Micromorphology of Pollen Grains, Trichomes of Sweet Basil, Egypt

Collection of pollen grains: Pollen grains were extracted from fresh flowers of plant specimens collected from the Royal Farms for Medicinal plants (Giza Governorate, Egypt) during spring season 2018, and compared with already well identified voucher specimens of the herbarium of Surveys of Natural Resources Department, Environmental Studies and Research Institute, University of Sadat City, Egypt. The studied pollen grains were prepared according to the method of [21].

Acetolysis

The collected pollen grains were acetolysed following the procedure of [21]. Fresh anthers were placed in a centrifuge tube and macerated in 70% ethyl alcohol stirring with a glass rod, this dehydrated the pollen grains, then centrifuged at the speed of 5,000 revolutions per minute for fifteen minutes. The alcohol was poured off, glacial acetic acid was added, and the mixture was centrifuged again at the same speed. Acetolysis mixture (sulphuric acid and acetic anhydride 1:9) was added next and the tube containing the pollen grains and the mixture was kept in water bath and heated from 70 °C to boiling point. The mixture was then stirred with a glass rod and centrifuged again at the same speed as above, after which the acetolysis mixture was decanted and glacial acetic acid added. The mixture was centrifuged again, distilled water added, followed by another centrifugation. Water was then decanted and the acetolysed pollen grains were mounted in dilute glycerin solution for glass slides permanent mount (Figure 1).

Figure 1:Anatomical characteristics of Ocimum basilicum L. leaves (a & b) Cross-sections of the root of Ocimum basilicum L. pr: pith rays. Bars=200μm.

Light microscopy examination

Acetolysed pollen grains were properly examined under the light microscope according to [22]. Pollen diameter (μm), Pollen wall thickness (μm), Depth of colpi (μm), Distance between colpi (μm), Pore diameter (μm), Number of pores were measured. Also, pollen grains size, Colpi length, pollen shape, tectum surface and colpi type of 20 pollen grains were taken with the aid of an ocular micrometer inserted in the eyepiece of the microscope. These measurements were later multiplied by the ocular constant with respect to the power under which they were taken. Photomicrographs of the acetolyzed pollen grains were taken, while pollen morphological descriptions are based on features recognized at x1000 magnifications and photographs were taken with an Olympus BX21 light microscope while, pollen slides prepared were kept in special slide holders at the herbarium of Surveys of Natural Resources Department, University of Sadat City, Egypt. Pollen grains were mounted on slides in glycerin jelly and photographed.

Pollen ultrastructure investigations

For scanning electron microscopy (SEM), pollen air dried using 95% ethanol was mounted on a glass coverslip attached to an aluminum stub and then coated with gold, to a thickness of 300A, using a Jeol JFC 1100 sputter coater, then viewed at 20KV in a JOEL JSM 5300SEM of the Central Laboratory, Faculty of Agriculture, Mansoura University, Egypt. The subsequent examination was with a Tesla BS 340SEM. Selected SEM micrographs were then digitized and classified using image analysis software. While analysis of variance and Scheffe’s test were used for statistical evaluation [23], results are expressed as mean±standard deviations. The measured polar axis and equatorial diameter were based on at least 25 samples and other characters on approximately 20 under the LM. All of the measurements were done using Caranoy 2 [24]. The U-test was applied using SPSS 10.0. Pollen terminology follows [25] and [26].

Trichomes investigations

Trichomes obtained from fresh plant material were made with the aid of a light microscope. Observations and measurements of the examined identified types of the trichomes followed the terminology according to [21,22].

