Isolation of High Purity Tissues from Developing Barley Seeds

Abstract

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.

Introduction

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.

Protocol

1. Growing plants

NOTE: Considering that a single barley plant usually has 5 to 6 tillers and only the middle 5 to 6 spikelets of each spike should be used for dissection, then a maximum yield per plant is 72 seeds for two-row and 216 seeds for six-row cultivars.

1. To germinate barley seeds, prepare a Petri dish padded with three layers of cellulose tissue paper covered with one layer of filter paper. Moisturize it with distilled water, so there is no excess water, put the seeds on the surface and close the Petri dish. Filtration paper avoids growing the roots through the cellulose tissue. Germinate the seeds for 3 days at 25 °C in the dark.
   NOTE: Alternatively, germinate seeds by putting them directly in a wet soil mixture (see step 1.2).
2. Transfer germinated seeds with a visible radicle and shoot of about 5 cm into 5 cm x 5 cm peat pots with a mixture of soil and sand (3:1, v/v). Water regularly. After 10 days, transfer the plants into 12 cm x 12 cm pots filled with the same soil mixture.
3. Grow the plants in a climatic chamber under the controlled long-day regime (16 h day 20 °C, 8 h night 16 °C; light intensity 200 µmol m^-2 s^-1; humidity 60%).
   NOTE: Spring barley requires approximately 8-10 weeks from sowing to the beginning of anthesis, with no requirement for vernalization. Winter barley needs 7-8 weeks of vernalization (short-day conditions, 8 h day 4 °C, 16 h night 4 °C; light intensity 200 µmol m^-2 s^-1; humidity 85%) to induce flowering.

2. Determination of pollination

NOTE: Precise determination of pollination is needed for proper estimation of developmental progression. Barley is a self-pollinating species. To define day of pollination (DOP), we monitored the day of self-pollination. This trait is cultivar specific, but starting protrusion of the awns from the leaf sheath is a good indicator of approaching DOP (Figure 1B).

1. Open the leaf-sheath covering the spike. Use fine-pointed tweezers to check anthers and the ovary inside the spikelets in the central part of the spike. Spikelets with yellow anthers and “fluffy” stigma will pollinate within few hours¹⁶ and are considered as DOP (Figure 1B).
2. Clip off the spike near the tip of the last spikelet, and remove the flag leaf and the upper part of the awns. Then, clip off the top 1/3 of hulls in each spikelet. This dries the anthers and leads to their more synchronized opening and release of pollen.
3. Cover the spike with a glassine bag with the spike ID, plant number and defined DOP date. This also prevents cross-pollination, which may compromise specific experiments.
4. Note the information to a tabular editor. Use the following formula to calculate the day after pollination (DAP) when tissue isolation should take place.
   xDAP = DOP + x
   x = expected DAP
   NOTE: The values should have ‘Data’ format.
5. For seed tissues dissection, collect the spikes at DAP according to the prepared tabular calendar.

3. Dissection of the seed tissues

NOTE: The following steps should be performed using a stereomicroscope. Remove the hulls before dissection using tweezers. Note that hulls become drier and more adherent from around 16 DAP. To keep physiological conditions and avoid drying of the plant materials during dissection, moisten the samples by putting them into a drop of 1x PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO_4, pH = 7.4). Use a new seed for dissection of each tissue to avoid DNA, RNA, or protein degradation due to extended sample collection time. For RNA isolation from dissected material, use only RNase-free materials and chemicals. Do not exceed the total dissection time 15 minutes for one sample consisting typically from tissues dissected from 5-10 seeds to minimize RNA degradation.

