Journal of Sciences 2016; 4(2): 29-36 http://www.sciencepublishinggroup.com/j/jps doi: 10.11648/j.jps.20160402.13 ISSN: 2331-0723 (Print); ISSN: 2331-0731 (Online)

Anatomical, Pollen Grains and Seed Exomorphic Studies on Five Species of L ( Bercht. & Presl) Collected from South West of Saudi Arabia

Wael Taha Kasem

Botany and Microbiology Department, Faculty of Science, Al-Azhar University, Cairo, Egypt

Email address : [email protected] 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

(Yadav and Sardesai, 2002). Khalifa and EL-Gohary (1982) 1. Introduction studied micro morphological attributes like anatomical features Cleome L, is the largest genus from family Cleomaceae of stem, leaves and petioles to evaluate their significance in comprising 180 to 200 species of herbaceous annual or differentiating the nine Cleome spp. of Egypt. Mark et al. perennial and shrubs widely distributed in tropical and (1993) have studied different seed plants for their phylogeny subtropical regions. The major diversity of Cleome is restricted and reported that the polyphyletics that occurs in capparidales to tropical regions (Raghavan, 1993). Cooke (1903) has is due to some members of capparaceae along with Cleome . described ten species of Cleome under Capparidaceae. Earlie Sanchez (2005) tried to investigate phylogenetic and floristic workers like Hooker and Thomson (1872) and Cooke morphological relationships within, South American Cleome (1903) put Cleome and other allied genera under the family species. Solomon et al. (1973) studied the pollen morphology Capparidaceae, however, recently all these genera are of Cleome serrulata L. and reported that Cleome L. pollen separated taxonomically and put under a separate family i.e. grains are prolate, tricolporate, tectum reticulate with Cleomaceae. Many of workers previously treated genus micropunctate lamina. Ruiz and Escale (1997) studied seed Cleome in family Capparaceae, until moden techniques which morphology of nininten species of Cleome L. from Venezuela revealed major Cleomaceae members are closer to using the scanning electron microscope and observed different Brassicaceae than Capparaceae (Stevens, 2001). Seven species characters. Richard et al. (1997), Nyananyo (1990), Mbagwu are found to be distributed in the region of Kolhapur district and Edeoga (2006) stated that pollen grains of the Cleome L. 30 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 either circular or oval in outline.and tricolpate with a porus acetolysed according to the Erdtman technique (Erdtman, aperture type and used pollen grain morphology to produce a 1952). Slides were examined using light microscope and more acceptable classification of this species. The present measurements were made of the length of the polar and work attempt to differences and specify of micromorphological equatorial axes (P and E) in optical section from ten pollen attributes between the examined species with the use of grains using a calibrated eyepiece graticule, pollen grains are modern statiscal analysis which in turn an attempts to photographed. Terminology of pollen grains are carried out understand the interrelationship among the Cleome species. according to Punt et al . (1994) and Erdtman, 1952. In the same way, dry mature seeds were cleaned, and examined by 2. Materials and Methods light microscope to show the different exomorphic parameters viz. shape, dimensions, colour and seed surface Fresh samples of the five species of the cleome obtained texture. Five to ten seeds for each species were taken to cover from different localities of south west of Saudi Arabia. the range of variations in seed exomorphic investigations by Specimens are identified according to Alfarhan et al ., 2005 SEM; the specimens fixed to stubs with an adhesive and and Masrahi 2012. For anatomical studies epidermal leaf and placed on the revolving discs of Joel fine coat ion sputter stem cross sections were done using freshly-collected (Joel, JFC 1100). Each seed was uniformly coated with gold. materials fixed in 70% ethanol, this material was cut free Specimens stubs were then fixed to the specimen holder of hand to make semi-permanent carried out according to the scanning electron microscope (Joel JSM 350) maintained at usual Johansen’s methods 1940. The slides were examined accelerating potential voltage of 15 Kv. and under the light microscope (LM) at magnification power of photomicrographed. The statistical analysis of the identified 40 × and photographed. Various anatomical features, such as data was carried out by multivariate cluster analysis using number and type of stomata, the stomatal index (SI) is carried Minitab 13.1 statistical program. out according to Salisbury (1927) equation (SI = S/S+ E×100) . For pollen grain preparations, one or two unopened 3. Results and Discussion ripe flower buds were removed from each fresh materials. The buds were dissected in wetting agent and pollen was Micromorphological attributes including stomata behavior, removed from the anthers and washed in deionized water. leaf and stem anatomy are recorded in the following Table. Pollen grains were then acidified using glacial acetic acid and Table 1. Studied aanatomical attributes in the five species of Cleome.

