Pleione 11(2): 187 - 194. 2017. ISSN: 0973-9467 © East Himalayan Society for Spermatophyte doi:10.26679/Pleione.11.2.2017.187-194 Studies on foliar architecture and micromorphology of Gynocardia odorata R.Br. (): a well-known medicinal of Assam, India

Dipjyoti Kalita2, Nilakshee Devi and Mohan Ch. Kalita1 Department of Botany, Gauhati University, Guwahati-781014, Assam, India 1Department of Biotechnology, Gauhati University, Guwahati-781014, Assam, India 2Corresponding author, e-mail: [email protected] [Received 31.10.2017; Revised 05.12.2017; Accepted 12.12.2017; Published 31.12.2017]

Abstract Gynocardia odorata R.Br. (Achariaceae) is a crooked, small to medium East Indian evergreen tree. The present investigation elucidated its foliar architecture and micro-morphological characteristics . Qualitative and quantitative characters of veins, vein order, areolation; epidermal cell, cell wall nature, length, breadth and number; stomatal distribution, type, frequency and index were taken into consideration for enhanced taxonomic comprehension. Present study is also an attempt to evaluate foliar epidermal features as aid to identification and delimitation of the genus Gynocardia R.Br. Key words: Foliar architecture, Micro-morphology, Gynocardia odorata

INTRODUCTION Commonly known as ‘Chaulmugra’, Gynocardia odorata R.Br. is belonging to Achariaceae (earlier in ) and the genus is monotypic (Rana & Ranade 2009; Bera et al. 2014). This is a small to medium East Indian evergreen tree, native to the moist forests of North East India (Bera et al. 2014) and it is fairly common in evergreen forests throughout the province Kanjilal et al. (1934). It is also known to grow in Terai and Duars regions of the adjacent state West Bengal (Clement 1991). The plant is used in various folk medicines and also in ayurvedic formulations (Khare 2004). The seeds of the plant are highly medicinal and are rich in oil content. They are grayish, irregularly ovoid, compressed, angular, smooth and possesses a peculiar nauseous aroma (Kurz 1877). Chaulmugra oil has long been applied for stiff joints and sprains, rheumatism, neuralgia and both internally and externally in leprosy, secondary syphilis, scrofula, phthisis, eczema, psoriasis and other inflammatory skin diseases (Cook 1896; Felter & Lloyd 1905; Felter 1922; Rai & Lalramnghinglova 2010). The juice from the paste of young shoots is drunk to combat jaundice (Shrestha & Dhillion 2003). The genus Gynocardia was first described by Robert Brown (1820) in the third volume of William Roxburgh’s “ of the Coast of Coromandel”. The generic name Gynocardia comes from the ancient Greek words “Gyne” means female or woman and “Kardia” means heart (directly referring to ovary), indicating its heart shaped ovary (Quattrocchi 1999; Patil 2007). So, the meaning of the generic name is “heart shaped ovary” or “heart shaped fruit”. This dioecious tree grows up to 30 m tall and has an untidy branching habit. Alternately arranged leaves are oblong- elliptic, rarely ovate-oblong or obovate-oblong, 13 – 20 cm x 5 - 188 Foliar architecture and micromorphology of Gynocardia odorata 10 cm, leathery. Pale yellow flowers are sweet scented that arises from old stem. Flowers have five rounded petals and a mass of long stamens at the center in males. Female flowers solitary and the fruits are slightly compressed-rounded. Taxonomic importance of foliar epidermal characters as well as the types of stomata and trichome in angiosperms has been widely emphasized by many authors including Stace (1961), Shah & Kothari (1975), Lavania (1990), Padmini & Rao (1995), Adedeji (2004), Kadiri (2006), Garg (2010), Paul & Devi (2013) and Kalita & Devi (2015). Not much has been elicited on the foliar architecture, micro-morphological and anatomical studies of G. odorata, and the present study will provide such characters as aid to its identification and to delimit the genus Gynocardia R.Br. in Achariaceae.

