International Journal of Basic & Applied Sciences IJBAS-IJENS Vol:19 No:03 15

Comparative Study of the Leaf Morphology and Anatomy of Selected Species: Strychnos Spinosa Lam., Strychnos Innocua Del. and Strychnos Usambarensis Gilg Found in Three Ecological Zones in

ASUZU, CHINWE U., NWOSU, MARIA O.

Abstract-- The morphological and anatomical study of three of literature on the detailed description of the anatomy of Nigerian species of Strychnos: S. spinosa, S. innocua and S. the leaf lamina, midrib and stomatal types in the three usambarensis was carried out. The leaves are dorsiventrally Nigerian species of Strychnos. This study aims at examining flattened, petiolate, with distinct palmate venation. The the morphological and anatomical variables of the leaf transverse section of leaf lamina revealed uniseriate epidermis lamina, midrib and stomatal types of the three Nigerian with double-layered palisade mesophyll and loosely arranged species of Strychnos. spongy mesophyll. Rosette crystals were found in the mesophyll of S. spinosa and S. usambarensis. Stomata are hypostomatic and the measurement of the length of guard cells MATERIALS AND METHODS of S. innocua differed from those of S. spinosa and S. The species of Strychnos were collected randomly usambarensis. Petiole anatomy revealed seven variously sized from three ecological zones in Nigeria where they grow. S. and separate vascular bundles in S. innocua and S. spinosa but innocua was collected from Rigasa village near Kaduna with a single arc shaped bundle in S. usambarensis. (Sudan savanna). S. spinosa was collected from Kuje village near Abuja ( savanna), while S. usambarensis was Index Term-- Strychnos spinosa, S. innocua S. usambarensis, collected from Ohebe-Dim village near Nsukka (Derived palmate venation, rosette crystals. savanna). Fresh leaves were collected from ten samples each. For the stomatal studies, freshly collected leaves of the INTRODUCTION three species were washed with distilled water to remove Strychnos belongs to the family and is any dust or dirt clinging on the leaves. The leaves were put made up of 200-400 species (Cruz, 2008; Rajesh et al. in three separate and well-labeled petri dishes. Fifteen to 2009). Strychnos is the largest genus of the family twenty ml of bleach (hydrogen peroxide) solution was Loganiaceae (Fraiser, 2011). Mwamba, 2006; Rajesh et poured into the petri dishes to completely submerge the al.2009 and Orwa et al.2009 all reported that, it was first leaves. The solution was left for 24-48 hours in order to described by Linnaeus based on S. nux-vomica which is the bleach the leaves. The bleached leaves were removed type species. carefully and rinsed thrice with distilled water to remove The genus Strychnos is well known for its alkaloid any trace of the bleach. The abaxial and adaxial surfaces of production, particularly strychnine from S. nux-vomica the leaves from each petri dish were scraped off. Viewing of which has been popularized for its potential nefarious uses. the adaxial and abaxial surfaces of the scraped leaves was Angenot (1988) observed that although investigation into done using the Zeiss light microscope. Another set of the genus Strychnos has been going on for some time, the leaves of freshly collected samples were cut into small African members suffered a long period of neglect. pieces and stored in well labeled bottles containing F.A.A. However, work on the uses of S. spinosa and S. innocua has (20 ml of 40% formaldehyde, 30 ml of distilled water, 30 ml been reported by Asesa et al.(2005); Mwamba (2006) of 100% ethanol and 20 ml of glacial acetic acid) in the Rajesh et al.(2009); Augustino et al.(2011); Kokwaro Anatomy Laboratory of the Department of Science (1976); F. A. O.(1983.); Mbuya et al (1994) and Orwa et al. and Biotechnology , University of Nigeria Nsukka. This (2009). Angenot (1988) and Cruz (2008) described the uses mixture helped to preserve the specimens and prevent any of S. usambarensis. Phytochemical studies on S. spinosa form of microbial growth. Transverse sections of the midrib has been carried out by Bisset (1970); Adebiyi and and petiole were made using a Reichert sliding sledge Sofowora(1978); Philippe et al. (2005); Morah (1982, microtome. The sections were cut at 10u thickness and 2011). Studies on the phytochemicals in S. innocua has stored in well-labeled petri dishes containing 70% ethyl been investigated by Corsaro et al. (1995); Philippe et al. alcohol. (2005) and Bello et al.(2008). There is however still paucity