Characters Stomata (leaf Stem characters Leaf characters SI (%) Species lower surface) Epi. Cu. V.B. Int. Hair Ep Hy. V.B. Mes. C. gynandra diacytic 36.4 one absent oval wide glan. one present conjoint absent C. viscosa anisocytic 28.5 one present circular wide pap. Two present separated present C. paradoxa diacytic 21.6 two present oval wide -- one present separated absent C. scaposa anomocytic 27.1 one present oval wide -- one absent separated present C. brachicarpa diacytic 33.8 one present oval narrow glan one absent separated Present

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. examinations (3 A, B & C).

Diacytic stomata is common. Stomatal index 36.4, glandular 3.2. Cleome viscosa L. hairs are observed on both leaf surface, midrib region is swollen, epidermis has single layered. Mesophyll is not Anisocytic stomara is common, stomatal index being 28.5%, differentiated into palisade and spongy tissues. Vascular glandular hairs are present on the leaf sides, midrib region is bundles are thick and conjoint in the medvain. Stem in swollen with lateral wings. Epidermis is round has single transvers sections shows single layere of round epidermal cells layered of round cells. Mesophyll tissue is differentiated into without cuticle. 6-7 cortical layers are observed and compactly palisade and spongy cells. Hypodermis has two layers. arranged stem hypodermis. Vascular bundles are oval shaped Vascular bundles are not conjoint and surrounded by bundle wide compact cells of 8-10 rows of the interfascicular region sheath. In stem anatomy one single layer of round cells found found. Xylem long radial multiples ranged from 4-6 rays. Pith on cuticle epidermis. 6-7 cortical layers of parenchyma and is occupied by parenchyma round cells, with intercellular collenchyma cells compactly arranged hypodermis region, spaces (Figs. 1 A, B & C). Pollen grains prolate, tricolporate, vascular bundles are rounded consist of interfascicular monad, polar axis (P) 15.3 µm, and equatorial diameter (E) cambium ring. The Interfascicular region is wide representing 14.4 µm. P/E ratio is 1.06 µm, colpi 12.6 µm long, 19.7 µm 6-8 rows of compact cells. Xylem vessels is oval and few. Pith wide, exine 1.54 µm thick. (Figs. 2 A). Seeds is browen in is composed of round thich walled cells without intercellular color, circular or curved ahaped, seed size being 1.0×1.5 mm, spaces (Figs. 1 D, E & F). Pollen grains is prolate, tricolporate, reticulate form with wax like materials appeared in SEM 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 Journal of Plant Sciences 2016; 4(2): 29-36 31

µm thick thicker than nexine (Fig. 2 B). Seeds is circular, size verrucate form is observed by SEM on the seed exomorphic 1.0×1.2 mm, browen in color, curved and nearly circular, (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.

Figure 2. Polar view of pollen grains of Cleome species by light microscope (LM; x=1500). A.. C. gynandra, B. C. viscosa, C. paradoxa, D. C. scoposa, E. C. brachycarpa. 32 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

