Comparative Study of the Leaf Morphology and Anatomy of

International Journal of Basic & Applied Sciences IJBAS-IJENS Vol:19 No:03 15

170501-1903-8484- IJBAS-IJENS @ June 2019 IJENS I J E N S

Comparative Study of the Leaf Morphology and

Anatomy of Selected Strychnos Species: Strychnos

Spinosa Lam., Strychnos Innocua Del. and

Strychnos Usambarensis Gilg Found in Three

Ecological Zones in Nigeria

ASUZU, CHINWE U., NWOSU, MARIA O. Abstract-- The morphological and anatomical study of three

Nigerian species of Strychnos: S. spinosa, S. innocua and S.

usambarensis was carried out. The leaves are dorsiventrally

flattened, petiolate, with distinct palmate venation. The

transverse section of leaf lamina revealed uniseriate epidermis

with double-layered palisade mesophyll and loosely arranged

spongy mesophyll. Rosette crystals were found in the

mesophyll of S. spinosa and S. usambarensis. Stomata are

hypostomatic and the measurement of the length of guard cells

of S. innocua differed from those of S. spinosa and S.

usambarensis. Petiole anatomy revealed seven variously sized

and separate vascular bundles in S. innocua and S. spinosa but

with a single arc shaped bundle in S. usambarensis.

Index Term-- Strychnos spinosa, S. innocua S. usambarensis,

palmate venation, rosette crystals.

INTRODUCTION

Strychnos belongs to the family Loganiaceae and is

made up of 200-400 species (Cruz, 2008; Rajesh et al.

2009). Strychnos is the largest genus of the family

Loganiaceae (Fraiser, 2011). Mwamba, 2006; Rajesh et

al.2009 and Orwa et al.2009 all reported that, it was first

described by Linnaeus based on S. nux-vomica which is the

type species.

The genus Strychnos is well known for its alkaloid production, particularly strychnine from S. nux-vomica

which has been popularized for its potential nefarious uses.

Angenot (1988) observed that although investigation into

the genus Strychnos has been going on for some time, the

African members suffered a long period of neglect.

However, work on the uses of S. spinosa and S. innocua has

been reported by Asesa et al.(2005); Mwamba (2006)

Rajesh et al.(2009); Augustino et al.(2011); Kokwaro

(1976); F. A. O.(1983.); Mbuya et al (1994) and Orwa et al.

(2009). Angenot (1988) and Cruz (2008) described the uses

of S. usambarensis. Phytochemical studies on S. spinosa

has been carried out by Bisset (1970); Adebiyi and Sofowora(1978); Philippe et al. (2005); Morah (1982,

2011). Studies on the phytochemicals in S. innocua has

been investigated by Corsaro et al. (1995); Philippe et al.

(2005) and Bello et al.(2008). There is however still paucity

of literature on the detailed description of the anatomy of

the leaf lamina, midrib and stomatal types in the three

Nigerian species of Strychnos. This study aims at examining

the morphological and anatomical variables of the leaf

lamina, midrib and stomatal types of the three Nigerian species of Strychnos.

MATERIALS AND METHODS

The species of Strychnos were collected randomly

from three ecological zones in Nigeria where they grow. S.

innocua was collected from Rigasa village near Kaduna

(Sudan savanna). S. spinosa was collected from Kuje village

near Abuja (Guinea savanna), while S. usambarensis was

collected from Ohebe-Dim village near Nsukka (Derived

savanna). Fresh leaves were collected from ten samples

each. For the stomatal studies, freshly collected leaves of the three species were washed with distilled water to remove

any dust or dirt clinging on the leaves. The leaves were put

in three separate and well-labeled petri dishes. Fifteen to

twenty ml of bleach (hydrogen peroxide) solution was

poured into the petri dishes to completely submerge the

leaves. The solution was left for 24-48 hours in order to

bleach the leaves. The bleached leaves were removed

carefully and rinsed thrice with distilled water to remove

any trace of the bleach. The abaxial and adaxial surfaces of

the leaves from each petri dish were scraped off. Viewing of

the adaxial and abaxial surfaces of the scraped leaves was done using the Zeiss light microscope. Another set of

leaves of freshly collected samples were cut into small

pieces and stored in well labeled bottles containing F.A.A.

