Abstract and Figures

In plants, liquid transport system does not participate in transportation of gases but the gaseous exchange is an essentially required for central processes like photosynthesis and respiration. Exchange of water, carbon dioxide, oxygen, other gases and compounds in plant takes place through stomatal opening called stomatal aperture or central slit. Stomata are mainly crescent or dumb bell in shape. They are made up of guard cells and subsidiary cells. Stomatal movement (opening and closing) is predominantly controlled by water availability and CO2 concentration and also by various endogenous factors in close association with environmental factors. Stomata are most commonly present on the surfaces of leaves, but they can also be present on inflorescence, fruits, herbaceous stems, petioles, tendrils and other parts of plant. Guard cell size and stomatal density (number of stomata/unit area) are determined by prevailing environmental conditions (water availability, light intensity, temperature and CO2 concentration) during the period of leaf growth. Consequently, stomatal density and size of stomatal aperture may strongly affect the rate of basic physiological processes such as photosynthesis, transpiration besides determining the rate and type of respiration as well. Variations in stomatal density and size of stomatal aperture can thereby cause great differences in light and water use efficiency. As a basis for causal analysis of data from stress physiology research, e.g. heat stress, water stress, elevated CO2 or ozone levels, studies on functional leaf anatomy are becoming more important for evaluating the performance of plants in a certain environmental conditions for tracing down its relation with eco-physiological adaptations and biosystematics as well.
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Manual of ICAR Sponsored Training Programme on “Physiological Techniques to Analyze the Impact of
Climate Change on Crop Plants” 16-25 January, 2017, Division of Plant Physiology, IARI, New Delhi
27
Measurements of Stomatal Density and Stomatal Index on Leaf/Plant Surfaces
Vijay Paul1*, Laxmi Sharma2, Rakesh Pandey1 and R.C. Meena1
1Division of Plant Physiology, ICAR- Indian Agricultural Research Institute (IARI), New Delhi-110 012
2ICAR-Central Research Institute for Jute & Allied Fibres (CRIJ&AF), Barrackpore, Kolkata-700120, West Bengal
*E-mail: vijay_paul_iari@yahoo.com
Introduction
In plants, liquid transport system does not participate
in transportation of gases but the gaseous exchange
is an essentially required for central processes like
photosynthesis and respiration. Exchange of water,
carbon dioxide, oxygen, other gases and compounds
in plant takes place through stomatal opening called
stomatal aperture or central slit. Stomata are found
in all the plants above the evolutionary level of the
saprophyte generation of mosses and the sporophyte
generation of ferns. Stomata in mosses are usually
non-functional and stomata of pteridophyta show
ra t h er sl u g g i sh mo v e ments. Stomata of
gymnosperms and angiosperms, on the other hand,
are highly versatile. Stomata are mainly crescent or
dumb bell in shape. They are made up of guard cells
and subsidiary cells. Stomatal movement (opening
and closing) is predominantly controlled by water
availability and CO2 concentration and also by various
endogenous factors in close association with
environmental factors. Stomata are most commonly
present on the surfaces of leaves, but they can also
be present on inflorescences, fruits, herbaceous
stems, petioles, tendrils and other parts of plant.
Guard cell size and stomatal density (number of
stomata/unit area) are determined by prevailing
environmental conditions (water availability, light
intensity, temperature and CO2 concentration) during
the period of leaf growth. Consequently, stomatal
density and size of stomatal aperture may strongly
affect the rate of basic physiological processes such
as photosynthesis, transpiration besides determining
the rate and type of respiration as well. Variations in
stomatal density and size of stomatal aperture can
thereby cause great differences in light and water
use efficiency. Stomatal density of selected species
is given in Table 1. As a basis for causal analysis of
data from stress physiology research, e.g. heat stress,
water stress, elevated CO2 or ozone levels, studies
on functional leaf anatomy are becoming more
important for evaluating the performance of plants
in a certain environmental conditions for tracing
do wn its rel ation wit h eco-ph y siolog y, eco-
physiological adaptations and biosystematics as well.
