INT. J. BIOL. BIOTECH., 14 (4): 539-559, 2017. MICROMORPHOLOGICAL STRUCTURE OF SEEDLINGS OF RHYNCHOSIA MINIMA (L.) DC. (PAPILIONACEAE) GROWING IN A DRY RUDERALIZED SITE IN KARACHI D. Khan1, M. Javed Zaki1 and Yousuf Khan2 1 2 Department of Botany, University of Karachi, Karachi-75270, Pakistan. Centralized Science Laboratory, University of Karachi, Karachi-75270, Pakistan. ABSTRACT The micromorphological structure of seedlings of Rhynchosia minima (L.) DC. is described, from a dry ruderalized site of Karachi, on the basis of optical (OM) and scanning electron microscopy (SEM). The seedling appeared to be “PhanerocotylarEpigeal-foliaceous” type. Several types of trichomes from cotyledons and leaves and structure of the resin gland (Brown-orange bulky capitate, glandular trichome) are described. Cotyledons are amphitrichomic and amphistomatic with paracytic arrangement of subsidiary cell. R. minima leaflets were amphistomatic with much larger number of stomata on ventral surface (283.3 ± 4.39 stomata per mm2, N = 100, CV = 38.6%) as compared to the dorsal surface (48.06 ± 1.85 stomata per mm2, N = 100, CV = 38.6%). The diversity of foliar stomata included, paracytic, anisocytic, staurocytic and anomocytic types. The stomatal size on abaxial surface leaf averaged to 18.53 ± 0.36 x 13.17 ± 0.30 µm. The predominating class of stomatal width (pore + guard cells) was 10.1 – 16 µm. Stomata on adaxial surface were comparable in size (19.76 ± 0.22 x 11.19 ± 0.18 µm). Quantitative element analysis based on elements detector system (EDS) of energy dispersive X-ray spectroscopy (EDS) is described. Key Words: Rhynchosia minima (L.) DC Seedling, micromorphology, trichomes, resin duct, stomata. INTRODUCTION Rhynchosia minima (L.) DC. is a pantropical species commonly known as least stout bean, Burn-mouth-vine, and Jumpy bean. It has long synonymy (Basionym: Dolichos minimus L. ) and is included in the red list of threatened species (Lopez-Poveda, 2012, IUCN, 2014). It occurs with grasses and shrubs in derelict areas, ruderalized land, and roadside distrurbed areas of Karachi. It is rated as good fodder with no known toxicities (Barnes, 1996; ANG, 2012) but leaves are shed in winter. It appears to be a potential medicinal species also. Jia et al. (2015) have reported the occurrence of some novel heteropolysaccharides from R. minima roots composed of arabinose, mannose, glucose and galactose of which PRM1 and PRM3 have exhibited strong in vitro anti-cancer activity. R. minima yields essential oil which has anti-oxidant and antibacterial activities (Gundidza et al, 2009) besides anthelmintic potential (Mali and Mahele, 2008). Bhattacharya and Maheshwari (1970) have investigated the extra-floral nectaries in Leguminales of Indian flora including Rhynchosia, Vicia and other genera and Vergas et al. (2015) have described the secretory structures and foliar anatomy of Rhynchosia spp. from herborized materials of Brazilian collection. R. minima appears to exhibit different ecotypes (ANG, 2012), we have undertaken to study the micro-structure of R. minima seedlings collected from Karachi University Campus, Karachi, arising after summer precipitation. A B Leaf Leaf Pod Pod Pod Cotyledon Cotyledon Fig. 1. Seedling of R. minima studied. A. Habit; B, Note that seedling under drought has produced two pods while still bearing green photosynthesizing cotyledons. 540 D. KHAN ET AL., MATERIALS AND METHODS Seedlings of R. minima were collected from a dry ruderalized site in August 2012 after some 30 days of light rains, from Karachi University Campus. The seedlings were studied for their morphological characters including stomatal types. Seedling type was described according to Vogel (1980) and Garwood (1996). Hickey (1979) and LWG (1999) were followed for description of cotyledon and leaf. To study stomatal types, leaflet epidermal impressions were made with clear nail polish (Wang et al., 2006) and studied under compound optical microscope. Stomatal nomenclature suggested by Prabhakar (2004) being simple and based upon structure of stomata and not their ontogenetic pathways was adopted to ascertain stomatal types. The data was analyzed statistically (Zar, 2010). For scanning electron microscopy (SEM), air-dried plant material (cotyledon and leaf) was mounted on brass stubs and coated with a 250 oA gold layer with JFC-1500 gold coater. SE micrographs were made at 15kV with JEOL JSM6380A electron microscope at various magnifications. The images were saved digitally on computer. The seedling employed in microscopic studies is shown in Fig. 1. Fig. 2A. Germination in R. minima. It appeared to be “Phanerocotylar-epigeal- foliaceous type” – Cotyledons aboveground due to Hypocotylar elongation, epigeal and leaf like. Testa remained subterranean. Fig. 2A. Cotyledon of R. minima (c. 269 mm2). Seedling. There arise five vascular nerves in the umbo region embayed in sinus. RESULTS AND DISCUSSION R. minima is twining or prostrate ruderal herb attaining height c 30-40cm, stem slender, densely pubescent, striate, and cylindrical. Leaves trifoliate. Inflorescence raceme. Calyx green, the lobes lanceolate, 2-3 mm long; corolla yellow Pods falcate to oblong ovate, flattened, minutely villous, slightly curved with a beak at the apex. Seeds are ovate-reniform, dark brown to almost black, 3- 4 mm long and 1-2 (3) in a pod. The seeds of R. minima in soil are exposed to high summer temperature which breaks the seed dormancy under arid field conditions of Karachi and allows seeds to germinate after showers of summer rains (Shaukat and Burhan, 2000). Seedling: “Seedling” is considered to be the final stage of the regenerative process of a plant from a seed. The use of this term is quite liberal. We have used this term as ecologists employ i.e. stage up to which the cotyledons are attached with the juvenile. According to Léonard’s (1957) classification, germination, in R. minima is of “type A” germination (cotyledons spreading above soil; no consideration to the presence or absence of the collet). Seedling of R. minima appeared to belong to “Type I” seedlings of Klebs (1885) i.e. cotyledons lie above soil due to hypocotylar growth and there is no collet. As per classification by Garwood (1996), it may be rated as “Phanerocotylar-Epigealfoliaceous” type of seedling (Fig. 2A) which is also seen in Anageissus latifolia, Cucumis sativus, Manilkara hexandra and Terminalia arjuna (Amritphale, 2004), Phaseolus vulgaris (Vogel, 1980) and in Luffa cylindrica as well (Khan et al., 2017). However, it appears to be characterized with the fact that seed coat remain subterranean. It may be emphasized that the terms, phanerocotylar and cryptocotylar proposed by Duke (1965) are synonymous to epigeal and hypogeal germination, respectively. However, the use of the two sets of terms appears quite logical (as employed by Garwood, 1996) in the light of a very rare cases of species (Rollinia saleifolia) where germination is INTERNATIONAL JOURNAL OF BIOLOGY AND BIOTECHNOLOGY 14 (4): 539-559, 2017. SEEDLINGS MORPHOLOGY OF RHYNCHOSIA MINIMA (L.) DC. (PAPILIONACEAE) 541 epigeal cryptocotylar (Franceschini, 2004). The morphometric characteristics of the seedling studied for surface ornamentation are given in Table 1. A B C D Fig. 3. A, venation in the basal part of a leaflet; B, Venation near the margin; C, Venation of a terminal leaflet drawn (Camptodromous venation with some brachidodromous loops); D, A sharply pointed trichome, the basal part of which is bulbous surrounded by nine sinuous epidermal cells arranged radially around.. Table 1. Morphometric parameters of the seedling studied for surface ornamentation. Seedling components Size Seedling Parameters Height (cm) 9.5 Lateral leaflet I area (mm2) Stem diameter (mm) 1.3 Lateral leaflet II area (mm2) Hypocotyl (cm) 4.2 Terminal leaf area (mm2) Number of leaves 3 Total leaf area (mm2) Cotyledonary petiole lengths, (cm) 1, 1 * Photosynthetic area (mm2) 2 Cotyledon I area (mm ) 269 α Number of pods Cotyledon II area(mm2) 257 α Pod I length (cm) Total cotyledonary area (mm2) 526 α Pod II length (cm) Foliar petiole (cm) 3.1, 4.1, 4.2** Pod I width (cm) Petiolule Length ( leaflet I) (mm) 1.5, 2.0, 2.0** Pod II width (cm) Petiolule Length (leaflet II) (mm) 1.4, 1.8, 2.0** Number of seeds Pod I Petiolule Length (terminal Leaflet) 7.0, 8.0, 8.0** Number of seeds Pod II (mm) α, One-sided area; *, for two cotyledons respectively; **, in order of lateral leaflet I, lateral leaflet of the three leaves, respectively. Size 112,229, 300**α 79, 417, 280**α 555, 741, 802**α 3515 α 4041α 2 1.5 0.5 0.4 0.2 2 leaflet II and terminal INTERNATIONAL JOURNAL OF BIOLOGY AND BIOTECHNOLOGY 14 (4): 539-559, 2017. 