3.3. Cleome paradoxa DC. found. Four to five heterogeneous collenchyma cells compactly arranged the cortical region. Several rounded Diacytic stomara is present, stomatal index 21.6 %. A vascular bundles appeared in the outline. Xylem vessels are few glandular hairs are observed on the adaxial surface. oval and few. The interfascicular region is narrow due to large Midrib region is not much swollen but shows presence of number of vascular bundles. Pith has round thin walled cells lateral wings. Epidermis is elongated single layered and without intercellular spaces (Figs. 1 J, K & L). Pollen grains covered with a thick cuticle. Palisade and spongy tissues subprolate, tricolporate, Polar axis is 17.2 µm and equatorial absent. Hypodermis is well distinguishable about three to diameter is13.8 µm. P/E ratio is 1.24 µm, colpi 18.2 µm long, five layers. Vascular bundles are complete and fully 16.8 µm wide, exine 12.6 µm thicker than nexine (Fig. D). developed, separated in midrib surrounded by bundle Circular seeds are noticed, seed size is 2.2×2.8 mm, bright sheath. In stem cross sections; two layer of round epidermal black in color. reticulate, raised form appeared by SEM on the cells found, with thick cuticle layer. five to six layers of surface (Figs. J, K & L). parenchyma and collenchyma cells are compactly arranged the stem hypodermis. They consist of distinct 3.5. Cleome brachycarpa Vahl ex DC. interfascicular cambium ring. Compact cells of 5-7 rows of the interfascicular region found. Xylem vessels is round, Diacytic stomata is observed, stomatal indexes 33.8%, more in number (Figs. 1 G, H & I). Pollen grains glandular hairs are present on the epidermis of both the leaf subprolate, tricolporate. Polar axis is 21.8 µm and surface. Midrib region is swollen, mesophyll tissue is equatorial axis 18.7 µm. P/E ratio is 1.16, colpi 13.6 µm differentiated into palisade and spongy cells. Epidermis one long, 22.7 µm wide, exine 1.54 µm thick (Fig. 2 C). Seeds layer of elongated cells, hypodermis is absent. Vascular of this species is oval, pale brown in color, size being bundles are separated in medvain. Single layered of 1.5×2.0 mm pressed ocellate showed on seed surface (Figs. epidermis without intercellular spaces and multilayered 3 G, H & I). cortical region are obvious. Circular vascular bundles are present in between a few of compact cells of 1-2 raw of the 3.4. Cleome scaposa B.BR. interfascicular region found. Oval xylem vessels are noticed. pith is composed of oval parenchyma cells (Figs. 1 Anomocytic stomata found, stomatal index represents M, N & O). Pollen grains are prolate, tricolpate, Polar axis 27,1%, no glandular hairs are present on the epidermis of both is 16.4 µm and equatorial axis is 17.5 µm. P/E ratio is 0.94, the leaf surfaces. Midrib region is swollen with lateral two colpi 14.6 µm long, 16.7 wide, exine 2.60 µm. Seeds are wings. Mesophyll tissue is not differentiated into palisade and oval, pale black in color (Fig. 2 E). Seed size is 1.1×1.4 spongy cells. Leaf epidermis has single layered of elongated mm. SEM reveals filled papillate form on their seed surface cells. Hypodermal layers is absent in the medvain. Vascular (Figs. 3 M, N & O). bundles showed to be discontinuous in the midrib region. In stem cross section two layers of elongated epidermal cells Table 2. Pollen grain characters in five species of Cleome.

Species C. gynandra C. viscosa C. paradoxa C. scaposa C. brachycarpa Data Pollen shape oval circular triangular triangular triangular Apertures type tricolporate tricolporate tricolporate tricolporate tricolpate Polar axis (P) (µm) 15.3 19.5 21.8 17.2 16.4 Equatorial axis (E) (µm) 14.4 18.9 18.7 13.8 17.5 P/E 1.06 1. 03 1.16 1.24 0.94

Table 3. Seed exomorhic attributes in five species of Cleome.

Species C. gynandra C. viscosa C. paradoxa C. scaposa C. brachycarpa Data Seed color browen browen pale brown pale black bright black Seed size (mm) 1.0×1.5 1.0×1.2 1.6×2.0 0,5-1.0 1.1-1.4 Seed shape circular circular oval circular rounded sculpture reticulate,waxed verrucate ocealate reticulate, raised ocealate Seed cleft ⃰ present present absent present present cleft. Aperture open open --- open closed

⃰ Cleft is small and narrow or large and spherical space in seed Journal of Plant Sciences 2016; 4(2): 29-36 33

Figure 3. Seed Exomorphic by light microscope (LM) and scanning electron micrographs(SEM) of Cleome. 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.).

