Trends1118 in Biosciences 11(7), Print : ISSN 0974-8431,Trends 1118-1122, in Biosciences 2018 11 (7), 2018

Studies on the Anatomy and Histochemistry of (Mart.) Solms LATHA SADANANDAN1, SOJA S.2 AND THUSHARA V.3 1PG and Research Department of Botany, Sree Narayana College, Kollam - 691001 2 PG and Research Department of Botany, University College,Thiruvananthapuram-695034 3PG Department of Botany, Sree Narayana College, Cherthala, Alappuzha – 688 582 email: [email protected]

ABSTRACT , and starves the water of oxygen, often killing The anatomical studies using hand sections of all parts of fishes. The plants also create a prime habitat for Eichhornia crassipes (Mart.) Solms were supported by mosquitoes, the classic vectors of diseases, Water micrometry. The histochemical localization of primary hyacinth is often problematic in man-made ponds if metabolites and secondary metabolites were carried out uncontrolled (Villamagna and Murphy, 2009). using suitable stains. The anatomical features of the Utilization of this plant to produce eco-friendly products showed absence of cuticle in all parts except , epidermis can help to reduce such environmental problems to on all parts consisted of a single layer of rectangular cells which is a characteristic feature of this species, presence some extent. A preliminary knowledge on the of gas filled chambers and passages in roots, and chemistry of the plant paves way to the discovery of rhizome, Raphide crystals (Calcium oxalate) in new potential drugs in pharmaceutical companies, eco- parenchyma cells of roots, petioles, rhizomes and leaves. friendly biofuel, or eco-friendly products in textile or The type of stomata on the leaves were of anomocytic type. paper industries. With this in view, the study aims to The stomatal indices and their frequency on the upper and understand the anatomy and histochemistry of the lower epidermis were almost the same i.e 47 and 1.6mm2 plant. respectively. The palisade ratio on the leaves was 4 palisade cells per epidermal cell. The histochemical localization MATERIALS AND METHODS for primary and secondary metabolites showed the presence of protein, reducing sugar and starch in all plant parts Anatomy of all parts of Eichhornia crassipes while lipid was found only on leaves. Among the secondary (Mart.) Solms viz., root, rhizome, petiole and leaf metabolites studied, only phenols were present while sections were studied using hand sections stained with alkaloids and flavonoids were absent. Proteins were Safranin and measured by micrometry. Microscopic localized on the vascular tissues while starch on the characters of the leaf such as stomatal type, stomatal palisade tissues in leaf and cortex in other parts. Reducing index on the upper epidermis and lower epidermis, sugar were present in the cortex. Lipid was present as thick cuticle on the leaf surface. Phenols were localized stomatal frequency, palisade ratio and features such in most tissues of all parts of the plant. A preliminary as Calcium crystals, sclerids and trichomes were understanding about the anatomical characters and studied. The values were found out using the formulae histochemistry of Eichhornia crassipes (Mart.) Solms (Wallis, 1997): helps one to find out any variations that occurred in them S X 100 by environmental or anthropogenic factors such as pollution. Stomatal Index = —————

Key Words Eichhornia crassipes, Anatomy, E+S Histochemistry Stomatal frequency = Stomatal frequency was calculated by multiplying the number of stomata by Eichhornia crassipes (Mart.) Solms, commonly the field area of 0.0346 mm2. known as water hyacinth, belonging to the family Palisade ratio = Average number of palisade is an (Plate 1A) native cells beneath one epidermal cell, using four contiguous to the Amazon basin (Brazil), and is often considered epidermal cells for the count. a highly problematic invasive species outside its native range. One of the fastest growing plants known, E. The localization of various phytochemicals such crassipes reproduces primarily by way of runners or as primary and secondary metabolites in the various stolon, which eventually form daughter plants. Each parts of plant tissues were carried out by taking the plant can produce thousands of seeds each year, and hand sections of the plant tissues viz., root, rhizome, these seeds can remain viable for more than 28 years. petiole and leaf, followed by their staining in suitable When not controlled, water hyacinth will cover lakes stains viz., Coomassie Brilliant Blue (Protein), and ponds entirely. This dramatically impacts water Dragendorff’s reagent (alkaloids), Fehling’s reagent flow, blocks sunlight from reaching native aquatic (reducing sugar), 5% alcoholic Ferric chloride SADANANDAN et al., Studies on the Anatomy and Histochemistry of Eichhornia crassipes (Mart.) Solms 1119