542 D. KHAN ET AL., A B Fig. 4. Surface of stem (A) – very few glands and Outer surface of calyx (B) - showing dense trichomes crop and orange-brown glands (10x10X). i iii ii iv Fig. 5A. Optical microscopic views of surface of various parts of seedling. i, Petiole (10x5 X); ii, Very young leaf (underside, 10x10 X); iii, Mature leaf (underside, 10x10 X); iv, Flower (5 x 10 X). Orange-brown bulky glands may be seen on all parts.. INTERNATIONAL JOURNAL OF BIOLOGY AND BIOTECHNOLOGY 14 (4): 539-559, 2017. SEEDLINGS MORPHOLOGY OF RHYNCHOSIA MINIMA (L.) DC. (PAPILIONACEAE) 543 ii i Fig. 5B. Optical microscopic views of surface of Young pod apex (i) and its enlarged view (ii) – showing orange-brown dots of glands (10x5 X). A B C D Fig.6. Dorsal surface of Cotyledon: A) A gland in a depression (c 100 µm in diameter), B) stoma (8.57 x 1.59 µm pore size), C and D) eglandular sharply-pointed trichomes (81.7 and 91.8 µm in length, respectively). INTERNATIONAL JOURNAL OF BIOLOGY AND BIOTECHNOLOGY 14 (4): 539-559, 2017. 544 D. KHAN ET AL., A B C D Fig. 7.Ventral surface of Cotyledon – A, Trichomes on the vein with two oppositely- placed glands; B, a bulky somewhat spherical but almost flat-topped gland in the depression, when seen from the top (the top tier is composed of three cells); C, a stoma (9.68 x 1.50µm pore size) and D, a trichome (106 µm in length). INTERNATIONAL JOURNAL OF BIOLOGY AND BIOTECHNOLOGY 14 (4): 539-559, 2017. SEEDLINGS MORPHOLOGY OF RHYNCHOSIA MINIMA (L.) DC. (PAPILIONACEAE) 545 Fig. 8. Dorsal leaf surface-Bulbous-based trichomes with sharply-pointed and elongated conical apical cells. Hypocotyl Long, cylindrical, pubescent. Epicotyl Epicotylar part of seedling is pubescent and parts of seedling (stem, petiole, leaves, calyces and pods bear trichome - glandular (resin glands) and eglandular both (Fig. 5A and B). The sinuous epidermal cells around the base cell of trichomes are arranged radially (Fig. 3D). Cotyledon Cotyledons are two, petiolate, wide elliptic or oblong-orbicular in shape, apex round with apex angle obtuse. The cotyledonary base is embayed in sinus with cotyledon base extension of c 0.25 cm. Five-nerved at base (Fig. 2B). Nerves are lighter in colour. Cotyledons are more or less equal in size (257 and 267 mm 2, respectively) and they stay with seedling for quit sometime. The seedling we studied had still borne cotyledons (and three leaves only) when entered the reproductive phase under drought conditions and produced two pods (Fig. 1). It indicated to the ruderal nature of R. minima. Curtailment of shoot under stress and diversion of resources into flowers, short life cycle, occurrence in disturbed areas and early production of flowers suggest to the ruderal nature of a species (Grime, 1977, 1979). Cotyledons of R. minima are amphitrichomic and amphistomatic. Conical trichomes of dorsal surface are bulbous at the base and pointed at apex. On dorsal surface the glands are present but somewhat infrequently (Fig. 6A). Stomata generally of paracytic type (Fig. 6B). Glands on ventral surface are stalked, situated in depression and provided with a bulky head measuring 51.3 µm (Fig. 7B). Stomata on ventral surface are also paracytic and are as large as on dorsal surface (Fig. 7C).Trichomes on ventral surface are relatively longer (Fig. 7D). Leaves Leaves alternate, trifoliolate, herbaceous; margins entire, upper surface dull, lower surface pale green. The leaflets are rhomboid, ovate or sub-orbicular. Leaves are amphitrichomic (Fig. 8 and 9). Trichomes are bulbousbased and apically straight-sharply-pointed, curved or round. They and much larger on the veins and margins of the leaf (Fig. 8-11). There are golden to orange-red glands (secretory trichomes) on the underside of leaf (Fig. 5Aii and 5Aiii, 10, 11, 12). In lateral leaflets apex is acute (apex angle 70-84o); the base of leaflets obtuse (base angle 91100o). In terminal leaflets the apex angle was mostly broad acute (82-88o) but in one terminal leaflet apex angle was larger than right angle (110o). The base angle of terminal leaflets was always obtuse (98–108o). Petiolules pubescent; stipules caduceus, densely pubescent; Stipels present. The lateral leaflets asymmetrical, elliptical-ovate, and significantly smaller than the terminal leaflet. In the seedling studied, the lateral leaflets varied in size from 79 mm2 to 417 mm2 whereas terminal leaflets varied from 555 to 802 mm2 (Table 1). INTERNATIONAL JOURNAL OF BIOLOGY AND BIOTECHNOLOGY 14 (4): 539-559, 2017. 