Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

Summary

We report an efficient and simple method to isolate embryos at early stages of development from Arabidopsis thaliana seeds. Up to 40 embryos can be isolated in 1 hr to 4 hr, depending on the downstream application. The procedure is suitable for transcriptome, DNA methylation, reporter gene expression, immunostaining and fluorescence in situ hybridization analyses.

Abstract

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a consequence, the isolation of plant embryos at early stages (1 cell to globular stage) is technically challenging due to their relative inaccessibility. Efficient manual dissection at early stages is strongly impaired by the small size of young Arabidopsis seeds and the adhesiveness of the embryo to the surrounding tissues. Here, we describe a method that allows the efficient isolation of young Arabidopsis embryos, yielding up to 40 embryos in 1 hr to 4 hr, depending on the downstream application. Embryos are released into isolation buffer by slightly crushing 250-750 seeds with a plastic pestle in an Eppendorf tube. A glass microcapillary attached to either a standard laboratory pipette (via a rubber tube) or a hydraulically controlled microinjector is used to collect embryos from droplets placed on a multi-well slide on an inverted light microscope. The technical skills required are simple and easily transferable, and the basic setup does not require costly equipment. Collected embryos are suitable for a variety of downstream applications such as RT-PCR, RNA sequencing, DNA methylation analyses, fluorescence in situ hybridization (FISH), immunostaining, and reporter gene assays.

Introduction

The embryo of flowering plants is surrounded by the endosperm, a nutritive tissue derived from a second fertilization event. Both embryo and endosperm are surrounded by several cell layers of the seed coat. Collectively these tissues form a seed, which develop inside the fruit. Thus, tissue- and cell-specific analyses of Arabidopsis embryos are strongly impaired due their inaccessibility. Nevertheless, embryos at the late-globular or later stages are relatively well amenable to manual dissection by using fine tungsten needles under the stereomicroscope, or by applying slight pressure on the seed using forceps to extract them. Such techniques were successfully used for transcriptome or epigenome profiling analyses such as microarray hybridization, bisulfite sequencing, or RNA sequencing (e.g. ^1-3). In contrast, studies of embryos at the zygote to early globular stage remain technically challenging. To date, only a few studies have reported transcriptome analyses on young embryos using either laser-capture microdissection (LCM) of embryonic tissues from fixed seed sections ⁴ or manual extraction of individual embryos from within seeds using fine tools ⁵. However, LCM equipment is not commonly available and manual embryo extraction at early stages is time consuming and requiring excellent dissection skills that are not easily transferable. In addition to genome-wide analyses, in situ gene expression analyses are also difficult to perform on young, whole-mount embryos of Arabidopsis. To some extent, young embryos can be released on microscope slides by gentle pressure on the seeds and used for reporter gene assays or protein detection by immunostaining (for example see ^6,7). This technique, however, does not allow high-throughput embryo isolation, thus hindering quantitative analyses.

Therefore, we developed an efficient and rapid protocol for early embryo isolation from Arabidopsis seeds that is simple to set up, easily transferable, and suitable for a variety of downstream applications. The basic principle is to gently crush seeds - dissected from young siliques in an Eppendorf tube using a plastic pestle in an appropriate isolation buffer. The seed extract is placed in droplets on a multi-well slide and is screened for the presence of released embryos at the desired stage using an inverted microscope. Embryos are collected using a glass microcapillary attached to a microinjector or a standard laboratory pipette. For molecular applications, embryos are washed twice by repeated release into drops of new isolation buffer before transferring them to the destination buffer in a minimal volume. For cytological applications (reporter assays, immunostaining, FISH), washing steps can be omitted.

The method offers several advantages: (i) it yields 25-40 embryos in ~45 min for cytological applications or in 3-4 hr for molecular applications (including the washing steps), (ii) it allows isolation of specific embryonic stages, (iii) it is easily transferable to other persons and laboratories due to its simple setup, (iv) it requires affordable equipment for the basic setup which is amenable to upgrades, and (v) it was successfully used for various downstream applications such as RNA sequencing ⁸, gene-specific DNA-methylation analysis ⁹, reporter assays (¹⁰ and Raissig et al., in prep.), and FISH (J. Jaenisch, U. Grossniklaus, C. Baroux unpublished, see Figure 5).

