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International Journal of Research in Pharmacy and Biosciences Volume 6, Issue 3, 2019, PP 22-26 ISSN 2394-5885 (Print) & ISSN 2394-5893 (Online)

Anatomical Studies on the of Some L. Species (Fabaceae-Papilionoideae)

Okeke, S.I.1, Edeoga, H.O.2, Nduche, M.U2*, Omosun, G2 1Department of Science Laboratory Technology, Federal Polytechnic, Oko, Anambra State, Nigeria. 2Department of Science and Biotechnology, Michael Okpara University of Agriculture, Umudike, Abia State, Nigeria *Corresponding Author: Nduche, M.U., Department of Plant Science and Biotechnology, Michael Okpara University of Agriculture, Umudike, Umuahia, Abia State, Nigeria, Email: [email protected]

ABSTRACT Anatomical studies on the roots of ten Crotalaria species namely C. aculeata, C. atrorubens, C. bamenda, C. cephalotos, C. cleomifolia, C. comosa, C. cuspidata, C. goreensis, C. harmsiana and C. retusa are reported in this study. An analysis of the features of the internal arrangement of the roots show that these taxa possess vital taxonomic characters that could be used to establish relationships among these taxa. An interesting aspect of the anatomy of these taxa is the variation in the number of vascular bundles and layers of cortex and piliferous layer as well as the number and size of metaxylem vessels among these taxa studied. The vascular bundles are very if numerous in C. harmsiana and few in C. aculeata and C. atrorubens. The metaxylem vessels are small and very few in C. atrorubens and C. cleomifolia, small and many in C. cephalotos and C. comosa and large and numerous in C. harmsiana and C. cuspidata. Eight of the species investigated possess one layer of piliferous layer whereas C. comosa has two-layered piliferous layer and it is three layers of cells thick in C. retusa. The roots of all species lack root hairs. These observations in the roots of Crotalaria species are relevant in the systematic and biological consideration of the Crotalaria species investigated. Keywords: Anatomy, Crotalaria, Fabaceae, systematics.

INTRODUCTION with stipules and sometimes a leaflet will be modified into a (Syllaet al., 1999). The genus Crotalaria L. is a member of the of most genera in this are of the family Fabaceae (Leguminosae). The well-known sweet or ―butterfly‖ type and legume family has such distinctive they are thus irregular, the five-parted corolla characteristics that its members can frequently forming a standard, two wings and a keel be recognized with but little experience. It is a (Brummit, 1992). are present, 5 and nine family with about 550 genera and 13, 000 of the ten are united to form a tube species (Sheahan, 2012. All sorts of plant habits around the : the tenth being free are represented: herbs with annuals and (Sangingaet al., 1992). The is perennials, , and (Heuzéet al., composed of a single carpel and the known 2018). The members of the family Fabaceae are as legume or pod, is the characteristic fruit of of worldwide distribution (Samba et al., 2002). this family (de Figueiredo and Lindoso Silva, It is a family of considerable importance in 2018). It forms from a single carpel and has one supplying food for man and his animals and locule and numerous and in many genera, many are used as ornaments, from it dehisces at maturity along both sutures shade trees to cut flowers, also lumber is (USDA 2006). obtained from some and many of the tropical species furnish wood for fine cabinet work Crotalaria species are mostly annual or (Chong et al., 2009). Association of the perennial herbs or shrubs with alternate, - fixing with roots of legumes lanceolate to obovate, simple or digitately 3-7 places this family in a unique position relative to foliate and usually petiolate (Hyde and Wursten, maintenance of soil fertility (Keener et al., 2002). The plant is erect, branched and 2018). Generally, the of legumes are variously pubescent (Okeke, 2008) and the prevailingly pinnately compound and quite often stems are angular or cylindrical, solid or hollow

International Journal of Research in Pharmacy and Biosciences V6 ● I3 ● 2019 22 Anatomical Studies on the Roots of Some Crotalaria L. Species (Fabaceae-Papilionoideae)