546 D. KHAN ET AL., Primary and secondary venation prominent. The venation in leaflets of R. minima appeared to be camptodromous. There were three veins arising from the base of lamina in lateral as well as terminal leaflet (Fig. 3A, B and C). The secondary adjacent veins from these veins were arching distally towards margin where they anstomose with each other. According to Weyland (1968), the venation pattern in tribe Phaseoleae is known to vary only slightly and the prominent venation is camptodromous. In Rhynchosia pringlej each lateral vein is prominent, arching distally towards the margin, where it joins with the distal adjacent lateral vein. Branching loops of these lateral veins form a series of diminishing loops. Some 26 out of 53 species of Papilionoideae (Leguminosae) had brachidodromous veins on the same lamina as well (Weyland, 1968). Fig. 9. Close up view of bulbous-based trichomes with sharp elongated conical apex on dorsal foliar surface and some scattered stomata. Fig. 10. Ventral surface of leaf -Straight and curved non-glandular trichomes on the veins are much larger in size than those on the laminar islands. Two glandular trichomes (glands) are also visible. INTERNATIONAL JOURNAL OF BIOLOGY AND BIOTECHNOLOGY 14 (4): 539-559, 2017. SEEDLINGS MORPHOLOGY OF RHYNCHOSIA MINIMA (L.) DC. (PAPILIONACEAE) 547 Fig. 11. Ventral surface of leaf. Long curved non-glandular trichomes arising from margins of the leaf (appressed with the margin) with some glands scattered near margin (ventral surface). Some trichomes are with curved apices (as shown in circles). Fig. 12. Ventral surface of leaf – General view showing glands scattered amongst the sharply pointed bulbous based trichomes. INTERNATIONAL JOURNAL OF BIOLOGY AND BIOTECHNOLOGY 14 (4): 539-559, 2017. 548 D. KHAN ET AL., Fig.13. Ventral surface of leaf - Close up view of gland on the lamina amongst the trichomes. The gland is provided with 8 cells arranged around a central cell. The gland admeasured 71.1 to 76.7 µm in diameter. The circles indicate bulbous-based trichomes with elongated but rounded or beaked apical cell (possibly glandular). Fig. 14. A dried collapsing gland showing basal attachment – c. 15 um in diameter. Trichomes with elongated apical cell, is possibly glandular (in circle). INTERNATIONAL JOURNAL OF BIOLOGY AND BIOTECHNOLOGY 14 (4): 539-559, 2017. SEEDLINGS MORPHOLOGY OF RHYNCHOSIA MINIMA (L.) DC. (PAPILIONACEAE) 549 Fig. 15. SEMs showing an umbrella-like gland (with 8 peripheral cells surrounding a central circular cell (shown in single-lined circle). It is presumably distal end of the stalk. A bulbous-based trichome with elongated terminal cells (presumably glandular) is shown in a double-lined circle). N = 120 Mean = 26.87 SE = 0.8606 Median = 29.487 CV = 35.08% G1= 0.154 Sg1 = 0.221 G2 = - 0.282 Sg2 = 0.478 Minimum = 9.83 Maximum = 49.14 KS-z = 2.204 P < 0.0001 Fig. 16. Trichome density per mm2 on ventral surface of lamina. Distribution is asymmetrical (platykurtic). INTERNATIONAL JOURNAL OF BIOLOGY AND BIOTECHNOLOGY 14 (4): 539-559, 2017. 550 D. KHAN ET AL., N = 100 Mean = 69.78 SE = 1.0522 Median = 68.88 CV = 15.08% G1 = 0.177 Sg1 = 0.241 G2 = 0.745 Sg2 = 0.478 Minimum = 39.36 Maximum = 98.40 KS-z = 0.906 P < 0.385 Fig. 17. Length of Trichomes (µm) distributed on ventral surface of lamina (exclusive those present on veins or margins.). Normal distribution. Trichomes Trichomes are present throughout the plant body of R. minima – on stem, cotyledons, leaves, petioles, calyces and fruits. Cotyledons and leaves both were amphitrichomic. The leaves of tropical species appear to be generally amphitrichomic. Khan et al. (2013) had studied 51 tropical species (no legume included) and found 38 spp. to