Protocol

The procedure is summarized in the flowchart shown in Figure 1. The microcapillaries and the instrumental setup are shown in Figure 2 and Figure 3, and typical steps of embryo isolation are shown in Figure 4.

1. Material and Buffer Preparation

1.1 Silicon coating of glass microcapillaries

1. Place the microcapillaries in a 15 ml Falcon tube with ~5 ml of Sigmacote (Sigma) and invert several times.
2. Remove the solution, place the Falcon tube containing the capillaries in an aluminum foil and bake them for 3 hr at 60 °C. Store at room temperature.

1.2 Obtain ~50-100 μm-diameter microcapillary tips

1. Pull 1 mm-diameter glass capillaries either manually over a Bunsen burner or by using a commercial puller (vertical filament puller or micropipette puller).
2. Use a diamond-tip pen or blade to cut the tip of the pulled capillary to create the desired opening. Select the best-shaped capillaries under a stereo microscope. The opening should be 50-100 μm (Figure 2).

1.3 Slide preparation

1. Siliconize clean slides by covering all the wells with Sigmacote (~1 ml/slide) for 5 min, remove. Bake 3 hr in aluminum foil. Store at room temperature.
2. Wash the multi-well glass microscopic slides for 10 min in 10% SDS, 2 x 2 min in nuclease-free water (autoclaved DEPC-ddH₂O), 2 min in 70% ethanol, 2 min in 100% ethanol, air-dry. All steps are done in autoclaved Coplin jars. Slides can be re-used multiple times providing thorough cleaning between each usage.
3. Just prior to embryo isolation spread ~0.5 μl of 10 mg/ml bovine serum albumin (BSA) with a pipette over the whole surface of each well and air-dry.

1.4 Microscope and capillary setup

1. Use an inverted microscope with a 10x and 20x magnification objective. Optimize the light contrast (embryos are quite transparent).
2. Place a micromanipulator to hold the glass capillary beside the microscope. The glass capillary is connected to a microinjector (Figure 3A) or to a regular P-200 pipette via a rubber tube (Figure 3B, see Discussion for a detailed description of this setup).
3. Place the capillary above the microscope slide at a ~70° angle (Figure 3) and adjust the position to have the opening in the field of view.
4. Just prior to embryo isolation take up and release ~5-10 μl BSA (1 mg/ml) to coat the capillary.

1.5 Buffers

Table 1 lists the isolation and destination buffers depending on the downstream applications.

1. Prepare ~1 ml isolation buffer per sample freshly before use and keep it on ice.
2. Prepare the destination buffer in a 0.5 ml Eppendorf low-binding tube on ice (molecular applications) or on a microscope slide (cytological applications) in a humid chamber.

2. Seed Dissection and Embryo Extraction

2.1 Synchronisation of Seed Development

1. Emasculate flowers and keep them 2 days in the growth chamber while avoiding contact of the exposed pistils with other flowers, then pollinate them (e.g. ¹¹).
2. Test the stage of development under your growth conditions by microscopic investigation of cleared seeds. With our growth conditions (16 hr light at 21 °C, 8 hr dark at 18 °C and 70% humidity) seeds collected 2.5 days after pollination (DAP) yielded mainly 2-4 cell embryos and seeds collected 3.5 - 4 DAP yielded globular embryos.

2.2 Seed Dissection and Rupture

1. Remove the seeds from 10-15 siliques (~2.5 DAP) under a stereomicroscope with forceps and insulin needles.
2. Immerse the seeds in 20 μl isolation buffer in a 2 ml round-bottom Eppendorf tube placed on ice.
3. Gently crush the seeds with a plastic pestle (pre-cleaned with 10% SDS, rinsed with DEPC-ddH₂O and washed with 70% ethanol) to release the embryos until the seed extract is cloudy. The force to apply is to be determined by every user upon trial.
4. Rinse the pestle with 300 μl of isolation buffer to wash the pestle and dilute the sample.
5. Spin-down the extract at 5,000 x g for 5 sec. Gently resuspend the pelleted extract by pipetting up-and-down 2-3 times.
6. Filter the extract with a 30 μm nylon mesh (mounted on tube adaptors, e.g. from PartecCelltricks). Rinse the mesh with an additional 200 μl isolation buffer.

