No. (4) NOV. (2014A.D/1435A.

H)

Comparative anatomical features of Lippia

nodiflora(L.)
Rich. and Verbena officinalis L., Verbenaceae
Zubaida A. Esmaeel1 and Ali H.AL- Musawi2
1- Department of Biology, College of Education, Al- Iraqia University,
Baghdad , Iraq
2- Department of Biology, College of Science, Baghdad University ,
Baghdad, Iraq

Abstract
In the present research , cuticular and epidermal anatomical
features were described , in addition to the anatomical features of
roots, stems and leaves for two species belonging to family
Verbenaceae. These species are Lippia nodiflora (L.) Rich. and
Verbena officinalis L. Anatomical comparison between them was
provided. The results showed that some of the anatomical
characters of roots, stems, leaves and foliar epidermis have good
taxanomic value to distinguish between these two species. These
characters are different in wood type of the root of these two
species, types of trichomes, stem shape, thickness of palisade and
spongy layers of leaf, in addition to the variation in the foliar
epidermal cell shapes and stomatal index.
Key words: Anatomical features; Lippia nodiflora;
Verbena officinalis; Verbenaceae.

21
 ‫)‪No. (4) NOV. (2014A.D/1435A.H‬‬

‫الخصائص التشريحيت الوقارنت لنباتي بربين جذاوي ‪Lippia‬‬

‫‪ nodiflora (L.) Rich.‬و ورد الوينا ‪، Verbrna officinalis L.‬‬
‫عائلت ورد الوينا‬
‫‪2‬‬ ‫‪1‬‬
‫علي حسين الووسوي‬ ‫و‬ ‫زبيذة عبذ اللطيف اسواعيل‬
‫‪ -1‬قسن علوم الحياة ‪ ،‬كليت التربيت ‪ ،‬الجاهعت العراقيت ‪ ،‬بغذاد ‪ ،‬العراق‬
‫‪ -2‬قسن علوم الحياة ‪ ،‬كليت العلوم ‪ ،‬جاهعت بغذاد‪ ،‬بغذاد‪ ،‬العراق‬

‫الخالصة‬
‫ذ‬ ‫مت يف هذ ا احث ذذا ة اخلذذئ االذرحيي احالدذ وائ ح ة ذذئ احثدذ ‪ ،‬ف الَذ‬
‫رحيوذذئ ة‬ ‫احالذذاقر‪ ،‬او اي ف حنذذىل ع ئلذذىلةا‪ ،‬ا‬ ‫ة اخلذذئ االذرحيي احالد ذ وائ حواذ‬
‫املانذ ذ ذذر ‪ .Verbenaceae‬ه ذ ذ ذ ذ ذ ذ ذ ذ ذ ذ ذ ذ ذ ذ ذ ذ ه اونذ ذ ذذىلا ه ذ ذ ذ نث ذ ذ ذ ذ ذ ذ ذ ذ ذ ذ ذ ذ ذ ذ ذرت ب ذ ذ ذ بع ذ ذ ذ ا‬
‫‪ Lippia‬نث ذ ذ ذ ذ ذ ذ ذ ذ ذ ذ ذ ذ ذ ذرت ة املانذ ذ ذ ذ ذ ذ ذ ذ ذ ذ ذ ذ ذ ذ ذ ذ ذ ذ ذ ذر‬ ‫‪nodiflora (L.) Rich‬‬
‫قر نذئ احنذىل ع حيدذ وارظ‪ .‬ت اذ ت احنالذرحي ا‪،‬‬ ‫‪ . Verbrna officinalis L.‬متذ‬
‫احالذذاقر‪ ،‬او اي يا ذذئ حيلذذنااائ اذ ‪ ،‬حوال ااذ بذذع‬ ‫حذذثلا احلذذارت احالدذ وائ حواذ‬
‫هذ ئ احنثذرحيع‬ ‫ه ئ احنذىل ع ‪ .‬هذ ه احلذارت هذ او ذال و يف نذىل اادذر يف ذ‬
‫يف انىلا احدلريات شكل احالري مسك احطثقئ احل رةئئ اوخلاناائ يف احىل يذئ ف الَذ ظ‬
‫ذ ذ ا ذذىل حي ذذرئ ات يف اش ذذكر ذ ذ ق احثدذذ ‪ ،‬يف احىل ي ذذئ احذ ذ حال اح ذذ ب ذذع احن ذذىل ع‬
‫امل كىل ئ ‪.‬‬
‫‪Introduction‬‬
‫‪Lippia nodiflora (L.) Rich and Verbena officinalis L.‬‬
‫‪belonging to family Verbenaceae which includes shrubs, trees or‬‬
‫‪woody climbers. This family includes trees of economic values‬‬
‫‪(Teak) which is highly prized for its wood, and some beautiful‬‬
‫‪garden plants (Verbena and Lantana), others were important in‬‬
‫‪medicinal uses[1]. The genus Lippia is extremely wide spread‬‬
‫‪encompassing about 240 species, many of which are aromatic [2].‬‬
‫‪L. nodiflora was trailing perennial herbs 12-60 cm long, branched‬‬
‫‪from the base upwards[3], known as Barbin Jidawi and Bulaiha [4].‬‬

