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Research Journal of Biology, 2: 73 - 83 (2014)
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RESEARCH ARTICLE
Open Access
Anatomical structure and development of reproductive organs of
Purple basil, Lamiaceae
Mastaneh Mohammadi1*, Ahmad Majd2, Taher Nejadsattari1 and Mehrdad
Hashemi3
1
Department of Plant Science, Faculty of Basic Sciences, Science and Research Branch, Islamic Azad University,
2
Tehran, Iran; Department of Biology, Faculty of Biological Sciences, Tehran North Branch, Islamic Azad
3
University, Tehran, Iran; Department of Genetics, Tehran Medical Branch, Islamic Azad University, Tehran,
Iran.
Abstract
Ocimum basilicum cv. Dark Opal is an ornamental aromatic and medicinal herb named “purple basil”. This study
aimed to characterize the anatomical structure and to make developmental analysis on vegetative and reproductive
organs of purple basil, cultivated in Iran, as anatomical characteristics are important taxonomic parameters for the
certification and quality control of medicinal plants. Cross sections of stem, petiole, leaf, and root and longitudinal
sections of blooms were fixed and prepared following standard techniques for light microscopy investigation. The
anatomical studies of vegetative organs showed the dicotyledonary-type, and the occurrence of two kinds of
trichomes is characteristic of Lamiaceae. Microsporogenesis is simultaneous. Anther wall initially with one middle
layer, and separation of tetrahedral tetrads are of dicotyledonary type. Pollen grains are zonocolpate. Ovule is
anatropous, and the embryo sac follows the monosporic polygonum type. This study is useful to identify this
cultivar, and contributing to the quality control of this medicinal plant. These investigations are even more
important for newly-identified varieties, to determine their anatomy, as in the case of purple basil.
Key Words: Anatomical structure, Megasporogenesis, Microsporogenesis, Purple basil.
(Received: 24/06/2014; Accepted: 12/07/2014; Published: 18/07/2014)
color is from anthocyanins and is considered as a potential
source of red pigments for the food industry and
antioxidant effect studies (Zoltec et al., 2011; Yesiloglu and
Sit, 2012).
Purple basil is described as having a variety of
medicinal and health benefits including having a general
restorative and warming effect, with a mild sedative
action. Like other basils, this herb has other health
benefits, such as improvement of digestive function,
relaxant for muscle spasms and cramps, improvement of
nausea, an antibacterial effect on infections, and
treatment for acne and insect bites (Horbowicz et al.,
2008; Verma and Kothival, 2012).
Considering that anatomical characteristics are
important taxonomic parameters for the certification and
quality control of medicinal plants, an investigation of
anatomical structures of the species organs is necessary
(Sinnott, 1960).
This study therefore provides an anatomicalhistological and developmental characterization of the
vegetative and reproductive organs of Ocimum basilicum
L. cv. Dark Opal.
Introduction
Lamiaceae is a large family, comprises 252 genera and
6800 species. The species grow in all types of
environments, at various altitudes, displaying a
cosmopolitan distribution (Watson and Dallwitz, 1992).
They are characterized by opposed or verticillate leaves,
indented or serrated margins with squared branches and
stem, glandular trichomes with aromatic oils (including
terpenoids) and tectorial ones, and small flowers arranged
in crests (Watson and Dallwitz, 1992; Mozaffarian, 2000).
The Ocimum genus belongs to the Lamiaceae family
includes approximately 150 species, which have variation
in phenotype, oil content, composition, and possibly
bioactivities (Omer et al., 2008; Liber et al., 2011).
Ocimum basilicum L. (sweet basil), is native to Iran,
India and other tropical regions of Asia and Africa. Basil
grows wild on some pacific islands, in warm or tropical
climates, and is cultivated in green houses or in the field in
many other regions (Rechinger, 1988).