1. Remove the rest of the hulls using fine-pointed tweezers before tissue dissection. Moisturizing with 1× PBS for 1 minute helps to remove dry residues of the spikelet.
2. Place the peeled seed on a Petri dish with a drop of 1× PBS and dissect individual parts using fine-pointed tweezers, fine-needle, and microcapillary pipette. A slightly different strategy is applied for dissection of individual tissues: the seed maternal tissues (step 3.3), the embryo (step 3.4) and the endosperm (step 3.5).
3. Dissection of seed maternal tissues
   1. Dissection from seeds up to 8 DAP
      1. Place a seed on the dorsal side and gently cut the seed along a longitudinal axis, and peel off seed maternal tissues except the last layer bordering endosperm from the apical to the basal part using tweezers.
      2. Collect seed maternal tissues from 5 to 10 seeds into a 1.5 mL tube with 50 µL of 1× PBS, discard the buffer using a pipette, rinse the tissue twice with 100 µL of PBS, remove excessive buffer by pipetting and close the tube and freeze in liquid nitrogen or use directly for flow cytometric ploidy measurement. The amount of material is sufficient for typically one downstream application (e.g., RNA isolation or flow cytometric ploidy measurement).
   2. Dissection from seeds after 8 DAP
      1. Place a seed on the dorsal side, gently cut in the middle of the ventral side of seed maternal tissues and gradually peel off the tissue around whole seed including nucellar projection. For each downstream application collect and wash the tissue from 5 to 10 seeds as described in step 3.3.1.
4. Dissection of embryo
   1. Dissection from seeds at 8 DAP and younger
      1. Place a seed on the dorsal side and cut basal 1/3 of the seed. Carefully split separated part in half and release the embryo. For each downstream application collect and wash the embryos from 10 to 20 seeds as described in 3.3.1.
   2. Dissection from seeds after 8 DAP
      1. Place a seed on the dorsal side and remove seed maternal tissues from the basal part of the ventral side. Gently disturb the thin layer of endosperm around the perimeter of the embryo by fine-needle or fine-pointed tweezers and peel out the embryo. For each downstream application collect and wash embryos from up to 5 seeds as described in 3.3.1.
5. Dissection of endosperm
   1. Dissection of syncytial endosperm from 4 DAP seeds.
      1. Place a seed on the dorsal side, and remove seed maternal tissues except the last layer of cells bordering endosperm. Gently puncture layer in the middle of the ventral side by a thin needle, and suck the syncytial endosperm by capillary action using a microcapillary pipette.
      2. For each downstream application collect liquid endosperms from 10 to 15 seeds into a new 1.5 mL tube with buffer suitable for the planned downstream analysis (i.e., 1x PBS, RNA isolation buffer, flow cytometry buffer). Buffer volume should reflect the protocol for the planned downstream aplication. Freeze in liquid nitrogen.
   2. Dissection of celullarizing endosperm from 5 to 8 DAP seeds
      1. Place a seed on the dorsal side, and remove all seed maternal tissues and embryo. For each downstream application collect and wash the endosperm from 10 to 15 seeds into a new 1.5 mL tube with 1x PBS and freeze in liquid nitrogen.
   3. Dissection of celullarized endosperm from seed after 8 DAP
      1. Place a seed on the dorsal side, remove all seed maternal tissues and embryo. For each downstream application collect and wash endosperm from a single seed per tube as described in step 3.3.1.
6. Store the tubes with isolated material at – 80 °C until use.
   NOTE: The protocol can be paused here.

4. Control of tissue purity using flow cytometry

NOTE: The sample purity can be checked using flow cytometry before RNA isolation. Proper instrument calibration is critical for the biological sample analysis. The flow cytometer/ploidy analyzer optics should be adjusted using calibration beads (fluorescently stained polystyrene microspheres highly uniform with respect to their size and fluorescence intensity) until the maximal peak sharpness, typically reaching the coefficient of variation (CV) < 2%. Cereal seed tissues contain mainly populations of G1, G2 and endoreduplicated nuclei; therefore, using a logarithmic scale is recommended. Start with a leaf tissue that contains mostly G1 nuclei and serves as a basal ploidy control.