Pollen grains characteristic features

Pollen types present were of hexacolpate and convex in shape (Figure 2). The mean diameter of the pollen grains was 65.80±0.94 um (Table 1), the apertures were situated in both the polar and equatorial views of the pollen grains. Pollen diameter, the wall thickness recorded 7.61±0.13um, the depth of colpi is 19.98±0.52um, while distance between the colpi 25.78±0.18um, the pore diameter is 7.37±0.59 um and the numbers of pores of the pollen grains 38.88±0.78um are shown in (Table1 and Figure 2 & 3). (Figure 2a & 2b is the polar view – c is the equatorial view), pollen shape: suboblate; Aperture: Stephanocolpate (hexa-zonocolpate); slit shaped, margo present, the sculpture: Pre-reticulate, reticulum continuous all over the surface. The grain size: 65um. Tectum: mega reticulate, (Figure 2a & 2b) and (Figure 3a & 3d). Data obtained from (Table 2, Figure 2a, 2b & 2c), it was clearly light microscopy photographs of pollen grains of the Ocimum basilicum L (a and b) polar view. (c) Equatorial view, while the pollen size about 65um with magnification power: (×=1000). The polar axis was 65.16um while the equatorial one 64.98μm, and the average P/E ratio was 1.01um (Table 2). Tectum examined by SEM showed bi-reticulate patterns and deep with regular rough granular particles of the inner part of substratum and enlargement part exine (Figure 2c & 2d).

Figure 2:ALight microscopy photographs pollen grains of the Ocimum basilicum L, (a and b) polar view. (c) Equatorial view. Magnification power: (x=1000), Scale bars = 10um. Tectum: (mega reticulate, photos a and b)

Figure 3:Scanning electron microscope micrographs of pollen grains. (a) Polar view; (b) Equatorial view; (c & d) enlargement part of exine (mega reticulate, black arrow)

Table 1:

Table 2:

Pollen Description

i. Shape: Suboblate.

ii. Aperture: Stephanocolpate, (hexa-zonocolpate), margo present.

iii. Sculpture: Mega-reticulate, reticulum continuous all over the surface.

iv. Lumina: Granulated

Anatomical characteristics of Ocimum basilicum L. leaves

(Figure 1a&1b): The leaf consists of adaxial concavity and abaxial prominent midrib with the lamina directed towards upper side (Figure 1a). The midrib is somewhat bowl shaped in sectional view (Figure 1b) it is 1.5mm wide and 60um thick. The ground tissue is parenchymatous and the cells are small, polygonal and compact. The vascular strand is single, wide and bowl shaped (Figure 1b), while the vascular strand consists of several short three or four cells long, angular narrow xylem elements with wide parenchymatous gaps in between. The phloem elements located along the lower end of xylem strand and the phloem is seen in small discrete masses.

Microscopical examination of leaf and flower powder

Microscopical examination of leaves and flower powders showed presence of numerous non- glandular trichomes of average length 90um and diacytic type stomata, whereas, T.S of leaf showed that the leaf has midrib and a thin lamina with uneven lower epidermis attached at the lateral sides of the upper side (Figure1a & 1b), also upper and lower epidermis showed simple covering uniseriate non glandular trichomes, as well as sessile short stalked glandular ones (Figure 4a-4d).

Figure 4:Micro morphological characteristics of Ocimum basilicum L. leaves: (a & d). Diacytic type Stomata (b). Glandular capitate trichomes (c & d), marginal non glandular trichomes (f), Parenchyma cells with ca- oxalate crystals.

Trichome micromorphology

Non-glandular trichomes: Non-glandular trichomes (Figure 4c & 4d): are non-secretory covering type, they occur on the surface or along margins of the lamina. The trichomes are either unicellular or multicellular, uniserate unbranched were observed on different parts of the plant, including the leaves and flowers. The observed trichomes are broader at the base and tapering at the tip, (arrect or bent) with smooth surface wall (Figure 4c & 4d). The glandular trichomes (Figure 2b): Peltate type is seen in the powder of the leaves, few glands containing essential oil are seen attached on the lamina (Figure 2b). The gland is circular plate with a central stalk; at the tip of the stalk occurs unicellular spherical or hemispherical glandular body and darkly stained secretory cells, the gland size measured 60um in diameter of peltate cup-shaped, (Figure 4b).

