Palynological investigation of lactiferous flora (Apocynaceae) of District Rawalpindi,Pakistan, using light and scanning electron microscopy

This was the first comprehensive pollen micromorphological investigation of lactiferous flora (Apocynaceae) of District Rawalpindi, Pakistan. The pollen morphology of 10 species of the family Apocynaceae was observed and documented using light microscopy and scanning electron microscopy. Pollen was found subspheroidal in shape in most of the species, however peroblate shape was noted in Vinca major. Exine sculpturing patterns (psilate, rugulate, scabrate, and microreticulate) were observed. The result indicated that exine ornamentation of Apocynaceae taxa is systematically informative at generic and species levels. Most of the species have tricolporate type pollen but tetraporate pollen was also observed in Trachelospermum jasminoides and tetracolpate in V. major. Minimum equatorial diameter was noted in Carissa edulis (27.13 μm) and maximum in V. major (108.25 μm). Similarly, maximum exine thickness was found in Cascabela thevetia (9.5 μm). In the present findings, the pollen morphological data are compared with available other pollen studies to evaluate the taxonomic value of pollen traits in Apocynaceae taxa by using multiple microscopic techniques. Furthermore, molecular and phylogenetic studies were recommended to strengthen the systematics of Apocynaceae taxa.     

Morphological and palynological assessment of taxonomically problematic genus Paspalum based on light and scanning electron microscopy

Present research work was carried out to clarify the variations among species of genus Paspalum morphologically and palynologically as this genus is taxonomically difficult due to having multiple similar morphologically overlapping characters which make it difficult to identify. Henceforth present research work was carried out to delimit taxa within the same genus by morphological and palynological tools through light microscopy (LM) and scanning electron microscopy (SEM). Both these tools are considered as the most useful taxonomic characters for taxonomically problematic genera. The results showed a lot of variations among morphological characters. In Paspalum dilatatum, the upper glume was ovate whereas in the other two species, the upper glume was elliptic. The upper glume apex found in P. dilatatum and Paspalum scrobiculatum was obtuse whereas in Paspalum distichum, upper glume apex was acute. Glume nerves showed variation in all three species. Paspalum distichum was 3 nerved, P. scrobiculatum was 5–7 nerved, and P. dilatatum was 5–9 nerved. All three species showed variation in lemma nerves. Paspalum scrobiculatum had 3 nerved lemma whereas in P. distichum 3–5 nerved and P. dilatatum 5–9 nerved lemma were present. In polar and equatorial view, pollen grains ranged from 25 (20–30) to 37.5 (30–45) μm. Paspalum distichum appeared to be the smallest in size whereas P. dilatatum was the largest. Exine thickness ranged from 0.75 (0.5–1) to 1.35 (1.2–1.5) μm. The higher value of pollen fertility was found in P. scrobiculatum as 87.69% and the lowest value was in P. distichum as 78.08%. Morphological keys were also given for correct identification.     

Identification of monocot flora using pollen features through scanning electron microscopy

Pollen used to track structural and functional evolution in plants as well as to investigate the problems relative to plant classification. Pollen characters including ornamentation, shape, apertural pattern, pollen symmetry, colpus length, width, and margins used to detect the similarities and dissimilarities between genera and also species of the same genus. In this study pollen features of 20 monocot species belonging to 15 genera of the Amaryllidaceae, Asparagaceae, Iridaceae, Ixioliriaceae, Liliaceae, and Xanthorrhoeaceae were studied using scanning electron microscopy (SEM) and light microscopy (LM). In this study two species that is Zephyranthes citrina and Tulbaghia violacea were reported for the first time from Pakistan. Pollen grains were visualized with LM. Non‐acetolyzed and acetolyzed pollen were examined using SEM. A taxonomic key was developed to highlight the variation in pollen features in order to make their systematic application for correct species identification.     

Seed morphoanatomy of the genus Passiflora L. (Passifloraceae) by scanning electron microscopy and light microscopy

