- Generate hypotheses about leaf and stomatal structure of plants based on transpiration lab results.
- Test those hypotheses by quantifying leaf internal anatomy and stomatal size and distribution.
Lab 4 Overview
I. Formulate Hypotheses about Plant Anatomy
II. Test Hypotheses about Plant Anatomy
a. Stomatal Anatomy Using Epidermal Peels
- Stomatal Distribution (top/bottom/total)
- Stomatal Counts
- Stomatal Length
b. Cross sectional Anatomy of Leaf Using Hand Microtomes
- Total Leaf Thickness
- Palisade Mesophyll Thickness
- Spongy Mesophyll Thickness
III. Data Analysis (See Statistics and Graphing for Tutorials)
a. Column graph of mean +/- standard deviations of upper and lower leaf stomatal densities for all plants
b. Stacked column graph of mean +/- standard deviations of total leaf thickness, palisade and spongy mesophyll thickness of all plants
c. Composite figure of stomatal peel and leaf cross section for all plants
d. Calculation of mean +/- stomatal conductance index for all plants
e. One-way Analysis of Variance of stomatal conductance index comparing all plants
Plant Morphology: Background
Today, we will study plant habit and leaf morphology/anatomy, which will help your understanding of various aspects of plant form and function. There are many aspects of plant and leaf morphology/anatomy that may affect the plant's ability to function in its environment.
The epidermal tissues of leaves serve a number of key functions: (a) to regulate gas exchange, (b) to prevent excessive water loss, (c) to defend against disease and predation (d) to screen out harmful ultraviolet light.
The epidermis is the outermost covering of the leaf. These cells generally lack chloroplasts. They are packed tightly together and sealed with a waxy coating called the cuticle. This tight packing and wax seal serve to prevent water loss and to stop bacteria and viruses from penetrating the outer surface.
During the period of leaf growth and maturation some epidermal cells differentiate into a complex of cells that regulates the flux of gases (CO2, H2O, O2) into and out of the leaf. Gas-regulating complexes of cells are referred to as stomatal complexes. The cells that compose these complexes are called guard cells and subsidiary cells. They work together to regulate the opening and closing of pores in the epidermis. The subsidiary cells are easily recognized because they are morphologically different from the majority of the other epidermal cells and directly border the guard cells. The pores are called stomata (singular: stoma) and it is through these pores that the movement of gases occurs.
The ground tissue of the leaf consists of parenchyma cells, which in this case have the specialized names, palisade and spongy mesophyll. Parenchyma is a general term used by plant biologists to refer to the thin walled living cells that form much of the ground tissues in all plant organs. It is between the leaf’s upper and lower epidermis that we find the chloroplast containing palisade and spongy mesophyll cells that perform the main function of the leaf, photosynthesis.
Making Epidermal Peels and Measuring Stomatal Densities
Duco cement epidermal peels are like impressions of the leaf surface topography and will include the surface of the epidermis and its stomata. Each person will apply a thin layer of cement (not more than 1-2 cm wide) to both the upper and lower surface of selected leaves while they are attached to the plants. Try to select a leaf from each plant that is mature and of similar age and size-not too young or too old. For each replica use a toothpick to place the duco cement on the leaf blade avoiding the mid-vein and the edge of the leaf. Allow the cement to dry for about 5 minutes. Peel off the transparent cement layer with forceps and mount a small section of it in water on a slide and place it under a cover slip.
When observing the stomatal imprints on the replicas under the compound microscope you will need to maximize image contrast. Do this by reducing light intensity and close down your iris diaphragm located on the substage condenser. Depending on how small the stomata are and their density, you may choose to observe them at a total magnification of 100X or 400X. Always start with the lower power. Your instructor will show you how to take photographs through the eyepiece of your microscope both with and without the grid. Be sure to record the magnification (x 100 or x 400) and share good images with your classmates.
Measurement of stomatal density (number of stomata mm-2):
In one of the oculars of your microscope you will view a square ocular grid superimposed on the microscopic image. The grid is composed of 100 identical small squares. At 100X total magnification each of these small squares has an area of 10,000 µm2 for a total grid area of 1 mm2.