Principle
Surface morphology/anatomical features can be
studied by using light microscope either by observing
the epidermal peel directly or initially making the
replica of leaf surface and then observing the same
under the microsco pe. Peel processing based
methods for investigating surface morphology
including stomata and lenticels are described by Paul
and Srivastava (2004), Paul et al. (2007) and Paul
et al. (2012). Replica based method was developed
by Meidner and Mensfield (1968) and Wolf et al.
(1979) and this is being used for measurements of
stomatal density and stomatal index on leaf/plant
surfaces (Mallick et al., 2016) either as such or with
minor modifications.
Materials Required
Leaves, replica fluid, brush, forceps, glass slides,
cover slips, needles, microscope, ocular meter, stage
micrometer etc.
Observations
Stomatal density, stomatal index and length & breadth
of size of stomatal aperture/cells or part of cells etc.
Methodology
Calibration of ocular micrometer
oStage micrometer (SM) is a calibrated scale
having 100 division of 1 mm and therefore 1
division if equal to 0.01 mm or 10 µm or micro-
Manual of ICAR Sponsored Training Programme on “Physiological Techniques to Analyze the Impact of
Climate Change on Crop Plants” 16-25 January, 2017, Division of Plant Physiology, IARI, New Delhi
28
me t er Place the SM on the stage of the
microscope and focus it at a given magnification
e.g. 40x.
oPlace the ocular micrometer (OM) in one of the
eye pieces of microscope. While observing
through the eye pieces, align the scale of OM
with that of SM.
oCount the divisions of OM coinciding with
divisions of SM.
For example: 4.0 divisions of OM = 45 divisions
of SM i.e., 450 µm
So, 1 division of OM = 11.25 µm
oBy following the above three steps, calibrate the
ocular scale for other magnifications as well i.e.
10x, 20x, 60x, 100x etc.
oOnce the ocular scale is calibrated for different/
required magnification(s), SM can be removed
and slide having sample material may be placed
for observations and measurements.
Preparation of replica
Commercially available quick fix (adhesive) has been
found to be the best option for replica preparation.
Celludin (8%), or cellulose acetate in acetone can
Table 1: Stomatal density of some selected species
Species Stomatal density (Number of stomata/mm2 of leaf surface)
Upper surface Lower surface
I. Grasses
Oryza sativa 931
Zea mays 52-94 68-158
Saacharum officinarum 59-167 176-351
Avena sativa 25-48 27-35
II. Shrubs and trees
Citrus sinensis (sub-tropic) 0-90 620
Fagus sylvatica (temprate):
Sun exposed leaves 130-295
Shade exposed leaves 94-177
Myrtus communis (sub-tropic) 0 158
Populus alba (temperate) 0 315
Quercus rubra (temperate) 0 680
Quercus triloba (sub-tropic) 0 1192
Sambucus nigra (temperate):
Sun exposed leaves 42-260
Shade exposed leaves 12-147
III. Herbaccous plants
Helianthus annuus 27-326 90-408
Phaseolus vulgaris 8-16 132-184
Solanum tuberosum 10-20 230
IV. Succulent plants
Sedum acre 21 14
V. Floating plants
Nymphea alba 400 0
Source: Bolhar-Nordenkampf and Draxler (1993); Woodward and Kelly (1995)
Manual of ICAR Sponsored Training Programme on “Physiological Techniques to Analyze the Impact of
Climate Change on Crop Plants” 16-25 January, 2017, Division of Plant Physiology, IARI, New Delhi
29
also be used for taking imprints. More recently,
imprints on silica rubber or poly-methyl-metacrylate
(PMMA) combined with replicas by cyanacrylate
adhensive are also being used.