Morphoanatomical analysis of seeds contributes to knowledge of the development of seedlings and identification of species, as well as supporting conservation studies. The conservation of the species belonging to the Passiflora genus is crucial due to of the threats to the genetic resources of these species. Thus, the objective of this study was to morphoanatomically characterize Passiflora seeds, verify possible injuries to the tissues after cryopreservation and thus contribute to the conservation strategies of the species of this genus. Initially, seeds of Passiflora coccinea, P. edulis, P. gibertii, P. maliformis, P. morifolia, P. setacea, P. suberosa, and P. tenuifila collected from the Passion Fruit Active Germplasm Bank of the Embrapa Cassava and Fruits research unit (Embrapa Mandioca e Fruticultura) were analyzed. Then, their length, width and thickness, shape of the base and tip, and ornamentations present on the body and edge of the seeds were evaluated. The seeds of the species were placed in cryotubes and immersed in liquid nitrogen to assess possible cryoinjuries. The tegument and tissues of the seeds were examined by scanning electron microscopy. The seeds had varied biometric data, with average values of 4.63 mm for length, 3.28 mm for width, and 1.51 mm for thickness. Six ornamentation types were observed: reticulate for the species P. coccinea; finely reticulate for P. edulis; foveolate reticulate for P. gibertii and P. setacea; alveolate reticulate for P. maliformis and P. tenuifila; coarsely reticulate for P. morifolia; and falsifoveolate reticulate for P. suberosa. Some seeds suffered tegument cracks due to the freezing in liquid nitrogen, but without physiological damages to the embryo and endosperm. The cryopreservation of the seeds in the presence of the tegument significantly reduced the cryoinjuries caused to the embryo. Cryopreservation can be promising for long-term conservation of passion fruit seeds.     

Light microscopy of Pakistani Berberis leaf cuticles and its taxonomic implications

Taxonomy of the genus Berberis is quite complex, due to overlapping morphological characters, making it very difficult to differentiate the species within the genus. In order to resolve this taxonomic complexity, the foliar anatomy of 10 Berberis L. species was carried out, for the first time from Pakistan, using light microscopy (LM). Significant variation in terms of epidermal cells shape, size, cell wall pattern, and stomata type was observed. B. baluchistanica has the largest epidermal cells, Adaxial: length = 45–(53.9 ± 3.6)–62.5 μm; and width = 22.5–(26.3 ± 1.3)–30 μm; Abaxial: length = 37.5–(43.25 ± 2.5)–50 μm; and width = 20–(22.6 ± 0.8)–25. The highest number of stomata was observed in B. glaucocarpa as 62 on the abaxial surface while the lowest number of stomata was recorded in B. baluchistanica as 8 on the adaxial surface. Of 10 investigated species, 6 possess anomocytic type stomata, while 2 species that is, B. aitchisonii and B. parkeriana have both anomocytic and anisocytic stomata while B. baluchistanica and B. calliobotrys have only paracytic type stomata. The highest number of cells per unit area was present on the adaxial surface of B. calliobotrys ranging from 245–(252.4)–260 followed by B. parkeriana with 209–(227.8)–250 on the abaxial surface. Stomatal index (SI) also varied considerably and was the lowest (2.6) percentage in B. baluchistanica and highest (31.9) percentage in B. kunawurensis. A taxonomic key based on micro‐morphological characters is provided for species identification.     

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.     

Taxonomic study of subfamily Nepetoideae (Lamiaceae) by polynomorphological approach

The present study is insight into pollen morphology for characterizing species and their utility in the taxonomic separation of certain taxa of subfamily Nepetoideae (Lamiaceae) from Pakistan. The pollen micromorphology of 11 species of the Nepetoideae was analyzed and documented using light microscopy and scanning electron microscopy (SEM) for both qualitative and quantitative characteristics. Most species have hexazonocolpate pollen grains but trizonocolpate and tetrazonocolpate pollen with circular and oval amb were also rarely observed in Mentha spicata. The basic pollen shape in most of the studied species was subspheroidal but prolate grains were also observed in M. spicata, S. coccinea, and S. plebeia. The exine sculpturing of Nepetoideae pollen was taxonomically very informative particularly at subfamily level. Observations of exine sculpturing with SEM revealed various types of pollen grains: reticulate, bireticulate, microreticulate, perforate, aerolate, and gammate. The bireticulate type further subdivided into three subtypes based on the number of secondary lumina in each primary lumen and is characterized by varying characteristics of the secondary reticulum and primary muri. A significant variation was observed in colpus surface ornamentation. The maximum polar diameter was found in O. americanum (58 ± 5.8 μm) and the maximum equatorial diameter observed in O. basilicum (50.25 ± 1.37 μm). Pollen features of the studied species were discussed and compared based on the current taxonomical concepts. The results showed that pollen traits of the subfamily Nepetoideae was found significant to classify the taxa. Furthermore, pollen features provide additional evidence to distinguish macromorphologically similar taxa from each other.     

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.     

