comparative (quantitative and qualitative) studies of ... · stomata, types of stomata present in...

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Int. J. LifeSc. Bt & Pharm. Res. 2012 Amal Kumar Mondal and Sayantan Tripathi, 2012

COMPARATIVE (QUANTITATIVE ANDQUALITATIVE) STUDIES OF STOMATA

OF SELECTED SIX MEDICINALLYVIABLE SPECIES OF CASSIA L.

Sayantan Tripathi1 and Amal Kumar Mondal1*

Research Paper

The Stomatal diversity (size, shapes, types and orientation) in the foliar epidermis has great

value in plant systematics studies. The present paper deals with comparative study of stomatal

structure of six species of Cassia which are used by rural people of South West Bengal as

medicine. Leaf epidermal studies mainly stomatal studies of the 6 species of Cassia of family

Caesalpiniaceae viz., Cassia alata L., Cassia fistula L., Cassia occidentals L., Cassia siamea

Lamk., Cassia sophera L., Cassia tora L. were made. Leaf clearings and cuticular preparations

were examined with light microscopy. The study is based on the presence and absence of

stomata, types of stomata present in the epidermal surface, stomatal count/cm2, stomatal index

and epidermal cell shape. The size and shapes of stomata are also varied in the tree species

which bear larger size of stomata in respect of other habits and habitats. Three types of stomata

were observed viz. paracytic, anisocytic and anomocytic. Among these 3 types of stomata the

paracytic type of stomata are more common and than other.

Keywords: Epidermis, Medicinal use, Cassia, Paracytic stomata

*Corresponding Author: Amal Kumar Mondal,[email protected]

INTRODUCTIONVarious plants and its parts are used in medicinal

purpose from ancient age. Now a days it is

observed that the African gorillas also use various

plants and its parts to cure their health. The

medicinal plants have been used by Hakims and

in folklore medicines as 80% of the population

ISSN 2250-3137 www.ijlbpr.comVol. 1, No. 3, July 2012

© 2012 IJLBPR. All Rights Reserved

Int. J. LifeSc. Bt & Pharm. Res. 2012

1 Plant Taxonomy, Biosystematics and Molecular Taxonomy Laboratory, Department of Botany & Forestry, Vidyasagar University.

lives in rural areas that mostly depend on Unani

system of medicines (Soomro et al., 1997). The

available literature shows that leaf epidermal

features are important in systematic botany

similar to the use of modern techniques and

chemical composition (Edeoga and Ikem, 2001;

and Mbagwu and Edeoga, 2006). Epidermal

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structures and stomatal ontogeny of some

Nigerian ferns have been found relevant in their

recognition (Gill and Karatela, 1985).

Many workers such as Edeoga (1991),

Edeoga and Osawe (1996), Mbagwu and Edeoga

(2006), Nwachukwu and Mbagwu (2006)

stressed that epidermal and cuticular traits of

plants epidermal cells, type and arrangement of

stomata, size and shape of trichomes and

number of vascular bundles could serve as vital

tools in solving taxonomic problems in

Angiosperms.

Stomatogenesis has long been studied by

morphologists, physiologists and taxonomist .The

morphology and ontogenies of taxa are important

in intrageneric systematics. Diversity in stomata

types, even on the same surface of an organ,

indicates the weakness in using stomata as a

taxonomic character. (Pant and Kidwai, 1964). In

spite of diversity, the most frequent stomata type

can be used as a taxonomic character (Gopal,

1970).

Caesalpiniaceae is a family of dicotyledonous

plant with about 160 genera and some 2,000

species. The genus Cassia is one of the genuses

of Caesalpiniaceae having about 692 species.

Some of the Cassia species is used as medicine

in Eastern India mainly South West Bengal. We

select Cassia alata L., Cassia fistula L., Cassia

occidentals L., Cassia siamea Lamk., Cassia

sophera L., Cassia tora L. which are frequently

used by the rural and tribal people of south West

Bengal.

Apart from physiognomic characters,

anatomical properties of plant parts are sources

for taxonomic inferences in different groups of

flowering plants (Edeoga et al., 2007; Guimeraes

et al., 2007; Kaplan et al., 2007; Keshavarzi and

Zare, 2006). Despite the immense economic

importance of the legumes and the physiological

importance of the stomatal apertures, reports on

the frequency and the structure of the stomata

are lacking or incomplete for many species.