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RESULT S. innocua lacks these lateral projections at the adaxial Leaf Morphology surface. The adaxial surface of S. spinosa and S. innocua are The morphology of leaves of S. spinosa, S. innocua and S. slightly undulating, while that of S. usambarensis is usamberensis is presented in Table 1. concave. Plates4 a-c Anatomical Evaluation Ground tissue: The ground tissue is homogenous, There were no visible stomata on the adaxial surface of the parenchymatous, thin walled, circular in shape and compact. three Strychnos species. Numerous stomata were found on The number of layers of parenchyma cells in the ground the abaxial surface and range in number from 25 to 40 per tissue of petiole is shown in Table 4. field of view. Comparison of the dimensions of the guard cells is shown in the Table 1. Vascular tissue: S. spinosa and S. innocua each has seven Transverse Section of the leaf distinct and separate vascular bundles of varying sizes, with Epidermis: The abaxial and adaxial epidermis in the three the middle one as the biggest S. usambarensis has one big species all have uniseriate layers. The adaxial epidermis is vascular bundle embedded in the ground tissue (Plates5a-c). rectangular in shape and has thin walls. The outer surface is In the three species, the xylem cylinder has closely arranged convex and is covered with a thin cuticle. radial files of vessel elements with phloem on the adaxial Mesophyll layer: The mesophyll in the three species is and abaxial surfaces. In S. spinosa and S. usambarensis, the differentiated into palisade and spongy. The palisade xylem elements are predominantly spherical to circular in mesophyll is double layered, rectangular shaped and longer shape, while in S. innocua, the xylem are angular. The than wide in the three species. There are seven to eight vessel elements range from eleven in the middle to six at the layers of cells that are large and loosely arranged in the right and left hand lateral corners in S. spinosa while in S. spongy mesophyll in the three species. Air spaces, rosette usambarensis, the rows of xylem elements are about crystals and bundle sheaths are also present. The abaxial twenty-eight in the single vascular bundle. In addition, there epidermis is not even because of the presence of stomata of is a pronounced region of protoxylem at the abaxial layer, anisocytic type. Plate 3a-c. which is not visible in S. spinosa and S. innocua. In S. Midrib: The midrib is plano convex in the three species innocua there are about eleven rows of xylem elements in with semicircular abaxial surface. Plates4a-c. the median bundle and one at the extreme left and right hand Adaxial surface: There is uniseriate layer of epidermal corners. In each of the seven bundles in the petiole of S. cells in the three species. The cells are rectangular with spinosa and S. innocua, there are sieve cells and phloem convex shape at the outer surface. There is a thin layer of cells at the adaxial and abaxial surfaces. In S. usambarensis, cuticle over the adaxial epidermis. the phloem tissue occurs at both adaxial and abaxial Ground tissue: The ground tissue is parenchymatous, thin surfaces. The abaxial epidermis is semicircular in shape and walled and the number of layers of cells in each of the uniseriate in the three species. species is in Table 2. DISCUSSION The palisade mesophyll does not extend across the adaxial Metcalfe and Chalk (1979) and Stace (1980) part of midrib and the vascular bundle is embedded in the recognized the use of leaf architectural design as an ground tissue. important tool in field . Thus the differences in leaf margin and apex in the three species is of taxonomic Vascular tissue: The vascular strand is single, semicircular interest in delimiting them. and bicollateral in S. spinosa, S. innocua and S. Significant difference was recorded in the dimensions of usambarensis. The xylem elements consist of radially the length of guard cells. S. innocua growing in Sudan oriented lines of xylem vessels and narrow bands of adaxial savanna with drier climatic conditions possessed guard cells and abaxial phloem. The xylem elements in S. spinosa and with the longest length. S. usambarensis growing in Derived S. innocua are predominantly polygonal in shape but a few savanna had the least length of guard cells. This is probably are circular. (See Table 3). a feature to enable each adapt to it’s particular vegetation Sclerenchyma sheath surrounds the vascular bundle zone and a response to the climatic conditions in that zone. in S. usambarensis and S. innocua. In the former, the James and Bell (1995) working on six clones of sclerenchyma layer is one to two celled thick at the adaxial Eucaliptus camaldalensis from five Australian locations surface. In the latter, the sclerenchyma layer is three to six found that there was a lack of correlation between leaf celled thick right round. characteristics and climatic data. They suggested that Abaxial epidermis: The abaxial epidermis is semicircular availability of ground water, root structure and internal and single layered in the three species. transport of water may have greater influence on leaf Petiole: structure than atmospheric demand. In the same vein Zu and Adaxial epidermis: The adaxial epidermis in the three Zhou (2008) opined that different effects of abiotic factors species is uniseriate. S. spinosa has two projections that are on stomatal size might depend on plant species varieties. bluntly conical with compact thin walled parenchyma cells. Martinez et al. (2007) however reported that under water