be amphitrichomic and 13 hypotrichomic. There were both types of trichomes – glandular and eglandular. Vergas et al. (2015) studied apical leaflets of seven species of genus Rhynchosia for trichome type in herborized materials. They also described two types of trichomes in Rhynchosia – Glandular or secretory and Eglandular. Besides eglandular trichomes, Vergas et al. (2015) have described four types of secretory trichomes from the two surfaces of leaflets of R. minima and referred to them as novelties in the secretory structures of the genus Rhynchosia. I. Spherical capitate trichomes with circular apical cells with stalk (unicellular) II. Ellipsoid capitate trichome (large terminal secretory cells and unicellular stalk. III. Bulky capitate trichome (large terminal secretary cells arranged in epidermal depressions. IV. Bulbous-based trichomes with cells apically elongated and terminal slightly rounded basal cells large. In our studies of R. minima seedling, we could find following types of trichomes. Type 1. Orange-brown spherical capitate glandular trichome with umbrella like head (top tier three celled) attached to the cotyledonary epidermis in a depression Cotyledons (Fig. 6 and 7). Type 2. Orange-brown bulky, capitate glandular trichome with umbrella like head composed with eight peripheral cells around the central cell, attaching to the surface through a unicellular stalk (ventral surface of leaflet Fig. 12, 13, and 14). They were also present on young stem, petiole, calyces and developing fruits (Fig. 4, 5 and 6). Type 3. Long trichome with bulbous base and apical cells elongated and terminal cell rounded (glandular - seen on leaflet, Fig. 13). Type 4. Long trichomes with bulbous base and apical cell elongated and sharply pointed (Eglandular seen on leaflet, Fig. 9, 10, 11). Presumably protective in function. Type 5. Short trichomes with bulbous base and straight sharply pointed apex. Eglandular, leaflet, Fig. 10). Presumably protective in function. INTERNATIONAL JOURNAL OF BIOLOGY AND BIOTECHNOLOGY 14 (4): 539-559, 2017. SEEDLINGS MORPHOLOGY OF RHYNCHOSIA MINIMA (L.) DC. (PAPILIONACEAE) 551 Density of bulbous-based protective eglandular trichomes as determined on ventral side of a large leaflet (of 802 mm2 in area) averaged to be 26.87 ± 0.861 trichomes per mm 2 (N = 120) varying 39.09% and distributed nonnormally (KS-z = 2.204, p < 0.0001, with little platykurtosis (Fig. 16). In 70.8 % of the observations, the trichome density varied between 11 and 30 trichomes per mm2. Frequently trichomes were attenuate and uniseriate. In 70% of the observations, length of the bulbous eglandular trichomes ranged between 61 and 70 µm. The base of trichome was around 20 µm in diameter. Trichome were found to be longer on the veins and much longer on the margins of the leaflets (Fig. 8, 9 10 and 11). Trichomes length in laminar islands averaged to 69.72 ± 0.105 um (N = 100) varying by 15.08% (39.4-98.4 um). It distributed normally (KS-z 0.906, P < 0.385) (Fig. 17). Special Glandular Trichomes or Nectaries or Resin glands Rhynchosia minima is a nectariferous plant. The brown orange or reddish glandular trichomes are present all over the plant surface but more copiously on the underside of leaf (Fig. 4, 5, 6 and 7) and the enlarged view of them is given in in Fig.13, 14 and 15). The glandular spherical capitate and stalked trichomes of R. minima are also described as gland (Ali, 1977), nectary (Bhattacharya and Maheshwari, 1970) or “Resin glands” (http:///www.tropicalgrasslands.asn.au/legume...). These nectaries (Type 1 and 2 trichomes as given above) appear as minute dots on the ventral surface of the leaflets. They are discoidal in shape and stalked. They may be seen on cotyledon, stem, petiole, calyx and developing fruits also (Fig. 4 and 5).The diameter of the foliar glands in head region averaged to 80.85 ± 1.55 µm varying 12.1%. Some 67.5% of the glands ranged in head size from 71to 90 µm. None of the gland observed was lesser than 60µm in head size (Fig. 18). Bhattacharya and Maheshwari (1970) have reported that these glands are not associated with vein endings of the leaflets and are covered with cuticular layer. In the early hours of the morning secretion occurs from