(20 ml of 40% formaldehyde, 30 ml of distilled water, 30 ml

of 100% ethanol and 20 ml of glacial acetic acid) in the

Anatomy Laboratory of the Department of Plant Science

and Biotechnology , University of Nigeria Nsukka. This

mixture helped to preserve the specimens and prevent any

form of microbial growth. Transverse sections of the midrib

and petiole were made using a Reichert sliding sledge

microtome. The sections were cut at 10u thickness and

stored in well-labeled petri dishes containing 70% ethyl alcohol.



RESULT

Leaf Morphology

The morphology of leaves of S. spinosa, S. innocua and S.

usamberensis is presented in Table 1.

Anatomical Evaluation

There were no visible stomata on the adaxial surface of the three Strychnos species. Numerous stomata were found on

the abaxial surface and range in number from 25 to 40 per

field of view. Comparison of the dimensions of the guard

cells is shown in the Table 1.

Transverse Section of the leaf Epidermis: The abaxial and adaxial epidermis in the three

species all have uniseriate layers. The adaxial epidermis is

rectangular in shape and has thin walls. The outer surface is

convex and is covered with a thin cuticle.

Mesophyll layer: The mesophyll in the three species is

differentiated into palisade and spongy. The palisade

mesophyll is double layered, rectangular shaped and longer than wide in the three species. There are seven to eight

layers of cells that are large and loosely arranged in the

spongy mesophyll in the three species. Air spaces, rosette

crystals and bundle sheaths are also present. The abaxial

epidermis is not even because of the presence of stomata of

anisocytic type. Plate 3a-c.

Midrib: The midrib is plano convex in the three species

with semicircular abaxial surface. Plates4a-c.

Adaxial surface: There is uniseriate layer of epidermal

cells in the three species. The cells are rectangular with

convex shape at the outer surface. There is a thin layer of cuticle over the adaxial epidermis.

Ground tissue: The ground tissue is parenchymatous, thin

walled and the number of layers of cells in each of the

species is in Table 2.

The palisade mesophyll does not extend across the adaxial part of midrib and the vascular bundle is embedded in the

ground tissue.

Vascular tissue: The vascular strand is single, semicircular

and bicollateral in S. spinosa, S. innocua and S.

usambarensis. The xylem elements consist of radially

oriented lines of xylem vessels and narrow bands of adaxial

and abaxial phloem. The xylem elements in S. spinosa and S. innocua are predominantly polygonal in shape but a few

are circular. (See Table 3).

Sclerenchyma sheath surrounds the vascular bundle

in S. usambarensis and S. innocua. In the former, the

sclerenchyma layer is one to two celled thick at the adaxial

surface. In the latter, the sclerenchyma layer is three to six

celled thick right round.

Abaxial epidermis: The abaxial epidermis is semicircular

and single layered in the three species.

Petiole:

Adaxial epidermis: The adaxial epidermis in the three species is uniseriate. S. spinosa has two projections that are

bluntly conical with compact thin walled parenchyma cells.

S. innocua lacks these lateral projections at the adaxial

surface. The adaxial surface of S. spinosa and S. innocua are

slightly undulating, while that of S. usambarensis is

concave. Plates4 a-c

Ground tissue: The ground tissue is homogenous,

parenchymatous, thin walled, circular in shape and compact. The number of layers of parenchyma cells in the ground

tissue of petiole is shown in Table 4.

Vascular tissue: S. spinosa and S. innocua each has seven

distinct and separate vascular bundles of varying sizes, with

the middle one as the biggest S. usambarensis has one big

vascular bundle embedded in the ground tissue (Plates5a-c).

In the three species, the xylem cylinder has closely arranged

radial files of vessel elements with phloem on the adaxial and abaxial surfaces. In S. spinosa and S. usambarensis, the

xylem elements are predominantly spherical to circular in

shape, while in S. innocua, the xylem are angular. The

vessel elements range from eleven in the middle to six at the

right and left hand lateral corners in S. spinosa while in S.

usambarensis, the rows of xylem elements are about

twenty-eight in the single vascular bundle. In addition, there

is a pronounced region of protoxylem at the abaxial layer,

which is not visible in S. spinosa and S. innocua. In S.

innocua there are about eleven rows of xylem elements in

the median bundle and one at the extreme left and right hand corners. In each of the seven bundles in the petiole of S.

spinosa and S. innocua, there are sieve cells and phloem

cells at the adaxial and abaxial surfaces. In S. usambarensis,

the phloem tissue occurs at both adaxial and abaxial

surfaces. The abaxial epidermis is semicircular in shape and

uniseriate in the three species.