170501-1903-8484- IJBAS-IJENS @ June 2019 IJENS I J E N S International Journal of Basic & Applied Sciences IJBAS-IJENS Vol:19 No:03 17 deficit, a decrease in cell size occurred indicating that an spinosa and S. innocua are plano convex with S. spinosa adaptation to drought is probable. The shapes of epidermal having lateral projections at the adaxial surface. S. and subsidiary cells at the adaxial and abaxial surfaces of usambarensis has petiole that the adaxial surface is concave. the leaves of the three species are markedly different such Thus the petiole outline can furnish information for the that if there is need to identify the species when they are not delimitation of the three species. S. nux-vomica is reported flowering, they could be successfully separated without any by Metcalfe and Chalk (1979) to have three separate mix up. vascular bundles. It is thus suggested that the organization Oladele (2002) pointed out the importance of plant of the vascular system in form of separate and variously stomata in humidification of the atmosphere. He opined that sized bundles is not diagnostic for the genus. the efficiency of the stomatal complex in carrying out this In conclusion, this study revealed that the three feature depends on factors like stomatal size, stomatal species studied are hypostomatic with presence of stomata density and the number of subsidiary cells per stoma. only at the abaxial surface. The varying thickness of Olafinobin and Oladele (1970) and Obiremi and Oladele sclerenchyma around the vascular bundle of the midrib (2001) stated that the higher the number of subsidiary cells suggests more strengthening. The presence of rosette per stoma, the greater the capacity of such stomatal complex crystals in the leaf mesophyll, midrib and petiole suggests in humidifying the atmosphere. According to Oladele additional physical protection and storage of calcium. (2002), the presence of high number of subsidiary cells per stoma enhances the presence of stomatal opening and REFERENCES consequently making humidification of the atmosphere to be [1] Adebiyi, O. O. and Sofowora, E. A. 1978. Phytochemical more rapid. The three species of Strychnos possessed a screening of some Nigerian medicinal . Lloydia 41: 234- minimum of four subsidiary cells around the stoma. From 236. [2] Angenot, L. 1988. Why further investigation of Brazillian the fore mentioned, it follows that there will be enhanced Strychnos? Acta Amazonica 18(1-2): 241-254. stomatal opening and rapid humidification of the [3] Asase, A., Oteng-Yeboa, A. A. Ddamtta, O. C. T., Simmonds, atmosphere by these species of Strychnos. M. S. T. 2005. Ethnobotanical study of some Ghanaian anti Bicollateral vascular bundles were clearly shown in malarial plants. Journal of Ethnopharmacology 99: 273-279. [4] Augustino, S., Hall, J. B., Makenda, F. B. S. and Ishegom, R. C. the midrib and petiole of leaf and stem of the three species. 2011. Medicinal resources of the Miombo woodlands of This feature is taxonomically important at the generic level Urumwa, Tanzania: Plants and its uses. J. Med. Plants Res,. since bicollateral vascular bundles were regarded as 527: 6352-6372. anomalous structure because of their restricted occurrence. [5] Bello, M. O., Falade, O. S., Adewusi, S. R. A. and Olawore, N. O. 2008. Studies on the chemical composition and anti-nutrients It was reported by Metcalfe and Chalk (1979) to occur in the of some lesser known Nigerian fruits. Afr. J. Biotechnol., 7(21): family Loganiaceae and other related families like 3972-3979. Asclepiadaceae, Rubiaceae and Apocynaceae. S. [6] Bisset, N. G. 1970. The African Strychnos usambarensis has a uniseriate layer of sclerenchyma at the species1:Ethnobotany. Lloydia 33: 291-343. [7] Corsaro,M. M.,Giudicianni. I., Lanzetta,R., Marciano, C. E., adaxial surface of the midrib but two to three at the abaxial Monaca ,P. and Parrilli, M. 1995. Polysaccharides from the surface, while S. innocua has three to six layers of seeds of Strychnos species. Phytochemistry 39: 1377-1380. sclerenchyma sheath right round the vascular bundle. Evert [8] Cruz, N. S. A. 2008. Strychnos usambarensis Gilg. ex Engl. In: (2006) stated that the presence of sclerenchyma around Plant resources of tropical Africa 2(1) Medicinal Plants Foundation. Backhuys Publisher/CTA Wageningen, vascular bundles performs the function of strengthening the Netherlands.1790pp. leaf. The variation in thickness of the sclerenchyma is of [9] Evert, R. F. 2006. Esau’s Plant Anatomy. John Wiley and Sons taxonomic interest in delimiting the species. Inc. NJ. 601pp Rosette crystals that are of taxonomic importance [10] FAO. 1983. Food and fruit bearing forest species-Examples from East Africa. Forestry Paper 44/1 FAO, Rome. 129pp. at the genus level as cited by Metcalfe and Chalk (1979) [11] Franceschi, V. R. and Niakata P. A. 2005. Calcium oxalate in were observed in the petiole, leaf, mesophyll and midrib of plants: formation and function. Annu. Rev. Plant Biol., 56: 41 – S. spinosa and S. usambarensis. Franceschi and Niakata 71. (2005) reported that the presence of small crystals in higher [12] Frasier, C. L. 2011. Evolution and systematic of the angiosperm order with in-depth focus on Loganiaceae and its plants is related to physical protection, storage of calcium species –rich and toxic genus Strychnos. Dissertation and regulation of light during photosynthesis for plants (unpublished). growing in the shade. In the species studied, the presence of [13] James, S. A. and Bell, D. T. 1995. Morphology and anatomy of rosette crystals confirms that they possess the characteristic leaves of Eucalyptus camaldulensis clones: Variation between geographically separated locations. Aust. J. Bot., 43: 415-433. feature of the family Loganiaceae as stated by Metcalfe and [14] Kokwaro, J. O. 1976. Medicinal plants of East Africa. East Chalk (1979). Metcalfe and Chalk (1950) considered the African Literature Bureau, Kampala. 217pp. petiole to be of taxanomic importance, as it is not influenced [15] Martinez, J. P., Silva, H., Ledent, J. F., Pinto, M. 2007. Effect of much by environmental changes. The organization of the drought stress on the osmotic adjustment, cell wall elasticity and cell volume of six cultivars of common beans Phaseolus vascular system in the petiole consisted of seven separate vulgaris L. Eur. J. Agron., 26:30-38. and biconvex bundles in S. spinosa and S. innocua but one [16] Mbuya, L. P., Msanga, H. P., Ruffo C. K., Birnie, A. and big bundle in S. usambarensis. The petiole outline in S. Tengnas, B. 1994. Useful trees and shrubs for Tanzania:

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Identification, propagation and management for agricultural and [23] Oladele, F. A. 2002. The only one we have. The 62ND Inaugural pastoral communities. Regional Soil Conservation Unit, lecture, University of Ilorin. Ilorin-nuc.edu.ng Swedish International Development Authority (SIDA).198PP. [24] Olafinobinu, O. E. and Oladele, F. A. 1997. Stomatal complex [17] Metcalfe, C. R. and Chalk, L. 1950. Anatomy of the types and transpiration rate in some afforestation tree species. dicotyledons. Vol 1. Oxford University Press. U.K. 1189PP. Biosci. Res. Comm., 9(2): 121-126. [18] Metcalfe, C. R. and Chalk, L. 1979. Anatomy of the [25] Orwa, C., Mutua, A., Kindt, R. Jamnadass, R., Simona, A. dicotyledons 2nd ed. Vol 1. UK: Oxford University Press, 2009. Agroforestree Database, a tree reference and selection 279PP. guide version 4.O (http/www.worldagroforestry. org/af/ireedbi). [19] Morah, F. N. I. 1982. Chemical studies of Strychnos spinosa [26] Philippe, G., Angenot, L., De Mol, P., Goffin, E., Hayette, M.- and Adenia cissampeloides. Ph.D Thesis of the University of P. et al. 2005. In vitro screening of some Strychnos species for Ibadan Nigeria. antiplasmodial activity. J. Ethnopharm., 97, 3: 535–539. [20] Morah, F. N. I. 2011. Medicinal Plants and the Health Care [27] Rajesh, P., Rajesh, K. V., Latha, S. and Salvamani, P. 2009. Delivery. The 45th Inaugural Lecture of the University of Photochemical and pharmacological profile of plants belonging Calabar, Calabar Nigeria. to Strychnos genus: A review. Curr. Biotica, 3(2): 171-181. [21] Obiremi, F. O. and Oladele, F. A. 2001. Water conserving [28] Stace, C. A. 1980. Plant taxonomy and biosystematics. London: stomatal systems in selected Citrus species. S Afr. J. Bot., 67: Hodder Arnold. pp 288. 258-260. [29] Xu, Z. and Zhou, G. 2008. Responses of leaf stomatal density to [22] Ogundipe, O. T. 1990. Morphological and anatomical studies of water status and its relationship with photosynthesis in a grass. some species of Brachiara (Trin.) Griseb. (Poaceae) Ph.D J. Exper. Bot., 59(12): 3317-3325. Thesis. Obafemi Awolowo University, Ife. 350p.