‫‪22‬‬
 No. (4) NOV. (2014A.D/1435A.H)

It is traditionally used as gastrointestinal and respiratory remedies,

it has antimalarian, antiviral, anti-inflammatory, analgesic,
antipyretic, antibacterial and antifungal activites [5, 6].
V.officinalis was perennial herbs, 30-100 cm long with
numerous stiffy erect tough stems, known as Batnig at siba on the
shatt al Arab[3], Vervain, holy herb and horse- whip[4]. It is found
in moderate climatic regions and in known for its anti-
inflammatory, diuretic, enteritis and expectorant properties. It was
used to treat acute dysentery, amenorrhea and depression [7.8]. The
root of this plant considered as a remedy for scrofula[1].
Anatomy of L. nodiflora and V.officinalis are rather limited
.Therefore, this study aims to investigate anatomic features for
these plants to evaluate the usefulness for systematic purpose.
Materials and Methods
Fresh specimens of L.nodiflora were collected from
different parts of University of Baghdad campus and specimens of
V.officinalis from Al.Taji farms (North of Baghdad) during March-
May 2012. The needed plant parts were cut into small pieces and
fixed in FAA (Formalin Acetic Alcohol) for 24 hrs[9]. Then , they
washed several times with 70% ethyl alcohol solution and
maintained in it [10]. Free hand thin transverse sections were made
and stained.

a- Preparing of transverse sectionings

The method of [11] and [12] were used with some
modifications. Some of sectionings were stained with
safranin:glycerine mixture 1:10.

b- Preparing of epidermal samples

Epidermis of both stem and leaf were peeled from fresh
plant specimens . The fresh stem and leaf were immersed in water
to prevent desiccation to procure epidermal cells. Pellicles from

23
 No. (4) NOV. (2014A.D/1435A.H)

stem and both abaxial and adaxial surfaces of leaf were obtained
with help of razor blade. Epidermal peels were stained with
safranin: glycerin mixture 1:10. After preparing , slides were
observed under compound light microscope for measurements.
Fifteen readings were taken by micrometer at random to obtain the
mean. Microphotographs were taken by using digital camera
(Model Sony Cyber-Shot T700) fitted on the light microscope.
These microphotographs were useful for identification and
differentiation cells of vegetative organs on microscopic base
features among different plant species.

C- Stomatal index (SI)

The stomatal index(SI) was calculated by using the
formula described by [13] that is :

X100

Where S indicates the number of stomata per unit and E the

number of epidermal cells in the same unit area.
Results
a- Epidermal cells and stomatal complexes
The data for the micromorphological features of stem and
foliar epidermis of the examined species were summarized in Table
(1) and illustrated in Figure (1) and Figure (2). Epidermal cells in
the examined species varied in their shapes. The shape of
epidermal cells on both surfaces that is , a baxial and adaxial, in
L.nodiflora seem to be polygonal , while they were highly
undulating to irregular shaped in V.officinalis. The shape of
epidermal cells of stem of both examined species were oblong with
straight walls. In both species leaves are amphistomatic bearing
anomocytic stomatal complexes. In addition to diacytic type in the
adaxial surface of L. nodiflora. There are no stomatal complexes in
the stem epidermis of both species. Stomatal index varied between
abaxial and adaxial surfaces of L. nodiflora, it was about 23.07%

24
 No. (4) NOV. (2014A.D/1435A.H)

and 35.71% on abaxial and adaxial surfaces, respectively ,while in

V.officinalis, stomata index were 22.22% on abaxial surface and
22.58% on adaxial surface.

