An Investigation of the Vegetative Anatomy of Piper sarmentosum , and a Comparison with the Anatomy of Piper betle ( Piperaceae )

This paper describes the detailed anatomy and micromorphology of the vegetative parts of Piper sarmentosum Roxb. (synonym, P. lolot C.DC.), which is a southeast Asian medicinal plant valued for its medicinal and culinary uses. The study also compares the anatomy of the leaf, petiole, stem and root of P. sarmentosum with those of the well-characterized P. betle L. The leaves of both the species are morphologically comparable and have similar culinary uses. The present study showed that the leaves of both the species possess more or less comparable anatomical features with a few differences; whereas, the microscopic features of the petiole, stem, and root of the two species are distinctive.


Introduction
Plants of the genus Piper L. (Piperaceae) are economically important because of their medicinal and culinary uses.The genus is represented by about 1050 species distributed mainly in the tropics [1].Piper sarmentosum occurs in the tropical forests in Cambodia, southern China, India (NE India and Andaman Islands), Indonesia, Laos, Malaysia, Philippines and Vietnam [2,3].
The leaves of P. sarmentosum are traditionally used as a condiment and also in many South Asian cuisines.In traditional medicine, the leaves are consumed for their carminative properties and the whole plant possesses anti-inflammatory, expectorant and anodyne properties.The plant is also used to cure skin diseases, rheumatism, headache, diarrhea, fever, indigestion and toothache.In addition, the roots can be administered for the treatment of cough, asthma and toothache [6].The dried fruits (infructescence) are occasionally used as spices and medicines [3].
Identification of the leaves of Piper sarmentosum is often difficult because of the possible confusion with the leaves of betel (P.betle L.) due to morphological resemblances as well as their similar usage as condiments.The anatomy of P. betle has been well studied previously [7][8][9][10][11][12], whereas, anatomical study of P. sarmentosum has been neglected.P. sarmentosum is also morphologically similar to and has been in the past confused with another species, viz., P. longum L. [2,3].Even though Gilbert [5] merged P. lolot C. DC. as a synonym under P. sarmentosum Roxb., many publications [1] and some online databases like www.ars-grin.gov;www.plantnames.unimelb.edu.au, www.wikipedia.org,etc. still treat the two species as distinct ones.
In spite of the prevailing confusions with other morphologically similar species, the anatomy of Piper sar-mentosum has been neglected.Therefore, the present work was undertaken to provide detailed anatomy and micromorphology of the species, by light and scanning electron microscopy (Figures 1-3).In addition, the anatomy of leaf, petiole, stem and root of P. sarmentosum was compared with those of P. betle (Figure 4 & Table 1), for proper identification.

Materials and Methods
Authentic samples of the fruiting plant of Piper sarmen-tosum (MPG # C004) and the male plant of P. betle (MPG # C006) grown in the green house at the University of Mississippi (UM)'s medicinal plants garden (MPG) were used for this study.Specimens of P. sarmentosum from live plants (NCNPR # 9860) as well as fresh twigs (NCNPR # 9861), purchased online from www.toptropicals.com and www.importfood.com,respectively, were also used for the study.Herbarium specimens of all the samples were deposited in the Repository of Botanicals at the National Center for Natural  Products Research (NCNPR), in the School of Pharmacy, University of Mississippi, MS, USA.

Sample Preparation for Light Microscopy
Freshly collected samples were fixed in FAA for two days and sections were prepared using razor blades.The sections were treated with chloral hydrate solution and stained with phloroglucinol/HCl, and photomicrographs were prepared using Nikon E600 and Nikon E600 POL microscopes equipped with Nikon DS˗Fiv camera systems and Nikon Elements imaging software (Nikon Inc., Tokyo, Japan).
The fixed specimens were also used for the preparation of permanent slides.The specimens were dehydrated using a series of alcohol solutions (10%, 20%, 30%, 50%, 70%, 95% and 100%) and passed through graded series of xylene: alcohol solutions up to 100% xylene.The specimens were then transferred to molten paraffin and specimen blocks were prepared using molds.Tissues em-bedded in blocks of paraffin wax were sectioned at thickness of 6 -15 µm using a Leica RM2255 fully automatic rotary microtome (Leica Microsystems, Wetzlar, Germany) and the sections were stained by safranin and counter stained with Fast Green using routine protocols [13,14].