DISCUSSION

Metcalfe and Chalk (1979) and Stace (1980)

recognized the use of leaf architectural design as an

important tool in field taxonomy. Thus the differences in

leaf margin and apex in the three species is of taxonomic interest in delimiting them.

Significant difference was recorded in the dimensions of

the length of guard cells. S. innocua growing in Sudan

savanna with drier climatic conditions possessed guard cells

with the longest length. S. usambarensis growing in Derived

savanna had the least length of guard cells. This is probably

a feature to enable each adapt to it’s particular vegetation

zone and a response to the climatic conditions in that zone.

James and Bell (1995) working on six clones of

Eucaliptus camaldalensis from five Australian locations

found that there was a lack of correlation between leaf characteristics and climatic data. They suggested that

availability of ground water, root structure and internal

transport of water may have greater influence on leaf

structure than atmospheric demand. In the same vein Zu and

Zhou (2008) opined that different effects of abiotic factors

on stomatal size might depend on plant species varieties.

Martinez et al. (2007) however reported that under water



deficit, a decrease in cell size occurred indicating that an

adaptation to drought is probable. The shapes of epidermal

and subsidiary cells at the adaxial and abaxial surfaces of

the leaves of the three species are markedly different such

that if there is need to identify the species when they are not

flowering, they could be successfully separated without any mix up.

Oladele (2002) pointed out the importance of plant

stomata in humidification of the atmosphere. He opined that

the efficiency of the stomatal complex in carrying out this

feature depends on factors like stomatal size, stomatal

density and the number of subsidiary cells per stoma.

Olafinobin and Oladele (1970) and Obiremi and Oladele

(2001) stated that the higher the number of subsidiary cells

per stoma, the greater the capacity of such stomatal complex

in humidifying the atmosphere. According to Oladele

(2002), the presence of high number of subsidiary cells per

stoma enhances the presence of stomatal opening and consequently making humidification of the atmosphere to be

more rapid. The three species of Strychnos possessed a

minimum of four subsidiary cells around the stoma. From

the fore mentioned, it follows that there will be enhanced

stomatal opening and rapid humidification of the

atmosphere by these species of Strychnos.

Bicollateral vascular bundles were clearly shown in

the midrib and petiole of leaf and stem of the three species.

This feature is taxonomically important at the generic level

since bicollateral vascular bundles were regarded as

anomalous structure because of their restricted occurrence. It was reported by Metcalfe and Chalk (1979) to occur in the

family Loganiaceae and other related families like

Asclepiadaceae, Rubiaceae and Apocynaceae. S.

usambarensis has a uniseriate layer of sclerenchyma at the

adaxial surface of the midrib but two to three at the abaxial

surface, while S. innocua has three to six layers of

sclerenchyma sheath right round the vascular bundle. Evert

(2006) stated that the presence of sclerenchyma around

vascular bundles performs the function of strengthening the

leaf. The variation in thickness of the sclerenchyma is of

taxonomic interest in delimiting the species.

Rosette crystals that are of taxonomic importance at the genus level as cited by Metcalfe and Chalk (1979)

were observed in the petiole, leaf, mesophyll and midrib of

S. spinosa and S. usambarensis. Franceschi and Niakata

(2005) reported that the presence of small crystals in higher

plants is related to physical protection, storage of calcium

and regulation of light during photosynthesis for plants

growing in the shade. In the species studied, the presence of

rosette crystals confirms that they possess the characteristic

feature of the family Loganiaceae as stated by Metcalfe and

Chalk (1979). Metcalfe and Chalk (1950) considered the

petiole to be of taxanomic importance, as it is not influenced much by environmental changes. The organization of the

vascular system in the petiole consisted of seven separate

and biconvex bundles in S. spinosa and S. innocua but one

big bundle in S. usambarensis. The petiole outline in S.

spinosa and S. innocua are plano convex with S. spinosa

having lateral projections at the adaxial surface. S.

usambarensis has petiole that the adaxial surface is concave.

