Anatomical Comparison of the Stem and Leaf in Some of Onobrychis Mill. (Fabaceae) Species in Kurdistan region-Iraq

Abstract

In the current research, the anatomical characteristics of stem and leaves cross sections of 11 species belong to the genus Onobrychis Mill. have been studied. From this study, some anatomical features, such as the outline of stem and leaves cross sections are described, and the differences were determined among them. The epidermis trichomes, collenchyma, cortex, xylem, and phloem layers of the stems were measured toward the pith and there was a variation between the studied species. The upper and bottom epidermal layers were labeled on the leaf. The variations of midrib outlines, main vascular bundles shapes and their characters among the studied taxa microscopically have been determined.

Full text

ZANCO Journal of Pure and Applied Sciences
The official scientific journal of Salahaddin University-Erbil
https://zancojournals.su.edu.krd/index.php/JPAS
ISSN (print ):2218-0230, ISSN (online): 2412-3986, DOI: http://dx.doi.org/10.21271/zjpas
RESEARCH
PAPER
Anatomical Comparison of the Stem and Leaf in Some of Onobrychis
Mill. (Fabaceae) Species in Kurdistan region-Iraq
FIRAS DHEIYA YUONUS AL-OTRAQCHE*
Department of Field Crops, College of Agricultural Engineering Sciences, Salahaddin University-Erbil, Kurdistan Region, Iraq
A B S T R A C T:
In the current research, the anatomical characteristics of stem and leaves cross sections of 11 species belong to the genus
Onobrychis Mill. have been studied. From this study, some anatomical features, such as the outline of stem and leaves cross
sections are described, and the differences were determined among them. The epidermis trichomes, collenchyma, cortex, xylem,
and phloem layers of the stems were measured toward the pith and there was a variation between the studied species. The upper
and bottom epidermal layers were labeled on the leaf. The variations of midrib outlines, main vascular bundles shapes and their
characters among the studied taxa microscopically have been determined.
KEY
WO
R
D
S
: Cross section, Midrib, Onobrychis, Outline, Paraffin, Stem, Taxonomy.
DOI: http://dx.doi.org/10.21271/ZJPAS.34.1.10
ZJPAS (2022) , 34(1);97-109 .
1. INTRODUCTION:
The Fabaceae L. is one of the great dicot families,
which consist of 730 genera and about 19500
species, it is important economically plants
(Simpson, 2006). Onobryehis Miller considered
one of the most difficult genus in the family,
within the tribe Hedysareae of subfamily
Faboideae. Yet, only 162 species were described
in the genus (Boissier, 1872; Sirjaev, 1925a;
Sirjaev, 1925b; Sirjaev, 1926; Bal1, 1968; Hayek,
1970; Hedge, 1970b; Mouterde, 1970; Schischkin
and Bobrov, 1972a; Bobrov, 1972b; Townsend,
1974; Meikle, 1977; Rechinger, 1984; Tan and
Sorger, 1986; Zohary, 1987; Davis et al., 1988;
Duman and Vural, 1990; Aktoklu, 1995). The
genus Onobrychis comprises about 170 species
that densely distributed in Irano-Anatolian
Caucasian, South West Asia and Mediterranean
and in temperate Europe in the world (Cronquist,
1981; Zohary, 1987; Aktoklu, 2001).
There are 23 species of Onobrychis are
recorded in the floramof Europaea (Ball, 1968),
while the major center of distribution ranges from
Central Asia. Ildis, (2005) confirmed 56 species,
which are 27 endemic. The genus Onobrychis is
restricted to the Palearctic region. Its distribution
began from the Mediterranean region to Caucasia,
the Zagros Mountains and central Asia. Onobrychi
is concentrated in different places as in Iran 53
species, Caucasia 39 species, Anatolia 52 species,
and only 8 species reach the Iberian Peninsula in
the west. So, Irano-Anatolian region is regarded
as one of the most prominent genetic diversity
centers. The anatomical characteristics are
considered as a piece of evidence that apply in the
taxonomic study from a century ago (Radford,et
al., 1974; Al-dabbagh and Saeed, 2019a). Stuessy,
(1990) was interpreted the anatomical information
is often quite helpful in solving taxonomical
problems, and the anatomical data can help in the
interpretation of evolution. Singh, (2010) stated
* Corresponding Author:
Firas Dheiya Yuonus Al-Otraqche
E-mail: firas.yuonus@su.edu.krd
Article History:
Received: 05/10/2021
Accepted: 06/12/2021
Published: 24/02/2022

