Determination of stomatal number

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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, as well as, the stomatal index and stomatal number are important diagnostic characteristics of dicot leaves.¹

Stomatal number is defined as the average number of stomata per sq mm of epidermis of the leaf. The actual number of stomata per sq mm may vary for the leaves of the same plant grown in different environment or under different climatic conditions. It is, however shown that the ratio of the number of stomata to the total number of epidermal cells in a given area of epidermis is fairly constant for any age of the plant and under different climatic conditions.

Stomatal index is the percentage which the number of stomata form to the total number of epidermal cells, each stoma being counted as one cell. 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, stomatal index is relatively constant and therefore, of diagnostic significance for a given species. It is employed for the differentiation of allied or closely related to species of same genus in air dried, as well as fresh conditions.

Timmerman (1927) and Rowson (1943) were amongst the first few to investigate leaf drugs for stomatal number and stomatal index.

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REQUIREMENTS

Compound microscope

Camera lucida

Drawing board

Micro slides

Cover glasses

Forceps

Spirit lamp

Small watch glass

Blade

Cello tape

Drawing sheet

Dark coloured pencil with sharp lead

Chloral hydrate solution

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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

1. Sometimes the epidermis can be easily peeled off in thick leaves by breaking it into pieces by sheering action. Separate the epidermis and treat with chloral hydrate.
   
2. Cut a number of 5mm pieces from the middle portion between the lamina and midrib.
   
3. Boil with chloral hydrate in a test tube placed 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
   

4. 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.
   
5. Trace the epidermal cells and stomata with the help of camera lucida.
   

Dry leaf

1. Heat the leaf with chloral hydrate in a test tube on a water bath for 30 min.
   
2. Cut the leaf into two pieces, observe under the microscope to see whether the stomata are present on both surfaces or one.
   
3. Place the cleared leaf with the veins facing down. Then the upper epidermis will be visible.
   
4. Place the other half with veins facing upwards. Then the lower epidermis will be visible.
   
5. Add two drops of glycerin and place a cover glass.
   
6. 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.

1. Clear the leaf with chloral hydrate as given in step no.1. cut the leaf into two halves.
   
2. Place one half with the upper surface facing downwards.
   
3. Carefully scrape off the upper tissue, with the edge of a razor blade, without disturbing upper epidermis. Clean it with a brush dipped in chloral hydrate solution.
   
4. The layer of cells remaining on the is the upper epidermis. Turn the layer upside to trace the cells.
   
5. Repeat the procedure with the second half, this time placing the lower surface facing downwards, proceed as given in step no. 3. And 4.
   
6. 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 dorsiventral leaf, almost the same number in isobilateral leaf.
   

In this experiment the number has to be determined per square millimeter.

1. Adjust the drawing board, if swift camera-lucida is used. It is not necessary to adjust angle with Abbe’s camera lucida.
   
2. With the help of stage micrometer, draw a line of 1mm using 10×10 magnification on a drawing sheet and draw a 10 cm square on that line (1 mm =10 cm) if magnification is correct.
   
3. Replace the stage micrometer with a prepared slide of the leaf as given for stomatal index.
   
4. With the help of camera lucida mark the number of stomata in the square.
   
5. Count the stomata. That gives the number of stomata per sq. mm.
   
6. Take 25 reading and calculate the average.
   

Note the side, for which the stomatal number is determined. Direct counting of stomata can also be done by a squared eyepiece micrometer, if the drawings are not required.

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CONCLUSION

Stomatal number and stomatal index of supplied leaf is ______________________

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REFERENCES

1. Kokate CK, Practical Pharmacognosy, 4^th edition, Vallabh Prakashan. Delhi; 1994: 117-118.
   
2. Joshi S, Aeri V. Practical Pharmacognosy, 1^st edition, Frank Bros. & Co. New Delhi; 2009: 208-09.