Determination of stomatal density
Collect the suitable leaf material/plant part, wash
them gently with running water to remove the dust
and debris and allow them to dry. If peeling of
epidermal layer is possible then it can be used directly
otherwise, apply the suitable replica fluid such as
quick fix in a thin and uniform film (by spreading a
drop or two of quick fix on the leaf surface) and
allow it to dry completely. Now, gently peel off the
replica with the help of forceps or fingers and place
it on the slide in a manner that imprinted surface
should be on upper side. Put one or two drops of
water/glycerol for proper spreading of replica and
cover it with cover slip. Similar preparations can be
made for different leaves for their upper and lower
surfaces. Now observations for number of stomata
present in microscopic view field can be recorded
for calculating the stomatal density that can be
expressed in terms of stomata/mm2. More details
are as follows:
At a given magnification count the total number of
stomata as visible in circular view field of microscope.
Calculate the diameter of view field by ocular scale.
Now the area of circle under microscopic view field
can be calculated by following formula: r2 where, r
is the radius of the circle (view field) i.e., ½ of the
diameter of circle. Now calculate the number of
stomata for mm2 of area for upper and lower surface
of leaves.
Determination of stomatal index:
Slides as prepared for stomatal density are also used
for determination of stomatal index by using the
formula, stomatal index (%) = (S/S+E) x 100 where,
S and E are the number of stomata and epidermal
cells respectively in microscopic view field. Like
st omat al den sity, stoma tal index (%) can be
calculated for both the surfaces of leaves. Data of
stomatal index for some crops are presented in
Table 2.
Length and breadth of cells or part of cells
Morphometric measurements for different cells
(epidermal, stomatal, subsidiary) can be taken under
suitable magnification by using calibrated ocular
micrometer.
Im p o r tant in s t ructions f o r the us e of
microscope
Never keep the lamp on when the microscope
is not in use or during the gap of observations.
Also, never keep the lamp on continuously for
long time.
While there is need to use high intensity light,
increase it gradually from low to high.
Never make rapid up movement of stage as the
slide kept on the stage can damage the lens of
the objective.
Limitations and improvements
Peeling method as well as replica with commonly
used replica fluids may not be suitable for leaves
with lots of hairs, appendages and thick waxy
depositions.
Leaf material can be preserved in suitable
fixative for recording the observations later.
Replica can also be prepared for the plants in
the field.
Waxy layer can be removed by treating waxy
leaves with acetone or chloroform and then
replica method could be applied.
With new imprinting material like cellulose-di-
acetate and polymethyl-metacrylate permanent
Table 2: Stomatal index for the leaves of few crops
Crop Number of Number of Stomatal
stomata epidermal index
ce lls ( % )
Cotton 6 32 15.8
Groundnut 9 36 20.0
Mung bean 5 12 29.4
Tomato 7 25 21.9
Sorghum 8 20 28.6
Manual of ICAR Sponsored Training Programme on “Physiological Techniques to Analyze the Impact of
Climate Change on Crop Plants” 16-25 January, 2017, Division of Plant Physiology, IARI, New Delhi
30
impression of epidermal surface can be created
and perfectly preserved for long-term storage.
In case of extremely sunken stomata, pleated
le a f s u rfaces, or conif e r ous n eedles,
cyanacrylate adhesive is found to give reliable
and reproducible results.
Drawing microscope and or imaging system
attached with microscope can be used for
effective and accurate data generation.
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... A cover slip was placed over the film after adding 1-2 drops of glycerol/water. The total number of stomata visible in circular-view field of microscope was counted at 40 × 10× magnification (Paul, Sharma, Pandey, & Meena, 2017). The diameter of the microscopic view-field was determined using ocular scale and area of circle was calculated using the following formula; ...
... The stomatal index-percentage proportional to the ultimate divisions of the leaf epidermis that has been converted to stomata-in the microscopic view-field (40 × 10×) was calculated using the following formula (World Health Organization, 2011;Paul et al., 2017): ...
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