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that the anatomical characteristics are observed by
using light microscope. The anatomy of some
species of Onobrychis was investigated in an
Atlas for trees, herbs, and shrubs (Schweingruber
et al., 2011).
Many studies conducted on the genus
Onobrychis in Iran morphological, palynological
and karyological traits were employed to estimate
the interspecific relationships ( (Karamian et al.,
2009, 2010; Ranjbar et al., 2009, 2010b.
However, there is no report on anatomy of the
genus. Anatomical characters are not always as
useful as morphological characters for plant
identifications. However, there is no record on
anatomy of the Onobrychis. Anatomical
characters are not constantly as useful as
morphological characters for identifications of
plant species. However, they are well-established
criteria and can offer significant assistance in
plant taxonomy (Güvenç & Duman, 2010;
Ranjbar et al., 2010a; Güvenç et al, 2011).
Moreover, genetic diversity in a worldwide
collection on Onobrychis using anatomical,
molecular and morphological was studied by
Zarrabian et al, (2013).
Particularly, the genus Onobryehis, that
covered by this study in Kurdistan Region-Iraq
and to enhance flora of Iraq. The aim of the
present study was focused on anatomical
characters on some perennial Onobrychis species
of Fabaceae family in Kurdistan region-Iraq for
the first time.
2. MATERIALS AND METHODS
Samples of Onobrychis were prepared from
herbarium specimens. In addition, to those were
collected specimens were stored in the herbarium
of the Department of Biology/ Sciences College/
Salahaddin University/ Erbil/Iraq.
For the anatomical study, herbarium stems,
and leaves put in the 1% NaOH for overnight,
then transfer to fixed solution as FAA (Formalin-
Acetic acid- Alcohol) for 24hr. After that the
samples were dehydrated by using series
concentrations of ethyl alcohol (80%, 90%, 95%,
100%) for 3-4-1hrs for each concentration, after
that the samples were placed in xylene
combinations (twice time) for 3-4hrs. for each
time for clearing processes. Then infiltrating the
samples by transfers them in paraffin and left in
the oven at 60ᴼC for a night.
The blocks of paraffin were made by using
a suitable manner for cutting. Slide sections have
been cut with the thickness of 8-10 micrometer
using the rotary microtome (Bright, LTD), the
ribbons placed and mounted on slides carefully,
then transferred to a hot plate for overnight. The
slides were stained by safranin and fast-green, and
covered by coverslips after adding a drop of DPX.
The prepared slides examined and imaged by
Light Microscopes (Olympus AC100 with a
camera, Japanese-made) (Najmaddin and
Mahmood, 2016); (Najmaddin, 2019).
3. RESULTS
3.1. General anatomical description of stems
The results showed that were different
outline shapes of the stems in studied onobrychis
species; the stem outline cross sections in studied
taxa are; triangular in O. caput-galli Lam , ovate
in O. aequidentata Sm. and O. schahuensis
Bornm, semi-circular in O. crista-galli Lam, O.
haussknechtii Boiss., O. megataphros Boiss , and
O. acaulis Bornm., circular in O. Susiana Nab.,
polygonal in O. kotschyana Feuzl., Rectangular in
O. ptolemaica Delile, and O. galegifoliaa Boiss
(Figure 1).
The epidermis consists of a single layer in
all studied taxa; the cells are triangle sub–circular
to sub rectangular in shape. The epidermis of most
species compasses trichomes except O. caput-
galli, O. aequidentata, and O. megataphros. The
multicellular glandular hairs are observed in O.
cristagalli and O. schahuensis, while the
multicellular non- glandular hairs were observed
only in O. haussknechtii. Nevertheless, the non-
glandular and glandular multicellular trichromes
are observed in the rest species (Figure 2 and
Figure 3).
Beneath the epidermis there is a
continuous ring of 12-13 cell thinned of
collenchymatous cells that are interrupted by
parenchyma tissue of cortex, the differences of
row numbers in conformity with the thickness of
the spaces between epidermis and pericycle. They