Sample Preparation for Scanning Electron Microscopy (SEM)
Fresh samples of various parts of the plant were fixed overnight in gluteraldehyde (2%) solution, washed with water and dehydrated by passing through increasing concentrations of acetone in water according to a standard method [15].The dehydrated specimens were then critical point dried in a Denton Vacuum critical point drier (Denton Vacuum, Moorestown, NJ, USA) using liquid CO 2 as a cryogenic fluid.The fully dried samples were mounted on aluminum stubs using glued carbon tapes and then coated with gold using a Hummer 6.2 sputter  Figures 1(c), (d)), of anisocytic, actinocytic, anomocytic, tetracytic and cyclocytic type [16], with 3 to 7 subsidiary cells arranged in one or more whorls, measuring 24 -30 µm long and 20 -27 µm wide; stoma about 20 µm long and 5 µm wide.Almost every cell in the adaxial epidermis contains prisms (10-13 µm × 3-6 µm), small druces and rods (8-13 µm × 4-5 µm) of calcium oxalate.Sand crystals of calcium oxalate were seen in almost every cell in the leaf, except vascular tissues.Minute prismatic crystals of calcium oxalate are seen in palisade and spongy cells; large silica crystals of varying size and shape are occasionally seen in hypodermal cells (Figures 2(a), (b)).Numerous hydathodes (Figure 1(a)) of 17 -22 µm in diameter, some with inclusions, are observed on both the epidermis (Figures 1(a)-(f)).Two types of trichomes are observed in P. sarmentosum: glandular (Figures 1(b), (e)) and non˗glandular (Figure 1(f)).Glandular trichomes [referred as "hydathode" by Chibber [11] and as "pearl-glands" by Vasuki et al. [9] in P. betle] present in the laminar region of the upper and lower surfaces; stalk 1-celled, narrow, sunken in the epidermis, surrounded by 5 -7 radiating epidermal cells; body unicellular, ovate-oblong, up to 58 µm long and 14 µm in diameter, often containing yellow substance, curved or nearly parallel to the leaf surface, smooth and thin-walled, obtuse at apex and narrowed at base.Non-glandular trichomes present usually on the veins, on both surfaces of leaf but more densely on the abaxial surface, most commonly 1-celled but also frequently 2-3-celled, up to 37 µm long and 14 µm across, usually straight or rarely slightly curved, with broad base and more or less pointed apex, rarely containing yellowish brown content, outer wall warty.

Leaf Midrib
Midrib is prominently raised abaxially and slightly raised adaxially.Trichomes mostly confined to the abaxial surface.In transverse section, both adaxial and abaxial epidermal layers covered externally by thin cuticle and most cells with papillae of growing trichomes.Hypodermal layers of adaxial epidermis 2 -  2(c),  (d)).

Petiole
Petiole grooved adaxially, heart˗shaped in cross section (Figure 3(b)).Epidermal layer made up of tabular or squarish cells measuring 15 -23 µm long and 10 -15 µm high, with dense cytoplasm, covered externally by cuticle of about 2 µm thick, most cells giving rise to 1-3-celled nonglandular trichomes (Figure 3(a)).Hypodermal layers 1 -2, consisting of polygonal, circular or slightly tangentially elongated parenchyma cells.This is followed by 6 -10 layers of angular collenchyma found as interrupted patches all around the petiole; cells polygonal with densely thickened walls and very narrow lumina.These collenchyma patches are separated from each other by thin-walled parenchyma of few to many cells wide.Vascular bundles collateral, few, arranged in a U-shaped arc in the ground tissue, larger and smaller bundles alternating with each other, and every bundle is positioned opposite to a collenchyma patch; phloem is facing outwards and the xylem facing the center of the petiole; xylem vessels 135 -385 µm long and 14 -31 µm wide, walls spiral or annular thickened, lignified, straight or obliquely pitted; some of the protoxylem elements slightly wavy; tracheids 165 -240 µm long and 16 -19 µm wide, with bluntly pointed tips, and spirally thickened and lignified walls.Vascular bundles are sheathed by 1-5-layered collenchyma with slightly thickened walls.Ground tissue made up of polygonal or circular parenchyma cells, most of them containing sand crystals of calcium oxalate, and abundant polyhedral or shell-shaped starch grains measuring 4.5 -6.5 µm; few cells contain silica crystals of varying shape and size and few others contain oil droplets.A large lysigenous mucilage canal, 90 -250 µm in diameter, is present at the center of the petiole.

