovery of radioactivity at the end of the nineteenth century played a key role in a series of historical landmarks that would lead to contemporary mutation breeding in agricultural crops. The aim of the earliest experiments was to test the effects of radiation on living organisms beginning with fruit flies. Exposure of plants to X-rays provided the first incontrovertible proof that phenotypic changes could be induced. Chemicals were a second type of mutagen tested from the 1940s and both forms are used today. This chapter is an overview of some of the historical developments that led to the use of mutagenesis in plants, with a focus on barley, a model species for mutation genetics and breeding as well as a major cereal crop. Perhaps the most well-known examples of mutant barley cultivars are Diamant, Golden Promise, and their hybrids.
- MeSH
- dějiny 19. století MeSH
- dějiny 20. století MeSH
- ječmen (rod) genetika růst a vývoj MeSH
- mutace genetika MeSH
- mutageneze genetika MeSH
- šlechtění rostlin ekonomika dějiny metody MeSH
- Check Tag
- dějiny 19. století MeSH
- dějiny 20. století MeSH
- Publikační typ
- časopisecké články MeSH
- historické články MeSH
- práce podpořená grantem MeSH
Uptake of water and nutrients by roots affects the ontogenesis of the whole plant. Nanoparticles, e.g. gold nanoparticles, have a broad range of applications in many fields which leads to the transfer of these materials into the environment. Thus, the understanding of their impact on the growth and development of the root system is an emerging issue. During our studies on the effect of positively charged gold nanoparticles on the barley roots, a hairless phenotype was found. We investigated whether this phenotype correlates with changes in symplasmic communication, which is an important factor that regulates, among others, differentiation of the rhizodermis into hair and non-hair cells. The results showed no restriction in symplasmic communication in the treated roots, in contrast to the control roots, in which the trichoblasts and atrichoblasts were symplasmically isolated during their differentiation. Moreover, differences concerning the root morphology, histology, ultrastructure and the cell wall composition were detected between the control and the treated roots. These findings suggest that the harmful effect of nanoparticles on plant growth may, among others, consist in disrupting the symplasmic communication/isolation, which leads to the development of a hairless root phenotype, thus limiting the functioning of the roots.
- MeSH
- buněčná diferenciace účinky léků MeSH
- buněčná membrána metabolismus MeSH
- epidermis rostlin cytologie účinky léků metabolismus MeSH
- ječmen (rod) účinky léků genetika růst a vývoj metabolismus MeSH
- kořeny rostlin cytologie účinky léků růst a vývoj metabolismus MeSH
- kovové nanočástice toxicita MeSH
- látky znečišťující půdu toxicita MeSH
- regulace genové exprese u rostlin účinky léků MeSH
- semenáček účinky léků růst a vývoj MeSH
- voda metabolismus MeSH
- živiny metabolismus MeSH
- zlato toxicita MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The plant hormones cytokinins are a convenient target of genetic manipulations that bring benefits in biotechnological applications. The present work demonstrates the importance of the subcellular compartmentalization of cytokinins on the model dicot plant Arabidopsis thaliana and monocot crop Hordeum vulgare. The method of protoplast and vacuole isolation combined with precise cytokinin analysis and recovery assay of a vacuolar marker protein were used to quantify the contents of individual cytokinin forms in the leaf extracellular space, cell interior and vacuole. The data obtained for wild type plants and in each case a specific mutant line allow comparing the effect of genetic manipulations on the hormone distribution and homeostatic balance of cytokinins in the modified plants.