(phenols), Iodine solution (starch), Lead acetate absent. The mesophyll is differentiated into palisade (flavonoids), Sudan III (oil, cuticle and suberin) and spongy mesophyll. Palisade layer is present in both upper and lower side of the epidermis. The mesophyll RESULTS AND DISCUSSION consist of a large number of air spaces surrounded by ANATOMY STUDIES thin walls of chloroplast. Smaller and larger vascular bundles are present in both upper and lower epidermis ROOT side. Each vascular bundle is collateral with xylem Root epidermis consisted of single layered towards the lower epidermis side and phloem towards compactly arranged rectangular cells (4.0µm). There the upper epidermis side (Plate 1E). Vascular bundles is no cuticle on the outside of the root epidermis. are surrounded by bundle sheath of parenchyma cells. Hypodermis is composed of 1 – 2 layers of thick walled Numerous raphides and sclereids are observed in the cells (3.5 µm). Beneath the hypodermis cortex is palisade layer (Plate 1H and 1I). differentiated into outer and inner cortex. The outer The stomatal type of Eichhornia crassipes was cortex is composed of 3 – 4 layered parenchymatous Anomocytic, i.e. the size of the subsidiary cells and cells (4.0 µm). Each air space (7.0 µm) has trabeculae. epidermal cells are equal (4 µm) . The average size of The inner cortex consists of 6~10 layers of guard cells was calculated to be 7 µm×4 µm, while parenchymateous cells. There is no sclerenchyma cell the average size of the stomatal pore was 4 µm×4 µm in the cortex. The stele is surrounded on the outside on both upper and lower epidermis (Plate 1F) by single layered endodermis (3.5 µm). Endodermis is .Comparison of average Stomatal Indices on the lower followed by a single layered pericycle. The stele and upper epidermis of the leaves showed that the consists of 7 to 10 xylem bundles alternating with leaf is Amphistomatic i.e. there is equal distribution of phloem bundles (3.5 µm). Each vascular bundle consist stomata on both epidermis (approx. 47) with an of a single metaxylem vessel (7.0 µm), surrounded by average of 47.15 on lower epidermis and 47.09 on smaller vessels. The root center is occupied by upper epidermis. This is due to the upright position of sclerified parenchyma cells (4.0 µm)(Plate 1B). the leaves above the water level, allowing equal distribution of sunlight on both surfaces of the leaf. RHIZOME The frequency of stomata on the upper and lower The single layered epidermis is made of epidermis are almost the same, i.e., 1.6 mm2 ( 1.631 compactly arranged rectangular cells (4.0 µm). The mm2 and 1.629 mm2on lower epidermis and upper cortex beneath the epidermis consists of 4 – 6 layered epidermis respectively). The average number of outer cortical cells (3.8 µm) having different size palisade cells per epidermal cell (palisade ratio) is four vascular bundles with patch of sclerenchyma to the (Plate 1G). outside of each bundle. Air space (3.4 µm) is also present in cortical region. Raphides are present in air HISTOCHEMICAL LOCALIZATION spaces. Xylem (5 µm) is V shaped. Phloem is present ROOT in between the arms of xylem. There is an inner portion of large air spaces separated from each other by a Presence of protein in the epidermis and stelar single cell layer of parenchyma. Air space are region. Reducing sugar was concentrated in the spherical. Vascular bundles are also present in the endodermis and inner cortex while starch was center. (Plate 1C). concentrated in the outer cortex and outside metaxylem. Presence of phenol was confirmed PETIOLE throughout the section viz., epidermis, hypodermis, Single layered epidermis composed of outer and inner cortex and stele. Alkaloid was present parenchyma cells (3.5 µm). Cuticle is absent. Vascular in the hypodermis. Flavonoids and lipids were totally bundles are embedded in outer parenchyma cells. absent (Plate 2 A-G). Each vascular bundle has a bundle cap of RHIZOME sclerenchyma cells. Hexagonal air spaces (47 µm ) are surrounded by bands of single layered parenchyma The stelar region showed the presence of protein cells. Raphides are present in aerenchyma. (Plate 1D) and Reducing sugar was observed throughout. Starch was concentrated to the ground tissue inner to cortex. LEAF Among the secondary metabolites phenol was present Epidermis consist of cuticle. Trichomes are in the vascular tissues while the others studied viz., 1120 Trends in Biosciences 11 (7), 2018

