Anatomical, Pollen Grains and Seed Exomorphic Studies on Five Species of Cleome L (Cleomaceae Bercht. & Presl) Collected from South West of Saudi Arabia
Wael Taha Kasem
Botany and Microbiology Department, Faculty of Science, Al-Azhar University, Cairo, Egypt
To cite this article:
Wael Taha Kasem. Anatomical, Pollen Grains and Seed Exomorphic Studies on Five Species of Cleome L (Cleomaceae Bercht. & Presl) Collected from South West of Saudi Arabia. Journal of Plant Sciences. Vol. 4, No. 2, 2016, pp. 29-36. doi: 10.11648/j.jps.20160402.13
Received: March 16, 2016; Accepted: March 28, 2016; Published: April 13, 2016
Abstract: Micromorphological attributes for five species of C. gynandra L., C. viscosa L, C. scaposa DC, C. paradoxa B.BR and C. brachycarpa Vahl ex DC. belonging to genus Cleome L. in south west of Saudi Arabia was studied by light (LM) and scanning electron microscope (SEM). Studies are interested by hair characters, stomata behaviour, stem and leaf anatomy, pollen grains and seed exomorphic attributes. Stem and leaf anatomy differentiation indicated good taxonomic tools to between species the species, the result of the stomata frequency was significant where the highest stomatal index (SI) of 36.4% was observed in C. gynandra while the lowest one (21.6%) was noticed in C. Paradoxa. However, pollen grains and seed exomorphic is significantly helpful at the specific level. Prolate, tricolpate, monad pollen grains are found in all studied species pollen grains differed in P/E (polar axis / equatorial axis) ratio and exine measurements. Also, every Cleome species has their own seed ornamentation which will be helpful information in differentiation of the studied species. On the basis of seed ultrastructure, three main types of seed ornamentation; reticulate, verrucate and oceallate were revealed. Analysed data by Minitab (Version 13.1) statistical program showed C. gynandra in single level while the remainders were grouped in one cluster in which C. viscocs and C. scaposa are excluded in a alone separated levels in addition the highest similarity percentage appered between C. paradoxa and C. brachycarpa.
Keywords: Cleome L., Anatomical Features of Stem and Leaf, Pollen Grains, Seed Exomorphic, Numerical Analysis
Cleome L, is the largest genus from family Cleomaceae comprising 180 to 200 species of herbaceous annual or perennial plants and shrubs widely distributed in tropical and subtropical regions. The major diversity of Cleome is restricted to tropical regions (Raghavan, 1993). Cooke (1903) has described ten species of Cleome under Capparidaceae. Earlie floristic workers like Hooker and Thomson (1872) and Cooke (1903) put Cleome and other allied genera under the family Capparidaceae, however, recently all these genera are separated taxonomically and put under a separate family i.e. Cleomaceae. Many of workers previously treated genus Cleome in family Capparaceae, until moden techniques which revealed major Cleomaceae members are closer to Brassicaceae than Capparaceae (Stevens, 2001). Seven species are found to be distributed in the region of Kolhapur district (Yadav and Sardesai, 2002). Khalifa and EL-Gohary (1982) studied micro morphological attributes like anatomical features of stem, leaves and petioles to evaluate their significance in differentiating the nine Cleome spp. of Egypt. Mark et al. (1993) have studied different seed plants for their phylogeny and reported that the polyphyletics that occurs in capparidales is due to some members of capparaceae along with Cleome. Sanchez (2005) tried to investigate phylogenetic and morphological relationships within, South American Cleome species. Solomon et al. (1973) studied the pollen morphology of Cleome serrulata L. and reported that Cleome L. pollen grains are prolate, tricolporate, tectum reticulate with micropunctate lamina. Ruiz and Escale (1997) studied seed morphology of nininten species of Cleome L. from Venezuela using the scanning electron microscope and observed different characters. Richard et al. (1997), Nyananyo (1990), Mbagwu and Edeoga (2006) stated that pollen grains of the Cleome L. either circular or oval in outline.and tricolpate with a porus aperture type and used pollen grain morphology to produce a more acceptable classification of this species. The present work attempt to differences and specify of micromorphological attributes between the examined species with the use of modern statiscal analysis which in turn an attempts to understand the interrelationship among the Cleome species.