- MeSH
- Arabidopsis genetika metabolismus MeSH
- biotechnologie MeSH
- cytokininy metabolismus MeSH
- extracelulární prostor metabolismus MeSH
- geneticky modifikované rostliny MeSH
- intracelulární prostor metabolismus MeSH
- ječmen (rod) růst a vývoj metabolismus MeSH
- kompartmentace buňky MeSH
- listy rostlin metabolismus MeSH
- membránové transportní proteiny genetika metabolismus MeSH
- mutace MeSH
- proteiny huseníčku genetika metabolismus MeSH
- protoplasty metabolismus MeSH
- regulátory růstu rostlin metabolismus MeSH
- vakuoly metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
Together with auxins, cytokinins are the main plant hormones involved in many different physiological processes. Given this knowledge, cytokinin levels can be manipulated by genetic modification in order to improve agronomic parameters of cereals in relation to, for example, morphology, yield, and tolerance to various stresses. The barley (Hordeum vulgare) cultivar Golden Promise was transformed using the cytokinin dehydrogenase 1 gene from Arabidopsis thaliana (AtCKX1) under the control of mild root-specific β-glucosidase promoter from maize. Increased cytokinin degradation activity was observed positively to affect the number and length of lateral roots. The impact on morphology depended upon the recombinant protein's subcellular compartmentation. While assumed cytosolic and vacuolar targeting of AtCKX1 had negligible effect on shoot growth, secretion of AtCKX1 protein to the apoplast had a negative effect on development of the aerial part and yield. Upon the application of severe drought stress, all transgenic genotypes maintained higher water content and showed better growth and yield parameters during revitalization. Higher tolerance to drought stress was most caused by altered root morphology resulting in better dehydration avoidance.
- MeSH
- aklimatizace genetika fyziologie MeSH
- biotechnologie MeSH
- cytokininy genetika metabolismus MeSH
- fenotyp MeSH
- fyziologický stres MeSH
- geneticky modifikované rostliny MeSH
- ječmen (rod) genetika růst a vývoj fyziologie MeSH
- kořeny rostlin fyziologie MeSH
- metabolické sítě a dráhy MeSH
- období sucha MeSH
- oxidoreduktasy genetika MeSH
- proteiny huseníčku genetika fyziologie MeSH
- regulátory růstu rostlin genetika metabolismus MeSH
- rekombinantní proteiny genetika metabolismus MeSH
- rostlinné geny MeSH
- rostlinné proteiny genetika MeSH
- stanovení celkové genové exprese MeSH
- upregulace MeSH
- Publikační typ
- časopisecké články MeSH
The shape of the maternal pericarp affects cereal grain mass and yield. Pericarp growth was analysed by magnetic resonance imaging (MRI), revealing topological maps of mobile water in developing pericarp of barley (Hordeum vulgare) and displaying tissue regions actively elongating in specific temporal-spatial patterns. Correlation analysis of MRI signals and growth rates reveals that growth in length is mediated by dorsal and also lateral rather than ventral regions. Growth in thickness is related to ventral regions. Switching from dorsal to ventral growth is associated with differential expression of axial regulators of the HD-ZipIII and Kanadi/Ettin types, and NPH3 photoreceptors, suggesting light-mediated auxin re-distribution. Auxin increases with the highest levels in the basal pericarp at 6 days after fertilization (DAF), together with transcriptionally up-regulated auxin transport and signalling. Gibberellin biosynthesis is transcriptionally up-regulated only later, and levels of bioactive gibberellins increase from 7 to 13 DAF, with higher levels in ventral than dorsal regions. Differential gene expression related to cell expansion indicates genes related to apoplast acidification, wall relaxation, sugar cleavage, water transport, and cell wall biosynthesis. Candidate genes potentially involved in pericarp extension are distinguished by their temporal expression, representing potential isoforms responsible for dorsal-mediated early growth in length or ventral-mediated late growth in thickness.