MATERIALS AND METHODS Extensive field studies were conducted during 2015 – 2017 and morphological characters of each individual were studied and documented in the Field Note Book following Hickey (1973). Voucher specimens were processed following standard herbarium techniques (Jain & Rao 1977) and were identified with the help of relevant literatures (Lemke 1988; e-Flora India) and previously identified specimens at GUBH, ASSAM, ARUN and CAL. Foliar architecture study was conducted using techniques suggested by Bersiar & Bocquet (1960) with slight modification. Small pieces of leaves (about 3 x 3 cm) were taken from the the apex, middle and basal portions of lamina on the mid-rib region and were treated with 5 % aqueous NaOH solution for overnight in an oven at 320 C and were later soaked in aqueous solution of HNO3 in various concentrations (30%, 50%, 70%, 90% and 100%). After repeated washing in distilled water the materials were dehydrated through the ethanol grade and kept there until the disappearance of chlorophyll. Permanent slides were prepared from the treated materials and microphotographs were taken. For descriptive terminology Hickey (1973), Dilcher (1974) and Melville (1976) were followed extensively. To study foliar epidermal characters fresh and mature leaves were cleared using techniques suggested by Boulos & Beakbane (1971) with slight modification. Peelings were taken out both from upper and lower epidermis by scrapping out with the help of blade using a 10% HNO3 solution. The peel was stained with 1% aqueous saffranine solution and was mounted with 1% glycerin after proper washing. The nature and distribution of stomata, epidermal cells, guard cells, subsidiary cells, cell size and stomatal index were worked out for both upper and lower epidermises. For description of stomata terminologies suggested by Hickey (1973), Stace (1984) and Prabhakar (2004) were followed. Stomatal Frequency: The stomatal frequency was calculated by following the formula of Ghosh & Davis (1973). Stomatal Frequency (S.F.) = S/A Where, S = Number of stomata per field A = Area of the field Stomatal Index: Stomatal index was calculated by following the formula of Dilcher (1974)and Salisbury (1927). Stomatal Index (S.I.) = Where, S = Number of stomata per unit area E= Number of epidermal cells per unit area Dipjyoti Kalita et al. 189 Guard Cell Area: The guard cell area were calculated by following the formula of Franco (1939). Guard Cell Area (G.C.A) = L×B×K Where, L = Length of the guard cell of the guard cell K = Franco’s Constant (K= 0.78524)

RESULTS Foliar Architecture: The leaves of investigated species were unicoastate reticulate and the major venation pattern was pinnate-brochidodromous or co-arcuate type (Plate I; Tables 1 & 2). The primary vein (1º) was moderate and straight, whereas 16 – 22 secondary veins (2º) were arranged alternately on either sides of primary veins forming smooth arch. The secondary veins (2º) were of weak brochidodromous type with basally increasing irregular spacing. 3 – 6 strong intersecondaries were also observed. The tertiary veins (3º) were alternate percurrent type with straight vein course, forming acute angle to primary (1º) which were inconsistent; quaternaries (4º) alternate percurrent type. Quineries (5º) are regular polygonal reticulate type and senaries (6º) were of thin dichotomizing type. The areoles were oriented, forming a contiguous field over most of the area of leaf which are 5 or more sided or sometimes incomplete. The free vein-ending were the senaries (6º) which were 3 times branched.

Table 1. Qualitative foliar architectural data of G. odorata R.Br.

Primary Secondary vein Tertiary Vein Vein

0 Size Size Size Angle Angle Course Course Spacing Spacing Category Category Category Category FEVS FEVS angle to 1 to angle 0 Areolation Areolation vein category category vein category vein 3 0 0 vein category category vein 4 5 0 6 Inter secondary vein vein secondary Inter vein angle variability angle vein

0 3 Acute Acute Strong Strong Pinnate Pinnate Straight Straight Irregular Irregular Moderate Moderate 3 branched branched 3 Inconsistant Inconsistant Dichotomising Dichotomising 5 or more sided sided more or 5 Moderate to Weak Weak to Moderate Alternate percurrent percurrent Alternate percurrent Alternate Weak Brochidodromous Brochidodromous Weak Regular polygonal reticulate reticulate polygonal Regular Abruptly increasing towards base base towards increasing Abruptly 190 Foliar architecture and micromorphology of Gynocardia odorata

A B

C D

E F

PLATE - I. Gynocardia odorata R.Br. (Achariaceae): A. a full grown tree; B. leafy twig; C. formation of coastal vein; D. detailed vein network; E. stomata in the lower part of the lamina; F. epidermis in the upper part of the lamina. Dipjyoti Kalita et al. 191 Table 2. Quantitative foliar architectural data of G. odorata R.Br. Prima Secondary Tertiary Quaternary Vein Pentanary Senary ry vein vein (40) vein vein Vein (20) (30) (50) (60) (10)