1. Use freshly prepared samples kept on ice (see step 3.3.1) or frozen tissue as described¹⁷.
   NOTE: Because the whole < 8 DAP sample is used for flow cytometry, this represents only an indirect control. We recommend researchers performing multiple isolations and measurements until reaching high proportion of pure samples (>90%) before proceeding to RNA isolation with < 8 DAP samples.
2. Release the nuclei from the 4 and 8 DAP embryo samples (for other samples see step 4.4) by homogenizing the tissues by 5 to 10 turns of the plastic pestle in 1.5 mL tube containing 300 µL of Otto I buffer (0.1 M citric acid monohydrate, 0.5% (v/v) Tween 20, filtered through a 0.22 µm filter)¹⁸.
3. Filter the crude suspension through 50 µm nylon mesh into a flow-cytometry analysis tube and add 600 µL of Otto II buffer (0.4 M Na₂HPO₄·12H₂O) containing 2 µg mL^-1 DAPI (4´,6-diamidino-2-phenylindole)¹⁸ to stain DNA.
4. Place all other tissues (including 16 DAP or older embryos) on a Petri dish containing 500 µL of Otto I buffer and homogenized by chopping with a razor blade. Filter the suspension as in step 4.3 and stain with 1 mL of Otto II buffer containing DAPI.
   NOTE: Manipulation with a sharp double edge razor blade requires special attention. To reduce the risk of injury, there are a single edge razor blades or special blade holders available.
5. Estimate the nuclear DNA content of the sample using a flow cytometer. At least 2000 particles per sample are required for analyzing the sample purity.

5. RNA isolation and quality measurement

1. Use frozen tissue to prevent RNA degradation by endogenous ribonucleases. From seed maternal tissues and embryo samples isolate RNA using commercially available kits or TRIzol reagent¹⁹. Due to a high starch content in endosperm tissues, isolate total RNA from all samples using commercial on-column RNA extraction protocols for problematic tissues (e.g., Spectrum Plant Total RNA Kit) with an on-column DNase I treatment²⁰.
2. Measure RNA concentration and integrity using a dedicated protocol for RNA gel electrophoresis or Agilent 2100 Bioanalyzer.
   NOTE: Intact total RNA has a clear 18S and 25S rRNA bands/peaks of size around 1.9 and 3.7 kb respectively. The 25S rRNA band should be approximately two times more intense than the 18S rRNA band.

Representative Results

To perform a tissue-specific transcriptomic analysis of barley seed development, we established a protocol for high purity tissue isolation. The protocol is based on the manual dissection of embryo, endosperm and seed maternal tissues from peeled (after manual hull removal) grains (Figure 1A). The protocol was successfully used for isolating materials from several two- and six-row spring barley cultivars, and the spikes were harvested at a given DAP and directly used for extraction without fixation (Figure 1D).

The definition of DOP was a critical parameter to be estimated before dissection. Barley spikes mature from the middle towards the edges. Therefore, the middle flower was used for DOP evaluation. At most, six seeds from the middle of the spikes were used for tissue dissection. The success rate of self-pollination was close to 100%. In comparison, manual pollination had much lower success.

During the selected developmental window (4 to 24 DAP), the difficulty of tissue dissection generally decreased over time. However, it has to be noted that the separation of tissues becomes again more difficult during and after seed dessication (> 24 DAP) due to tissue adherence. During the dissection of the tissues from 4 DAP seeds, a critical part was not to collapse the squashy syncytial endosperm (Figure 2A). Therefore, we removed seed maternal tissues by gentle cut and peel off. The embryo had to be protected against drying by adding buffer. At 8 DAP grain, the isolation strategy was analogous to younger seeds, but the nucellar projection (a part of seed maternal tissues on the dorsal side of the seed) appeared at this stage and its careful removal was required to avoid contamination of endosperm tissues. Conversely, this tissue is an important part of seed maternal tissues and should not be forgotten during isolation of this tissue. At later stages (16 and 24 DAP) seed maternal tissues were more cohesive. Our practical experience was that seed maternal tissues can be dissected and harvested in strips without damaging the endosperm (Figure 2C). The perimeter of the embryo should have a clear round shape and its original position in the seed should be clean of any rests of embryo tissues (Figure 2C).