Taxonomic importance of pollen morphology in Veratrum L. (Melanthiaceae) using microscopic techniques

Previous studies reported that intrageneric relationships of genus Veratrum of family Melanthiaceae are controversial and hard to delimit. Therefore, we observed the pollen morphological features of six species in the genus Veratrum in detail using both light microscopy and scanning electron microscopy and investigated their significance for Veratrum taxonomy. Among them, five were studied for the first time. The results demonstrated that pollen grains of Veratrum are medium in size with P/E being 0.31–0.60. Three types of shape in polar view have been observed elliptic, long‐elliptic, or wide‐elliptic with blunt or rounded at both ends. Two types of width of colpus reported narrow or wide, two types of depth of colpus reported deep or flat, and two types of length of colpus reported extend almost or do not extend to the ends, whereas two types of colpus membranes reported absent or obvious. One type of surface ornamentation was noted as reticulate. These results support species Veratrum album and Veratrum lobelianum as well as Veratrum grandiflorum and Veratrum oxysepalum as two independent species, respectively, rather than classifying Veratrum into two sections. Overall, we demonstrated that the ratio of polar axis length to equatorial axial length, pollen characteristics at the polar view, the colpus morphology, and the surface ornamentation of pollen grains of genus Veratrum have important systematic significance in identification and delimitation of species.     

Pollen morphology of Aletris L. (Nartheciaceae) and its systematic significance

In this paper, we studied pollen morphologies of seven species in genus Aletris in detail by light microscopy and scanning electron microscopy. Of these, six species were reported for the first time. The palynological characteristics do not support the infrageneric classification into two clades. The results indicated that pollen grains of Aletris are small or medium with the P/E ratio of 0.36–0.59. They are elliptic or long‐elliptic in the polar view with blunt, round or acute ends and bilateral symmetric with a monosulcate, narrow or wide, deep colpus that has length extending to the ends of pollen grains, obvious or absent colpus membranes. The pollen ornamentation is gemmate, perforated, or reticulate. The sexine is slightly or quite thicker than the nexine.     

Pollen morphology of subfamily Caryophylloideae (Caryophyllaceae) and its taxonomic significance

The Pollen morphology of subfamily Caryophylloideae (Caryophyllaceae) from different phytogeographical region of Pakistan has been evaluated. In this research, 16 species belong to 6 genera of subfamily Caryophylloideae have been studied using light (LM) and scanning electron microscopy (SEM) for both qualitative and quantitative characters. Different palyno‐morphological features were observed including; pollen ornamentation, pore ornamentation, echini arrangement, echinidensity, number of pori, size of polar and equatorial diameter, P/E ratio, exine thickness, and size of pore were studied. The palyno‐morphological characters of subfamily Caryophylloideae have taxonomically significant in identification and delimitation of species. Two pollen types, i.e., subspheroidal (15 species) and prolate (one species Vaccaria hispanica) were observed. Microechinate‐punctate and microechinate‐perforate pollen ornamentation were examined. Two pollen types of pori, i.e., prominent (5 species) and sunken (11 species) ornamentation have been reported. Three types of echini arrangement have been reported irregular, regular and rather regular, while three type of echinidensity (i.e., medium, dense and sparse) were observed. Pori numbers were found different in different species range from 5 in Silene apetala to 19‐35 in Silene vulgaris. Based on different palyno‐morphological characters, taxonomic key was develop for quick and accurate identification. The quantitative data were processed using SPSS software for average, minimum, maximum, and standard error.     

Taxonomic study of Triblemma by using light and scanning electron microscopy

Triblemma Ching is a genus proposed by Ching Ren‐Chang in 1978, and it is composed of two species, Triblemma lancea (Thunb.) Ching and Triblemma zeylanica (Hook.) Ching. There has been much debate on the systematic position of Triblemma, and this genus has always been included in Diplazium. Here, we have described new features of tracheary elements, epidermis, spore, scale, and rachis of Triblemma revealed using light and scanning electron microscopy and proposed that Triblemma is closely related to Athyriopsis, which conflicts with the traditional viewpoint.     

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.     

Leaf and stem micromorphology of Byrsonima sericea DC. by light and scanning electron microscopy

Micromorphological studies were carried out using multiple microscopic techniques on the leaves and stem bark of Byrsonima sericea DC. (Malpighiaceae), a species popularly known as “murici” and used medicinally, in order to identify both qualitative and quantitative features of leaf and stem anatomy and histochemistry as differential parameters to support both the quality control of its ethnodrugs and the taxonomy of the genus. The study was conducted using traditional techniques of plant anatomy, histochemical tests, and the stomatal index (SI). Byrsonima sericea has hypostomatic leaves, anomocytic stomata, and its epidermal walls are anticlinal and straight on the adaxial and curved on the abaxial faces. T‐shaped trichomes were observed mainly on the abaxial surface. The leaf epidermis showed waxes syntopism on both surfaces, with the occurrence of different crystalloid forms on a single phylloplane. The mesophyll is dorsiventral, with 3‐4 collateral vascular bundles. Phenolic compounds, starch, and proteins were identified in the petiole and stem. The SI was 14.5 ± 0.53% (p < .05), but did not showed significant variations. A set of characters were found to be distinctive for the studied species, however, constituting parameters that could be used to separate B. sericea from other species of the genus.     