0 0 0

0 Angle Angle Angle Angle Angle Number angle with 4 Length (mm) Length (mm) Length (mm) Length (mm) Length (mm) Length (mm) angle with 2 angle with 3 0 0 0 angle with 1 6 4 5 0 3 157.0 22 52.0 490 36.0 610 58 21.0 690 630 10.0 740 670 3.0 890 520 ± ± ± ± ± ± 0 ± ± ± ± ± ± ± ± ± 0.2 0.18 0.24 0.43 0.11 1.4 ± 0.36 0.4 1.3 0.2 0.13 0.26 0.1 0.52 0.36 1 0.8 1

Table 3. Qualitative foliar morphological data of G. odorata R.Br. Leaf Petiole Lamin Blade Laminar Apex Base Lobatio Marg attachment features & a size class shape and angle angle n in and attachment range symmetry and and type organizatio (mm2) shape shape n Alternate; Pulvinate; 4523- Mesophy Oblong; Acute; Acute; Unlobed Entire even- marginal 25376 ll symmetric acuminat rounde pinnate al e d Table 4. Qualitative foliar micro-morphological data of G. odorata R.Br. Surface Epidermal cells Coastal Trichome Stomatal Guard Type of Cell Cell Wall Area Size Cell Stomata Shape Upper Irregular Prominent Distinct - - - and arched Lower Irregular Prominent Distinct - Equal Unequal Diacytic and and sinuous undulate

Foliar Micro-morphological Characteristics: Epidermis: Qualitative and quantitative micro-morphological characteristics of epidermis were presented in tabular form (Tables 3 & 4). Epidermis was covered with non-uniform single layered cuticle with undulate and slightly sinuous prominent anticlinal walls. There was no definite pattern of arrangement of epidermal cells. The coastal area was distinct. The size of epidermal cell ranges from 30.38 – 32.97 μm × 50.82 - 53.44 μm on lower surface whereas on upper surface it ranges from 30.49 - 31.72 μm × 51.67 - 54.34 μm. The L/B ratio of epidermal cells on both surfaces was ±0.62. Stomata: Leaves hypostomatic, stomata were non-uniformly distributed in intercoastal areas but occasionally a few stomata may also present in the coastal areas. The type of stomata was diacytic, and they were widely separated from each other by epidermal cells. The average size of the stomata was 39.66 – 41.03 μm × 49.73 – 50.58 μm. Size of stomatal 192 Foliar architecture and micromorphology of Gynocardia odorata aperture on the lower surface was ± 0.72 µm x 0.25 µm. The L/B ratio of stomata and stomatal aperture were 0.91 and 2.88 respectively. The stomatal frequency ranged from 19.54 to 36.56 per sq mm whereas the stomatal index on the lower surface ranged from 186.42 to 257.70 per sq mm [Table 5]. Table 5. Quantitative foliar micro-morphological data of G. odorata R.Br.

SS EC SA Ratio (L/B) GCA

2 2 mm Surface SI (µm) SF (µm) L (µm) B (µm) L (µm) B (µm) L (µm) B (µm) Stomata Epi. Cell No. of Stomata per No of EC per mm Stomatal Aperture

- 987 ------0.62 - - Upper 51.67 ± 1.44 30.49 ± 0.44

59 905 186 19.5 0.91 0.62 2.88 0.44 .42 4 Lower 0.72 ± 0.04 39.66 ± 1.3 0.25 ± 0.01 50.82 ± 0.44 30.38 ± 0.98 49.73 ± 1.09

DISCUSSION The investigated species showed mesophyll type of leaf blade. Lamina oblong, symmetrical, unlobed, acute. Foliar micro-morphological studies revealed that the epidermal cells are usually irregular in shape on the upper surface, whereas on the lower surface they were with a combination of undulate and irregular shaped sinous cells. The stomata were completely absent on the upper surface, whereas on the lower surface the number of stomata varies from leaf to leaf (59 – 65 per mm2). Foliar architecture of G. odorata showed that the major venation pattern of the leaves was pinnate brochidodromous type where the primaries were moderate and secondaries were weak to moderate. Intersecondary veins were strong. The tertiary, quaternary, quinaries and senaries formed the minor venation pattern. Senaries were the ultimate vein order and variation in areole size was also recorded in the present investigation. It is now essential to compare these derived data with other members of Achariaceae and other related taxa, may be also in Flacourtiaceae, to evaluate their importance and characterization of the species.

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