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M N O P Plate 1.A- Eichhornia crassipes (Mart.) Solms, B- Root T.S., C- Rhizome T.S., D- Petiole –T.S, E- Leaf- T.S, F- Anomocytic stomata, G – Palisade tissue, H – Raphides, I- Sclerides, J- P:Histochemical localization in leaf alkaloids, flavonoids and lipids were absent (Plate 2 LEAF H-N). Presence of protein in the upper and lower PETIOLE epidermis, stele and palisade tissues. Reducing sugar was concentrated in mesophyll parenchyma while The presence of protein was found in stellar starch was observed in the upper and lower palisade region of the petiole. Reducing sugar was observed in tissues. Phenol was concentrated in the palisade the outer and inner aerenchyma while starch was found tissues. Lipid was present in high concentration on in the hypodermis. Among the secondary metabolites the upper and lower epidermis in the form of cuticle. studied, only phenol was observed in epidermis while Flavonoids and alkaloids were absent (Plate 1 J-P). alkaloids, flavonoids and lipids were absent (Plate 2 O-T). The type of stomata on the leaf was of SADANANDAN et al., Studies on the Anatomy and Histochemistry of Eichhornia crassipes (Mart.) Solms 1121

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Plate 2.Histochemical Localization A-G : Root, H- N: Rhizome, O – T : Petiole 1122 Trends in Biosciences 11 (7), 2018

Anomocytic type though it was reported to be histochemical localization for primary and secondary Paracytic type by Mahmood et al., 2005. Anatomical metabolites showed the presence of protein, reducing studies are supported by earlier workers (Nasir and sugar and starch in all plant parts while lipid was found Ali, 1977, Barnabas, 1996). The absence of alkaloids, only on leaves. Among the secondary metabolites anthraquinones, tannins and saponins in this study is studied, only phenols were present while alkaloids and supported by the Ph.D work of Nehal (1985), while flavonoids were absent. Proteins were localized on they found the presence of sterol. The presence of the vascular tissues while starch on the palisade tissues terpenoids, flavonoids, phenols and cardiac glycosides in leaf and cortex in other parts. Reducing sugar were are supported by Kurup and co-workers (2013). present in the cortex. Lipid was present as thick cuticle on the leaf surface. Phenols were localized in most SUMMARY AND CONCLUSION tissues of all parts of the plant. The work serves a Anatomical characterization of all parts of basic knowledge about the plant and paves way for Eichhornia crassipes (Mart.) Solms were carried it’s exploitation in many ways for the production of out using hand sections of fresh material with Saffranin eco-friendly products thus reducing the environmental aided with Micrometry. Histochemical localization of problems caused by them. primary and secondary metabolites were carried out using suitable stains in all parts of the plant. The LITERATURE CITED anatomical features of plant studied showed many Barnabas, A.D. 1996. Casparian band like structures in the root hydrophytic adaptations such as absence of cuticle in hypodermis of some aquatic angiosperms. Aquatic Bot., 55:217–225. all parts except leaf. Epidermis on all parts of water Kurup, A.R., Divya, R., Jency, B., Sruthy, C., Thampatty, A.R. hyacinth consisted of a single layer of rectangular cells and Veena, P.V. 2013. Detailed Analysis on Phytochemicals, which is characteristically a constant feature of this Antioxidants, Antimicrobial Activity of Eichhornia crassipes. species. A very thin cuticle and thin cellulose walls of International Jour of Scientific Res., 2 (2): 17-19. epidermal cells in a typical hydrophyte facilitate steady Mahmood, Q., Ping, Z., Siddiqui, M.R., Islam, E., Azim, M.R. absorption from surrounding water. The most and Yousaf, H. 2005. Anatomical studies of water hyacinth pronounced anatomical feature of this plant is the (Eichhornia crassipes (Mart.) Solms under the influence of textile wastewater. J Zhejiang Univ. Sci. B, 6(10): 991 -998. presence of gas filled chambers and passages in roots, Nehal, A. A. 1985. Phytochemical and pharmacological studies leaves and rhizome. There were many Raphide on Beta vulgaris and Eichhornia crassipes plants. PhD thesis. crystals (Calcium oxalate) in parenchyma cells of Zagazig University. pg 215 roots, petioles, rhizomes and leaves. The type of Nasir, E. and Ali, S.I. 1977. Flora of West Pakistan.. Islamabad, stomata on the leaves were of Anomocytic type. The Pakistan: National Agriculture Research Council, 114: 1–4. Stomatal Indices and their frequency on the upper and Villamagna, A. and Murphy, B. 2009. Ecological and socio-eonomic lower epidermis were almost the same i.e 47 and impacts of invasive water hyacinth (Eichhornia crassipes): A review. Freshwater Biology, 55 (2): 282–298. 1.6mm2 respectively. The Palisade Ratio on the leaves Wallis, T.E. 1997. Textbook of Pharmacognosy .5th ed.CBS was 4 palisade cells per epidermal cell. The Publishers and Distributors, Delhi.

Received on 13-02-2018 Accepted on 16-02-2018