Purple basil, a cultivar of Ocimum basilicum, is an
annual species, and has white or cerise pink flowers,
arranged in a terminal spike. This basil variety grows to
about 45 cm high and 25 cm wide, depending on
conditions. With deep purple, sometimes mottled leaves,
purple basils are highly marketable herbs, not only for
culinary purposes but also for their ornamental value
(Simon et al., 1999; Phippen and Simon, 2000). The purple
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*Corresponding author: Mohammadi_918@yahoo.com
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Mohammadi et al., 2014
large bundles have radial ranges of ligneous vessels
separated by uni- or multi-seriate areas of
parenchymatous cells (fig. 2a). The sclerenchyma and
fibers, at the end of the large phloem vascular bundles,
have less thickened but still cellulosic walls, which will be
thicker and lignified from the base to the top of the stem
(fig. 2a, b). The number of glandular and tectorial
trichomes per surface unit is decreased from the top to
base of the stem. The tectorial trichomes are uniseriate,
consisting of three cells, with an acute apex and a bi- or
multicellular basis (fig. 2c). The glandular ones are
capitate, show short unicellular stalk and uni- or bi-cellular
secretory head (fig. 2d).
Materials and methods
Plant material
Individual cultivated samples of Ocimum basilicum L. cv.
Dark Opal, Lamiaceae, were collected, from a farm at
Shahr e Ray city nearby Tehran. The examined samples
collected from these individual samples, were washed
with the fresh water.
Cross section studies
The upper third and the lowest third part of root, stem,
petiole and leaf blade samples were cut in 1cm pieces and
fixed in mixture of ethanol-glycerin (50-50%) for about 1
week. Then after using a double-edged razor blade, thin
slices of tissue were obtained. The cross sections were
soaked in 5% sodium hypochlorite solution (10 min), then
transferred to 3% Acetic acid for 10 min. Sections were
stained in methylene green for 2-3 seconds and then in
Carmine for 15 min. After each part, they were rinsed two
to three times with sterile distilled water. Finally the light
microscopy (Olympus) and photography was done.
Leaf anatomical properties
Lamina has uniseriate epidermis with sinuous contour
cells, in surface view (fig. 3b). Its structure is heterofacialbifacial (fig. 3a). The lamina presents amphistomatic
dyacytic stomata with unequally sized subsidiary cells (fig.
3b). Mesophyll is differentiated as a very elongated cells,
uni- or bi-stratified palisadic, and a multi-stratified
lacunous tissue (fig. 3a). Collateral vascular bundles,
encountered in the middle of the mesophyll, and encircled
by a parenchymatic sheath (fig. 3a).
There are glandular and tectorial trichomes on both
surfaces (figs. 3a, e). The glandular trichomes are two
types. 1- Capitate glands are smaller and have two large
spherical basal epidermal cells, and one or two short
rectangular stalk cell. On the top of the stalk is one or two
celled spherical secretory body (fig. 3e). 2- Peltate glands
have tetra-cellular basis, one or two celled stalk, and a
circular plate of secretory body. The stalk of the gland
arises from four epidermal basal cells which are
surrounded by many radiator rosette of cells (fig. 3b, e).
Longitudinal section studies
Blooms were prepared and fixed in FAA (2 ml 37%
Formaldehyde, 17 ml Absolute ethanol and 0.7 ml Acetic
acid glacial). Samples were dehydrated in 30% to 100%
ethanol, and soaked in distinct ratio mixtures of tolueneethanol. The dehydrated specimens were paraffinized and
embedded in melted paraffin wax, then sectioned by
microtome at a thickness of 6-7 μm. Sections were placed
on slides and deparaffinized, then hydrated and stained in
hematoxyline and eosin, cleared in toluene and mounted
in Enthalen. Longitudinal sections were then studied by
light microscopy (Olympus) and finally photography was
done.
Results
Root anatomical properties
The upper third and the lowest third parts of root was
studied. The structure of the root is secondary type.
Epidermis has made one layer, continued with
exodermis consists of compressed cells. There is
cortical parenchyma under exoderm, which includes
cells with thin walls and irregular aspect with spaces
between them (fig. 1a). Casparian ring in endodermis
layer, pericycle layer and vascular tissues are observed
(fig. 1a). Cambium cells are 1-2 layered, flat and
distinguishable, while it is up to 6 layers in the upper
third part of the root (fig. 1a, b). The pith is Lignificated.
In sections of the highest length, the secondary xylem
which is entirely lignified forms visible 2 annual rings
(fig. 1b). The fibers and sclerenchyma cells have thick
and lignified walls. Parenchymatic rays are uni- or
biseriate and homogenous (figs. 1a, b).
