COMPARATIVE ANATOMICAL STUDY OF SOME · PDF fileThe seeds were identified by plant taxonomist Ustaz Hasan AL-Bager (Soba ... (Leitz1512 West Germany), ... (Esau,1960), the palisade

European Journal of Advanced Research in Biological and Life Sciences Vol. 5 No. 1, 2017 ISSN 2056-5984

Progressive Academic Publishing, UK Page 36 www.idpublications.org

COMPARATIVE ANATOMICAL STUDY OF SOME ACACIA TAXA

SEEDS GROWN IN CENTRAL SUDAN

Eiman Mohammed Ali Mustafa, Hatil Hashim Alkamali

& Ahlam Salih Eltahir

1Department of Botany, Faculty of Science and Technology

Omdurman Islamic University, Omdurman, SUDAN

ABSTRACT

This study deals with the anatomical characters of eight species and subspecies of Acacia

(Acacia mellifera, A.nilotica ssp. adansonia, A. nilotica ssp. nilotica, A. orefota, A.

polyacantha ssp. cambylacantha, A. Senegal var. Senegal, A. seyal var. seyal, and A.

siberiana var. , siberiana) which were collected from central Sudan. Wax methods were

adopted to make transverse sections in the seeds, the prepared slides were investigated and

compared. It is found that the seeds of the studied plants are more or less similar in their

general internal structure The epidermis is formed of uniseriate cells covered by cuticle,

followed by lignified cells of different thicknesses , they are 6-7 layers in A. mellifera, A.

niloticassp, adansonia, A. nilotica ssp nilotica, and A. sieberiana var sieberiana, and 3-4

layers in A. orefota, A. polycanthas sp campylacantha, A. Senegal var Senegal and A. seyal

var seyal. Many layers of thick walled lignified parenchyma tissue ranging from 3-25 layers.

The embryos formed of two elongated cotyledons the structure of the embryo is different in

A. mellifera, A. niloticassp, adansonia, A. nilotica ssp nilotica and A. seyal var seyal . The

outermost layer of the embryo is formed of one layer of small compact cells surrounding

parenchyma cells filled with reserved food materials their nature and quantities differ from

one species to another.

Keywords: Anatomy , Seeds, Acacia, Seed coat, Embryo.

INTRODUCTION

The genus Acacia is quite abundant diverse comprising approximately 1356 species, which is

currently divided into three subgenus: subg. Acacia (c.161), subg. Acuiferum (c. 235 species)

and subg. Phyllodineae (c. 960 species).

Most species of Acacia tolerate extreme dryness and adopt well to degraded soils, and they

play an important role in the conservation and improvement of soil fertility by means of

nitrogen fixation (Allen and Allen,1981; Karlin et al; 1997). Their wood is hard, heavy, and

digable. Gum Arabic is obtained from some Acacia species (Lean de Pinto et al., 1998), and

Acacia flowers are important for the perfume industry (Allen and Allen,1981). Some species

are grown as ornamental and /or shade trees, while armed shrubs are suitable for fences.

In Sudan Acacia represented by 34 species (AL-Amin,1990). A numerical taxonomy review

on morphology of northern Sudanese Acacia species has been carried out by Kordofani and

Ingrouille (1991). Several studies emphasize seed morphology as an important tool for the

taxonomic work. In spite of the importance and stability of seed characters in systematic,

very little work seems to have been done on seed anatomy of Acacia, especially no studies

have been conducted so far regarding the seeds of Acacia mellifera, A.nilotica ssp.

adansonia, A. nilotica ssp. nilotica, A. orefota, A. polyacantha ssp. cambylacantha, A.

Senegal var. Senegal, A. seyal var. seyal, and A. siberiana var. , siberiana. (AL-Gohary and



Mohamed, 2007). Nahed et al 2012 outlined the morphology and anatomy of the seeds of

elven species and sub species of Acacia and constructed an artificial key indicating that these

characters of the seed anatomy contribute in the classification of Acacia species. Little

studies on the seed anatomy of Acacia have been reported. Various seed microscopical

studies of leguminous taxa have been performed from time to time (Sharma et al., 1977; Buth

and Narayan, 1986; Sahai, 1999; Murthy and Sanjappa, 2002, Mallick and Sawhney 2003,

Salimpuvret al 2007, ( E-Khamali and Rehab, 2010).