2. Materials and Methods
Fresh samples of the five species of the cleome obtained from different localities of south west of Saudi Arabia. Specimens are identified according to Alfarhan et al., 2005 and Masrahi 2012. For anatomical studies epidermal leaf and stem cross sections were done using freshly-collected materials fixed in 70% ethanol, this material was cut free hand to make semi-permanent carried out according to the usual Johansen’s methods 1940. The slides were examined under the light microscope (LM) at magnification power of 40 × and photographed. Various anatomical features, such as number and type of stomata, the stomatal index (SI) is carried out according to Salisbury (1927) equation (SI = S/S+ E×100). For pollen grain preparations, one or two unopened ripe flower buds were removed from each fresh materials. The buds were dissected in wetting agent and pollen was removed from the anthers and washed in deionized water. Pollen grains were then acidified using glacial acetic acid and acetolysed according to the Erdtman technique (Erdtman, 1952). Slides were examined using light microscope and measurements were made of the length of the polar and equatorial axes (P and E) in optical section from ten pollen grains using a calibrated eyepiece graticule, pollen grains are photographed. Terminology of pollen grains are carried out according to Punt et al. (1994) and Erdtman, 1952. In the same way, dry mature seeds were cleaned, and examined by light microscope to show the different exomorphic parameters viz. shape, dimensions, colour and seed surface texture. Five to ten seeds for each species were taken to cover the range of variations in seed exomorphic investigations by SEM; the specimens fixed to stubs with an adhesive and placed on the revolving discs of Joel fine coat ion sputter (Joel, JFC 1100). Each seed was uniformly coated with gold. Specimens stubs were then fixed to the specimen holder of scanning electron microscope (Joel JSM 350) maintained at accelerating potential voltage of 15 Kv. and photomicrographed. The statistical analysis of the identified data was carried out by multivariate cluster analysis using Minitab 13.1 statistical program.
3. Results and Discussion
Micromorphological attributes including stomata behavior, leaf and stem anatomy are recorded in the following Table.
SI = stomatal index, Epi= epidermis, Cu= cuiticle, V.B= vascular bundles, Int= intrafascular region, Mes= mesophyll, Hy = hypodermis, glan=glandular, pap.= papillose
3.1. Cleome gynandra L
Diacytic stomata is common. Stomatal index 36.4, glandular hairs are observed on both leaf surface, midrib region is swollen, epidermis has single layered. Mesophyll is not differentiated into palisade and spongy tissues. Vascular bundles are thick and conjoint in the medvain. Stem in transvers sections shows single layere of round epidermal cells without cuticle. 6-7 cortical layers are observed and compactly arranged stem hypodermis. Vascular bundles are oval shaped wide compact cells of 8-10 rows of the interfascicular region found. Xylem long radial multiples ranged from 4-6 rays. Pith is occupied by parenchyma round cells, with intercellular spaces (Figs. 1 A, B & C). Pollen grains prolate, tricolporate, monad, polar axis (P) 15.3 µm, and equatorial diameter (E) 14.4 µm. P/E ratio is 1.06 µm, colpi 12.6 µm long, 19.7 µm wide, exine 1.54 µm thick. (Figs. 2 A). Seeds is browen in color, circular or curved ahaped, seed size being 1.0×1.5 mm, reticulate form with wax like materials appeared in SEM examinations (3 A, B & C).
3.2. Cleome viscosa L
Anisocytic stomara is common, stomatal index being 28.5%, glandular hairs are present on the leaf sides, midrib region is swollen with lateral wings. Epidermis is round has single layered of round cells. Mesophyll tissue is differentiated into palisade and spongy cells. Hypodermis has two layers. Vascular bundles are not conjoint and surrounded by bundle sheath. In stem anatomy one single layer of round cells found on cuticle epidermis. 6-7 cortical layers of parenchyma and collenchyma cells compactly arranged hypodermis region, vascular bundles are rounded consist of interfascicular cambium ring. The Interfascicular region is wide representing 6-8 rows of compact cells. Xylem vessels is oval and few. Pith is composed of round thich walled cells without intercellular spaces (Figs. 1 D, E & F). Pollen grains is prolate, tricolporate, monad. Polar axis is 19.5 µm and equatorial axis is 18.9 µm. P/E ratio is 1.03 µm, colpi 11.6 µm long, 17.6 µm. Exine 2.6 µm thick thicker than nexine (Fig. 2 B). Seeds is circular, size 1.0×1.2 mm, browen in color, curved and nearly circular, verrucate form is observed by SEM on the seed exomorphic (Figs. 3 D, E & F).
