Palynological investigation of some selected species of family Fabaceae from Pakistan,using light and scanning electron microscopy techniques

Pollen morphology of 11 species of family Fabaceae that is, Trifolium alexandrinum, Trifolium resupinatum, Arachis hypogaea, Lathyrus aphaca, Medicago lupulina, Vicia sativa, Lathyrus odoratus, Pongamia pinnata, Melilotus indicus, Medicago polymorpha, Medicago sativa from Pakistan has been investigated by light and scanning electron microscopy. Pollen were generally tricolporate, radially symmetrical, isopolar, elliptic in equatorial view and triangular in polar view under LM. Results showed that pollens were per‐prolate (T. alexandrinum), prolate (T. resupinatum, V. sativa, L. odoratus, Melilotus indicus, M. polymorpha, M. sativa) and sub‐prolate (A. hypogaea, L. aphaca, M. lupulina, P. pinnata). The larger polar/equatorial (P/E) ratio was found in T. alexandrinum (2.26 μm) and the smallest was found in M. lupulina (1.21 μm). The exine of T. resupinatum was 3.00 μm in thickness while others posses smaller exine thickness. The larger pore diameter was found in P. pinnata (16.01 μm) while others have smaller. The length of colpi was larger in Arachis hypogaea (32.24) while others posses smaller. Eight types of surface ornamentation (Psilate, faintly rugulate). Perforate and rugulate to verrucate have been observed under SEM. The pollens were europalynous type. Pollen morphology proved to be useful for the specific delimitation and serve as a tool for the identification and classification of taxa at specific and generic levels and can also be used as a key for the taxonomic features. Diversity in exine sculpture is helpful indicative characters for the isolation of closely related species. Hence, it is clear that both qualitative and quantitative characters of pollen can be useful for differentiating between taxa at specific level.     

Light and scanning electron microscopy-based foliar morpho-anatomical comparison of selected family Rosaceae members distributed in District Lahore,Punjab, Pakistan

In the present study morpho-anatomical characterization of selected Rosaceae members distributed in District Lahore was performed. Light and scanning electron microscopy was used for systematic characterization of the selected 19 species. Distinguished morpho-anatomical features such as size and shape of epidermal cells, size and type of stomatal cells, size and shape of trichomes, oil droplets, and silica bodies were contrasted. Results reported remarkable variations which could be taxonomically useful in identification of these members. Polygonal epidermal cells were observed in Eriyobotraya japonica, Potentilla bifurca, Potentilla supina, and Prunus amygdalus. However, Prunus cerasus possessed irregular-shaped epidermal cells that can be distinguished from hexagonal epidermal cells of Prunus persica. Similarly, stomatal type varied among some members. Paracytic or perisocytic stomata were observed in E. japonica whereas P. bifurca observed paracytic and anisocytic stomata. Lengths of guard cells were also of variable sizes. The average length of guard cells ranged from 53 (52–54) μm to 74 (73–75) μm in abaxial view. Potentilla supina had biggest, while Rosmarinus officinalis had tiny guard cells. Trichomes were tubular, stellate, cylindrical, ribbon-like, glandular, and nonglandular. Silica bodies in the present investigation were bilobed, rounded, and oval-shaped. It is inferred that diverse anatomical features proved to be valuable taxonomic tools that could be fruitfully helpful in identification of plants at specific as well as generic level.     