Figure 2: Flow cytometric estimation of the purity of dissected seed tissues. (A) Morphology of dissected seed tissues from 4 DAP seeds. The groups correspond to the whole seeds (WS) photographed as hulled (left) and peeled from ventral and dorsal sides (middle and right, respectively); the next are dissected seed maternal tissues (SMTs), endosperm (END) and embryo (EMB). Scale bar = 5 mm, except for the inset to which 100 µm scale bar applies. (B) Representative histograms of nuclear DNA content obtained from described tissues. The histograms show marked C-value peaks for diploid EMB and/or seed maternal tissues (2C, 4C, 8C) and/or triploid END tissues (3C, 6C, 12C). The x-axis shows DNA content (relative fluorescence on log₃ scale) and the y-axis the number of measured particles. NOTE: The scale on the y-axis should not be compared between the histograms as it varies depending on the starting amount of material and duration of the measurement. Only the presence/absence of a peak and relative height within one histogram should be evaluated. (C, D) Morphology and flow cytometric profiles of 16 DAP seeds. The figures are organized as in A and B. The flow cytometric measurement reveals also 16C and 24C nuclei, from diploid and triploid tissues, respectively. Scale bar = 5 mm. Please click here to view a larger version of this figure.

To test the purity of isolated tissues, we estimated nuclear DNA content using flow cytometry (Figure 2B and D). We used fresh barley leave to establish the position of the peak corresponding to diploid (2C) nuclei (Figure 3A). This tissue contained > 95% nuclei with 2C and 4C DNA content, corresponding to G0/G1 and G2 phases of the cell cycle, respectively and < 5% nuclei with 8C and 16C DNA content, corresponding to endoreduplicated nuclei. Next, all subsequent seed-tissue samples were measured with the same flow cytometer settings. Flow cytometric histograms of the whole seeds contained C-value peaks for diploid seed tissue (a mixture of an embryo and seed maternal tissues; 2C, 4C, 8C and 16C) and triploid endosperm tissues (3C, 6C, 12C and 24C)²¹. In properly dissected seed tissues, only C-value peaks for either the diploid or the triploid tissues were present (Figure 2B and D). Samples mixing tissues were identified based on the presence of contaminant diploid or triploid peaks (Figure 3B and C).

Figure 3: Examples of control and contaminated seed tissue samples as revealed by flow cytometry. (A) Representative histogram of nuclear DNA content from 10 days old barley leaf representing somatic tissue control. (B) Example histogram of dissected 16 DAP endosperm (3C, 6C, 12C and 24C peaks) contaminated by a diploid tissue (2C peak – red-labeled). (C) Example histogram of 8 DAP dissected seed maternal tissues contaminated by endosperm tissues as indicated by the presence of 3C and 6C peaks (red-labeled). The x-axis shows DNA content (relative fluorescence on log₃ scale) and the y-axis the number of measured particles. NOTE: The scale on the y-axis should not be compared between the histograms as it varies depending on the starting amount of material and duration of the measurement. Only the presence/absence of a peak and relative height within one histogram should be evaluated. Please click here to view a larger version of this figure.

As an example of downstream use of the samples, we isolated RNA from separated seed tissues using either the commercial RNA isolation kits or TRIzol reagent. However, due to high starch content in endosperm of older seeds (after 16 DAP), we used a commercial column-based kit for RNA isolation from problematic tissues. RNA isolation from endosperm samples older than 16 DAP using TRIzol resulted in unsuficient RNA quality and high level of protein contamination. To remove a residual DNA contamination, we performed on-column DNase I treatment that is an optional step in the commercial kits. The amount of isolated total RNA per sample was 200 – 3,000 ng for endosperm, 600 – 15,000 ng for embryo and 1,500 – 15,000 ng for seed maternal tissues. Next, we assessed the quality of isolated RNA using a bioanalyzer. Although the pattern can differ between tissues, two sharp peaks/bands representing the large and the small ribosomal RNA subunits should be normally present in the spectra/gel (Figure 4). The presence of additional peaks and high background in the fast and the inter-region indicates RNA degradation, whereas the 5S rRNA region includes various types of small rRNAs and those peaks do not affect the quality of the isolated RNA. A signal in the precursor region can indicate residual genomic DNA contamination. The samples with RNA integrity number (RIN) ≥ 7 are considered of sufficient quality for analysis including reverse transcription PCR or RNA-sequencing.