The is usually receme, flowers 95% and 100% for dehydration. The dehydrated typically yellow, variously marked, less often materials were infiltrated with wax by passing white or blue, usually with bracts zygomorphic through different proportions of alcohol and or actinomorphic, bisexual, hypognous, chloroform (3:1, 1:1:1:3, v/v). As the complete and papilionaceous (Okeke, 2008) The chloroform gradually replaced alcohol, pure are typically, dark brown to at chloroform and wax were put in the bottles maturity, glabrous, manyseeded and inflated as containing the plant materials to gradually they mature (Unamma, 1999; Flores et al., infiltrate the tissues with wax to make them hard 2009). Crotalaria species are common weeds of enough for sectioning. cultivated crops, bush regrowths, waste areas The bottles were left on a hot plate (37 – 40o C) and roadsides (Akobundu and Agyakwa, 1998). for 24 hours before transferring to the oven (50o Crotalaria is one of the most wide-spread – 60o C) in order to evaporate the chloroform. genera of the family Fabaceae (Papilionoideae) The wax at its melting point completely and consists of over 500 species, spreading over infiltrated the tissues in it. After a period of 2 -3 tropical and subtropical areas (Ferro et al., 2006) days with constant addition of wax, the and at least 500 species are known in . specimens were embedded in paraffin wax. This The species of the genus Crotalaria are selected was achieved by quick orientation of the for this study because of their immense specimens in the melted wax in moulds with a economic importance. They are of great hot mounted needle and forceps and quick medicinal value (Gill, 1992; Njorogeet al., cooling in ice-blocks. The metal moulds were 2004; Kong et al., 2003), with outstanding later removed and the specimens within the wax agricultural and agronomic importance (Muller cubes were trimmed and sectioned using a – Samann and Kotschi, 1994; Cook and White, Reichert rotary microtome at 20-24um 1996; Jourandet al., 2004). Series of scanty following a slightly modifies method of documented descriptions of the morphological Olowokudejo (1990).The root sections came out characteristics of some of these are found in ribbons of wax. in the literature (Wagner et al., 1999). There The ribbons were placed on clean slides was no information on the anatomical features smeared with a thin film of Haupt’s albumen, of the vegetative organs of these Nigerian allowed to dry and drops of water added. The species of Crotalaria and hence the present slides were placed on a hot plate at 40oC for a study. few minutes to allow the ribbons to expand and The significant role of vegetative anatomy in were stored overnight. The slides were botanical and systematic reconstruction of the immersed in pure xylene for 2-5 minutes, then origin, natural distribution, extent of cultivation in a solution of xylene and absolute alcohol with and cultivars within species of plants dates back 1:1 ratio (v/v) for 5 minutes. The slides were to many centuries ago. They include the works then transferred to another solution of xylene of Edeoga and Okoli (1997) in Costaceae, and absolute alcohol in the ratio 1:3 (v/v) for a Edeoga (2002) in Discoreaceae, Edeoga and few minutes and then to 95%, 90%, 70% and Ogbebor (1991) in Aneilema, Edeoga and 50% alcohol. Drops of alcian blue were added to Osuagwu (2006) in Phyllantus species and a the specimens for 5 minutes, washed off with of others. The aim of the present study water and counterstained with safran in for 2 includes the provision of basic root anatomical minutes, then dehydrated in a series of alcohol – information that will be added to data from 50%, 70%, 80%, 90 and pure xylene at intervals other fields of study as morphology, cytology, of a few seconds and mounted in Canada balsam palynology and histochemistry for possible and subjected to microscopic observations. taxonomic review of the classification of these Coloured photomicrographs were taken with a Crotalaria species. Leitz Wetzler Ortholux microscope fitted with a MATERIALS AND METHODS Vivitar –V- 335 Camera. Mature and fresh roots of the Crotalaria species RESULTS were obtained from living samples from The results of anatomical characteristics of the different parts of Eastern Nigeria. Small roots of the ten Crotalaria species studied are portions of the roots of the plants under study illustrated in figs. 1-3 and Table 1. The roots of were fixed in FAA (1:1:18, v/v) for 48-72 hours. all the species investigated are composed of an These were then rinsed in several changes of outer piliferous layer that is one layer of distilled water when needed for study and parenchyma cells thick in eight of the species. It passed through alcohol series – 30%, 50%, 70%, is two layers of cells thick in C. comosa (fig.