them in the form of glistening drops. We observed these glands in R. minima to be very closely arranged in young leaves (Fig. 5Aii), as also observed by Bhattacharya and Maheshwari (1970). The nectaries later get dispersed with some distances among them. In our studies, the density of these glands on young leaf of c 1cm2 was observed to be 914.9 glands per cm2. In 62% of the observations, gland density ranged from 700 to 1000 glands per cm2 and this parameter distributed normally (KS-z: 1.047, p < 0.229, NS). On larger leaf of 6 cm2 in size, the glands were quite lesser in number (132 glands per cm2) (Fig. 20 A and B). In 61% of the observations the density on larger leaf ranged between 151 and 200 glands per cm2. The distribution of gland density was, therefore, symmetrical in young leaf and asymmetrical in larger leaf. Such a pattern of distribution may be due to foliar expansion and removal of glands with maturity of leaf owing to random events. Single-celled stalk of the glands (Fig. 14, 15) should make them quite vulnerable to detach from the foliar surface. However, the secretion of these glands coupled with the sharply- pointed non-glandular trichomes should provide considerable protection to younger leaves from insects. The glands sometimes impart a reddish shade to the surface of cotyledons. Both surfaces of cotyledons were observed to have such bulky glands in depressions (Fig. 6 and 7). Such glands on the ventral surface of cotyledon appeared to have bulky spherical head of diameter c 51 µm and composed of three cells in apical tier when seen from the top. It greatly resembles the three-tiered gland (3 cells in each tier) described by Bhattacharya and Maheshwari (1970, Fig. 2 (Y, page 14) and reproduced here as Fig. 19 (A) from the foliar surface. They have not investigated the cotyledons. The foliar glands were larger (80.85 ± 1.55 µm) than cotyledonary glands (c 51.3 µm). Foliar glands are bulky capitate and their head region is composed of eight peripheral cells surrounding a central circular cell (possibly stalk cell or the distal end of the stalk) (Fig. 13-15). The development and structure of foliar nectaries in R. minima and some other genera of Fam. Papilionaceae are described by Bhattacharya and Maheshwari (1970). The full-grown nectary in R. minima was also reported by them to show eight cells in its apical region in Transverse section (Fig. 2 (Za, page 14 of Bhattacharya and Maheshwari, 1970; reproduced here as Fig. 19(B). The absence of the central cell in the mid of the apical head of the nectary in their studies may presumably be attributed the lower order of magnification employed in their studies (X 768) as compared to that employed in the present studies by SEM (X 1100/1200). Bhattacharya and Maheshwari reported while senescing the upper tier cells lose cytoplasm and cell walls are disorganized (Fig. 19 C and D). The structure of nectaries of R. minima is reported to be somewhat similar to Vicia faba. They are non-vascularized and do not contain crystals. Extra-floral nectaries unlike Mimosaceae and Caesalpiniaceae are considered to be uncommon in Papilionaceae. They occur in tribes Vicieae, Phasoleae, Galegeae, Trifolieae, Hedysaraceae, Delbergieae, Genisteae and Loeae. But they are absent in the tribes Podalyrieae, Sophoreae and Swartzieae. Such nectaries are taxonomically very important in genus Rhynchosia, which has been divided into three groups by Bhattacharya and Maheshwari (1970). Nectaries are absent in R. filipes Benth. and R. pseudo-cajan Cambers. Nectaries are present on the lower surface only in R. acutissima Twaites, R. capitate DC., R. INTERNATIONAL JOURNAL OF BIOLOGY AND BIOTECHNOLOGY 14 (4): 539-559, 2017. 