The present study provides useful information on the micromorphology, palynology of pollen grains, leaves anatomy and trichomes morphology of sweet basil (Ocimum basilicum L.) which consider the main of all basil species used as pharmacopeial medicinal plants in Egypt [27]. Data and observations of pollen ultrastructure revealed that, mega reticulated form pollen sculpture and granulated lumina in the studied Ocimum basilicum L. in accordance with those of [9], it was clearly from light microscopy photographs of pollen grains of the Ocimum basilicum L. (Figure 2a & 2b) polar view and (c) Equatorial view, with pollen size about 65um. The polar axis recorded 65.16 um while the equatorial one 64.98 μm, and the average P/E ratio was 1.01um with aperture: Stephanocolpate, (hexa-zonocolpate), six colpi, (Table 2). On the other hand, tectum examined by SEM showed bi-reticulate patterns and deep with regular granular particles of the inner part of substratum and enlargement part exine (Fig. 2c & 2d). In a similar study, [28] used pollen grain sizes to group some species O. basilicum var. purpurascens and O. gratissimum are more closely related based on pollen grain attributes, since they clustered together at a higher similarity level than that of O. basilicum and O. canum. From the main characteristic element founded (mega reticulation sculpture) which more specific to Ocimum basilicum L of all Ocimum genus. The results of the present study revealed that the pollen characters of the Ocimum basilicum L. are valuable in taxonomic applications and may be useful in quality control (microscopic examinations of medicinal plants). [29,30] reported that, aerial parts of the aromatic plants belonging to the family Lamiaceae are covered with trichomes, including non-glandular and glandular or secretory trichomes. In the present study, the results recorded two types of non- glandular and one type of glandular trichomes of the leaves and flowers powder. The non-glandular trichomes are traditionally known as plays an important role in the physical protection of plants against biotic and abiotic stresses, on the other hand, these trichomes acting as a mechanical barrier against, high light intensity and temperatures, and feeding activities of different types of harmful insects. [31] stated that, for the Lamiaceae family, there are different plants species can have different densities of nonglandular and glandular trichomes, and different morphological features which could be of high important taxonomic values. The observed non glandular trichomes with average length 90um are broader at the base and tapering at the tip, (arrect or bent) with smooth surface wall (Figure 4c & 4d). In present study, one distinct glandular trichomes, short-stalked capitates, and peltate glandular trichomes were found (Figure 4b). While, the glandular trichomes (Figure 2b): Peltate type is seen in the powder of the leaves, few glands containing essential oil are seen attached on the lamina, at the tip of the stalk occurs unicellular spherical or hemispherical glandular body and darkly stained secretory cells, the gland size measured 60um in diameter of peltate cup-shaped, (Figure 4b). In this connection, [32] stated that glandular trichomes are the primary sites of secondary metabolite biosynthesis and plays an important role in secretion and storage biological substances and essential oils [33]. The findings of [29] who reported that capitates trichomes are variable in stalk length, glandular head shape and secretions so can be classified into various types. The short-stalked capitates trichomes with size measured 60um in diameter (Figure 4b) are represented in the studied plant Ocimum basillicum L. [34] stated that the short-capitate trichomes are the commonest type of capitates trichome found in Lamiaceae. Previous studies reported that, many species of Lamiaceae family have a broad head of peltate glandular trichomes with four to twelve cells [35,36]. Microscopical observations and anatomy of leaf showed that, the leaf stomata were of the diacytic type (Figure 4e) and the size (29um±2) while the guard cells with two subsidiary cells [37]. Findings of this study revealed that, the stomata of diacytic type and more characteristic of Ocimum basilicum L. leaves they play important role as a potential element as indicators of such aromatic plant [38].

In the present study we can concluded that, O. basilicum L (sweet basil) belonging to the Lamiaceae family known as the ‘King of Herbs’ has been used in traditional medicine. The leaves are rich in essential oil and secondary metabolites of biological and therapeutic importance to cure different diseases. This study is the first of its kind that primarily focuses on the pollen micromorphology and leaves trichomes, stomata of O. basilicum L (sweet basil) in Egypt.

a) The present study demonstrated uneven pollen sculpturing (mega reticulate tectum) of Ocimum basilicum L. (sweet basil).

b) Uneven distribution of glandular, non-glandular trichomes and diacytic stomata on the leaves.

c) We distinguished two types of non-glandular trichomes and one type of glandular trichomes. The short capitate glandular trichomes showed high morphological differentiation, the non-glandular trichomes of these species participate in the chemical interaction of the plants with the environment.

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