Thus the petiole outline can furnish information for the

delimitation of the three species. S. nux-vomica is reported

by Metcalfe and Chalk (1979) to have three separate vascular bundles. It is thus suggested that the organization

of the vascular system in form of separate and variously

sized bundles is not diagnostic for the genus.

In conclusion, this study revealed that the three

species studied are hypostomatic with presence of stomata

only at the abaxial surface. The varying thickness of

sclerenchyma around the vascular bundle of the midrib

suggests more strengthening. The presence of rosette

crystals in the leaf mesophyll, midrib and petiole suggests

additional physical protection and storage of calcium.

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Plate 1a: S. spinosa adaxial no stomata present x400

Plate 2a: S spinosa abaxial x400

Plate 1b: Adaxial leaf surface of S. innocua, no

stomata present x400

Plate 2b: Abaxial leaf surface of S. innocuax400



Plate 3a: Leaf lamina S. spiniosa x 200

1 – Adaxial epidermis, 2 – Palisade mesophyll, 3 –

Spongy mesophyll

1

2

3

Plate 3b: Leaf lamina S. innocua x 200

1 – Adaxial epidermis, 2 – Palisade mesophyll, 3

– Spongy mesophyll



Plate. Leaf Usamba 3 x200

Plate 3c: T.S leaf of S. usambarensisx200 1 – Adaxial epidermis, 2 – Palisade mesophyll, 3 – Spongy mesophyll, 4 – crystal

4 – Crystal,

1

2

3

4

1

2

3

5

4

Plate 4a: Midrib of leafS.spinosax40

1 – adaxial epidermis, 2 – internal phloem,

3 – xylem elements, 4 – external phloem, 5 – ground tissue



Plate 5a: Petiole of S. spinosa x40 Plate 5d: Bicollateral vascular bundle of

petiole of S. spinosa x200

Plate 5e: Bicollateral vascular bundle of

petiole of S. innocua x200

Plate 5b: Petiole of S. innocua x40

1

2

3

4

5

Plate 4b: Midrib of leaf S. innocua x40

1 – adaxial epidermis, 2 – internal phloem, 3 – xylem elements, 4 – external phloem,

5 – ground tissue



Table I

Stomatal dimensions of S. spinosa, S. innocua and S. usambarensis

Name of plant Length of

guard cell

(mm)

Width

guard cell

(mm)

No of

stomatal/field of

view

No of subsidiary

cell/field of view

Cell wall thickness

of epidermal cells

(mm)

Stomatal

index

S. spinosa 0.176 0.027 10.9 66.2 0.016 14.13

S. innocua 0.187 0.029 12.2 92 0.037 11.70

S. usambarensis 0.158 0.040 11.6 97.8 0.014 10.60

Table II

Number of layers of ground tissue in midrib of the three species of Strychnos

S. spinosa S. innocua S. usambarensis

Layers of cells at adaxial surface 11.7±0.82 6.5±0.52 4.3±0.48

Layers of cells at abaxial surface 7.5±0.51 8.5±0.53 8.5±0.53

Table III

Number of xylem elements in the vascular bundle of midrib

S. spinosa S. innocua S. usambarensis

Number of xylem elements at median

part

4.34±0.61 7.5±0.50 2.3±0.64

Number of xylem elements at left and

right hand corners

1.5±0.52 2.3±0.45 4.5±0.50

Table IV

Number of layers of parenchyma cells in the ground tissue of petiole

Name of plant Median Adaxial Extreme Left &

Right adaxial

Median Abaxial Extreme Left and

Right Abaxial

S. spinosa 21.5 ±1.56 9.50±2.00 18.5±3.55 8.5±1.33

S. innocua 13.9±2.34 9.87±0.98 11.45±0.87 6.50±0.55

S. usambarensis 13.55±0.65 16.50±1.87 10.55±1.76 9.50±0.50

Plate 5c: Petiole of S. usabarensis x40 Plate 5f: Bicollateral vascular bundle of

petiole of S. usabarensis x200

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Comparative Study of the Leaf Morphology and Anatomy of