Here we present a protocol for high purity manual isolation and quality control of embryo, endosperm and seed maternal tissues during entire barley seed development.
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.
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 reproduction1,3.
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 stages5,6,7. 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 stages1,10. 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 coats10,12. 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.
Here, we present a protocol that allows high purity isolation of barley seed tissues. Although it was developed and tested for barley, it can be easily adopted to other members of the Triticeae tribe such as wheat, oat, rye or triticale27. The initial part of the protocol, focusing on seed tissue dissection, does not require any non-standard or expensive equipment and therefore should be accessible to many scientists. A highly specialized instrument such as a flow cytometer is required fo…
The authors have nothing to disclose.
We thank Dr. Jan Vrána and Dr. Mahmoud Said for the maintenance of flow cytometers, Eva Jahnová for preparation of buffers Marie Seifertová for list of materials and Zdenka Bursová for plant care. This work was supported primarily from the Czech Science Foundation grant 18-12197S. A.P. was further supported by the J. E. Purkyně Fellowship from the Czech Academy of Sciences and the ERDF project "Plants as a tool for sustainable global development" (No. CZ.02.1.01/0.0/0.0/16_019/0000827).
0.22 um filter | Merck | SLGSV255F | |
1.5 ml Eppendorf tube | Sarstedt | 72.690.001 | |
4',6-diamididno-2-phenylindole | Invitrogen | D21490 | |
50 um nylon mesh | Silk a Progrers | uhelon 120 T | |
Agilent 2100 Bioanalyzer | Agilent | G2939BA | |
Bulb Assembly | Drummond Scientific Company | 1-000-9000 | |
Calibration beads | Invitrogen | A16502 | |
Cellulose tissue paper | |||
Citric acid monohydrate | Penta | 13830-31000 | |
Climatic chamber | Weiss Gallenkamp | ||
DNase I | Sigma Aldrich | DNASE70 | |
Filter paper | Fagron | ||
Fine-pointed tweezers | Fine Science Tools | 11254-20 | |
Flow cytometer | Sysmex-Partec | ||
Flow cytometry tube | Sarstedt | 55.484 | |
Freezer | |||
Glassine bag | |||
KCl | Lachner | 30076-AP0 | |
KH2PO4 | Litolab | 100109 | |
Liquid nitrogen | Linde | ||
Microcapillary pipette | Fivephoton Biochemicals | MGM 1C-20-30 | |
Minutien Pins | Fine Science Tools | 26002-20 | |
Na2HPO4 | Lachema | ||
Na2HPO4.12H2O | Lachner | 30061-AP0 | |
NaCl | Lachner | 30093-AP0 | |
Peat pots | Jiffy | 5×5 cm | |
Petri dish | |||
Pin Holder | Fine Science Tools | 26016-12 | |
Plastic pestle | p-Lab | A199001 | |
Pots | 12×12 cm | ||
Razor blade | Gillette | ||
RNAse zap | Invitrogen | AM9780 | |
Sand | |||
Scissors | Fine Science Tools | 14060-11 | |
Soil | |||
Spectrum Plant Total RNA Kit | Sigma Aldrich | STRN50 | |
Stereomicroscope | Olympus | ||
Tween 20 | Sigma Aldrich | P2287 | |
TRIzol reagent | Invitrogen | 15596026 | |
RNA 6000 Pico Kit | Agilent | 5067-1513 |
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