Stem
Surface view of the epidermal layer of the stem exhibits polygonal cells with straight and slightly thickened anticlinal walls.Stomata present, similar to those of leaf.Nonglandular trichomes scattered, 1-2-celled.In transverse section (Figure 3

Root
The Transverse section of the primary main root is circular in outline (Figure 3(f)).Epidermis made up of 1-layer of tabular cells containing brown contents; some of the cells giving rise outwards to slender, unicellular rhizoids or root hairs.This is followed by 1 -2 layers of meristematic parenchyma (phellogen

Conclusion
In the current contribution, a detailed microscopy of the leaf, petiole, stem, aerial root, and root of Piper sarmentosum is provided (Figures 1-3).In addition, a comparative study of anatomy of leaf, petiole, stem and root of P. betle is provided (Table 1, Figure 4).The leaf anatomy of both the species are more or less comparable except that P. betle usually has more hypodermal layers in the adaxial epidermis and more of the secretory cells in the mesophyll region.Capitate glandular trichomes with stalk sunken in the epidermis; cortical fibers embedded in collenchyma; additional mucilage canals opposite to major vascular bundles; and numerous rod-shaped crystals of calcium oxalate in ground tissue are observed in the petiole only in P. betle.Of the two species, P. betle stem can easily be distinguished by the presence of cortical fibers as well as an additional ring of mucilage canals while these features are not observed in P. sarmentosum.The root anatomy in P. sarmentosum shows cortical fibers in outer cortex; parenchymatous pericycle; a continuous ring of xylem with 1-3-cell wide shallow medullary rays; and well defined parenchymatous pith.Whereas, in P. betle, cortical fibers are not observed; pericycle consists of incomplete ring of lignified and pitted sclereids; xylem forming 7 -9 irregularly-shaped arches separated by 9-25-cells wide medullary rays; and the pith is replaced by lignified and thickened-walled xylem parenchyma.

Figure 3 .
Figure 3. Micromorphology and anatomy of Piper sarmentosum.(a: SEM; b-f: LM; b, d: stained in safranin/fast green; c, e, f: stained in phloroglucinol/HCl). a: surface of petiole showing parallely elongated epidermal cells and abundant nonglandular trichomes; b: TS of petiole showing U-shaped arrangement of vascular bundles and a central mucilage canal; c: TS of stem showing discontinuous ring of collenchymatous outer cortex, cortical and medullary whorls of vascular bundles and a large central mucilage canal; d: TS of a collateral medullary vascular bundle of stem surrounded by pith parenchyma; e: TS of aerial root showing wide cortex and pith separated by a narrow ring of vascular tissue; f: TS of root showing collenchymatous ring of outer cortex with sporadic fibers, wide parenchymatous inner cortex, wide ring of excentric xylem and a central pith.(Ca: cambium, Cb: cortical bundle, Cf: cortical fibers, Ck: cork, Co: collenchyma, Cx: cortex, Mb: medullary bundle, Mc: Mucilage canal, Pa: parenchyma, Ph: phloem, Pi: pith, Vb: vascular bundle, Xy: xylem).Scale bars: a, d = 100 µm; b, e, f = 500 µm; c = 250 µm.

Table 1 . Comparative anatomy of Piper sarmentosum and P. betle.
-cells wide, made up of radially elongated thinwalled parenchyma cells.The stele is occasionally eccentric which imparts anomalous structure to the root anatomy.Phloem occurs as patches between medullary rays; cells polygonal, thin-walled, 3 -12 µm long, 2 -10 µm high.Cambium indistinct.Xylem elements forming a continuous ring of 190 -335 µm wide, consisting of vessels, fibers and xylem parenchyma-all with lignified walls.Vessels 15 -50 µm in diameter, with spirally thickened, pitted and lignified walls; xylem parenchyma consists of polygonal cells, with moderately thickened, pitted and lignified walls, radially arranged and somewhat elongated, measuring 6 -31 µm in length, 6 -27 µm in breadth.Pith occupies the central part of the root; cells polygonal or circular, 12 -40 µm in diameter, filled with starch grains which are of two types: large, polyhedral grains measuring 8 -10 µm in diameter and smaller shell-shaped grains measuring 2 -5 µm in diameter.Mucilage canals absent.