552 D. KHAN ET AL., cyanosperma Benth., R. densiflora DC. R. falconeri Baker, R. minima DC, R. nummularia DC, R. rufescens DC. R. suaveolens DC. and R. viscosa. Nectaries are present on both surfaces of leaf e.g., R. arvensis Bath. Ex Baker. Large elongated glands are also known to occur on Cercideae leaf surfaces. Bauhinia (55 taxa), Cercis (1sp.), Phanera (1 sp.), Piliostigma (2 spp.), Schnella (19 spp.) and Tylosema (1 sp.) were studied by Duarte-Almeida et al. (2015). Forty two Bauhinia taxa had glands on abaxial surface. From humid forests Bauhinia spp. had no gland surface. Density of glands was higher on species from “Cerrado” (a savanna ecosystem) and “Caatinga” (a semi-arid ecosystem from Northwest Brazil. N = 40 Mean = 80.852 SE = 1.552 Median = 80.362 CV = G1 = 0.363 Sg1 = 0.374 G2 = - 0.320 Sg2 = 0.733 Minimum = 62.32 Maximum = 101.68 KS-z = 0.812 P < 0.524 Fig. 18. Diameter (µm) of glands in head region on the ventral surface of leaf. Fig. 19. Morphological structure of foliar nectary of R. minima as given by Bhattacharya and Maheshwari (1970) at magnification: X 768. Mature nectary showing three tiers of three cells each subtended by a single cell (A). A transverse section of nectary at this stage shows 8 cells (B). C, Mature nectary showing remnants of cytoplasm in the upper tier. The cell walls have become disorganized. D, Enlarged view of a nectary. INTERNATIONAL JOURNAL OF BIOLOGY AND BIOTECHNOLOGY 14 (4): 539-559, 2017. SEEDLINGS MORPHOLOGY OF RHYNCHOSIA MINIMA (L.) DC. (PAPILIONACEAE) A B 553 N = 100 Mean = 914.933 SE = 16.1289 CV = 17.63% G1 = 0.279 G2 = 0.023 KS-z = 1.040 P < 0.229 Symmetrical N = 100 Mean = 132.48 SE = 7.5584 CV = 57.05% G1 = 0.312 G2 = -0.247 KS-z = 1.505 P < 0.022 Asymmetrical Fig. 20. Frequency distribution of density of glands per cm2 on the ventral surface of young (A, around 1 cm2 in size) and mature (B, around 6 cm2 in size) leaf of R. minima. G1 = skewness and G2 = kurtosis. The errors of skewness and kurtosis, Sg1 and Sg2, in each case were 0.241 and 0.478, respectively, for N = 100. Stomata In present studies, R. minima showed a variety of stomatal types. Paracytic stomata were found on adaxial and abaxial surfaces of cotyledons (Fig. 6 and 7). There were, however, paracytic, anisocytic, staurocytic and anomocytic types of arrangements of subsidiaries in foliar stomata (Fig. 21-23). Parveen et al. (2007) reported only anomocytic stomata in R. minima. Metcalfe and Chalk (1979) have reported paracytic, anomocytic and parallelocytic but not anisocytic stomata from Papilionaceae. According to Tripathi and Mondal (2012) three stomatal types of Leguminales are paracytic, anisocytic and anomocytic – found in various combinations in Caesalpiniaceae, Mimosaceae and Fabaceae. The basic type of stoma in R. minima in our studies, however, appeared to be paracytic one which is considered to give rise to other stomatal types. Stace (1966) opined that many of the genera may have basically paracytic subsidiary cells but that extra walls may develop and thus giving the appearance of an anomocytic state. According to Stace (1966) the occurrence of anomocytic, anisocytic and paracytic stomata on one leaf of Anopyxis (Boodle and Fritsch in Metcalfe and Chalk, 1950) is probably explicable in this way. The studies into the seedling structure of Cassia fistula (D. Khan, unpublished) are supportive to the phenomenon of transformation of a paracytic stoma into anisocytic one by formation of a cell wall in a subsidiary cell. Paracytic stomata are, however, the most frequent in several Papilionaceous plants (Metcalfe and Chalk, 1950, 1979; Bokhari and Dasti, 199; Freire et al., 2005; Almeida, 2010, 2011; Tripathi and Mondal, 2012; Khan et al., 2014, 2015a and b). INTERNATIONAL JOURNAL OF BIOLOGY AND BIOTECHNOLOGY 14 (4): 539-559, 2017. 554 D. KHAN ET AL., A B C * * * Fig. 21. Stomatal types of R. minima. A, Two paracytic stomata are clearly visible. B, a paracytic stoma (bottom) and the other paracytic stomata turning into an anisocytic stoma due to a wall development in the upper subsidiary cell (shown by an arrow). C, An anisocytic stoma. Subsidiaries and ground epidermal cells are sinuous in their anticlinal contours. There were also some abnormal stomata in R. minima. A half stoma associated contiguously with a paracytic stoma (Fig. 24). The stomatal abnormalities may be the result of environmental perturbations as suggested by several workers (Car and car, 1990; Gan et al., 2010). It has been suggested by Croxidale (2000) that structure, INTERNATIONAL JOURNAL OF BIOLOGY AND BIOTECHNOLOGY 14 (4): 539-559, 2017. SEEDLINGS MORPHOLOGY OF RHYNCHOSIA MINIMA (L.) DC. (PAPILIONACEAE) 555 development and patterning of stomata on the leaf surface is the function of