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2B) but three layers thick in C. retusa (fig. 3B). and metaxylem is larger than the protoxylem. There is total absence of root hairs in the roots The vascular bundles and the metaxylem varied of all the species studied. Beneath the piliferous in number among the species studied. The layer is the cortex which is conspicuous in all vascular bundles ranged in number from 102 in the species and composed of thin-walled, C. harmsiana, 98 in C. cephalotos, 88 in C. colourles sparenchyma cells with vascular cuspidata, 85 in C. comosa and to 59 in C. bundles at the centre. The layers of the cortex aculeate and C. atrorubens (Table 1). The varied markedly among the ten Crotalaria metaxylem vessels showed great variation species investigated. There are 7-12 layers of among the ten Crotalaria species studied and the cortex in C. retusa, 4-10 in C. cephalotos, 5- ranged from 84 in C. harmsiana to 36 in C. 9 in C. aculeata, 4-9 in C. comosa, 8 in C. bamenda. The metaxylem vessels also varied in atrorubens and C. cleomifolia, 7 in C. size among the species studied and were small harmsiana and 2-5 in C. bamenda (Table 1). in four of the species, large in C. cuspidate (Fig. The inner-most layer of the cortex which is the 2C) and C. harmsiana (Fig. 3A) but very large endodermis, is well-developed and one layer of in C. goreensis (Fig. 2D), C. bamenda (Fig. 1C). cells thick in all the ten species studied. The Aggregates of calcium oxalate crystals were cells of the endodermis are barrel-shaped and present in the root vessels of C. goreensis (Fig. thick-walled. The vascular bundles are made up 2D), C. aculeate (Fig. 1A), C. comosa (Fig. 2B), of the xylem and phloem with xylem being C. retusa (Fig 3B), C. cuspidate. (Fig 2C), C. more prominent. However, the xylem is bamenda (Fig. 1C) and C. harmsiana (Fig. 3A) composed of the metaxylem and protoxylem but absent in other species. Table 1: Anatomical characteristics of the roots of the ten Crotalaria species studied Species/ C. C. C. C. C. C. C. C. C. C. Features aculeata atrorub bamenda cephalotos cleomif comosa cuspidata goreens harmsiana retusa ens olia is Outer Present Present Present Present Present Present Present Present Present Present piliferous layer of root Root hairs Absent Absent Absent Absent Absent Absent Absent Absent Absent Absent Layer of 5-9 8 2-5 4-10 8 4-9 7 5-9 7 7-12 cortex Layer of 1 1 1 1 1 1 1 1 1 1 endodermis Number of 59 59 36 98 88-90 85 88 56 102 56 vascular bundles

Fig2. T. S. of root of Crotalaria species studied (A) Fig1. T. S. of root of Crotalaria species studied (A) small metaxylem vessels of C. cleomifolia (B) very large metaxylem vessels of C. aculeata with double-layered piliferous layer and small metaxylem calcium oxalate crystals (arrowed) (B) small vessels with calcium oxalate crystals (arrowed) in C. metaxylem vessels of C.atrorubens(C) very large comosa (C) large metaxylem vessels with calcium metaxylem vessels of C. bamenda with calcium oxalate crystals (arrowed) in C.cuspidata (D) very oxalate crystals (arrowed) (D) small metaxylem largemetaxylem vessels with calcium oxalate crystals vessels of C. cephalotos x 250 (arrowed) in C. goreensis x 250

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calcium oxalate crystals in the vessels of seven of the taxa and absent in others provide important chemical criterion that could be used in the description, separation and identification of these Crotalaria species investigated. From this investigation, the similarities in the root anatomical structures showed reasons for the unification of these taxa into the same genus while differences in structures showed reasons for these taxa being in different species.

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Citation: Okeke, S.I., Edeoga, H.O., Nduche, M.U et al. "Anatomical Studies on the Roots of Some Crotalaria L. Species (Fabaceae-Papilionoideae)", International Journal of Research in Pharmacy and Biosciences, vol. 6, no. 3, pp. 22-26, 2019. Copyright: © 2019 Nduche, M.U et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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