Stem anatomical properties
The stem has a primary structure only in the upper third
part, and a secondary structure in the other two. The
epidermis layer is uniseriate, and has been covered by a
thin cuticle layer (fig. 2b). The parenchymatous cortex is
collenchymatic in a hypodermal position (fig. 2b-d). The
Figure 1: Cross sections of root. a: Lower third part of root (×100),
b: Upper third part of root (×20); Ep: Epidermis ،Ex: Exodermis,
Ms: Mesodermis, En: Endodermis, Ca: Casparian ring, Pr:
Pericycle, Ph: Phloem, PX: Protoxylem, Mxy: Metaxylem, Sxy:
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Secondary xylem, Sph: Secondary phloem, Cam: Cambium, Ray:
Parenchymatic Ray, Sc: Sclerenchyma, F: Fiber; the arrows show
the intercellular spaces.
Figure 3: a: Transection of a young leaf (×20), b: Abaxial surface
view of dyacytic stomata and the peltate gland with tetra-cellular
basis (×10), c: Peltate and Capitate glands (×40); Ad: Adaxial, Ab:
Abaxial, Sp: Spongy parenchyma, Pp: Palisade parenchyma, Rc:
Rosette cells, Gp: Ground parenchyma, Ep: Epidermis, St:
Stomata, Vb: Vascular bundle , TR: Tectorial trichome, Cg:
Capitate gland, Pg: Peltate gland , Sh: Parenchymatic sheath.
TR
Figure 2: Cross sections of stem: a: A large vascular bundle (×20),
b: Stem in secondary growth; Details of a corner (×20), c: Tectorial
trichome (×40), d: Glandular trichome (×100). Ep: Epidermis, Coll:
Collenchyma, Co: Cortex, F-Sc: Fiber-Sclerenchyma, Ph: Phloem,
PX: Protoxylem, Xy: Xylem, Sxy: Secondary Xylem, Sph: Secondary
phloem, Cam: Cambium, M: medulla, Gl: Glandular trichome, TR:
Tectorial trichome, Bf: Bi- cellular basis of gland, H: Head of the
gland.
Vb
Gp
Ep
Petiole anatomical properties
The petiole has nearly plain convex contour in transection,
in which the abaxial side is semicircular. A continuous
strand of angular collenchyma, formed by one or two
rows, encircles the ground parenchyma. The vascular
system includes one or two large crescent shaped main
bundles, and two pairs of small, marginal ones, all are
distributed collaterally. The main bundle consists of
several short straight lines of xylem, small clusters of sieve
elements, and the collenchymatic cells which situated at
the abaxial side of the vascular bundle (fig. 4a). Ep
Figure 4: a: Cross section of an older petiole (×20); Ep: Epidermis,
Coll: Collenchyma, Gp: ground parenchyma, Vb: Vascular bundle,
TR: Tectorial trichome, Mb: Marginal vascular bundle.
Microsporogenesis stages
In the beginning of reproductive phase, apical meristem
becomes flat and slightly wide, morphologically. The
medullar meristem includes vacuolated large cells, twolayered tunica and the corpus consist of condensed
protoplasmic cells are observed. Development of floral
organs begins, centripetally (fig. 5a-c).
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Mb
Coll
Mohammadi et al., 2014
Figure 5: Longitudinal sections of apical meristem: a: vegetative meristem (×20), b, c: Reproductive meristem development (×20), d:
Shows a very young sepal, petal primordia, and two stamen primordia (×20), e: Stamen primordium development, and carpel
primordium initiation (×20), f: Growth of stamen primordium, and development of anther and filament (×20), T: Tunica, C: Corpus,
Mm: Medullar meristem, S: Sepal, P: Petal, Sp: Stamen primordium, Cp: Carpel primordium, A: Anther, F: Filament.
Stamen primordia are produced by periclinal divisions
of meristematic cells (fig. 5d). After initiation of stamen
primordium, production of carpel primordium will be start,
which is observed in figure 5e. While continuation of
divisions causes the expansion of stamen primordia (fig.
5e), alteration of the top part of primordium, forms the
immature anther, so that the immature filament which
includes elongated parenchymatic cells is differentiated
(fig. 5f). The immature anther consists of ground
meristematic cells, surrounded by protoderm layer, and
will be labiated through the development stages.