Start with a peel from the lower leaf surface. At 100X count the number of stomata in the entire square grid, or, if you find the stomata too numerous, count the number of stomata in 20 contiguous small squares of the grid, and multiply by 5 to get an estimate for the entire grid. Repeat on the upper surface peel.
- Calculate the number of stomata per mm2 for lower and upper leaf surface.
- Sum the upper and lower to record total stomatal density in 1 mm2 of leaf area.
- Record the data for upper and lower stomatal density in the data table below and on the class data sheet.
NOTE: The stomata included under the whole grid at 100X total magnification represent the number of stomata in 1mm2. If you count the stomata under the whole grid at 400X total magnification multiply by 16 for the final number of stomata in 1mm2. If, at 400X, you count only 20 small squares of the grid you need to multiply by 5 and by 16 for the number of stomata per mm2.
Each person is responsible for recording the mean upper and lower stomata on 1 leaf from each of the different plants in the class data sheet.
Measuring Stomatal Length Using Epidermal Peels
We can effectively make measurements of the relative sizes or quantities of some of the structures and tissues present within the leaf. Because the measurements are of very small structures the light microscopes in the laboratory are equipped with ocular micrometers. An ocular micrometer looks like a small ruler with uniform intervals on the scale (Fig. 1). Depending on your microscope you will find the micrometer along one edge of the ocular grid or in the other ocular.
The smallest interval on the scale represents a specific distance. The exact distance depends on the objective lens that is being used to observe the specimen. The distance equivalent to the smallest interval on the micrometer for the 10X objective lens (100X total magnification with the 10X ocular) is 10 µm and for the 40X objective lens (400X total magnification) the distance is 2.5 µm. Therefore, if you are observing your structure at a total magnification of 100X and the distance spanned is 20 small intervals, the thickness of the mesophyll layer is calculated as:
10 µm x 20 intervals = 200 µm
Measurement of stomatal aperture length using lower leaf stomatal peels:
Use the micrometer in the eyepiece. (In some of the microscopes the eyepiece(s)can be turned while others require that you turn the head of the microscope to move the micrometer).
Using the lower leaf Duco cement replicas, measure the length of five different stomata on the surface of the leaves using the ocular micrometer and calculate the average and standard deviation of these 5 measurements.
Calculating Stomatal Conductance Index
Stomatal conductance, or the ability of materials to pass through the stomata of a leaf, is dependent on both the size of the stomata and the total density of stomata on the leaf (Holland and Richardson, 2009). This variable should relate to the rate at which water passed through the stomata of each plant during the transpiration lab.
Each student should calculate the stomatal conductance index for one leaf on their plant using the following formula:
Stomatal conductance = (guard cell length)2 X total stomatal density X 10 -4 .
Making a Cross Section of a Leaf Using a Hand Microtome
Cut a piece of leaf about 1 cm long and 0.5 cm wide, so that it will fit into the microtome well when it is fully expanded.
Pour a small amount of liquid paraffin in the bottom of the well, and place the leaf specimen vertically in the paraffin. Tweezers help.
When the original paraffin cools, add more hot paraffin to completely fill the well and surround the specimen. Cool the microtome in the refrigerator for about 5 minutes, or until the paraffin plug is hard.
Using the microtome knife, shave the top of the plug to give a flat surface. Turn the bottom dial of the microtome to elevate the plug and gently cut a section. Thinner sections are less apt to break. When you have a section, place it on a microscope slide, add a drop of water, and cover with a cover slip.
Use the micrometer in the ocular of your microscope to measure the entire thickness of the leaf, the thickness of the palisade mesophyll, and the thickness of the spongy mesophyll.
- Prepare a composite photomicrograph of a representative epidermal peel and cross section of your plant with an appropriate figure caption.
- Write a brief section of results text for the one-way analysis of variance (ANOVA) comparing the stomatal conductance index among all plant species, including the appropriate text citation for ANOVA test results.
- In preparation for next week's lab, read section 10.2 in Chapter 10, Biological Science by Freeman.