Figure 1. Stomata (x= 400) and transver sections of stem and leaf (x= 100) of Cleome species. C.gynandra (A., B & C.), C. viscosa (D., E & F), C. paradoxa (G., H & I.), C. scoposa (. J., K. & L.), C. brachycarpa ( M., N & O), column A for stomata, column B for leaf anatomy and column C for stem anatomy.
3.3. Cleome paradoxa DC
Diacytic stomara is present, stomatal index 21.6 %. A few glandular hairs are observed on the adaxial surface. Midrib region is not much swollen but shows presence of lateral wings. Epidermis is elongated single layered and covered with a thick cuticle. Palisade and spongy tissues absent. Hypodermis is well distinguishable about three to five layers. Vascular bundles are complete and fully developed, separated in midrib surrounded by bundle sheath. In stem cross sections; two layer of round epidermal cells found, with thick cuticle layer. five to six layers of parenchyma and collenchyma cells are compactly arranged the stem hypodermis. They consist of distinct interfascicular cambium ring. Compact cells of 5-7 rows of the interfascicular region found. Xylem vessels is round, more in number (Figs. 1 G, H & I). Pollen grains subprolate, tricolporate. Polar axis is 21.8 µm and equatorial axis 18.7 µm. P/E ratio is 1.16, colpi 13.6 µm long, 22.7 µm wide, exine 1.54 µm thick (Fig. 2 C). Seeds of this species is oval, pale brown in color, size being 1.5×2.0 mm pressed ocellate showed on seed surface (Figs. 3 G, H & I).
3.4. Cleome scaposa B.BR
Anomocytic stomata found, stomatal index represents 27,1%, no glandular hairs are present on the epidermis of both the leaf surfaces. Midrib region is swollen with lateral two wings. Mesophyll tissue is not differentiated into palisade and spongy cells. Leaf epidermis has single layered of elongated cells. Hypodermal layers is absent in the medvain. Vascular bundles showed to be discontinuous in the midrib region. In stem cross section two layers of elongated epidermal cells found. Four to five heterogeneous collenchyma cells compactly arranged the cortical region. Several rounded vascular bundles appeared in the outline. Xylem vessels are oval and few. The interfascicular region is narrow due to large number of vascular bundles. Pith has round thin walled cells without intercellular spaces (Figs. 1 J, K & L). Pollen grains subprolate, tricolporate, Polar axis is 17.2 µm and equatorial diameter is13.8 µm. P/E ratio is 1.24 µm, colpi 18.2 µm long, 16.8 µm wide, exine 12.6 µm thicker than nexine (Fig. D). Circular seeds are noticed, seed size is 2.2×2.8 mm, bright black in color. reticulate, raised form appeared by SEM on the surface (Figs. J, K & L).
3.5. Cleome brachycarpa Vahl ex DC
Diacytic stomata is observed, stomatal indexes 33.8%, glandular hairs are present on the epidermis of both the leaf surface. Midrib region is swollen, mesophyll tissue is differentiated into palisade and spongy cells. Epidermis one layer of elongated cells, hypodermis is absent. Vascular bundles are separated in medvain. Single layered of epidermis without intercellular spaces and multilayered cortical region are obvious. Circular vascular bundles are present in between a few of compact cells of 1-2 raw of the interfascicular region found. Oval xylem vessels are noticed. pith is composed of oval parenchyma cells (Figs. 1 M, N & O). Pollen grains are prolate, tricolpate, Polar axis is 16.4 µm and equatorial axis is 17.5 µm. P/E ratio is 0.94, colpi 14.6 µm long, 16.7 wide, exine 2.60 µm. Seeds are oval, pale black in color (Fig. 2 E). Seed size is 1.1×1.4 mm. SEM reveals filled papillate form on their seed surface (Figs. 3 M, N & O).
