Determination of stomatal index
BACKGROUND
Stoma (plural-stomata) is a minute epidermal opening covered by two kidney-shaped guard cells in dicot leaves. These guard cells, in turn, are surrounded by epidermal (subsidiary) cells. Stomata perform the functions of gaseous exchange and transpiration in plants. The nature of the stomata and the stomatal index and stomatal number are important diagnostic characteristics of dicot leaves.
The stomatal number is defined as the average number of stomata per sq mm of the epidermis of the leaf. The actual number of stomata per sq mm may vary for the leaves of the same plant grown in different environments or under different climatic conditions.
However, it is shown that the ratio of the number of stomata to the total number of epidermal cells in a given area of the epidermis is fairly constant for any age of the plant and under different climatic conditions. The Stomatal index is the percentage in which the number of stomata forms to the total number of epidermal cells, each stoma being counted as one cell. The Stomatal index can be calculated by using the following equation:
Stomatal Index = S x 100/E+S
Where S= Number of stomata per unit area E= Number of epidermal cells in the same unit area. Whilst stomatal number varies considerably with the age of the leaf and due to changes in environmental conditions, the stomatal index is relatively constant and therefore, of diagnostic significance for a given species
REQUIREMENTS
Compound microscope Camera lucida Drawing board Micro slides Cover glasses Forceps Spirit lamp Small watch glass Blade Cello tape Drawing sheet, Dark colored pencil with sharp lead Chloral hydrate solution.
PROCEDURE
1. Preparation of lamina
Take a mature leaf. If the leaf is small, the whole leaf may be taken and if the leaf is large cut 5 mm square pieces from the middle portion between the lamina and midrib
Fresh Leaf
- Sometimes the epidermis can be easily peeled off in thick leaves by breaking it into pieces by sheering action. Separate the epidermis and treat it with chloral hydrate.
- Cut a number of 5mm pieces from the middle portion between the lamina and midrib.
- Boil with chloral hydrate in a test tube and place in a water bath. The epidermis separates out. Carefully place the epidermis on a slide with the help of a brush along with 1-2 drops of chloral hydrate; cool and then place a cover glass. OR
- Prepare an imprint of the epidermis: Take a little piece of gelatin gel (50%) with the help of a needle. Smear it on a hot slide, place a fresh leaf, and slightly press the leaf. Invert the slide and
- cool it under a water tap till the gel is solidified. Then the leaf is removed. This leaves an imprint of the stomata and epidermal cells on the gel. Trace the epidermal cells and stomata with the help of camera lucida.
Dry leaf
- Heat the leaf with chloral hydrate in a test tube in a water bath for 30 min.
- Cut the leaf into two pieces, and observe under the microscope to see whether the stomata are present on both surfaces or one.
- Place the cleared leaf with the veins facing down. Then the upper epidermis will be visible.
- Place the other half with veins facing upwards. Then the lower epidermis will be visible.
- Add two drops of glycerin and place a cover glass.
- Label the slides as “upper” and “lower” and trace the epidermal cells and stomata.
If the leaf is too thick and dark, separate the epidermis are given below
- cut the leaf into two halves.
- Place one half with the upper surface facing downwards.
- Carefully scrape off the upper tissue, with the edge of a razor blade, without disturbing the upper epidermis. Clean it with a brush dipped in chloral hydrate solution.
- The layer of cells remains on the upper epidermis. Turn the layer upside to trace the cells.
- Repeat the procedure with the second half, this time placing the lower surface facing downwards, proceed as given in steps no. 3. and 4.
- Usually, herbs and small shrubs have stomata on both surfaces. In tree species, stomata are present on the lower surface. More stomata are present on the lower surface in the dorsiventral leaf, almost the same number in the isobilateral leaf.
2. Tracing of cells
- Draw a square of about 8-10 cm square on a drawing sheet or any unit area.
- Place the prepared slide on the stage of the microscope.
- Focus epidermal cells and the stomata first with 10×10 and later focus with 10×40 or 10×20.
- With the help of camera lucida, trace the stomata and the epidermal cells in the square.
- Trace epidermal cells and the stomata outside the square to completion on two adjacent sides of the square, for counting purposes.
- Number the complete epidermal cells and the stomata within the square.
- Then continue numbering the cells that are more than half on two adjacent sides.
Calculation
Stomatal index = no. of stomata x 100 No. of stomata + epidermal cells
REFERENCES
- Kokate CK, Practical Pharmacognosy, 4 edition, VallabhPrakashan. Delhi; 1994: 117-118.
- Joshi S, Aeri V. Practical Pharmacognosy, 1 edition, Frank Bros. & Co. New Delhi; 2009: 208-09
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