The aim of this study is to identify and differentiate selected Acacia taxa seeds. In order to

achieve this goal:

The most important characters in the micro morphological differentiation of studied Acacia

taxa (viz ; A. mellifera (Val) Benth, A. nilotica ssp. adansonia, A. nilotica ssp. nilotica,

A.oerfota L., A. Polyacantha Willd ssp. campylacantha (Hochst.exA.Rich.) Brenan, A.

senegal (L.)Willd.var. .senegal Brenan, A. seyal Del. and A. sieberana DC. var A.

sieberiana). Micro morphological characters of studied taxa through anatomical studies by

Light Microscope(LM).

MATERIALS AND METHODS

Plant Materials

Acacia seeds of (8 taxa) : Acacia mellifera, A. nilotica ssp. adansonia, A. nilotica ssp.

nilotica, A. oerfota, A. polycanthas sp. Campylacantha, A. Senegal var senegal, A. seyal

var seyal and A. sieberiana var sieberiana were collected from various areas of West Sudan.

The seeds were identified by plant taxonomist Ustaz Hasan AL-Bager (Soba Research

Forests, Ministry of Science and Technology, Khartoum.). The identification of the studied

taxa was also done according to AL-Amin, 1990. Voucher specimens were kept at Herbarium

of Botany Department, Faculty of Science and Technology Omdurman Islamic University.

Methods

The anatomical investigation was achieved through transverse sections of seed by a hand

microtome and stained with safranine and light green stains (Alexander, 1940).

Preliminary treatment of the plant material

Imbibing

The seeds were soaked in running water not more than 24 hour to avoid germination, seeds

coat were partially by using filter paper.

Fixation

The seeds were sliced befor fixation so as to facilitate the passing of chemical and wax later

on.Transferred seeds into specimen bottle filled with a fixative of (F. A. A.) Formaldehyde:

Acetic acid: Alcohol (5:5:90v/v) fixed for 24 hours.

Washing

The fixed seeds were washed with distilled water threetimes interval.



Dehydration

Dehydration by using serial concentrations of ethyl alcohol 70%, 90%, 95% and absolute

alchohol respectively, 24 hour for each step.

Clearing

For clearing the seeds they were transferred every three hours from a mixture of 1:1 cedar

wood oil: absolute alcohol, into pure cedar wood oil followed by mixture of cedar wood oil

and xylene then left over night in pure xylene.trimmed then put the specimen bottle in an

oven adjusted at 60˚C left to half hour, then pure melted wax to half hour, then again pure

wax to half hour.

Blocking

After preparing 9 bath provided with cool water-spatula then took the plant matrrial by the

spatulathen locate it in the wax to give the desired section.after that the plant segments were.

Sectioning

Seeds were sectioned using a Rotatory Microtome (Leitz1512 West Germany), with

Specimen Holder and Disposable Blade Holder. All controls were placed on the front of the

microtome ensuring convenient operation during the setting of section thickness, the

controlling sectioning of the object, and the rapid adjustment of the knife. For precise

alignment of the specimen and the knife, the specimen clamp can be tilted through 10degrees

in any direction by means of two knurled screws.

The microtome adjusted at 7 microns. Using a brush, the ribbons of sections were collected

on glassed slides, which had been wetted with egg albumin to keep the sections attached to

the slides. The slides were left overnight on a hot plate to give maximum expansions of the

seeds.

Staining

Dewaxing of seeds was done by immersing the slides with their sections in pure xylene for 3

minutes. The sections were then dehydrated by transferring then into series of ethyl alcohol

concentration 100% ,95% ,90% 70%, and 50% respectively in 3 minutes for each step and

was stained by flooding them with safranin stain dissolved in 50% ethyl alcohol. They were

then dehydrated back into 50%, 70%, 90%, 95% and 100% respectively in two minutes for

each step and then was stained with safranin stain about two minutes then was stained fast

green stain afew seconds, then washed it in absolute alcohol then xylene washed in mounted

in a drop of D.P.X, and was covered with a cover slip.

Drying

The prepared slides were left to dry in an oven adjusted at 60˚C for at least three days.



Microscopical examinations

The prepared permanent slides were examined using (Leitz Dialux20), Microscope with large

field of view at critical sharpness to the very edge of the picture, by more than 23%

compared with conventional microscopes, using power X20, X100 and X400.

