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 control1,2. Arabidopsis embryos are a useful model to study the control of morphogenesis because the sequence of cell divisions during embryogenesis follows an expected pattern3,4. 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, indicating the key role of those genes in embryogenesis. The characterization of pattern formation in the embryo-lethal mutants provides a basis for understanding the molecular mechanism whereby essential genes regulate embryo development.
A detailed method is described here for the characterization of embryo pattern formation in the model plant Arabidopsis by direct microscopic observation. The method involves ovule clearance followed by the microscopic observation of an embryo. The characterization of an embryo-lethal mutant is also described.
The morphology of embryos at different developmental stages can be determined directly by microscopy using the ovules that have been cleared with Hoyer's solution6,7. Such ovules exhibit a high refractive index; thus, this ovule clearing method is especially advantageous for specimens that must be observed by differential interference contrast (DIC) microscopy.
In addition, the genes that are expressed in a particular cell or a limited number of cells during embryogenesis can be used as markers to identify the cells in an embryo. Therefore, the cell identity specification during embryogenesis can be annotated by monitoring the expression of GFP-tagged marker proteins using confocal laser scanning microscopy. Conversely, observing the expression pattern of an unknown functional gene-GFP fusion during embryogenesis can provide a link between embryo patterning and the expression of that gene, and facilitate the identification of new genes that are required for embryogenesis in Arabidopsis.
The method described here can also be used to characterize the phenotype of an embryo-lethal mutant, and to determine the impact of the affected gene on embryogenesis at the cellular level. Furthermore, in combination with a comparison of the expression pattern of marker genes during embryogenesis between wild-type and mutant plants, the method can yield clues about the molecular mechanisms and signaling pathways involved in embryogenesis8,9. Indeed, the method was applied to naa10 plants, and it was found that the abnormal division of the hypophysis in the mutant may be caused by a change in the auxin distribution during embryogenesis5.
Protocol
Representative Results
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 microscopy5,12. 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. …
Divulgations
The authors have nothing to disclose.
Acknowledgements
We thank Dr. Jessica Habashi for critically reading of the manuscript. We thank Dr. Xianyong Sheng of the Imaging Center, College of life Sciences, Capital Normal University (Beijing, China), for performing the localization of DR5-GFP assay. This work was supported by grants from the Beijing Municipal Government Science Foundation (CIT&TCD20150102) and from the National Natural Science Foundation of China (31600248).
Materials
| 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 | ||
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