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appear peltate of 3-7 cell thickness in O. caput-
galli; 5-9 cell thickness in O. aequidentata; 7-9
cell thickness in O. haussknechtii; O.
megataphros and O. kotschyana; 3-4 cell
thickness in O. susiana; 5-8 cell thickness in O.
ptolemaica; 3-7 cell thickness in O. acaulis and
O. galegifoliaa, laqunar of 6-7 cell thickness in O.
cristagalli, and angular of 9-12 cell thickness in
O. schahuensis.
The prismatic crystals and tannins are
observed stems of all taxa, whereas the secretory
cells observed only in cortex of O. schahuensis
stem. Cup-shaped 13-24 cell thickness of fibers
are arranged in tangential layers and are located
above phloem of each vascular bundles,
surrounded by parateacheal and marginal
parenchyma.
Opened vascular bundles are arranged in
continuous close ring-like inward surrounding the
pith, there were two groups are separated
according to the vascular bundles size, first the
uniform sizes as in O. caput-galli, O.
aequidentata, O. cristagalli, O. haussknechtii, O.
megataphros, O. susiana, O. kotschyana., and O.
galegifoliaa, and the second the vascular bundles
are vary in sizes such as O. schahuensis, O.
ptolemaica, and O. acaulis. The pith is innermost
of the stem toward the center is a mass of
parenchymatous tissue with different sizes of thin
wall, with large cell spaces parenchyma cells,
which almost become hollow at maturity.
3.2. General anatomical description of leaves
The leaves are consisting of a single
layered in all examined taxa. Upper epidermis
composed of rectangular, oval, square or
sometimes irregular shaped cells in O.
galegifoliaa, while rectangular, occasionally
square or irregular shaped cells in O. megataphros
and O. ptolemaica, but oval, rectangular
sometimes irregular shaped cells in O.
schahuensis. It is depressed square or occasionally
irregular shaped cells in O. megataphros, O.
acaulis, O. aequidentata and O. caput-galli, it is
depressed rectangular oval shaped cells in O.
kotschyana, O. susiana, and oval, rectangular
occasionally regular shaped cells in O.
haussknechtii (Figure 4).
The trichomes were noticed in both
epidermis in most studied taxa except O. caput-
galli, O. aequidentata, and O. crista-galli. There
are non-glandular unicellular trichomes are
noticed in O. kotschyana, O. megataphros, O.
galegifoliaa, O. haussknechtii, O. acaulesent, O.
schahuensis and O. susiana. Whereas the non-
glandular multicellular trichomes are observe in
O. kotschyana , O. susiana, O. galegifoliaa and O.
ptolemaica. Sessile unicellular-glandular hairs
observe in O. susiana, O. haussknechtii, and O.
schahuensis. In addition, the glandular
multicellular trichomes are present in O.
kotschyana , O. susiana, O. haussknechtii, and O.
megataphros. Nevertheless, the O. haussknechtii
was unique in possessing the non-glandular
branched multicellular trichomes (Figure 4).
The leaves are amphistomatic, and the
mesophyll is isobilateral in all examined taxa,
whilst, they differ in the length, size, distribution
and abundance. Underneath of upper epidermis,
there are palisade parenchyma cells. Palisade
parenchyma is 3–5 layers and cells are rectangular
or cylindrical oval shaped in Onobrychis acaulis,
Onobrychis aequidentata and Onobrychis caput-
galli. Palisade are arranged in 4–6 layers of cells
and sometimes rectangular oval or irregular
shaped in O. haussknechtii, O. megataphros, O.
schahuensis, O. kotschyana and O. ptolemaica.
Whilst arranged in 5–6 layers cells and are usually
cylindrical or irregular shaped in O. cristagalli, O.
galegifoliaa and O. susiana (Figure 4).
The margin outlines of leaves showed
different outline shapes in studied Onobrychis
species, the margin is straight rounded as in O.
caput-galli, O. cristagalli, O. schahuensis, O.
susiana, O. ptolemaica, O. acaulis and O.
galegifoliaa. While rounded upward in O.
aequidentata, O. megataphros, O. kotschyana .
When rounded slightly downward in O.
haussknechtii (Figure 5).
The midrib outlines of studied species are
various in shapes, the midrib outline is
approximately eminent in abaxial, and convex in
adaxial as in O. caput-galli, while the abaxial is