Comparative pollen and foliar micromorphological studies using light microscopy and scanning electron microscopy of some selected species of Lamiaceae from Alpine Zone of Deosai Plateau,Western Himalayas

The study was conducted to highlight a detailed account of morphology of pollen chosen species of Lamiaceae through scanning electron microscopy, and the anatomical characteristics of leaf epidermis of seven species using simple light microscopy. In results, Anisomeles indica and Otostegia aucheri belong to subfamily Lamioideae because it has tricolpate pollen while the rest eight species belong to subfamily Nepetoideae (hexacolpate pollen). The exine sculpturing of pollen of studied species was found to be reticulate. In the family Lamiaceae, four kinds of stomata were found anomocytic, anisocytic, diacytic, and actinocytic, respectively. The cell wall patterns of epidermal cells were irregular or polygonal with straight or undulate walls. It was noted that the variety of the epidermal trichomes seems of taxonomically important for the identification of species of Lamiaceae. Both nonglandular and glandular trichomes were analyzed. The nonglandular trichomes were characterized with long, thin, and pointed apical unicellular cells. The nonglandular trichomes were A‐shaped in Thymus linearis. In Perovskia abrotanoides, stellate glandular trichomes were observed whereas in A. indica and Mentha royleana both glandular and nonglandular trichomes were found. In A. indica, the nonglandular trichomes were sessile and peltate in M. royleana. For the first time in this study, pollen and foliar micromorphological features of selected species of this area are carried out. These taxonomic characters were found to be important in discrimination of species from each other. In future, the detailed study with comprehensive morphology coupled with other important characters is required for delimitation of taxa at various levels.     

Microscopic investigation of pollen morphology of Brassicaceae from Central Punjab‐Pakistan

The intention of the current study is to provide an account on the palynological features of Brassicaceae from Central Punjab‐Pakistan as a basis for future studies. Different morpho‐palynological characteristics both qualitative and quantitative were analyzed during this research which includes shape of pollen, diameter of pollen, P/E ratio, exine sculpturing, thickness of exine, type of pollen, shape and size of lumens, and thickness of murus. Taxonomic keys were also constructed based on pollen morphological characters for correct identification of species. This study aims to provide detailed information of pollen diversity and their exine structure based on both qualitative and quantitative characters by using Light microscopy and Scanning electron microscopy. Shape of pollen is mostly prolate, but some species also have sub‐prolate to spheroidal prolate types. Exine ornamentation in most species was reticulate, whereas micro reticulate (one species) and coarsely reticulate (one species) exine also observed in some pollen. All the pollen mentioned in this study have tricolpate apertures. Variation found in thickness of exine and other characters proved to be helpful at generic and specific level. The results reinforced the significance of pollen morphological features of family Brassicaceae and aid for valuable taxonomic tool in plant systematics.     

Palynological characteristics of some selected species of family Cucurbitaceae (Juss.) using light and scanning electron microscopy techniques

The pollen morphology of 11 species distributed in seven genera belonging to family Cucurbitaceae has been studied using light microscopy (LM) as well as scanning electron microscopy. Pollen morphology of this family is diverse and is almost europalynous. Nearly all types of pollen belonging to different size class and shape class with a variety of exine sculpturing are found in the family. Pollen grains are usually triporate to tricolporate, prolate, oblate‐spheroidal, spheroidal, to suboblate, radially symmetrical, and isopolar. The common exine pattern is reticulate of various forms ranging from finely reticulate to coarsely reticulate with some species having echinate type of exine ornamentation, for example, Cucurbita pepo and Praecitrullus fistulosus. Most of the studied species are somewhat triangular with respect to equatorial outlook and circular to elliptic in polar outlook. The palynological characteristics of the members of the family are useful in identification of taxa at generic level mostly. Based on the present study it is not recommended to wholly solely depend upon the pollen morphology for the identification of taxa especially at specific levels because very minor differences regarding pollen characters exist among the species of the same genera.     

