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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

This protocol presents how to live image and analyze the shoot apical meristems from different plant species using laser scanning confocal microscopy.

Abstract

The shoot apical meristem (SAM) functions as a conserved stem cell reservoir and it generates almost all aboveground tissues during the postembryonic development. The activity and morphology of SAMs determine important agronomic traits, such as shoot architecture, size and number of reproductive organs, and most importantly, grain yield. Here, we provide a detailed protocol for analyzing both the surface morphology and the internal cellular structure of the living SAMs from different species through laser scanning confocal microscope. The whole procedure from the sample preparation to the acquisition of high resolution three-dimensional (3D) images can be accomplished within as short as 20 minutes. We demonstrate that this protocol is highly efficient for studying not only the inflorescence SAMs of the model species but also the vegetative meristems from different crops, providing a simple but powerful tool to study the organization and development of meristems across different plant species.

Introduction

The plant meristem contains a pool of undifferentiated stem cells and continuously sustains the plant organ growth and development1. During the postembryonic development, almost all aboveground tissues of a plant are derived from the shoot apical meristem (SAM). In crops, the activity and size of the SAM and its derived floral meristems are tightly associated with many agronomic traits such as shoot architecture, fruit production, and seed yield. For example, in tomato, an enlarged SAM causes an increase in the shoot and inflorescence branching, and thus results in generating extra flower and fruit organs2. In maize, an ....

Protocol

1. Media and imaging dishes preparation

  1. MS plates: Add 0.5x Murashige & Skoog MS medium, 1% agar into deionized water and then adjust pH to 5.8 using potassium hydroxide solution (Optional: Add 1% sucrose for the long-term plant growing). Autoclave and pour plates.
  2. Imaging dishes: Fill plastic Petri dishes (6 cm wide, 1.5 cm depth) to 0.5-0.8 cm with 1.5% molten agarose.

2. Plant growth

  1. Arabidopsis growth
    1. Sow sterilized seeds on.......

Representative Results

To evaluate the efficiency of our protocol and to explore the morphology of the meristems from different species, we have performed the confocal live imaging experiments on the inflorescence meristem from Arabidopsis and the vegetative meristems from both tomato and soybean. In this study, Arabidopsis ecotype Landsberg erecta, tomato cultivar Micro-Tom and soybean cultivar Williams 82 have been used as examples.

Discussion

Here, we describe a simple imaging method that can be applied to the study of shoot apical meristems from different plants with minor modification, opening a new avenue to study the meristem regulation at both vegetative and reproductive stages in model plants and crops. In contrast to the SEM and histological staining methods, this protocol can help reveal both surface view and internal cellular structures of the SAMs, without the need for labor-intensive sample fixation and/or tissue sectioning steps. This protocol is .......

Acknowledgements

The authors acknowledge Purdue Bindley Bioscience Center Imaging Facility for accessing the laser scanning confocal microscope and for the technical support, and the authors appreciate the help from Andy Schaber in the Purdue Bindley Imaging Facility. This activity was funded by Purdue University as part of AgSEED Crossroads funding to support Indiana’s Agriculture and Rural Development.

....

Materials

NameCompanyCatalog NumberComments
Agar PhytoDot Scientific Inc.DSA20300-1000
AgaroseDot Scientific Inc.AGLE-500
Forceps ROBOZRS-4955Dumont #5SF Super Fine Forceps Inox Tip Size .025 X .005mm, for dissecting shoot apices.
LSM 880 Upright Confocal Microscope Zeiss
Murashige & Skoog MS mediumDot Scientific Inc.DSM10200-50
Plan APO 20x/1.1 water dipping lensZeiss
Plastic petri dishes 100 mm X 15 mmCELLTREAT Scientific Products229694Use as making MS plates
Plastic petri dishes60 mm X 15CELLTREAT Scientific Products229665Use as imaging dishes
Propagation MixSungro Horticulture
Propidium iodideAcros Organics4403002501 mg/mL solution in water, to stain the cell walls
Razor bladePERSONNA 62-0179For cutting shoot apex from plants
StereomicroscopeNikonSMZ1000
TissueVWR82003-820
Zen blackZeissImage acquisition software

References

Explore More Articles

Confocal Live ImagingShoot Apical MeristemPlant SpeciesArabidopsisTomatoSoybeanPropidium Iodide StainingDissectionSample PreparationConfocal Microscopy

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