Inside the immature anther, four archespores consist
of a large nucleus and condensed cytoplasm, are observed
in the corners (fig 6a). Periclinal divisions of each
archespore, finally generate primary partial cells and
sporogenous cells that form the pollen sac wall and pollen
mother cells, respectively (figs. 6b, c).
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Figure 6: Development of anther and initiation of pollen mother cells division. a: Immature anther with three observed archesporic
cells (×40), b: Anther development and production of partial and sporogenous cells (×20), c: Anther and filament are observed (×20),
d, e: Details of sac wall and pollen mother cells (×100), f: Formation of callose, degeneration of toppy cells (×40). Arc: Archespore,
Gm: Ground meristem, D: Dermis, Pc: Partial cells, Sc: Sporogenous cells, C: Procambium, A: Anther, F: Filament, Aw: Anther wall, Ep:
Epidermis, En: Endothecium, Ml: Middle layer, Tp: Tapetum layer, M: Pollen mother cells, CI: Callose, N: Nucleus. The asterisk in
figures ‘d’ and ‘e’ shows fibrous appearance of tapetum wall.
The anther wall typically consists of epidermis,
endothecium, middle layer-initially with one layer and the
tapetum layer (figs. 6d, e).The tapetum is of the secretory
type, and the cytoplasm of tapetal cells is dense, that
indicate a high cellular activity. All tapetal cells, initially
uni-nucleate, become bi- or multi-nucleate before meiosis
(fig. 6f). Shortly before the formation of the special pollen
mother cell wall, tapetum cell walls start to acquire a
slightly fibrous appearance, which will be degenerate and
finally disappear, through the formation of callose (figs.
6d-f).
Microsporogenesis is simultaneous, and pollen
mother cells make the tetrahedral tetrad, by meiotic
division (figs. 7a-d). The tetradial cells separate and the
immature microsporous cells are formed, in which there is
large central nucleus (fig. 7e). In mature microsporous
cells, the nucleus is in lateral situation (fig. 8a). Entine and
exine layers and the ornamentation of exine are going to
be formed, and development of immature pollens starts
(fig. 8a-c). In this stage, division of the nucleus causes the
formation of a large round vegetative, and a small crescent
reproductive nuclei (fig. 8c).
The purple basil mature pollens shed as single grains,
and the ornamentation of pollen is reticulate and
zonocolpate. The colpi are fissure or slit like aperture, and
at the colpi the exine is reduced. The pollens are spherical,
light yellow to yellowish in color, and 20 μ diameter (figs.
8d-g). So that according to classification of pollen grains
depending on their sizes, by Erdtman (1952), the pollen
grains of purple basil could described as minuta.
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Mohammadi et al., 2014
Mc
Figure 7: a: Formation of dyad and tetrad in mother pollen cells (×40), b: Dyad meiospores are obviously observed (×100), c, d:
The forth nucleus in tetrad is observed (The arrow) (×200), e: Separation of tetrad cells, called immature microspores, in which
there is a central large nucleus (×40). M: Pollen mother cell, D: dyad meiospores, T: tetrad meiospores, Mc: Microspores. The
arrows in figures ‘c’and ‘d’ shows the forth meiosporous cell, Mm: pollen mother cells make the tetrahedral tetrad, by meiotic
division .
Mc
Figure 8: Formation of Microspore and pollen grain. a: Development of mature Microspore, includes a lateral nucleus (×100),
b: A stage shortly before formation of pollen (×100), c: Zonocolpate pollen (×100), d-g: mature pollen grains (×100). Mc:
Microspore, P: Immature pollen, MP: Mature Pollen, N: Nucleus, Ex: Exine, En: Entine, V: Vegetative nucleus, G: Reproductive
nucleus, S: Co: Colpi.
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The two carpel primordia join together through
ontogenic fusion, and make gamocarpous gynoecium (figs.
10a-c). Undergoing next development process, the two
styles will grow separately (fig. 10d).
Megasporogenesis stages
Gynoecium primordium is initiated by divisions of
meristematic cells (fig. 9a). Then after, by subsequent
unequal divisions of apex part, primordium becomes
concave, forms ovary hole and carpel primordia (fig. 9b).