Figure 4: Quality control of isolated total RNA. (A) A representative spectrum of high quality (blue) and partially degraded (red) total RNA from 8 and 16 DAP seed maternal tissues respectively produced using Agilent 2100 Bioanalyzer with the RNA integrity number (RIN) 8.00 and 6.40 respectively. The graph shows the intensity of the peaks of the ribosomal RNA subunits: nuclear large-25S, small-18S and 5S RNA. nt = number of estimated nucleotides based on ladder; FU = relative fluorescence units. (B) The electrophoretic gel-like view of high quality (blue) and partially degraded (red) RNA indicating the subunit bands. Please click here to view a larger version of this figure.

To test the purity of tissues prepared using this protocol at the molecular level, we performed RNA-sequencing (Kovacik, Nowicka and Pecinka, unpublished data) and analyzed transcript levels for several well known marker genes of embryo, endosperm and seed maternal tissues development (Figure 5). As embryo markers, we selected barley homologs of maize LEAFY COTYLEDON 1 (HvLEC1; HORVU.MOREX.r2.6HG0506770) and GLOBULIN 2 (HvGLB2; HORVU.MOREX.r2.5HG0430450), which are important genes for embryogenesis and production of storage protein, respectively^22,23. HvLEC1 transcript was highly abundant in emryo tissues at 8 DAP and its amount strongly decreased at 16 DAP and was absent at 24 DAP embryo and all stages on other analyzed tissues. In contrast, HvGLB2 transcript started low at 8 DAP embryo, but greatly increased at 16 and 24 DAP embryos. HvGLB2 showed also low level of transcript in endospem and seed maternal tissues. Endosperm marker genes were represented by barley homologs of maize ALEURONE 9 (HvAL9; HORVU.MOREX.r2.1HG0010310) and GLUTENIN SUBUNIT (HvGS; HORVU.MOREX.r2.1HG0001010) which are related to aleurone differentiation^24,25,26 and energy storage, respectively. The transcripts were highly specific for endosperm tissues, and HvAL9 peaked at 8 DAP while it was 16 and 24 DAP for HvGS, which is consistent with endosperm tissue differentiation and energy accumulation. Our markers for seed maternal tissues were represented by barley BETA AMYLASE (HvBA; HORVU.MOREX.r2.2HG0113950) and CHLOROPHYLL A/B BINDING PROTEIN (HvCAB; HORVU.MOREX.r2.1HG0073450). BETA AMYLASE is connected with utilization of first storage protein deposition in seed maternal tissues at early seed development¹. As the seed maternal tissues are the only green tissue in the seed, CAB proteins are important for photosynthesis. Highly tissue-specific profiles of all selected marker genes demonstrate that our protocol has potential for genetating samples without or with only minimal contaminoation from surrounding tissues.

Figure 5: Examples of expression from marker genes. PolyA enriched mRNAs from barley seed tissues were sequenced in three biological replicates using Illumina platform. The graphs show an average fragments per kilobase per million reads (FPKM) at different days after pollination (DAP) in embryo (orange lines), endosperm (yellow lines) and seed maternal tissues (green lines). Standard deviation between biological triplicates is indicated by the gray field. Two examples of early (top row) and late (bottom row) marker genes are shown for (A) embryo, (B) endosperm and (C) seed maternal tissues. Please click here to view a larger version of this figure.

Discussion

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 for the thorough quality control analysis. However, many plant research institutions have a flow cytometer or ploidy analyzer operated by a trained research staff.

For plant pollination, we make use of the barley’s ability to self-pollinate and rely on a set of simple morphological parameters that define the exact day of natural pollination. Hence, the protocol avoids manual flower emasculation and pollination that is a common approach applied to many plant species. We have initially tried both methods and the manual pollination method resulted in much smaller rate of successfully developing seeds (< 40%). Although the monitoring of spontaneous pollination requires experience in estimating maturity of stigma and anthers, it can be very reliable method with a reduced hands-on time, and can produce higher numbers of seeds needed for dissections.

