A Method for Characterizing Embryogenesis in Arabidopsis

Summary

This protocol outlines a method for observing embryogenesis in Arabidopsis via ovule clearance followed by the inspection of embryo pattern formation under a microscope.

Abstract

Given the highly predictable nature of their development, Arabidopsis embryos have been used as a model for studies of morphogenesis in plants. However, early stage plant embryos are small and contain few cells, making them difficult to observe and analyze. A method is described here for characterizing pattern formation in plant embryos under a microscope using the model organism Arabidopsis. Following the clearance of fresh ovules using Hoyer's solution, the cell number in and morphology of embryos could be observed, and their developmental stage could be determined by differential interference contrast microscopy using a 100X oil immersion lens. In addition, the expression of specific marker proteins tagged with Green Fluorescent Protein (GFP) was monitored to annotate cell identity specification during embryo patterning by confocal laser scanning microscopy. Thus, this method can be used to observe pattern formation in wild-type plant embryos at the cellular and molecular levels, and to characterize the role of specific genes in embryo patterning by comparing pattern formation in embryos from wild-type plants and embryo-lethal mutants. Therefore, the method can be used to characterize embryogenesis in Arabidopsis.

Introduction

Embryogenesis is the earliest event in higher plant development. A mature embryo forms from a zygote through cell division and differentiation under strict genetic control^1,2. Arabidopsis embryos are a useful model to study the control of morphogenesis because the sequence of cell divisions during embryogenesis follows an expected pattern^3,4. However, an Arabidopsis embryo containing one to several cells is too small to be observed and analyzed. In addition, the mutation of certain genes can cause embryo lethality⁵

Protocol

NOTE: This protocol has three parts: 1) observing the pattern formation in wild-type Arabidopsis embryos using DIC microscopy; 2) characterizing the embryo pattern formation via the observation of marker protein expression using confocal laser scanning microscopy; and 3) determining the role of a specific gene in embryogenesis (using the naa10 mutant as an example).

1. Ovule Clearing

1. Plant material preparation
   NOTE: The protocol described here is an example on how to grow healthy plants; other ways to grow healthy plants also work.
   1. Add 100 seeds to a 1.5 mL tube, and....

Results

The method described in this paper can be used to observe embryogenesis directly under a microscope, annotate the cell identity specification during embryogenesis with specific markers, and characterize the role of a particular gene during embryogenesis.

Representative results from the analysis of embryo patterning (from the elongated zygote stage to the mature walking-stick stage) in the wild-type Arabidopsis are shown.......

Discussion

Ovule clearance is a useful method for detecting cell division and assessing morphology during embryogenesis in Arabidopsis; using this technique, embryos can be observed directly under DIC microscopy^5,12. The critical step for ovule clearance is step 1.3.4. The time required for ovule clearance in step 1.3.4 is variable. Embryos at the 2/4-cell stage can be clearly observed after 2 h in the Hoyer's solution, whereas they look indistinct after 12 h. .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Chloral Hydrate	Sigma	15307
Murashige and Skoog Basal Medium	Sigma	M5519
Phytagel	Sigma	P8169
Hygromycin	Roche	10843555001
Growth chamber	Percival	CU36L5
Fluorescence microscope	Zeiss Oberkochen	Zeiss Image M2	camera: AxioCam 506 color
Confocal Laser Scanning Microscopy	Zeiss Oberkochen	Zeiss LSM 5	filters: BP 495 - 555 nm
Stereoscope	Zeiss Oberkochen	Axio Zoom V16	camera: AxioCam MRc5
nutrient-rich soil	KLASMANN, Germany
50-mL tube	Corning Inc.
1.5-mL tube	Corning Inc.
10% sodium hypochlorite solution			Domestic analytical reagent
sucrose			Domestic analytical reagent
KOH			Domestic analytical reagent
glycerol			Domestic analytical reagent
culture dish			Domestic supplies
vermiculite			Domestic supplies
glass slide			Domestic supplies
syringe needle			Domestic supplies
fine-tipped tweezers			Domestic supplies
super clean bench			Domestic equipment