On the basis of arrangement of epidermal all

neighbouring the guard all, more than 25 main

types of stomata in dicots have been recognized

(Metcalfe and Chalk, 1979). Stace (1980) reported

31 different types of stomata among cotyledonous

plants. But the present study is based on the

paper of Metcalfe and Chalk (1950) who described

the 4 types – i.e. Anisocytic, Anomocytic, Diacytic

and Paracytic.

The aim of present study is to use stomatal

characters as aid in taxonomy of these selected

medicinally useful Cassia species. The study

would help in the identification and authentication

of these medicinal plants on the basis of

stomatogenesis.

MATERIAL AND METHODSPlant Material: The medicinal plant specimens

Cassia alata L., Cassia fistula L., Cassia

occidentals L., Cassia siamea Lamk., Cassia

sophera L. and Cassia tora L. belonging the family

Caesalpiniaccae under order Leguminales were

taken for stomatal study.

Cassia alata L. is popular in traditional

treatment of asthma, leprosy and it is used for

controlling of ringworm (Paria, 2005).

Role of Cassia fistula L. is used as purgative

and also used to treat skin disease. Its leaves

are used to treat skin disease and ulcer. Powder

of Cassia fistula L. root is given to cure menstrual

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disorder and its pulp of seed is used in high blood

pressure (Paria, 2005).

Cassia ossidentales L. is popularly used as

purgative. It is also used to treat hysteria,

whooping cough and urinary troubles (Paria,

2005).

Cassia sophera L. is an annual erect shrub of

important medicinal properties. The whole plant

extracts and leaves have expectorant properties,

cures cough, asthma and acute bronchitis. They

are specific to eliminate ring worms and also

useful in the treatment of gonorrhoea and syphilis.

The bark is used in the treatment of diabetes; the

roots in elephantiasis, wounds. (Kirtikar and

Basu,1935).

The leaves of Cassia siamea Lamk. are used

in the treatment of diabetes, disturbances in the

bodily functions, lymph node swelling, urine

stones, general deficiency conditions, beri beri ,

classic deficiency of avitaminose by lack of

vitamin B1 (thiamine) in gastrointestinal disorders

- malabsorption - meals taken with polished rice

etc., antihypertensive, insomnia (sleeplessness),

against dysentery and disorders of the large

intestine. (Kirtikar and Basu, 1935).

Cassia tora L. is popularly known for the

treatment of dysentery and eye disease. Leaves

are also used in boils, ring worm, leprosy, bronchitis

and liver complaints. Pest of fresh pods is applied

to cure lentigo. Seed powder is taken to cure

intestinal problems caused by intestinal worms.

Seed pest mixed with boiled rice water is applied

in case of one sided headache (Paria, 2005).

The species identification of the selected

material was determined according to standard

literature. It was done in the months from February

to April. The foliar epidermal peals were taken

from the middle of both surfaces of mature leaves.

Isolation of Epidermis: Epidermis of leaf is

isolated from both fresh and dry plant specimen.

The mature leaves were fixed in FAA solution

(acetic acid: alcohol: formalin: water = 2:5:1:12)

for 24 hours and washed in 70% ethanol. Three

circular disk samples were cut from an area

adjacent to the midrib of each leaf. Disk sample

was boiled in 5% aqueous solution of KOH for

5-10 minute. Epidermal peals were stripped and

stained with 1% in 50% aqueous ethanol,

saffranin and temporary mount in glycerine.

Stomatal frequency counts made and camera

lucida were drawn. The numbers of stomata were

counted in each field (0.001386 cm2). The

stomatal frequency was based on average

obtained from observations of 3 microscopic

fields. Stomatal index (I) was calculated by the

following formula using the no. of stomata(s)

and epidermal cells was present in a unit area

[I=S/(S+E)].

After preparing, slides were observed under

light microscope [(40x)Leica DM 1000] and phase

contrast microscope for detailed analysis and

obtaining better picture as well as measuring the

length and breadth of stomata including guard

cells.

RESULTS AND DISCUSSIONThe results in this investigation were summarised

in tables including stomata count, stomata type,

stomatal index, stomatal complex.

The entire selected species share a number

of common characters, i.e., shape of epidermal

cell, presence of stomata, stomatal index and

stomatal complex which are useful in

distinguishing these species.