b- Root anatomy
Root epidermis of L.nodiflora and V.officinalis, as shown
in transverse sections, consist of single layered of rectangular cells
reached to 25m and 28.56m thickness in L.nodiflora and
V.officinalis, respectively. Cortex of L. nodiflora comprised of 10-
14 layers of semi-rectangular parenchymatous cells, the average
thickness reached to 185.8m. In V.officinalis, the cortex consist an
elongate rectangular parenchymatous cells which become ovate
when reached to the root center. The average thickness reached to
313.33m. The phloem of both species comprised of rectangular
and cuboidal cells arranged in the form of rings .The average
thickness of the phloem reached to 142.34m in L.nodiflora and
35.32m in V.oflicinalis. The xylem of L.nodiflora was irregular
diffused Porous wood with a half diameter 45696 m; the outer
part of the wood showed dense tracheary elements, as a distinct
thick ring, 127.33m in thickness; while in V.officinalis it was
regular diffused porous wood with a half diameter 850m (Figure
3).

C- Stem anatomy
The anatomical features of the stem of L.nodiflora
and V.officinalis are summarized in Table(2) and illustrated in
Figure(4) and Figure (5). In transverse sections, the stem showed
circular to subcircular shape in L. nidiflora; and quadrangular in
V.officinalis. Epidermis of both species was single layered; the
epidermal cells were circular- ovate in L.nodiflora and semi-
rectangular in V.officinalis. In the outer surface, the epidermis
covered with cuticle which shows minute projections in
L.nodiflora, while it was smooth in V.officinalis. The cortex of

25
 No. (4) NOV. (2014A.D/1435A.H)

L.nodiflora consists of 5-8 layers of lamellar collenchyma then 13-

15 layers of Parenchymatous cells in the inner regions of the cortex
. These Parenchymatous cells enlarged in size when reached to the
stele. In V.officinalis , the cortex consist of angular then lamellar
collenchyma cells in the angles and 13-16 layers of chlorenchyam
cells in the rib regions. The innermost layer of the cortex of V.
officinalis was distinguished as starch sheath. In both species , the
stele was in the form of continuous cylinder . The phloem
accompanied by stone cells in L.nodiflora . In V.officinalis , bundle
caps fiber thickness in the angles regions were more than their
thickness in the ribs regions . It was about 15om and 45m
thickness in angles and ribs regions ,respectively. Tracheary
elements of wood resembled by vessels and trachieds , are in radial
rows in both studied species . wood arms projected clearly towards
the pith . The pith cells of both species are big , with thin walls ,
these cells increased in size towards the stem center and their walls
become thinner . These cells are similar in their shape, usually
spherical and Polygonal.
Stem of L.nodiflora was covered with two types of
trichomes (Figure 6):
a- Calcified unicellular- non glandular hairs with average
length reached to 99.73m and average width 37.17m.
b- Sessiled unicellular glands, with average length of the
head reached to 60.74m and average width 43.52m.

d- Leaf anatomy
The anatomical features of the leaf of L.nodiflora and
V.officinalis are summarized in Table (3) and illustrated in
Figure(7) and Figure (8). A single layer of semi- rectangular cells
of epidermis was observed in the leaf of both species. The
epidermis covers with cuticle vary in thickness in both species. The
mesophyll was Bifacial (Dorsiventral) in both species , consist of
2-3 layers of palisade parenchyma and 5-7 layers of spongy
Parenchyma in L.nodiflora, while it consisted of 3-4 layers of

26
 No. (4) NOV. (2014A.D/1435A.H)

Palised Parenchyma and 6-8 layers of spongy Parenchyma in

V.officinalis. The Palisade cells were elongated with straight walls
in both studied species, while spongy cells were polygonal and
tuberculate in L.nodiflora and irregularly lobed in V.officinalis. The
midrib composed of single arch like collateral vascular bundle in
both species, but in L.nodiflora , it was accompanied by
sclerenchyma in the form of stone cells. The tracheary elements
arrange as rows in both studied species. Leaves of L.nodiflora and
V.officinalis covered with different types of hairs. Features of these
hairs are summarized in Table (4) and illustrated in Figure (9) and
Figure (10).