3. Embryo Isolation

3.1 Slide preparation

1. Place a clean, BSA-coated and siliconized multi-well slide on the stage of the inverted microscope, resuspend the filtered seed extract by pipetting gently up and down and pipette 2 droplets of 40-50 μl seed extract into 1 or 2 wells. Screening only 1 or 2 drops at a time prevents evaporation of the sample.
2. Place 50 μl of fresh isolation buffer (1^st wash drop) in a well of a different slide prepared as before. Keep this slide in a covered, humid chamber to prevent evaporation.

3.2 Screen, clean, collect

1. Screen the droplets of seed extract for embryos at the desired stage with the 10x magnification objective. If necessary, confirm the stage with the 20x magnification. The embryos usually sink to the bottom of the slide.
2. Manually remove debris around the embryo with a tungsten needle, an insulin needle or similar equipment.
3. Move the glass capillary near the embryo using the micromanipulator, take up the embryo with as little solution as possible.
4. Collect several embryos (e.g. all of one droplet) and release them in the 1^st wash drop(molecular application) or in destination buffer (cytological applications). Each collecting round should be kept within 5-10 min and embryos should be collected in a minimal volume (the total volume of all collecting rounds should be <5 μl).
5. Repeat the screening and collection until the desired amount of embryos is gathered in the 1^st wash drop (centrally, if possible, to facilitate recollection).
6. Recollect all embryos at once from the wash drop (if debris are carried over, remove them with a needle before recollection).
7. Release the embryos into a 2^nd wash drop of 50 μl. Repeat 3.2.6.
8. Release the embryos in the destination buffer. The transfer should involve only a contact, and not immersion, of the capillary tip.
9. Replace the microcapillary for the next sample.

Results

Our embryo isolation procedure (Figure 1) allows isolation of up to 40 embryos in 4 hr if washes are performed, e.g. for molecular applications, or in less than an hour if washes are omitted, e.g. for cytological applications. Figure 2 displays high and low quality microcapillary tips and Figure 3 shows the setup of the embryo isolation machine. Figure 4 displays the process of embryo isolation on the inverted microscope.

Discussion

We developed an embryo isolation protocol that is rapid, effective, and can be easily transferred to other laboratories.

The equipment described here consists of an inverted microscope, a micromanipulator, glass microcapillaries, a vertical filament puller and a microinjector (Figure 3A). The setup is similar to the one described for single animal cell isolation for transcriptomics analyses ¹⁷. We also successfully worked with a more basic setup where glass microcap...

Materials

Name	Company	Catalog Number	Comments
REAGENTS
Sigmacote	SIGMA	SL2-100 ml
RNAse OUT	Invitrogen (life technologies)	10777-019
First- strand buffer	Invitrogen (life technologies)	18064-022	contained in Superscript II package
DTT	Invitrogen (life technologies)	18064-022	contained in Superscript II package
Bovine serum albumin (BSA) 100x =10 mg/ml	New England Biolabs Inc.		Different suppliers will also work
Thin wall Capillaries 1.0 mm	World Precision Instruments	TW100F-4
DNA LoBind tube 0.5 ml	Vaudaux-Eppendorf	0030108.035
CellTricsΔ 30 μm	PARTEC	04-0042-2316
5wells 10 mm diameter slides	Electron Microscopy Sciences	63421-10
Formaldehyde Solution	Sigma-Aldrich	F1635
Superfrost Plus slide	Thermo Fisher	J1800AMNZ	Menzel-Gläser
Tris	Amaresco	0497
EDTA	Applichem	A2937
Glycin	Fluka	50050
SDS pellets	Roth	CN30.3
Micro Pestle	VWR	431-0094
Microfine insulin syringes	BD	U-100
DEPC	Sigma-Aldrich	D5758
Ethanol	Schaurlau	ET00102500
Forceps N5	Dumont	0108-5
Bioanalyzer Pico Chip	Agilent Technologies	5067-1513
EQUIPMENT
Inverted microscope	Nikon TMS (Japan),
Micromanipulator	Leitz		Leica
Micomanipulator Post mount LH1 probe	Leica microsystems	39430101	Different brand will also do the work
Vertical filament puller	Sutter instrument	P-20 model	Other model are also suitable
Cell Tram vario	Vaudaux-Eppendorf	5176.000.033
Bioanalyzer	Agilent Technologies	2100
Qubit Fluorometer	Invitrogen (life technologies