Epidermal Cell: The shape of foliar epidermis is

one of the significant taxonomic characters.

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Taxonomic studies of a number of families are

based on leaf epidermis anatomy (Bhatia, 1984,

Jones, 1986). In this investigation it was found that

the epidermal cell shape varies slightly with growth

habit. The shrub species posses pentagonal to

polygonal shaped epidermal cells. The epidermal

cell shapes of tree species are variously shaped

or undulating to irregular (Table 1).

In these six species only Cassia siamea Lamk.

(Figure 2E, F) bears hypostomatic leaves i.e.

stomata present only at the abaxial surface of

leaves. Here it was found that the shape of

epidermal cells varies in their surfaces. The

adaxial surface of the leaf shows tetragonal to

polygonal shaped epidermal cells but the abaxial

surface i.e. stomata bearing surface shows

undulating to irregular shaped epidermal cells.

Stomata: Three different types of stomata

anomocytic, anisocytic, paracytic were observed

in these selected species. It was found that

abundant stomata were present on abaxial

surface as compared to adaxial surface.

All these selected species bear paracytic

stomata except the adaxial surface of Cassia

alata L. (Figure 2A). The adaxial surface of Cassia

alata L. (Figure 2A) bears anisocytic stomata.

Anisocytic and paracytic stomata were also found

in the abaxial surface of Cassia occidentals L.

(Figure 1B), Cassia sophera L. (Figure 1F) and

Cassia tora L. (Figure 1D). From this investigation

it was found that all the anisocytic stomata

bearing species are shrub in growth habit.

Anomocytic stomata are found only in the abaxial

surface of Cassia siamea Lamk. (Figure 2F). All

the investigated species showed paracytic

stomata, a mixture of stomata in same species

were also observed. Cassia ocidentales L., Cassia

sophera L. and Cassia tora L. are closely related.

They showed paracytic stomata in adaxial surface

and in the abaxial surface they showed paracytic

stomata along with anisocytic type (Table 1).

StomataPresence Stomata Type

S. No. Taxa Habit Surface Leaf Epidermal Cell Shape of Anomo- Aniso- Para- Dia-Stomata cytic cytic cytic cytic

1. Cassia alata L. Shrub Ad. Pentagonal to hexagonal + – + – –

Ab. Pentagonal to hexagonal + – + + –

2. Cassia fistula L. Tree Ad. Variously shaped + – – + –

Ab. Variously shaped + – – + –

3. Cassia occidentals L. Shrub Ad. Pentagonal to polygonal + – – + –

Ab. Pentagonal to polygonal + – + + –

4. Cassia siamea Lamk. Tree Ad. Tetragonal to polygonal – – – – –

Ab. Undulating to irregular + + – + –

5. Cassia sophera L. Shrub Ad Pentagonal to polygonal + – – + –

Ab Undulating to irregular + – + +

6. Cassia tora L. Shrub Ad. Pentagonal to polygonal + – – + –

Ab. Pentagonal to polygonal + – + + –

Table 1: Qualitative Foliar Stomatal Characteristics of the Selected Species of Cassia

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S. No. Taxa Habit Surface Stomatal Complex. Legth × Width (with guard cell) µm2

1. Cassia alata L. Shrub Ad 23.89(21.28-25.77)×20.27(18.43-22.41)

Ab 18.48(17.46-19.31)×12.36(10.47-14.12)

2. Cassia fistula L. Tree Ad 13.74(13.04-14.65)×11.66(11.32-12.59)

Ab 15.22(12.5-17.5)×10.25(7.75-12.5)

3. Cassia occidentals L Shrub Ad 23.11(22.31-23.81)×16.49(15.67-17.11)

Ab 22.98(21.65-25.48)×17.09(14.28-20.99)

4. Cassia siamea Lamk. Tree Ad –

Ab 23.49(21.34-25.40)×15.32(14.49-16.71)

5. Cassia sophera L. Shrub Ad 24.85(23.61-26.47)×15.24(14.47-16.10)

Ab 22.54(21.24-24.55)×15.29(14.54-16.35)

6. Cassia tora L. Shrub Ad 22.10(19.99-23.16)×13.06(11.84-13.92)

Ab 23.28(23.27-23.29)×15.66(15.17-16.38)