⃰ Cleft is small and narrow or large and spherical space in seed
Diacytic stomata are common in C.gynandra, C. paradoxa and C. brachycarpa. Anisocytic stomata are restricted to C.viscosa whereas anomocytic is found in C. scaposa. Stomata density and stomata index are found higher on the lower leaf surface than that on upper leaf. On the basis of arrangement of the epidermal cells near the guard cell, more than 25 main types of stomata in dicots have been recognized (Metcalfe and Chalk, 1983). Stace (1980) reported 31 different types of stomata among dicotyledonous plants. It is evident that, variations in stomatal number and index in studied species indicated that responseof these species to different ecological conditions in which they grow.
The maximum number of stomata index was recorded in C. gynandra (SI-36.4%) because of this species grows mostly near water rich area or along water reservoir while the lowest one (21.6%) was noticed in C. Paradoxa. Leaf and stem anatomical features and different measurements between the species are recorded in Table 1 and showed in Figure. Micromorphological features such as size, shape and characters of seeds have indicated the presence of variations among the examined species. Dendrogram of Figure 4 obtained from all studied data of Table 4 showed that, Cleome gynandra is separated in a single level which supported by the anatomical and seed coat microstructures results. The second group included the remainders. In the second group C. viscocs and C. scaposa are excluded in a separated levels also, the highest similarity percentage was C. paradoxa and C. brachycarpa.The transverse sections of the stem and leaves in five Cleome species were externally bounded by the epidermis, which has one to two elongated cell layers in C.scoposa and C.paradoxa while rounded thin in the three reminders. Two to three layers of parenchyma cells are present in the studied species are thin walled and mostly hexagonal except C.scoposa is rounded shape. Cortical zone has collenchyma cells differed in row number; ranged from 4 -5 layers in C. scoposa, C. brachycarpa and C. pardoxa. But ranged from 6 -7 layers in C.gynandra and C.viscosa. Vascular bundles are varies in number and form between the taxa. Conjoint vascular bundles found in C.brachycarpa whereas separated found in the remainders. Irregular phloem bands are found in all studied taxa. Interfascicular rays appeared homogenous in C. viscosa heterogeneous in the remainders. All studied species show much similarity with their pollen characters like prolate or lobate shape, monad unit, tricolpate aperture. These characters may be helpful to strengthen their taxonomic position under same taxa. Pollen grain data and different measurements by light microscope (LM) between the taxa are recorded in Table 2 and showed in Figure 2. All the five species of Cleome showed distinct variations in their pollen shape and structure and were differing from earlier studies particularly in exine and aperture measurements. Prolate, tricolporate are common in all taxa except C. brachycarpa, such results in accordance with Solomon et al. (1973) and Thulin (2002) and Mitra (1970). It appears that P/E ratio of all Cleome ranged from 0.9 μm to 1.36 μm. The highest P/E found in C.scaposa while the lowest one found in C. brachycarpa which is agree with other present results. A scanning electron microscope (SEM) considers high magnification capacity is used to study minute biological material like pollen grains and seed exomorpic which can be a good additional tool in taxonomic discrimination and classification of critical plant species. LM studies for five Cleome species revealed different aperture types and ornamentation patterns. Also seed surfaces exhibited diversity in the surface ornamentation (Figure. 3 and Table. 3). SEM studies have shown every Cleome species have their own ornamentation characteristics which will be helpful in identification of plant species. It is evident that seeds of all the Cleome species studied are strongly curved and nearly circular, more or less horseshoe shaped. Interestingly, however, the seed ornamentation differed in some species than results of Lupita (2005) which studied seed morphology of several Cleome species and their ornamentation. Seeds of Cleome also exhibited a small and narrow or large and spherical space referred to as (cleft). Such results in accordance with Aparadh and Karadge (2010). The obtained seed exomorphic data between the studied species reveals a great variation which will be helpful in identification of the studied species. Such results are agreement with results of Kasem et al., 2011 on 32 taxa of Brassicaceae.