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rounded and concave in adaxial such as O.
aequidentata and O. galegifoliaa. In O.
haussknechtii, O. kotschyana and O. megataphros
the abaxial is rounded and straight in adaxial. The
midrib among studied taxa, which is V-shaped,
has been observed in O. cristagalli. In O.
schahuensis the abaxial is U-shaped and convex in
adaxial, whilst in O. susiana is convex in abaxial
and adaxial. In O. ptolemaica, elongate rounded
in abaxial and straight in adaxial, however, the
midrib in O. acaulis is straight in adaxial and
semi-circle dish-shaped in abaxial (Figure 6).
Midribs main vascular bundles are single
closed in all studied taxa, surrounded by a single
layer of parenchymatous bundle sheath. But they
are differing in size and shapes, the vascular
bundle is small, circular, ovate shape in O. caput-
galli, O. cristagalli, O. haussknechtii and O.
kotschyana , while large, circular, ovate shape in
O. susiana and O. ptolemaica. Small semi-
circular crescent shaped in O. megataphros, the
rest are crescent semi-circular in the rest such as
O. aequidentata, O. schahuensis, O. acaulis and
O. galegifoliaa (Figure 6).
4. DISCUSSION
The epidermis comprise of a single layer in all
studied taxa, the cells are triangle, sub–circular to
sub-rectangular, ring-shaped, polygonal, or elliptic
with a reticular or smooth border in shape
(Butnariu, et al, 2005; 2006, Rodino, et al, 2014).
The lamina mesophyll of leaves in all studied of
Onobrychis species are heterogeneous. The
prismatic crystals, secretory cells with tannins are
observed in all taxa, as has been reported by
(Shweingruber et al., 2011). Onobrychis
represents a triangle shape in stem outlines. The
palisade cells are oblong in shape, and the spongy
cells have different irregular shapes with large
intercellular spaces, the palisade and spongy cells
are arranged in to 8-9 layers depended on the
location and lamina thicknesses as mentioned by
(Al-dabbagh and Saeed, 2019b). The cortex is
consists of 4 layers of semi–circular to sub-
rectangular parenchymatous tissue (Butnariu and
Giuchici, 2011; Ianculov, et al., 2004; Butu, et
al., 2014).
Onobrychis aequidentata, Onobrychis
cristagalli and Onobrychis haussknechtii shows
elliptic shape in stem outlines. The stem outlines
in cross sections are circular or circular to
elongate, they relatively have a similar shape in
four species Onobrychis megataphros,
Onobrychis schahuensis, O. susiana, and O.
kotschyana . Whereas, O. ptolemaica, O. acaulis,
and Onobrychis galegifoliaa have hexagonal
shaped stem outlines covered with hairs reticular
or smooth on their surface.
The tannins and crystals present in
parenchyma, vascular bundle and pith of species.
Tannin and prismatic crystals are cited by various
authors for the family. The collenchymatous
(lacunar collenchyma), consists of 2-6 layers of
collenchyma tissue, the differences of row
numbers in conformity with the thickness of the
spaces between epidermis and pericycle.
The endodermis is 1-6 layers of large well-
developed parenchyma cells, which the numbers
of row are depended on the endodermis
thicknesses and conversely with numbers of
hypodermal cells.
This study investigated that the trichomes are
unicellular glandular, non-glandular, and sessile
multicellular glandular and non-glandular in the
stem of the genus taxa. However, they are vary in
dense and abundance in addition to their size,
length. Obviously, the multicellular glandular
trichomes are terminating a head which is either
globose or clavate, besides, the trichomes walls
are impregnated with plentiful crystalline
granules. These glandular trichomes usually
excrete an extremely sticky substance (Struwig et
al., 2011).
Under upper epidermis, between palisade
parenchyma cells there are voluminous cylindrical
shaped secretory cells in all examined taxa as
confirmed by (Najmaddin and Khalid, 2017). The
secretory cells are presence in all examined taxa.