Table 2: Quantitative Foliar Stomatal Characteristicsof the Selected Species of Cassia

S. No. Taxa Habit Surface of Leaflet Stomatal Index (SI) Percentage Stomata Count/cm2

1. Cassia alata L. Shrub Ad 8.3 6475

Ab 12.5 7914

2. Cassia fistula L. Tree Ad 13.3 8734

Ab 16.8 11511

3. Cassia occidentals L Shrub Ad 21.7 12950

Ab 28.7 14388

4. Cassia siamea Lamk. Tree Ad – –

Ab 22.6 13670

5. Cassia sophera L. Shrub Ad 18 11511

Ab 23.1 19424

6 Cassia tora L. Shrub Ad 23.3 10072

Ab 24.3 12950

Table 3: Stomatal Index Percentage and Stomatal Count/cm2

of the Selected Species of Cassia

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Figure 1: Leaf Epidermis Showing Stomata ofA. Cassia occidentalis (adaxial surface); B. Cassia occidentalis (abaxial surface);

C. Cassia tora (adaxial surface); D. Cassia tora (abaxial surface);E. Cassia sophera (adaxial surface); and F. Cassia sophera (abaxial surface)

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Figure 2: Leaf Epidermis Showing Stomata ofA. Cassia alata (adaxial surface); B. Cassia alata (abaxial surface);

C. Cassia fistula (adaxial surface); D. Cassia fistula (abaxial surface);E. Cassia siamea (adaxial surface); and F. Cassia siamea (abaxial surface)

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Quantitative analysis of stomatal complex in

the selected species shows great variation (Table

2). Here it was found that except Cassia fistula L.

Cassia alata L., Cassia occidentals L., Cassia

siamea Lamk., Cassia sophera L., Cassia tora

L. share almost equal size of stomata which

shows close relationship. In these 5 species

Cassia sophera L. and Cassia occidentals L.

share almost equal size of stomata and the ratio

between size of stomata of adaxial and abaxial

surface is almost equal. Smallest stomata were

found in the adaxial surface of Cassia fistula L.

(13.74×11.68 µm2).

Stomatal index is one of the useful tools in order

to distinguish species. It was found that stomatal

index has low value on adaxial surface as

compared to abaxial surface (Table 3). According

to stomatal index Casia alata L. and Cassia fistula

L. are closely related. Cassia occidentals L.,

Cassia siamea Lamk., Cassia sophera L. and

Cassia tora L. showed relationship among them.

Highest value of stomatal index was observed

on abaxial surface of Cassia occidentals L. (28.7),

where lowest value was observed on the adaxial

surface of Cassia alata L. (8.3).

Stomatal count is a significant character to

differentiate various species. Highest stomata

count per unit area was found on the abaxial

surface of Cassia sophera L. (194245/cm2).

Lowest stomata count was found in adaxial

surface of Cassia alata L. (6475/cm2). All the

investigated Cassia species share more or less

equal number of stomata which shows they are

closely related.

CONCLUSIONThe study and investigation mainly focused on

comparative study of quantitative and qualitative

stomatal characters of 6 species of Cassia

belonging to family Caesalpniaceae under the

order Leguminales, used in medicinal purpose in

the South West Bengal region.

From the study it can be concluded that there

is no relationship between the stomata size and

growth habit. Size of stomata and epidermal cell

shape prove that all the investigated species are

closely related. All the investigated species

showed paracytic stomata, a mixture of stomata

in same species were also observed. Cassia

ocidentales L., Cassia sophera L. and Cassia tora

L. are closely related. They showed paracytic

stomata in adaxial surface. In the abaxial surface

they showed both anisocytic and paracytic type

of stomata. Shape of epidermal cells, presence

of stomata, stomatal type, stomatal complex,

stomatal index were investigated. These

parameters are helpful to differentiate the species.

However, the stomatal features may prove to be

a little taxonomic value unless the developments

of different stomata types were studied. A greater

number of information on taxa will be helpful to

understand the taxonomic value of stomata type

and distribution.

ACKNOWLEDGMENTWe are grateful to our honourable Vice Chancellor

of Vidyasagar University, Prof. Ranjan

Chakraborti, and we are also thankful to our

friends (Santanu Dash and Dulal Chandra Bera).

Without their help we cannot complete our work.

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