The difficulty of tissue dissection changes over the time of seed development. The most challenging is the isolation of tissues from the youngest (0 to 8 DAP) seeds, which are minute and easy to damage due to their soft texture and liquid character (endosperm). Therefore, fine tools are needed. Using the presented protocol, we were able to manually isolate seed maternal tissues and endosperm from 4 DAP or older seeds in sufficient amount and quality for complex assays. Dissection of embryo before 8 DAP was problematic and we were not able to collect sufficient amount of tissue for downstream analyses. We envisage that further improvements could be achieved with a micromanipulator. An alternative method could be tissue sectioning followed by a laser microdissection²⁸. Although this method offers great resolution it usually brings a very small amount of material that needs to be extensively PCR amplified before analysis. This may introduce certain biases or redundancy. The presence of cell walls in plant tissues prevents separation of intact cells and their simple isolation by fluorescence-activated cell sorting (FACs) as performed for animal or fungal cell cultures. In plants, fluorescence-activated nuclei sorting (FANs) based on the nuclei C-values is feasible²⁹. Although FANs is highly sensitive method, it represents only part of cellular information (e.g., cytoplasmic RNA and proteins are largely missing), generates small amounts of sample, and requires highly advanced instruments. It has to be emphasized that the protocol presented here provides relatively large amounts of material and does not involve PCR-based amplification before library preparation. Dissection of tissues older than 8 DAP with a cellularized endosperm is considerably easier, but drying of seed parts at later stages may decrease tissue separability. A simple solution is moisturizing the tissues with a physiological buffer.

An important factor affecting all downstream analyses is the purity of extracted tissues. In highly sensitive experiments such as RNA-sequencing, tissue contamination results in decreased resolution and false information. Therefore, we implemented a flow cytometry-based purity control step in the protocol that is based on different ploidies of seed tissues. Distribution of nuclei within the tissue is not always homogenous and some parts can be absent of nuclei while other part of the same tissue may still contain nuclei (e.g., central starchy endosperm and aleurone layer, respectively). We were able to detect nuclei in each completely dissected tissue in the selected developmental window. Based on their triploid nature, the endosperm tissues can be easily distinguished from the diploid seed maternal tissues and embryo. The most common type of contamination observed in the samples was between seed maternal tissues and endosperm, possibly originating from the nucellar projection. However, when the sample contained less than 5% of different ploidy level, this could not be visible as a separate ploidy peak. It is also obvious that the purity control cannot distinguish contamination between seed maternal tissues and embryo. However, this type of contamination is less likely as both tissues do not adhere to each other and can be easily manually separated. In addition, to reduce tissue contamination after dissections, we applied multiple washes using a physiological buffer.

Since RNA can be easily destroyed by the activity of endogenous RNases, the quality of dissected tissue was determined based on RNA degradation level. As an index of quality we used RIN calculated automatically by a bioanalyzer. Degradation is also a natural process caused by aging and together with unproper handling during dissection is the main factor affecting the quality of dissected tissues. This limitation did not allow isolation of good quality RNA from seed maternal tissues older than 24 DAP.

The isolated material is suitable for various types of downstream analyses including isolation of nucleic acids, proteins and other cellular compounds. We have already successfully used the tissues to isolate RNA and perform RNA-sequencing experiments. This is significant experimental improvement because until now, barley seed transcriptomic studies were done using either the mixture of embryo and endosperm³⁰ or even whole seeds³¹. Application of the optimized strategy will greatly increase the resolution and specificity of the RNA-sequencing data as also demonstrated on the expression values of several tissue-specific genes (Figure 5).

In conclusion, this protocol provides means for detailed analysis of individual seed tissues. This will help unravelling the mechanisms controlling seed development in barley and other cereals.

Materials

Name	Company	Catalog Number	Comments
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		5x5 cm
Petri dish
Pin Holder	Fine Science Tools	26016-12
Plastic pestle	p-Lab	A199001
Pots			12x12 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