Diacytic stomata are common in C.gynandra, C. paradoxa than 25 main types of stomata in dicots have been recognized and C. brachycarpa . Anisocytic stomata are restricted to (Metcalfe and Chalk, 1983). Stace (1980) reported 31 C.viscosa whereas anomocytic is found in C. scaposa . different types of stomata among dicotyledonous plants. It is Stomata density and stomata index are found higher on the evident that, variations in stomatal number and index in lower leaf surface than that on upper leaf. On the basis of studied species indicated that responseof these species to arrangement of the epidermal cells near the guard cell, more different ecological conditions in which they grow. 34 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

Table 4. Micromorphological attributes used in Statiscal program between the studied species.

Species C. gynandra C. viscosa C. paradoxa C. scaposa C. brachycarpa Data Hair types 1-glandular hair, 2-papillose 1 2 1 1 1 Stomata type 1-diacytic, 2-anisocytic, 3-anomocytic 1 2 1 3 1 Leaf anatomy Hypoderm 1- two layers to three layers 2 1 2 3 3 2-more, 3-absent Medvain Vascular bundles 1-conjoint 2- separated 1 2 2 2 2 Mesophyll 1-differeniated,2-udifferentiated 2 1 2 2 1 Leaf bundle sheath 1-present, 2- absent 2 1 1 2 2 Stem anatomy Cuticle layer 1-present, 2- absent 1 1 2 2 2 Vascular bundles 1-oval, 2-circular 1 2 1 1 1 Intrafasicular region 1-wide, 2- narrow 1 1 1 1 2 Pollen grain characters Size 1-Less than 1 µm, 2- from 1-1.2 µm, 3-more than 1.2 µm 2 2 2 3 1 Shape 1-oval, 2-circular, 3-triiangular 1 2 2 2 2 Aperture type 1- tricolporate, 2- tricolpate 1 1 1 1 2 Seed exomorphic characters Seed size 1-≥1.5mm 2-≤ 1.5mm 1 1 2 1 1 Seed form 1- oval, 2- round 1 2 1 1 1 Seed colour 1- browen, 2- black 1 1 1 2 2 Seed Clift 1-present , 2- Absent 2 2 1 1 1 Clift aperture 1-open, 2- closed 1 1 2 2 1 Seed ornamentation 1-reticulate, 2-ocealate,3- verrucate 1 3 2 1 2

Figure. 4. The relationships among the studied Cleome based on the micromorphological data.1. C. gynandra,2. C. viscosa, 3. C. paradoxa,4. C. scoposa, 5. C. brachycarpa. Journal of Plant Sciences 2016; 4(2): 29-36 35

The maximum number of stomata index was recorded in helpful in identification of plant species. It is evident that C. gynandra (SI-36.4%) because of this species grows mostly seeds of all the Cleome species studied are strongly curved near water rich area or along water reservoir while the lowest and nearly circular, more or less horseshoe shaped. one (21.6%) was noticed in C. Paradoxa . Leaf and stem Interestingly, however, the seed ornamentation differed in anatomical features and different measurements between the some species than results of Lupita (2005) which studied species are recorded in Table 1 and showed in Figure. seed morphology of several Cleome species and their Micromorphological features such as size, shape and ornamentation. Seeds of Cleome also exhibited a small and characters of seeds have indicated the presence of variations narrow or large and spherical space referred to as (cleft). among the examined species. Dendrogram of Figure 4 Such results in accordance with Aparadh and Karadge obtained from all studied data of Table 4 showed that, (2010). The obtained seed exomorphic data between the Cleome gynandra is separated in a single level which studied species reveals a great variation which will be supported by the anatomical and seed coat microstructures helpful in identification of the studied species. Such results results. The second group included the remainders. In the are agreement with results of Kasem et al ., 2011 on 32 taxa second group C. viscocs and C. scaposa are excluded in a of Brassicaceae. 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 References bounded by the epidermis, which has one to two elongated cell layers in C.scoposa and C.paradoxa while rounded thin [1] Alfarhan, A., Al-Turky, T. and Basahy, A. (2005). 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