PLANT ANATOMY LABORATORY IV

HISTOLOGY OF PLANT EXTRACELLULAR MATRIX

 Objectives

      Characterization of plant tissue types depend on attributes of cell walls, cytoplasm and cell geometry.  This exercise will familiarize you with the various features of primary and secondary plant cell walls resolvable with the light microscope.  Learn to recognize these features quickly for future success in cell and tissue characterization.

Description

     In an intact plant each cell has its own wall, and the walls of adjacent cells are separated by the middle lamella.  The wall adjacent to the middle lamella is the primary or original wall of the cell.  It is this wall which is subjected to various changes during the growth of the cell.  In many cells only this primary wall is present.  In others, however, additional wall substance is deposited to produce a thickened structure.  This later-formed wall is the secondary wall.  These two components of the cell wall may be contrasted as follows.
 
 
Primary Wall Secondary Wall
Capable of extension in growth of cells. Not capable of extension.
Undergoes reversible changes in thickness.  Does not undergo reversible changes in thickness.
Primary pit fields present.  True pits present.
Plasmodesmata usually present. No plasmodesmata present.
Wall continuous across pit field. Wall not continuous across pit.

The various layers and components of plant cell walls are tabulated below:

HISTOLOGY OF PLANT EXTRACELLULAR MATRIX

COMPONENT
FOUND IN
MAJOR CHEMICAL COMPONENT
SPECIFIC HISTOCHEMICAL TESTS
EFFECT ON POLARIZED LIGHT
Middle Lamella All Cells Pectin Ruthenium Red Isotropic
Primary Wall All Cells
Meristematic Cells
Epidermal Cells
Sieve Cells
Sieve Tube Elements
Parenchyma
Collenchyma		 Small Randomly Oriented Cellulose (B1-4 Glucose) 1.  Neutral Red

2.  Acidulated 
Potassium Iodide

 Anisotropic
Secondary Wall Ray Parenchyma
Vessel Elements
Tracheids
Fiber-tracheids
Fibers
Sclereids
 Large Parallel Oriented Cellulose that change orientation between layers 1.  Neutral Red

2.  Acidulated 
Potassium Iodide

 Anisotropic
Lignin 1.  Acidulated Phloroglucinol

2.  Light Blue Autofluorescence

 Isotropic
 
Callose Sieve Cells
Sieve Tube Plates		 B 1-3 Glucose
1.  Analin Blue 
- Fluoresces Green
 		 Isotropic
Suberin Cork Cells
Guard Cells		 Suberin Sudan IV Anisotropic
Cuticle Endodermal Cells Cutin Sudan IV Anisotropic
Waxes Endodermal Cells Wax Soluble in Xylene Anisotropic

   Polychromic stains are useful for delimiting various cytoplasmic and cell wall features using one stain solution.  This technique is often used when less specificity of component analysis is needed.  Available ploychromic stains that work well with fresh material include Fabil and Toluidine Blue O.   

    There are three basic planes in which round organs can be sectioned that are useful in reconstructing their three dimensional anatomy.  One of these, the transverse or cross section, is made at right angles to the longitudinal axis of the organ.  The other two are longitudinal sections and are made parallel to the longitudinal axis of the organ.  Radial longitudinal sections are prepared parallel to a radii of the organ, whereas tangential longitudinal sections are made at an angle to a radii of the organ.  An exact radial longitudinal section is referred to as a median longitudinal section and is quite instructive in studies of meristems, but extremely difficult to make via free hand sectioning.

    There are also three basic planes in which flat plant material can be sectioned.  The transverse or cross section is made orthogonal to the longitudinal axis.  The sagittal or longitudinal section is made parallel to the longitudinal section and at right angles to the epidermal layers.  The paradermal section is made parallel to the epidermis of the organ and is rather difficult to achieve via free hand methods.

     Maceration is another very useful technique for study plant cells in which the middle lamella is dissolved resulting in separation of the individual cells.  You will employ maceration during the next the next section of the course.  Since the process takes a few days you will begin maceration of several plant organs and tissues today so that these will be ready for next week. 

<>   By the end of this exercise you should have mastered the ability to distinquish the different components of the cell wall using appropriate histochemicalmethods and the polarized light microscope. Note in linked dye pages which times give you the best results for specific material.  This will speed up your future observations on similar material in future exercises.

Exercises

1.  Devise an efficient way to stain your sections to locate cells with  A) Pectin,  B)  Primary Cell Walls,  C) Secondary Cell Walls,  D)  Callose and E) Suberin.  You may wish to collaborate with your colleagues on staining material.

2.  Make free hand-sections of the fresh Apium (celery) petioles in the transverse, radial longitudinal and tangential longitudinal planes .   Use your staining procedure to test for A) Pectin,  B)  Primary Cell Walls,  C) Secondary Cell Walls, and  D)  Callose.   Record your staining scheme and your observations in your exercise sheet.

3.  Make transverse, sagittal, and paradermal sections of Ficus leaves.   Look for a cuticle layer overlying the epidermis and a layer of wax overlying the cuticle.  Test  for Cutin with Sudan IV.  Dissolve the wax from the surface using  xylene.  Record your observations in your exercise sheet.

4.  Make free hand sections of Pseudotsuga twigs in the transverse, radial longitudinal, and tangential longitudinal planes.    Stain your sections to locate cells with A)  Primary Cell Walls,  B) Secondary Cell Walls, C)  Callose, and D) Suberin.   Record your observations in your exercise sheet.

5.  Record any modifications to your initial staining procedure that will make it more efficient in the future in your exercise sheet.

6.  Begin maceration of Apium petioles, Ficus leaves, and Pseudotsuga twigs

Material