Plate 1a: S. spinosa adaxial no stomata present x400 Plate 2a: S spinosa abaxial x400

Plate 1b: Adaxial leaf surface of S. innocua, no Plate 2b: Abaxial leaf surface of S. innocuax400 stomata present x400

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1

2

3

Plate 3a: Leaf lamina S. spiniosa x 200 1 – Adaxial epidermis, 2 – Palisade mesophyll, 3 – Spongy mesophyll

Plate 3b: Leaf lamina S. innocua x 200 1 – Adaxial epidermis, 2 – Palisade mesophyll, 3 – Spongy mesophyll

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1

2

3

Plate 3c: T.S leaf of S. usambarensisx200 4 1 – Adaxial epidermis, 2 – Palisade mesophyll, 3 – Spongy mesophyll, 4 – crystal 4 – Crystal, Plate. Leaf Usamba 3 x200

1

2

3 4

5

Plate 4a: Midrib of leafS.spinosax40 1 – adaxial epidermis, 2 – internal phloem, 3 – xylem elements, 4 – external phloem, 5 – ground tissue

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1

2

3 4

5

Plate 4b: Midrib of leaf S. innocua x40 1 – adaxial epidermis, 2 – internal phloem, 3 – xylem elements, 4 – external phloem, 5 – ground tissue

Plate 5a: Petiole of S. spinosa x40 Plate 5d: Bicollateral vascular bundle of petiole of S. spinosa x200

Plate 5e: Bicollateral vascular bundle of Plate 5b: Petiole of S. innocua x40 petiole of S. innocua x200

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Plate 5c: Petiole of S. usabarensis x40 Plate 5f: Bicollateral vascular bundle of petiole of S. usabarensis x200

Table I Stomatal dimensions of S. spinosa, S. innocua and S. usambarensis Name of plant Length of Width No of No of subsidiary Cell wall thickness Stomatal guard cell guard cell stomatal/field of cell/field of view of epidermal cells index (mm) (mm) view (mm)

S. spinosa 0.176 0.027 10.9 66.2 0.016 14.13 S. innocua 0.187 0.029 12.2 92 0.037 11.70 S. usambarensis 0.158 0.040 11.6 97.8 0.014 10.60

Table II Number of layers of ground tissue in midrib of the three species of Strychnos S. spinosa S. innocua S. usambarensis Layers of cells at adaxial surface 11.7±0.82 6.5±0.52 4.3±0.48 Layers of cells at abaxial surface 7.5±0.51 8.5±0.53 8.5±0.53

Table III Number of xylem elements in the vascular bundle of midrib S. spinosa S. innocua S. usambarensis Number of xylem elements at median 4.34±0.61 7.5±0.50 2.3±0.64 part Number of xylem elements at left and 1.5±0.52 2.3±0.45 4.5±0.50 right hand corners

Table IV Number of layers of parenchyma cells in the ground tissue of petiole Name of plant Median Adaxial Extreme Left & Median Abaxial Extreme Left and Right adaxial Right Abaxial S. spinosa 21.5 ±1.56 9.50±2.00 18.5±3.55 8.5±1.33 S. innocua 13.9±2.34 9.87±0.98 11.45±0.87 6.50±0.55 S. usambarensis 13.55±0.65 16.50±1.87 10.55±1.76 9.50±0.50

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