Morphofunctional structure of the lingual papillae in three species of South American Camelids: Alpaca,guanaco, and llama

The aim of this study was to compare the anatomical and functional characteristics of the lingual papilla among the Camelidae. For this purpose, tongues of alpaca, guanaco, and llama were used. Numerous long and thin filiform papillae were located in the median groove and none were detected on the rest of the dorsal surface of the lingual apex in alpaca. Secondary papillae originated from the base of some filiform papillae on the ventral surface of alpaca tongue. The bases of some filiform papillae of the lateral surface of the lingual apex were inserted into conspicuous grooves in guanaco and tips of filiform papillae on the dorsal surface of the lingual body were ended by bifurcated apex. On the dorsal surface of the lingual apex of llama, there were no filiform papillae but there were numerous filiform papillae on both the lateral margins of the ventral surface of the lingual apex. Fungiform papillae were distributed randomly on dorsal lingual surface and ventral margins of the tongues of all camelid species. Lenticular papillae were located on the lingual torus and varied in size and topographical distribution for each species. Circumvallate papillae had irregular surfaces in llama and alpaca, and smooth surface in guanaco. In conclusion, llama and alpaca tongues were more similar to each other, and tongues of all camelid species displayed more similarities to those of Bactrian and dromedary camels in comparison with other herbivores and ruminants. Microsc. Res. Tech. 79:61–71, 2016. © 2015 Wiley Periodicals, Inc.     

Foliar epidermal anatomy of some selected wild edible fruits of Pakistan using light microscopy and scanning electron microscopy

The present study is focused on the detailed foliar epidermal anatomy of some selected wild edible fruits (WEFs) from Pakistan using light microscopy (LM) and scanning electron microscopy (SEM). The studied species are Ficus racemosa L., Solanum nigrum L., Capparis spinosa L., Physalis divaricata D.Don, Rosa moschata Herrm. and Ribes orientale Desf. collected from various localities of Pakistan. The objective of the present study is to investigate qualitative and quantitative anatomical characters for the identification and differentiation of collected wild edible fruits. The characters studied are shape and size of epidermal cells, anticlinal wall pattern, trichome type and shape, average number of stomata, length and width of stomata and pore. The detailed microscopic investigation and variations in the characters recorded have a key role in the determination and authentication of wild edible fruits. This study possesses great potential for plant taxonomists to further evaluate the species at molecular and genetic levels.     

Quality assurance and authentication of herbal drug (Argyrolobium roseum and Viola stocksii) through comparative light and scanning electron microscopy

The quality assurance and authentication of crude herbal drugs play important role in the effective therapeutic effect of herbal drug and their products. There are many reported problems in quality assurance of herbal crude drugs concerning to their correct identification. The present study was designed with the aim to document the authentication and quality assurance of the herbal crude drugs (Argyrolobium roseum and Viola stocksii) thorough light microscopy (LM) and scanning electron microscopy (SEM). The detailed foliar anatomical studies showed polygonal epidermal cells having anticlinal walls in Argyrolobium roseum while rounded epidermal cells were observed in Viola stocksii. The anomocytic stomata type was observed in Argrolobium roseum while actinocytic was noticed in Viola stocksii. The pollen of studied species appeared as tricolporate showing reticulate exine sculpturing in Argrolobium roseum while fine perforations were recorded in Viola stocksii. Furthermore, quantitative analysis of phenols, flavonoids, and antioxidant activity showed high flavonoid and phenol content in Argyrolobium roseum as compared with Viola stocksii. It was observed that Argyrolobium roseum was discriminated from the Viola stocksii based on the leaf and pollen micromorphological traits by using LM and SEM techniques. It was concluded that LM and SEM techniques were found useful for the quality assurance of botanicals and their authentication.     

Taxonomic investigation of selected rust fungi using scanning electron microscopy from Khyber Pakhtunkhwa,Pakistan

