Isolation of High Purity Tissues from Developing Barley Seeds

Seeds are complex structures composed of several tissues of maternal and filial origin¹. Cereal grains represent a special type of seed, with the largest part being formed by endosperm, a specialized triploid tissue that protects and nourishes the embryo. Cereals provide around 60% of global food resources and are the most valuable output from plant production². The knowledge of molecular processes controlling cereal seed development is important due to their economic prominence and central role in plant reproduction^1,3.

Cultivated barley (Hordeum vulgare subsp. vulgare; 2n = 2x = 14; 1C = 5.1 Gbp) is the fourth most important cereal crop worldwide. It is used for animal feed, food, and biotechnology⁴. Besides that, it is also a classical temperate zone cereal crop model species of growing importance⁵. Barley genomic resources include genetic maps, collections of cultivars, landraces and mutants, high-quality genome assemblies and annotations as well as transcriptomic data of the major developmental stages^5,6,7. Also, barley genes are used for genetic improvements of other cereals. Resistance to abiotic stresses such as drought and salinity, specific pathogens, and high content of beneficial compounds (e.g., β-glucan) make barley a valuable source of traits for wheat breeding⁸.

Seed development is initiated by fertilization on the day of pollination (DOP). DOP is defined by evaluation of the morphology of stigma and anthers according to the Waddington scale (W10.0)⁹. The spikes containing non-pollinated flowers were characterized by compact (unbranched) stigma and green anthers, whereas pollinated spikes contained extended spiklets, extended and widely branched stigma, swollen ovule, opened anthers and free pollen. The flowers at DOP represented an intermediate phenotype. The anthers had a yellow color, disrupted easily and then released pollen. Stigma had widely spread sigmatic branches of the pistil (Figure 1C).

Barley seed development includes three partially overlapping stages^1,10. The stage I (0 – 6 days after pollination; DAP) is launched by double fertilization, typified by cell proliferation and the absence of starch synthesis; stage II (7 – 20 DAP) comprises differentiation and great biomass gain accompanied by the production of starch and protein storage molecules; stage III (after 21 DAP) corresponds to seed maturation, weight reduction by desiccation and the onset of dormancy. Alternatively, the phases are called early, middle and late, respectively¹¹.

Barley grain is covered by hulls, which consist of the lemma, palea, and glumes¹². In most barley genotypes, the hulls tightly wrap dry seeds. The seed itself is formed by the embryo, endosperm and seed maternal tissues (Figure 1A). The diploid embryo originates from the fertilization of the egg cell by one sperm cell nucleus. In the fully developed seed, the embryo consists of the embryonic axis with the coleorhiza surrounding the radicle, the coleoptile enclosing the shoot meristem and primary leaves, and the scutellum (cotyledon)^1,10,13,14. The triploid endosperm is the result of fertilization of the diploid central cell by the second sperm cell nucleus. The proliferation of endosperm begins with the syncytial (coenocyte) stage, where the dividing nuclei are pushed to the periphery by the central vacuole. At the end of the syncytial phase, microtubules form a radial network around the nuclei and indicate the anticlinal cell wall formation and the onset of endosperm cellularization. Endosperm differentiation occurs simultaneously with the cellularization and results in five major tissues: the starchy endosperm, the transfer cells, the aleurone and subaleurone layers, and the embryo surrounding region. Seed maternal tissues are a multi-layered diploid structure of maternal origin containing pericarp and seed coats^10,12. Seed maternal tissues include a nucellar projection on the dorsal side of the grain that has a transport-related function, and becomes embedded in endosperm at later stages of seed development¹⁵.

Figure 1: Developing barley seeds. (A) The schematic drawing of cereal grain at the sagittal plan with indicated seed maternal tissues (SMTs, green), endosperm (END, yellow), embryo (EMB, orange) and hulls (H, grey). (B) Morphology of barley spike close to the anthesis. Scale bar = 1 cm. (C) Morphology of stigma and anthers at the stages before, during and after pollination. Inset shows detail of the stigma with pollen grains (arrowheads). Scale bar = 5 mm, inset bar = 200 µm. (D) Sagittal and transverse sections of 4, 8, 16 and 24 DAP seeds. (NP, nucellar projection) Scale bar = 5 mm. Please click here to view a larger version of this figure.

Recent progress in high-throughput genomics provides the tools for the study of individual seed tissue development. However, the major obstacle of this purpose is the compact structure and tight adhesion of the seed tissues¹. We developed a protocol for high purity dissection of seed tissues from developing barley seeds with possibility to subsequent use for highly sensitive analyses, such as RNA-sequencing. In addition, the presented protocol can be easily adapted to other cereals.