complex processes. This should be viewed from evolutionary, physiological, ecological and organ point of view. Local flora need to be investigated from such point of view. A * * B * * * * * * Fig. 22. Rare staurocytic type of arrangement of subsidiaries in R. minima. Fig. 23. Two anomocytic stoma on mature leaflet – ventral surface. Magnification: 45 x15Xand zoom 2X. R. minima leaflets are amphistomatic with much larger number of stomata on ventral surface (283.3 ± 4.390 stomata per mm2, N = 100, CV = 38.6%) as compared to the dorsal surface (48.06 ± 1.853 stomata per mm 2, N = 100, CV = 38.6%). On dorsal surface the stomatal density was distributed asymmetrically (positively skewed and leptokurtic, KS-z = 1.667, p < 0.008). Around 76% of observations on stomatal density fell in the size class of 21-60 stomata per mm2. On ventral surface, in 74% of the observations the stomatal density fell between 250 and 300 stomata per mm2 (Fig. 25). The stomatal size on abaxial surface of R. minima leaf, based on observations of 60 stomata, averaged to 18.53 ± 0.36 x 13.17 ± 0.30 µm. In more than 72% of the observations, stomatal length (pore + guard cells) fell between INTERNATIONAL JOURNAL OF BIOLOGY AND BIOTECHNOLOGY 14 (4): 539-559, 2017. 556 D. KHAN ET AL., 15.1 and 20 µm. The predominating class of stomatal width (pore + guard cells) was 10.1 – 16 µm. Stomata on adaxial surface were comparable in size (19.76 ± 0.22 x 11.19 ± 0.18 µm, N = 60) to that on the abaxial surface. * * * * * Fig. 24. Besides a normal paracytic stoma in right lower corner, an abnormal stoma may be seen in the upper left corner. It is surrounded with three subsidiaries and provided with a half stoma (single guard cell) besides an adjacent normal stomatal pore. A N = 100 Mean = 48.064 SE = 1.8528 Median = 49.145 CV = 38.55 G1 = 0.716 Sg1 = 0.241 G2 = 0.929 Sg2 = 0.478 Minimum = 9.83 Maximum = 108.12 KS-z = 1.617 P < 0.008 B N = 100 Mean = 283.28 SE = 4.3906 Median = 276.88 CV = 15.50 G1 = 0.244 Sg1 = 0.241 G2 = - 0.241 Sg2 = 0.478 Minimum = 190.4 Maximum = 415.3 KS-z = 1.254 P < 0.086 Fig. 25. Frequency distribution of stomatal density per mm2 on dorsal and ventral surface of mature leaf of R. minima. INTERNATIONAL JOURNAL OF BIOLOGY AND BIOTECHNOLOGY 14 (4): 539-559, 2017. SEEDLINGS MORPHOLOGY OF RHYNCHOSIA MINIMA (L.) DC. (PAPILIONACEAE) 13000 557 scan 1 12000 Acquisition Parameter Instrument : 6380(LA) Acc. Voltage: 20.0 kV Probe Current: 1.00000 nA PHA mode : T3 Real Time : 41.44 sec Live Time : 30.00 sec Dead Time : 27 % Counting Rate: 6389 cps Energy Range: 0 - 20 keV 10000 9000 CKa 11000 7000 OKa 5000 4000 ClKa 3000 2000 1000 KKa CaKa KKb CaKb 6000 ClKb Counts 8000 0 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 keV ZAF Method Standardless Quantitative Analysis Fitting Coefficient : 0.6366 Element (keV) mass% Error% At% Compound C K 0.277 53.42 0.26 61.60 O K 0.525 42.78 1.39 37.04 Cl K 2.621 0.64 0.37 0.25 K K 3.312 2.29 0.52 0.81 Ca K 3.690 0.87 0.61 0.30 Total 100.00 mass% Cation 100.00 JED-2300 AnalysisStation Fig. 26. Elemental analysis of gland and the adjoining area of R. minima leaf. INTERNATIONAL JOURNAL OF BIOLOGY AND BIOTECHNOLOGY 14 (4): 539-559, 2017. K 56.3506 35.7435 1.3525 4.7238 1.8296 558 D. KHAN ET AL., EDS Determination of Elements Elements detector system (EDS) based on Energy dispersive X-ray spectroscopy (EDS) attached to SEM was employed for element detection and quantitative elemental analysis. The result of scan for the foliar region containing an extra-floral nectary is presented in Fig.26. In scan, the dominating element was carbon (53.42 ± 0.26%), followed by oxygen (42.78 ± 1.39%), Calcium (0.87 ± 0.34%), Potassium (2.29 ± 0.52%), Magnesium (2.27 ± 0.29%) and Chlorine (0.64 ± 0.37 %). Barnes (1996) has presented data on nitrogen and macro- and micronutrients from the harvested herbage of R. minima in Nyankpala trials (Ghana) and analyzed according to AOAC (1970) methods and expressed as mg/kg biomass dry weight. The content of Nitrogen was 1.81mg/kg, that of Ca (7171 mg /kg), P (899 mg/kg), Na (103.96 mg/kg), K (3198 mg/ kg), Mg (326 mg/kg), Mn (395 mg/kg), Zn (39 mg/kg), Cu (7 mg/kg), and Fe (760 mg/kg). 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