Rusts comprises the largest natural group of plant pathogens including approximately 8% of all described Fungi. Rust fungi are extremely plant pathogens responsible for great losses to agriculture productivity. Rust species belong to several genera among which more than half are Puccinia species. In Pakistan, rust causes severe damage to agriculture crops. Current study was carried out to identify and characterize different rust species common in the research area through microscopy and Scanning electron microscopy (SEM) in Khyber Pakhtunkhwa, Pakistan. Morpho-anatomical investigation of each collected rust species was carried out using different standard protocols. The dimensions of spores were measured and snapped under a stereomicroscope. SEM was used to examine the shape, size, and ornamentation of the spores of each rust fungus. Results revealed documentation of seven rust fungi, that is, Melampsora euphorbiae, Phragmidium barclayi, Puccinia nepalensis, P. exhausta, P. menthae, Uromyces capitatus, and Uromyces decorates belong to four different genera, were recorded. SEM revealed that spermogonia and Aecia were missing in most of the rust fungus studied. Uredinia was found in a scattered, irregular, lengthy, and epidermis-enclosed form. Urediniospores were found to be ovulating, elongated, echinulate, globose to sub-globose, ellipsoid to ovoid, and globose to sub-globose. Telia was found as sub-epidermal, amphigenous, dispersed, minute, and spherical cells. Teliospores ranged in form from cylindrical to oblong. The germ pores were detected in both apical (top cell) and basal (bottom cell) idiosyncratic and pedicel-attached cells. The techniques used in the current investigation will aid mycologists in rust identification and microscopic characterization.     

Microscopic investigations of some selected species of Papilionaceae through SEM and LM from Skardu valley,northern Pakistan

Pollen morphology of 10 species and foliar epidermal anatomy of eight species of Papilionaceae from Skardu valley, northern Pakistan has been estimated for the first time. The present study was commenced with an aim to provide a detailed account of the pollen morphology by scanning electron microscopy and foliar epidermal anatomy by light microscopy. The pollen aperture was tricolporate with reticulate exine in the selected species. Stomata types are actinocytic, paracytic, and anomocytic. Irregular or polygonal with undulate or straight walls, epidermal cells were reported. A unique diversity was observed in the foliar trichomes that show the taxonomic significance of the discrimination of taxa. Non‐glandular trichomes were observed in the selected species which are unicellular with thin, long and pointed apical cells. Pollen and foliar micro morphological characters proved to be helpful for the identification of taxa at a specific level.     

Potential of CLSM in studying some modern and fossil palynological objects

We have tested possibilities and limitations of confocal laser scanning microscopy to study the morphology of pollen and spores and inner structure of sporoderms. As test objects, we used pollen grains of the modern angiosperm Ribes niveum (Grossulariaceae) and Datura metel (Solanaceae), fossil angiosperm pollen grains of Pseudointegricorpus clarireticulatum and Wodehouseia spinata dated to the Late Cretaceous, fossil gymnosperm pollen grains of Cycadopites‐type dated to the Middle Jurassic, and fossil megaspores Maexisporites rugulaeferus, M. grosstriletus, and Trileites sp. dated to the Early Triassic. For comparative purpose, we studied the same objects with application of conventional light, scanning electron (to entire pollen grains and spores or to semithin sections of their walls), or transmission electron microscopy. The resolution of confocal microscope is much lower than that of electron microscopes, as are its abilities to reconstruct the surface patterns and inner structure. On the other hand, it can provide information that is unreachable by other microscopical methods. Thus, the structure of endoapertures in angiosperm pollen grains can be directly observed. It is also helpful in studies of asymmetrical pollen and pollen grains bearing various appendages and having complicated exine structure, because rotation of 3‐D reconstructions allows one to examine all sides and structures of the pollen grain. The exact location of all visible and concealed structures in the sporoderm can be detected; this information helps to describe the morphology and inner structure of pollen grains and to choose necessary directions of further ultrathin sectioning for a transmission electron microscopical study. In studies of fossil pollen grains that are preserved in clumps and stuck to cuticles, confocal microscope is useful in determining the number of apertures in individual pollen grains. This can be done by means of virtual sections through 3‐D reconstructions of pollen grains. Fossil megaspores are too large and too thick‐walled objects for a confocal study; however, confocal microscope was able to reveal a degree of compression of fossil megaspores, the presence of a cavity between the outer and inner sporoderm layers, and to get some information about sporoderm inner structure.     