- MeSH
- gibereliny metabolismus MeSH
- ječmen (rod) genetika růst a vývoj metabolismus MeSH
- kyseliny indoloctové metabolismus MeSH
- magnetická rezonanční tomografie MeSH
- regulace genové exprese u rostlin * MeSH
- regulátory růstu rostlin metabolismus MeSH
- rostlinné proteiny genetika metabolismus MeSH
- semena rostlinná genetika růst a vývoj metabolismus MeSH
- stanovení celkové genové exprese MeSH
- vývojová regulace genové exprese MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Reduced plant height and culm robustness are quantitative characteristics important for assuring cereal crop yield and quality under adverse weather conditions. A very limited number of short-culm mutant alleles were introduced into commercial crop cultivars during the Green Revolution. We identified phenotypic traits, including sturdy culm, specific for deficiencies in brassinosteroid biosynthesis and signaling in semidwarf mutants of barley (Hordeum vulgare). This set of characteristic traits was explored to perform a phenotypic screen of near-isogenic short-culm mutant lines from the brachytic, breviaristatum, dense spike, erectoides, semibrachytic, semidwarf, and slender dwarf mutant groups. In silico mapping of brassinosteroid-related genes in the barley genome in combination with sequencing of barley mutant lines assigned more than 20 historic mutants to three brassinosteroid-biosynthesis genes (BRASSINOSTEROID-6-OXIDASE, CONSTITUTIVE PHOTOMORPHOGENIC DWARF, and DIMINUTO) and one brassinosteroid-signaling gene (BRASSINOSTEROID-INSENSITIVE1 [HvBRI1]). Analyses of F2 and M2 populations, allelic crosses, and modeling of nonsynonymous amino acid exchanges in protein crystal structures gave a further understanding of the control of barley plant architecture and sturdiness by brassinosteroid-related genes. Alternatives to the widely used but highly temperature-sensitive uzu1.a allele of HvBRI1 represent potential genetic building blocks for breeding strategies with sturdy and climate-tolerant barley cultivars.
- MeSH
- alely MeSH
- aminokyseliny MeSH
- brassinosteroidy metabolismus MeSH
- fenotyp MeSH
- ječmen (rod) genetika růst a vývoj metabolismus MeSH
- jedlá semena MeSH
- mapování chromozomů MeSH
- modely strukturální MeSH
- molekulární sekvence - údaje MeSH
- mutace MeSH
- počasí MeSH
- počítačová simulace MeSH
- regulace genové exprese u rostlin * MeSH
- sekvence nukleotidů MeSH
- sekvenční analýza DNA MeSH
- signální transdukce MeSH
- teplota MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
Reverse transcription coupled with real-time quantitative PCR (RT-qPCR) is a frequently used method for gene expression profiling. Reference genes (RGs) are commonly employed to normalize gene expression data. A limited information exist on the gene expression and profiling in developing barley caryopsis. Expression stability was assessed by measuring the cycle threshold (Ct) range and applying both the GeNorm (pair-wise comparison of geometric means) and Normfinder (model-based approach) principles for the calculation. Here, we have identified a set of four RGs suitable for studying gene expression in the developing barley caryopsis. These encode the proteins GAPDH, HSP90, HSP70 and ubiquitin. We found a correlation between the frequency of occurrence of a transcript in silico and its suitability as an RG. This set of RGs was tested by comparing the normalized level of β-amylase (β-amy1) transcript with directly measured quantities of the BMY1 gene product in the developing barley caryopsis. This panel of genes could be used for other gene expression studies, as well as to optimize β-amy1 analysis for study of the impact of β-amy1 expression upon barley end-use quality.
- MeSH
- beta-amylasa genetika metabolismus MeSH
- exprese genu MeSH
- glyceraldehyd-3-fosfátdehydrogenasy genetika MeSH
- ječmen (rod) enzymologie genetika růst a vývoj MeSH
- kvantitativní polymerázová řetězová reakce normy MeSH
- referenční standardy MeSH
- regulace genové exprese enzymů MeSH
- regulace genové exprese u rostlin MeSH
- reprodukovatelnost výsledků MeSH
- rostlinné geny * MeSH
- rostlinné proteiny genetika MeSH
- semena rostlinná enzymologie genetika růst a vývoj MeSH
- stanovení celkové genové exprese * MeSH
- vývojová regulace genové exprese MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- validační studie MeSH
