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....

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....

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

List of materials used in this article

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