Microscopic diagnosis of the leaf and stem of Piper solmsianum C.DC

Piper solmsianum C.DC., which is popularly known as pariparoba, is a shrub that measures 1–3 m in height and it inhabits areas with wet tropical soils. The objective of this study was to analyze the leaf and stem anatomy using light microscopy, scanning electron micrographs, and energy‐dispersive X‐ray spectroscopy in order to provide information for species identification. The anatomical profile showed the following main microscopic markers: hypostomatic leaf; hypodermis layer on both sides; pearl glands; biconvex midrib shape; five collateral vascular bundles in open arc with the central bundle larger than the others; circular stem shape; collateral vascular bundles arranged in two rings; sinuous sclerenchymatic sheath in the pith; secretory idioblasts; and starch grains in the mesophyll, in the ground parenchyma of the midrib, petiole, and in the stem; and six morphotypes of calcium oxalate crystals (styloids, cuneiform, tabular crystal rosettes, cuneiform crystal rosettes, elongated square dipyramids, as well as very elongated square dipyramids).     

Charging in scanning electron microscopy "from inside and outside"

This paper is an attempt to analyse most of the complicated mechanisms involved in charging and discharging of insulators investigated by scanning electron microscopy (SEM). Fundamental concepts on the secondary electron emission (SEE) yield from insulators combined with electrostatics arguments permit to reconsider, first, the widespread opinion following which charging is minimised when the incident beam energy E0 is chosen to be equal to the critical energy E(o)2, where the nominal total yield delta(o) + eta(o) = 1. For bare insulators submitted to a defocused irradiation, it is suggested here that the critical energy under permanent irradiation EC2 corresponds to a range of primary electrons, R, and nearly equals the maximum escape depth of the secondary electrons, r. This suggestion is supported by a comparison between published data of the SEE yield delta(o) of insulators (short pulse experiments) and experimental results obtained from a permanent irradiation for EC2. New SEE effects are also predicted at the early beginning of irradiation when finely focused probes are used. Practical considerations are also developed, with specific attention given to the role of a contamination layer where a negative charging may occur at any beam energy. The role of the various time constants involved in charging and discharging is also investigated, with special attention given to the dielectric time constant, which explains the dose rate-dependent effects on the effective landing energy in the steady state. Numerical applications permit to give orders of magnitude of various effects, and several other practical consequences are deduced and illustrated. Some new mechanisms for the contrast reversal during irradiation or with the change of the primary electron (PE) energy are also suggested.     

Histomicroscopy and normal anatomy of the adult killifish Aphanius hormuzensis (Teleostei; Aphaniidae) from the Persian Gulf coastal environment

The histomicroscopy and normal anatomy of the major body organ systems were investigated in the adult killifish, Aphanius hormuzensis using histological examination, X‐ray imaging, double staining, light microscopy and scanning electron microscopy (SEM). Based on the histomicroscopic observations, the kidney, liver and swim bladder in the studied species were comparable to other fish models. The anterior portion of the kidney is bulbous, while the posterior portion is narrow and elongated; the liver has a single lobe and the swim bladder is a single‐chambered organ with no connection to the digestive tract (physoclistous). X‐ray imaging and double staining examination showed 12 abdominal and 15 caudal vertebrae and a single hypural plate in the caudal skeleton. According to light microscopy, the scales were rounded to pentagonal in shape with three types of radii (primary, secondary and tertiary), and the urohyal bone was elongated. SEM microscopy showed a single row of tricuspid teeth on the upper and lower jaw, respectively, each tooth has two lateral cusps that are shorter than the middle one. The number of teeth was 17–18 in the upper jaw and 19–20 in the lower jaw. The saccular otoliths were rounded‐trapezoid in shape with a moderately incised and V‐shaped excisura. The members of killifishes are an important group for biologists because of their evolutionary properties, regeneration capacity and usefulness as biological control and also for the ecotoxicological assessment of environmental pollution. The outcomes of this study may provide a useful basis for future research on the genus Aphanius.     