Photographing

The prepared slides were photographed using (Olympus VANOX AHBT3), Manual/Semi –

automatic Camera System with Focusing magnifier and Polaroid Camera back with Leitz

0.8X relay lens.

RESULTS AND DISCUSSION

The seeds of studied eight taxa of Acacia species were anatomically investigated. The present

work deals with the following taxa: Acacia mellifera, A. niloticassp, adansonia, A. nilotica

ssp nilotica, A. orefota, A. polycanthas sp campylacantha, A. Senegal var senegal, A. seyal

var seyal and A. sieberiana var sieberiana .

Seed sections were obtained and used to establish tissue and cell features. The internal

structure of the seed is differentiated into two distinct regions , the seed coat and the embryo.

The seed coat

An anatomically complex structure of the seed-coat is a general characterstic of all members

of the Leguminasae. Variations may be useful at the generic level.

The seed coat of Acacia species have a rough cuticle surface and rigid structure. The structure

of the seed coat shows that there are significant differences among taxa based on the layers

and cell sizes of seed coat. The seed coat is formed of:

The epidermis

The outermost layer of the seed coat, it is appeared in small regions in the sections of A.

nilotica ssp. adansonia, A. orefota, A. polycanthas sp campylacantha, and A. seyal var seyal

(plate 1 (2,4,5 and 7)) and it is not appeared in the rest of the studied species it was cut

(plate1( 1,3,6 and 8)). The epidermis is formed of uniseriate cells covered by cuticle. This

layer is called palisade layer it is formed of malphigian cells with an un evenly thickened

walls, and as stated by (Esau,1960), the palisade layer characteristic of leguminous seeds and

its structure in hard legume seeds is connected with their high degree of impermeability and

thus their germinability.

The sub epidermal layer

It followed the epidermis (plates 2-9), it is formed of two regions;

i. Lignified cells of different thicknesses , they are 6-7 layers in A. mellifera, A. niloticassp,

adansonia, A. nilotica ssp nilotica, and A. sieberiana var sieberiana, and 3-4 layers in A.

orefota, A. polycanthas sp campylacantha, A. Senegal var Senegal and A. seyal var seyal .

ii.Many layers of thick walled lignified parenchyma tissue ranging from 3-7 layers in all the

studied species except in the two sub species of A. nilotica where it is very wide (20-25

layers).



The endosperm

Is the innermost layer of the seed coat region, it is formed of one layer of small parenchyma

cells in all the species and sub species studied Leguminous seeds are exalbuminous. The

food materials are stored in the cotyledons of the embryo.

The embryo

Dicotyledonous embryos formed of two elongated cotyledons (plates 1) the structure of the

embryo is different in A. mellifera, A. niloticassp, adansonia, A. nilotica ssp nilotica and A.

seyal var seyal but this may be due to different stages of development. The outermost layer

of the embryo is the epidermis and it is formed of one layer of small compact cells

surrounding ground tissue of parenchyma cells filled with reserved food materials their nature

and quantities differ from one species to another (plates 2-9).

1 2

3 4

5 6

7 8

Plate1 Transverse sections of the seeds (X20) of (1) Aacia mellifera,

(2) A. nilotica ssp adansonia, (3) A. nilotica ssp. Nilotica, (4) A. oerfota, (5) A. polyacantha

spp camplyacantha (6) A. Senegal var senegal (7) A. seyal var. seyal, (8) A. sieberiana

var sieberiana



Plate2 Aacia mellifera(X 100)

Plate 3 Acacia nilotica ssp. Adansonia (X 100)

Plate 4 Acacia nilotica ssp. Nilotica (X 100)



Plate 5 Acacia oerfota(X 400)

Plate 6 Acaciapolyacanthasppcamplyacantha(X400)

Plate 7 Acacia Senegal var Senegal (X100)



Plate 8 Acacia seyal var. seyal (X 100)

Plate 9 Acacia sieberiana var sieberiana (X 100)

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COMPARATIVE ANATOMICAL STUDY OF SOME · PDF fileThe seeds were identified by plant taxonomist Ustaz Hasan AL-Bager (Soba ... (Leitz1512 West Germany), ... (Esau,1960), the palisade