Pollen morphological investigations of family Cactaceae and its taxonomic implication by light microscopy and scanning electron microscopy

The family Cactaceae is the diversified group of angiosperm plants whose pollen statistics has been used for taxonomic identification. In this article, we present the pollen morphology of eight species belong to seven taxonomically complex genera of Cactaceae including Astrophytum, Cylindropuntia, Echinocereus, Echinopsis, Mammillaria, Opuntia, and Thelocactus using light and scanning electron microscopy. The pollen grains were acetolyzed, measured, described, and electron photomicrographs were taken. Cactaceae can be characterized by presenting different palynomorphological features including pollen type, sculpturing, polar and equatorial diameter, aperture orientation, exine thickness, P/E ratio, and echini features. Four types of pollen shapes, that is, prolate spheroidal (three species), subprolate (two species), prolate (two species), and oblate spheroidal in Echinocereus reichenbachii were observed. The polar and equatorial diameter observed maximum in O. ficus indica 116.95 and 112.27 μm while minimum in M. compressa 38.42 and 21.05 μm. Pollen of two types, tricolpate in members of subfamily Cactioideae and pantoporate in the Opuntioideae were examined. The fertility percentage has been observed maximum in Opuntia macrocentra (83.84%) and minimum in Opuntia ficus‐indica (57.89%). Exine sculpturing showing great variations such as granulate, reticulate, granulate perforate and micro‐echinate foveolate ornamentation was examined only in Echinopsis eyriesii. A key to species, based on pollen micromorphological attributes, has been constructed for correct identification of complex cactus species.     

Using microscopic techniques for taxonomic implications of seed and fruits of Delphinium L. (sensu lato) (Ranunculaceae)

In present research, micromorphological characters of the genus Delphinium L. (sensu lato) Ranunculaceae seeds and fruits were studied using microscopic techniques. A total of 37 species were studied using light (LM) and scanning electron microscopy (SEM) to evaluate seeds and fruits micromorphological characteristics for the taxonomic identification and species relationship in the genus. For each taxon, 33 characters were observed and studied. All characters were analyzed using MVSP software and UPGMA method by using dissimilarity index (average taxonomic distance). Considerable differences were observed in trichomes shape of fruit and seed color features. The results revealed that all studied taxa are separated into three clades. The first clade consists of D. pallidiflorum and D. semibarbatum, which have black seed and two types of glandular and crispate trichomes in fruit. The second clade consists of D. speciosum, D. szowitsianum, D. dasystachyson, D. aquilegifolium, D. ursinum, C. trigonelloides, and C. camptocarpa, which had a brown color seeds, two types of glandular and villous trichomes on fruits also have been observed. The remaining species were classified as third clade having various colors including yellow, black, and brown color seeds, the trichome shapes included one of the following forms strigose, villous, trichomes less, glandular, hooked, and crispate trichomes in fruits. The aim of the present study is to use micromorphological characters of seeds and fruits for the taxonomic identification and species delimitation in the genus Delphinium.     

Application of microscopy in authentication and distinguishing of 11 Paris species in West Sichuan

In the trade and raw drug market, the medicinal plants of genus Paris are available in the form of rhizomes without any vouchers, thus making it difficult to identify and distinguish the different species of Genus Paris. Recent studies have shown that the species of Paris possess different chemical constituents, pharmacological activities, and efficiencies in clinical application. To distinguish 11 species of Paris collected from the western Sichuan province of China and ensure their safety and efficacy, in the present work, the microscopic characteristics of rhizomes and the crude drug powder of the 11 species of Paris were compared using a light microscope according to the usual microscopic techniques. The results of the microscopic features were systematically described and illustrated. The differences among these species are great enough that the identity of most material can be easily determined. Also, semiquantitative and quantitative micrographic parameter tables were simultaneously presented. Further, the key authentication parameters based on these anatomic characteristics analyzed was drawn up and presented for the Paris species studied. The study indicated that light microscopy and related techniques could provide a method that is convenient, feasible, and can be unambiguously applied in the authentication of species of Paris. This information is required not only for the identification procedures that guarantee the utilization of the appropriate raw material, but also for the quality control standards demanded. Microsc. Res. Tech. 2009. © 2009 Wiley‐Liss, Inc.