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Spongy parenchyma cells are cylindrical, oval
or circular shaped in all taxa Spongy parenchyma
consists of (2-5) layered in all species.
Crystalliferous cells frequently form a sheath
along the outer boundary of the pericyclic
sclerenchyma (Metcalfe and Chalk, 1950).
Solitary prismatic crystals are recognized to be
distributed in the cortex, rarely in the phloem and
xylem. (Bakr and Fthulla, 2017). These Tanins
cells are circular or oval or occasionally irregular
shaped present in the cortex, palisade parenchyma
and spongy parenchyma and vascular bundle cells.
Midrib outline showed different shapes and
different vascular size, these variations might have
taxonomical value (Al-dabbagh and Saeed,
2019b).
5. CONCLUSION
In this investigate concluded the species differ in
the outline shapes of the stem, leaves midrib and
margin. Prismatic crystals, fibers, and tannins are
found in stems present. in stems of all studied
taxa. Trichomes are presented which are
multicellular or unicellular and glandular or non-
glandular. The leaf mesophyll has no such great
variation of taxonomic value.
Conflict of Interest (1)
There is no conflict of Interest.

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Fig. 1. Variations of stem cross section outlines of the studied Onobrychis Species, A- O. caput-galli, B- O.
aequidentata, C- O. cristagalli, D- O. haussknechtii , E- O. megataphros, F- O. schahuensis, G- O. susiana
, H- O.
kotschyana. I- O. ptolemaica, J-O. acaulis, K- O. galegifoliaa (4X).
vb= vascular bundle, lvb=large vascular bundle

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Fig. 2. Variations of stem cross sections of the studied Onobrychis Species, A- O. caput-galli, B- O. aequidentata,
C- O. cristagalli, D- O. haussknechtii , E- O. megataphros, F- O. schahuensis (40X).
ep= epidermis, t= tannin, col= collenchyma, fi= fiber, ph= phloem, xy= xylem, cr= crystal, sc= secretory cells, tr=
trichome.

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Fig. 3. Variations of stem cross sections of the studied Onobrychis Species, (A-E); A- O.susiana, B- O.
kotschyana. C- O. ptolemaica, D-O. acaulis, E- O. galegifoliaa; and (F-G) trichomes, F- glandular hair, G-
non-glandular hairs (40X).
ep= epidermis, col= collenchyma, fi= fiber, ph= phloem, xy= xylem, cr= crystal, t= tannin, sc= secretory cells,
gtr= glandular trichome, ntr= non-glandular trichomes

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Fig. 4. Variations of lamina cross sections of the studied Onobrychis Species, A- O. caput-galli, B- O.
aequidentata, C- O. cristagalli, D- O. haussknechtii , E- O. megataphros, F- O. schahuensis, G- O. susiana
, H-
O. kotschyana. I- O. ptolemaica, J-O. acaulis, K- O. galegifoliaa (4X) and L- O. haussknechtii, magnified
portion of lamina(40X).
Uep= upper epidermis, lep= lower epidermis, pa= palsied, sp= spongy cells, st= stomata, vb= vascular bundle, ta=
tannins, tr= trichomes, cr= crystal.

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Fig. 5. Variations of leaves margin cross sections of the studied Onobrychis Species, A- O. caput-galli, B- O.
aequidentata, C- O. cristagalli, D- O. haussknechtii , E- O. megataphros, F- O. schahuensis, G- O. susiana
,
H- O. kotschyana. I- O. ptolemaica, J-O. acaulis, K- O. galegifoliaa (4X).

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Fig. 6. Variations of midrib cross section outlines of the studied Onobrychis Species, A- O. caput-galli, B- O.
aequidentata, C- O. cristagalli, D- O. haussknechtii , E- O. megataphros, F- O. schahuensis, G- O. susiana
H- O.
kotschyana. I- O. ptolemaica, J-O. acaulis, K- O. galegifoliaa (10X).
vb= vascular bundle, ta= tannins, ph= phloem, xy= xylem, tr= trichome, cl= collenchyma.
,m

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