Discussion
Anatomical studies have been used successfully to clarify
taxonomic status and help in identification of different species
[14,15]. The results obtained from this study showed that, the
anatomical characters can separate taxa. Leaf epidermal tissue
characters have an important role in taxonomy and determination
number of plant genera and species. The variation in the foliar
epidermal cells which are Polygonal in L.nodiflora and highly
undulating to irregular shaped in V.officinalis could be used to
separate these two species. In addition, the study found that
epidermal cell walls on the adaxial surface were more undulating
than the cell walls of the abaxial side. This observation was ensured
by [16], this may be due to the sudden growth of cells during leaf
differentiation [17,18]. The undulation patterns have an important
taxonomic value [19,20]. [21] noted that undulating of cell walls is
a mesomorphic character and that environmental conditions such as
humidity which play a significant role in determining the pattern of
anticlinal cell walls. Straight or curved walls were identified as
characteristics of species growing in drier conditions, while
undulate walls were found mostly in species growing in area of
high humidity.
Leaves are amphistomatic bearing, anomocytic type of both
abaxial and adaxial surfaces of L.nodiflora and V.officinalis, in

27
 No. (4) NOV. (2014A.D/1435A.H)

addition to diacytic type on the adaxial surface of L.nodiflora. [22]

and [23] pointed out the presence of diacytic and anisocytic type of
stomatal complexes in L.nodiflora foliar epidermis, which were not
noticed in our findings; but the study of [22] agreed with our results
by the presence of anomocytic type. [1] pointed out the presence of
anisocytic type of stomatal complexes in V.officinalis foliar
epidermis, which were not agreed with our results. The root
anatomy shows wide range of variations in the thickness of all its
parts between the studies species. As well as, the xylem was
irregular diffused in L.nodiflora and regular diffused in
V.offcinalis. L.nodiflora differs from V.officinalis in the stem
anatomy, it was hairy and has circular to subcircular shape, while it
was glabrous with quadrangular shape in V.officinalis. The
quadrangular stem shape is a typical characteristic of Verbenaceae
family[24].Trasverse sections of stem of L.nodiflora show nearly
quadrant in the study of [22],while it was distinctly bilobed with
deep furrows in the study of [23]. The cortex consists of 5-8 layers
of lamellar collenchyma then 13-15 layers of parenchyma cells in
L.nodiflora, while it consists of angular collenchyma then lamellar
collenchyma in the angles and 13-15 layers of chlorenchyma in the
rib regions in V.officinalis. [22] showed that the cortex of
L.nodiflora is made up of chlorenchymatous and collenchymatous
tissue, while [23] study showed that, it consist of 7-8 layers of
parenchymatou cells. In L.nodiflora the phloem accompanied by
stone cells. This result show a parallelism with the record of [19].
Stem trichomes of L.nodiflora were calcified unicellular non-
glandular and sessiled glands type. Sessile glands were also noted
in the study of [23]. Leaves of L.nodiflora and V.officinalis covered
with different types of trichomes. Trichomes considered relevant in
corporative systematic investigation[19]. They can be used as an
important taxonomic tools .L.nodiflora leaves covered with non
glandular unicellular branched hair type, while V.officinalis leaves
covered with glandular hairs, non glandular unicellular and
bicellular hair types. This results showed a parallelism with [19].
However,two-celled non-glandular hairs were not mentioned by

28
 No. (4) NOV. (2014A.D/1435A.H)

[19]. This study, therefore, is based on the principles that, root,

stem and leaf anatomy has play an important role in the
identification, characterization and delimitation of plants ,as an
additional taxonomic characters.
Epidermal cells Stomatal Complexes
Species Surface
Length (m) width(m) Length (m) width (m)
ab (54.4-68)61.65 (27.2-40.8)33.54 (24.48-27.2)25.84 (16.32-19.04)17.68
L.
ad (48.96-57.12)53.49 (21.76-35.36)29.01 (21.76- 24.48)23.12 (16.32-19.04)17.68
nodiflora
St (108.8-157.76)134.18 (27.2-68)46.24 ----- -----
ab (54.4-81.6)65.28 (16.32- 38.08)26.29 (32.64-33.36)34 (21.76-24.48)23.12
V.officin
ad (27.2- 54.4)38.98 (10.88-19.04)14.5 (24.48-27.2)25.84 (16.32-19.04)17.68
alis
St (27.2- 40.8)33.54 (19.04-32.64)26.29 ------- ------