Figure 9: Longitudinal sections: a: Gynoecium primordium (×40), b: Ovary, carpel primordia, ovule primordium are observed (×20).
Gp: Gynoecium primordium, Op: Ovule primordium, Ov: Ovary, Cp: Carpel primordium.
Figure 10: Longitudinal sections from Ovule formation stages, a: Ontogenic fusion of carpel primordia and inception of ovule
primordia (×20), b: Figure ‘a’ from other angle (×20), c: Cross section of Gynoecium shows two locular ovary and the 2 ovule
primordia in per loculus, d: Formation of ovule primordia have been completed, and style is obvious (×10), e: Anatropous ovule is
formed and is growing. The circle shows a number of cells that are differentiating (×20), f: Formation of ovule integuments have
been initiated. Micropyle and the differentiated cells are obvious (×40). CP: Carpel Primordium, Op: Ovule primordium, Ov:
Ovary, C: Carpel, O: Ovule, St: Style, M: Micropyle, F: Funiculus, Oi: Outer integument, Ii: Inner integument, NC: Nucellus, S:
Sepal.
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Mohammadi et al., 2014
Figure 11: a: Development of nucellus tissue and ovule integuments (×40), b: Nucellus tissue surrounded by the differentiated
endothelium cells (×100), c: Megaspore mother cell differentiated among other nucellus cells (×100), d: Ovule from other angle,
megaspore mother cell forms a dyad (×60). O: Ovule, Oi: Outer integument, Ii: Inner integument, NC: Nucellus, E: Endothelium cells, MM:
Megaspore mother cell, Pc: Partial cells, M: Micropyle, D: Dyad. The asterisk in figure d shows the chalazal cell.
By more divisions and growth, ovary hole becomes
more concave, and ovule primordia arise from the
placental tissue of the ovary (figs. 9b, 10 a, b). Ovary is 2
locular, and there are two ovules in per loculus, in an
axillary placentation (fig. 10c). Anatropous ovules with
ventral raphe, and the micropyle directed downwards, are
observed (fig. 10e). In ovule, a number of cells, form
nucellus tissue mass, which is partially covered by the
developing integuments, except in the micropylar region
(figs. 10f, 11a).
The inner and outer integuments are two- layered,
and are created by periclinal divisions of protoderm (fig.
11a). The cells that cover the nucellus, will be
differentiated and form the endothelium (fig. 11b).
In nucellus tissue, an archesporial cell divides into
some partial cells and one sporogenous cell, which
enlarges and becomes the megaspore mother cell (fig.
11c). Megaspore mother cell, undergoes the first meiotic
division to form a dyad, contained chalazal cells and a
smaller micropylar one (fig. 11d). Each cells of dyad seems
to undergo the second meiotic division that resulting a
tetrad. After degeneration process of the three micropylar
pole cells, the functional megaspore remains, which
undergoes the differential stages of embryo sac formation
(fig.12a).
In the beginning of embryo sac initiation,
degeneration in the nucellar cells in contact with the
embryo sac begins (fig. 12d). The functional megaspore
undergoes three successive mitotic divisions to produce an
8-nucleate megagametophyte. The first two nuclei divide
to form a 4-nucleate embryo sac, and these four nuclei
finally divide to produce an eight-nucleate embryo sac,
with a large central vacuole (fig. 12b-g). During the whole
of this process, the embryo sac enlarges and the eight
nuclei finally undergo reorganization and cellularization
(figs. 12f).
Purple basil shows a monosporic and initially
seven/eight-nucleate polygonum-type of embryo sac.
Embryo sac consists of a number of cells, vacuolar
expansion and nuclear migration, are observed (fig. 12f).
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Figure 12: Longitudinal and cross Sections from embryo sac. a: Embryo sac is formed (×20), b: Embryo sac includes 1 nucleus (×100),
c: Embryo sac includes 2 nuclei (×100), d: Cross section from Embryo sac includes 2 nuclei (×100), e: Embryo sac includes 4 nuclei,
and vacuolization are observed (×40), f: Embryo sac consists of 8 nuclei (×40), g: Cross section of Embryo sac includes 8 nuclei, and
the central vacuole (×100). O: Ovule, Oi: Outer integument, F: Funicule, Es: Embryo sac, E: Endothelium, V: Vacuole.