Macroanatomic,light, and electron microscopic examination of pecten oculi in the seagull (Larus canus)

The present study was conducted to determine macroanatomic characteristic as well as light and electron microscopic examination (SEM) of pecten oculi and totally 20 bulbus oculi belonging to 10 seagulls (Larus canus) were used. Pecten oculi formations consisted of 18 to 21 pleats and their shape looked like a snail. Apical length of the pleats forming pecten oculi were averagely measured as 5.77 ± 0.56 mm, retina‐dependent base length was 9.01 ± 1.35 mm and height was measured as 6.4 ± 0.62 mm. In pecten oculi formations which extend up to 1/3 of the bulbus oculi, two different vascular formations were determined according to thickness of the vessel diameter. Among these, vessels with larger diameters which are less than the others in count were classified as afferent and efferent vessels, smaller vessels which are greater in size were classified as capillaries. Furthermore, the granules which were observed intensely in apical side of the pleats of pecten oculi were observed to distribute randomly along the plica.     

Leaf epidermal micromorphology and its implications in systematics of certain taxa of the fern family Pteridaceae from Northern Pakistan

The present study is insights into foliar epidermal anatomy for characterizing clades, and their utility in taxonomic segregation of certain species of Pteridaceae from Northern Pakistan. The leaf epidermal anatomy of 10 species of Pteridaceae representing four genera were examined using light and scanning electron microscope. A micromorphological matrix was constructed for eight qualitative and 12 quantitative characters. unweighted pair group method with arithmetic means and principal components analysis statistical analysis were performed to test the validity of foliar epidermal anatomical features as method of separating species and genera, and phylogenetic clusters among species are constructed using qualitative and quantitative traits. The qualitative characters described here are shape of epidermal cells, stomata, guard cell and subsidiary cells, anticlinal wall pattern, and trichomes types which is helpful in defining groups within Pteridaceae. In addition, the size of stomata, guard cells, subsidiary cells, stomatal pore epidermal cells, and trichomes are quantitatively analyzed. All species have hypostomatic leaves. Two types of stomata were observed in studied species, anomocytic and polocytic. Anomocytic stomata were observed in three genera namely: Adiantum, Onychium, and Chielanthes whereas Pteris can be discriminated from other genera by its polocytic stomata. On the basis of multivariate analysis present study does provides sufficient information on the taxonomic importance of foliar anatomy which validate its efficacy in species and genera discrimination. From result obtained here it is further possible to use leaf micromorphologic data in ferns phylogeny and providing basis for future taxonomic delimitation in other taxa.     

Postembryonic development of hard jaws (trophi) in a species belonging to the Brachionus plicatilis complex (Rotifera, Monogononta): a morphometric analysis

The presence of hard jaws (trophi), with species-specific shape and size, is a taxonomic feature of Rotifera, a group of microscopic metazoans. Since trophi are used to discriminate among species, it is important to know whether these structures change in taxonomically important ways during postembryonic development. Using both SEM and optical images, we analyzed more than 100 individuals of a single clonal lineage of a monogonont rotifer, Brachionus plicatilis, in order to describe body and trophi development after hatching. Body size, expressed as lorica width and length, was isometrically related to age of the animals only during preadult stages. Trophi size, expressed as length of the different parts, was unrelated to either age or body size. Therefore, trophi elements do not grow after hatching in B. plicatilis. Despite the dimensional invariance with age, some differences in trophi size among individuals of the same clone were recorded. No difference in left-right asymmetry of the trophi was shown; the asymmetric elements of the trophi named rami consistently had the right ramus longer than the left. This constancy is in contrast to the reported trophi asymmetries in bdelloid rotifers, in which left-right asymmetries are not constant within clonal lineages. In conclusion, we suggest that also trophi size, constant within the analyzed clone, may be used as an additional taxonomic feature to help in the discrimination of taxa within the B. plicatilis complex of cryptic species.