research article open access anatomical structure and...

Research Journal of Biology, 2: 73 - 83 (2014) www.researchjournalofbiology.weebly.com

73 *Corresponding author: [email protected] Copyright © 2014 RJB

RESEARCH ARTICLE Open Access

Anatomical structure and development of reproductive organs of Purple basil, Lamiaceae Mastaneh Mohammadi1*, Ahmad Majd2, Taher Nejadsattari1 and Mehrdad Hashemi3 1Department of Plant Science, Faculty of Basic Sciences, Science and Research Branch, Islamic Azad University,

Tehran, Iran; 2

Department of Biology, Faculty of Biological Sciences, Tehran North Branch, Islamic Azad University, Tehran, Iran;

3Department of Genetics, Tehran Medical Branch, Islamic Azad University, Tehran,

Iran.

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

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 anatomical-histological and developmental characterization of the vegetative and reproductive organs of Ocimum basilicum L. cv. Dark Opal.

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)

Mohammadi et al., 2014

74 Copyright © 2014 RJB

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.

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 toluene-ethanol. 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

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).

Leaf anatomical properties Lamina has uniseriate epidermis with sinuous contour cells, in surface view (fig. 3b). Its structure is heterofacial-bifacial (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).

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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TR

Secondary xylem, Sph: Secondary phloem, Cam: Cambium, Ray: Parenchymatic Ray, Sc: Sclerenchyma, F: Fiber; the arrows show

the intercellular spaces.

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.

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 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.

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, two-layered tunica and the corpus consist of condensed protoplasmic cells are observed. Development of floral organs begins, centripetally (fig. 5a-c).

coll

par

xy ph

Gp

Ep

Coll

Vb

Mb



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.



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 .

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.

Mc

Mc

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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.

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).

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).



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 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

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 3-cellular 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



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.

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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