Discussion
environments as recorded by Faria (2008). The occurrence
of wide interfascicular regions with distinguishable
collateral bundles in purple basil, has been mentioned for
herbaceous dicotyledons (Esau K 1977).
In leaf anatomy investigation, the family exhibits
predominantly dyacytic stomata on both surfaces
(Metcalfe CR, L Chalk 1988). Purple basil, presents both 3cellular tector trichomes and glandular trichomes of the
capitate and peltate types. The occurrence together of
diverse kinds of glandular trichomes, of the peltate and/or
capitate type consist of a secretory head, and simple
tector trichomes is characteristic of Lamiaceae (Werker et
al., 1993; Svoboda et al., 2001). Trichomes are considered
relevant in comparative systematic investigations and
morphodiagnosis (Johansen, 1940).
Concerning
the
reproductive
anatomy,
microsporogenesis is simultaneous. The initial microspores
are tetrahedral tetrads. Anther wall is initially with one
middle layer, of the dicotyledonary type. The tapetum of
purple basil is of the secretory type, as is common in
Lamiaceae (Johri et al., 1992). It can therefore be expected
that the tapetal cell wall becomes modified for releasing
cytoplasm and its products. In fact, before callose
formation begins, the tapetal cell walls swell and acquire a
fibrous appearance, which will be followed by the
In root anatomy investigation, we observed lignified pith in
the center of the organ. The secondary xylem which is
entirely lignified forms two annual clearly visible rings.
Therefore, structure of purple basil root is secondary, as a
single result of the cambium activity, as has been recorded
by previous studies, in Ocimum basilicum. Also
intercellular spaces between root cortical cells was
observed, which belongs to Ocimum genus, and is
considered of diagnostic value (Pandey, 2005; Nassar et
al., 2013).
The caulinar secondary growth of purple basil is of
the ordinary type, as observed in many herbaceous
dicotyledons (Esau, 1977). The quadrangular transection is
frequently described for Lamiaceae (Metcalfe and Chalk,
1988; Barroso, 1991), as well as the evident collenchyma
in the four angles (Cronquist, 1981), which is considered of
diagnostic value according to Metcalfe and Chalk
((Metcalfe and Chalk, 1988). However the arrangement of
the collenchyma is not restricted to the angles, which is
presented in interfascicular regions. Metcalfe and Chalk
(1972) also determined some scleranchymatous tissue
surrounds the phloem groups of vascular bundles, as can
be seen the same in purple basil. The sclerenchyma is the
predominant support tissue for species that grow in dry
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Mohammadi et al., 2014
reduction to a thin, fragile layer, in the early tetrad stage.
Similar features were shown for Rosmarinus oficitialis
(Hidalgo et al., 2010). Another noteworthy feature of
purple basil is number of colpi on pollen grains, since it has
been a useful tool in tracing evolutionary relationship
among the species of a genus. The advanced dicotyledons
have more colpi than the primitive ones, with either a
colpus (monocolpate) or none at all (acolpate) (Walker,
1976; Arogundade and Adadeji, 2009). Thus it can be
affirmed that the zonocolpate pollen grains observed in
purple basil is a mark of recent evolutionary development
in the basil species.
Finally, the embryo sac formation in purple basil
follows the monosporic polygonum-model without any
variation from megaspore to eight-nucleate phase, which
is the most common developmental pattern of the
megagametophyte in Lamiaceae (Walker, 1976). From the
beginning of embryo sac formation we observe symptoms
of degradation in the neighboring nucellar cells in contact
with it, as has been observed by other workers (Foster,
1939; Gupta and Bhambie, 1978; Nikiticheva, 2002). It
seems that energy coming from this degradation is used
for embryo sac development stages.
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Conclusion
Anatomical characters of diagnostic value in purple basil
were recorded in the present study. Furthermore, ordinary
characters of the Lamiaceae family and the purple basil
were found. This information is important, as it helps in
the identification of this variety and contributes to its
quality control, and evaluation. This investigation helps to
differentiate from the closely related other species and
varieties of Ocimum basilicum.
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