Table(1) Quantitative data of foliar and stem epidermis

of L.nodiflora and V.officinalis
ab: abaxial surface of leaf; ad: adaxial surface of leaf;
St: stem epidermis
Cortex
Vascular bundles
Cuticle
Epidermis
Species thickness Angular Lamellar
thickness (m) Parenchyma Phloem thickness Xylem
(m) collenchyma collenchyma
thickness(m) (m) thickness (m)
thickness (m) thickness(m)
L. (8.16-10.88) (21.76-29.92) (95.2- 103.36) (280.16-29.04) (68-89.76) (100-130)
_____
nodiflora 9.52 25.38 97.92 284.69 79.78 113.33
V.officina 2.72-5.44 (8.16-13.6) (44 -600) (300-400) (122.4- 136) (50-70) (100-190)
lis 4.08 10.88 500 350 127.84 60 153.33

Table(2): Quantitative data of stem anatomy of L.

nodiflora and V.officinalis
Upper Palisad Spongy Lower Midrid
Blade Cuticle layer layer epidermis
epidermis
Species thickness thickness thickness thickness thickness Rows No. of Element
thickness Vascular bundle
(m) (m) (m) (m) (m) tracheary No. of
(m) thickness (m)
element each row
(8.16- (7.92-116.9)
L. (220-250) (19.64-21.76)
10.88) 107.88 (68- 89.76) (16.32-19.04) (130-200) 160 25-33 4-7
nodiflora 233.33 20.4
9.52 76.16 17.68
(211.52-
V.officinali (5.44- (32.64- (81.6-92.48) (78.88- (13.6-21.76)
257.2) (180-220) 196.66 18-23 3-6
s 8.16) 6.8 38.08)35.36 86.13 89.76)85. 18.13
223.65
22

Table(3): Quanitative data of leaf anatomy of L.

nodiflora and V.officinalis

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 No. (4) NOV. (2014A.D/1435A.H)

Non- glandular
Glandular
Stalk Head Type of Trichomes Species
width Length width Length width (m) Length (m)
(m) (m) (m) (m)
(27.2-43.52) (261.12-353.6)  Non-glandular unicellular branched
- - - - L. nodiflora
34.45 319.14 hairs
(43.52-78.88) (163.2 -315.52)  Non- glandular unicellular
- - - -
64.37 262.02
(89.76-100.64) (261.12-285.6)
- - - -  Non-glandular bicellular
96.1 272.9 V.officinalis
(32.64- (244.8- (27.2- (38.8-
 Glandular cell with one celled
35.36) 252.96) 29.92) 40.8) - -
head and one celled stalk
34.14) 248.42 28.58 39.8

Table(4): Types of leaf Trichomes of L. nodiflora and

V.officinalis

a b c
b
Figure(1): Types of stomatal complexes observed in
L.nodiflora
a- Abaxial surface (anomocytic type)(2000X) ; b- Adaxial
surface (anomocytic type)(2000X)
c- Adaxial surface (diacytic type)(1040X) ; d- Stem epidermis
(2000X)

30
 No. (4) NOV. (2014A.D/1435A.H)

a
b
Figure(2): Types of stomatal complexes observed in
V.officinalis
a- Abaxial surface (anomocytic type)(2000X) ; b- Adaxial surface
(anomocytic type) (2000X)
c- Stem epidermis (1680X)

a b
Figure (3): C.S in root
a- L.nodiflora (500X) ; b-V.officinalis (310X)

31
No. (4) NOV. (2014A.D/1435A.H)

Figure (4) : C.S. in L.nodiflora stem

a
a- (124X) ; B- (310X) b

starch
sheath

Figure (5): C.S. in V.officinalis stem

a
a-(88X) ; b- (420X) b

32
No. (4) NOV. (2014A.D/1435A.H)

Figurea (6): Stem trichomes of L.nodiflora b

a- calcified unicellular non glandular type (1440X)
b- sessiled unicellular gland (2000X)

a b
Figure (7): C.S. in L.ndiflora leaf
a- leaf blade (760X) ; b- leaf midrib (500X)

33
No. (4) NOV. (2014A.D/1435A.H)

Figure (8): C.S. in V.officinalis leaf

a- leaf blade (1040 X) ; b- leaf midrib (420 X) b
a

Figure (9) :leaf trichomes of L.nodiflora (unicellular branched )

a- a(190X ) ; b- (760X) b

a (10) :leaf trichomes of V. officinalis

Figure
a- non glandular unicellular (400X) ; b- non glandularbbicellular
(640X) ; c- glandular hair (2000X)

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 No. (4) NOV. (2014A.D/1435A.H)

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