A proper understanding of the structural organization of the plant body is essential to any study in plant biology. Experimental studies in vivo and in situ will lead to structural, physiological, and cellular changes of the experimental material. To study macroscopic and microscopic changes, different histological methods and microtechniques can be used as they provide valuable information of the experimental system. In addition, the observed structural changes allow investigators to set hypothesis for further studies based on one's own observation. Thus, proper selection and utilization of microtechniques are a must for the success of a research program. At present, an up-to-date collection of protocols are not readily available in the literature. The latest work in plant microtechniques was published in 1999 by Ruzin but many others are no longer in print [e.g., Jensen (1964); O'Brien and McCully (1981)]. Furthermore, a majority of published works focus on techniques related to general processing and staining procedures. A comprehensive treatment that encompasses broader applications of microtechniques to other disciplines is lacking [e.g., archeology, wood science, etc.]. There is a need to create a comprehensive volume of botanical methods and protocols which includes traditional and novel techniques that can be used by researchers in plant science and investigators in other disciplines that require plant microtechniques in their research and teaching. This book covers a wide variety of applications and brings them up-to-date to make them understandable and relevant, especially to students using the methods for the first time. It is our intention to create a useful reference for plant histology and related methods that will serve as a foundation for plant scholars, researchers, and teachers in the plant sciences.¿

Edward C. Yeung Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada

Claudio Stasolla Department of Plant Science, University of Manitoba, Winnipeg, Manitoba, Canada

Michael J. Sumner Department of Biological Sciences, University of Manitoba, Winnipeg, Manitoba, Canada

Bing Q. Huang Mayo Clinic, Rochester, Minnesota, USA

Contents

Preface

Contributors

Part I   Fixation, Processing, Embedding and Staining of Botanical Specimens

1.      A Guide to the Study of Plant Structure with Emphasis on Living Specimens

Edward C. Yeung   

2.      Chemical and Physical Fixation of Cells and Tissues: an Overview

Bing Q. Huang and Edward C. Yeung 

3.      Paraffin and Polyester Waxes

Claudio Stasolla and Edward C. Yeung

4.      The Glycol Methacrylate Embedding Resins - Technovit 7100 and 8100

Edward C. Yeung and Colin K. W. Chan

5.      Epoxy Resins for Light and Transmission Electron Microscopy

Michael J. Sumner

6.      LR White Acrylic Resin

Edward C. Yeung and Bing Q. Huang

7.      High Pressure Freezing and Freeze Substitution of In vivo and In vitro Cultured Plant Samples

Jose M. Seguí-Simarro

8.      Three-Dimensional Imaging for Electron Microscopy of Plastic-embedded Plant Specimens

Jose M. Seguí-Simarro

9.      Fluorescent Staining of Living Plant Cells

Sarah Schoor, Shiu-Cheung Lung, Dustin Sigurdson, and Simon D. X. Chuong

10.  Improved Methods for Clearing and Staining of Plant Samples

Alexander Lux, Marek Vaculík, and Ján Kovác

Part II  Microtechniques related to Cell and Molecular biology studies

11.  Whole Mount Immunofluorescence Staining of Plant Cells and Tissues

Masaki Shimamura                                                 

12.  Protoplast Isolation and Staining

Shiu-Cheung Lung, Sarah Schoor, Dustin Sigurdson, Makoto Yanagisawa, Kelly Yeung, Mi Qi Liu and Simon D. X. Chuong

13.  Guiding Principles for Live Cell Imaging of Plants using Confocal Microscopy

Subramanian Sankaranarayanan and Marcus A. Samuel

14.  Immunogold Labeling for Electron Microscopy: Strategy and Problem Solving

Fengli Guo and Bing Q. Huang

15.  Abscisic Acid Immunostaining

Yung-I Lee and Mei-Chu Chung

16.  Plant Chromosome Preparations and Staining for Light Microscopic Studies

Subhash C. Hiremath and C.C. Chinnappa

17.  Chromosome techniques and FISH

Mei-Chu Chung

18.  Detection of S-phase of Cell Division Cycle in Plant Cells and Tissues by

Soujanya Kuntam and Ferhan Ayaydin

19.  Staining Methods for Programmed Cell Death

Owen S. D. Wally and Claudio Stasolla

20.  Laser Microdissection of Plant Tissues

Jenna L. Millar, Michael G. Becker, and Mark F. Belmonte

21.  RNA In situ Hybridization

Shalini Mukherjee, Claudio Stasolla, and Belay Ayele

Part III   Preparative Methods for the Study of Wood Anatomy

22.  Microscopic Examination of Wood: Sample Preparation and Techniques for Light Microscopy

Jacques

23.  Collecting and Processing Wood Microcores for Monitoring Xylogenesis

24.  Three-dimensional Imaging of Cambium and Secondary Xylem Cells by Confocal Laser Scanning Microscopy

Satoshi Nakaba, Peter Kitin, Yusuke Yamagishi, Shahanara Begum, Kayo Kudo, Widyanto Dwi Nugroho, and Ryo Funada

Part IV   Botanical Techniques and Protocols for Archeology and Herbarium Collection

25.  Collection of Plant Remains from Archaeological Contexts

Alessandra Celant, Donatella Magri, and Francesca Romana Stasolla

26. 

Alessandra Celant and Gemma Coccolini

27.  Archaeopalynological Preparation Techniques

Donatella Magri and Federico Di Rita

28.  Phytoliths: Preparation and Archaeological Extraction

Brian Kooyman

29.  Starch Granules: Preparation and Archaeological Extraction

Brian Kooyman      

30.  Plant Collection, Identification, and Herbarium Procedures

Bonnie Smith and C. C. Chinnappa

Index