MICROSCOPIC CHARACTERISATION OF ROOT, STEM AND LEAVES OF AN UNRESOLVED SPECIES: RUBUS INDICUS (ROSACEAE)

MICROSCOPIC CHARACTERISATION OF ROOT, STEM AND LEAVES OF AN UNRESOLVED SPECIES: RUBUS INDICUS (ROSACEAE)

Bhavadas Narayanan ^{* 1}, Harinarayanan Cheruppoyilathu Mana ² and Umesh Balakrishnan Thara ³

Research and Development Centre ¹, Bharathiar University, Coimbatore - 641046, Tamil Nadu, India.

Pharmacognosy Division ², Centre for Medicinal Plants Research, Arya Vaidya Sala, Kottakkal, Malappuram, Kerala - 676503, Kerala, India.

Department of Bioscience ³, MES College, Marampally, Aluva - 683107, Kerala, India.

ABSTRACT: The genus, Rubus is considered as highly potent plant crop and possesses economical, medicinal and ecological importance. Rubus plant species have been utilized inbreeding programme and has identified various pharmaceutical compounds. However, the taxonomy of the genus Rubus has been in a state of flux, because of the absence of comprehensive work and associated taxonomic disputes. Among the genus, most of them are referred to as ‘unresolved name” in the scientific database. The present study discussed the microscopic characterization of one unresolved species, ‘Rubus indicus’ and the examination were carried out by using their vegetative parts (root, stem and leaf). The systematic study of this high potent wild crop (R. indicus) has not been reported so far; hence, the resolved characters may be useful for proper species identification and for meaningful bioprocessing.

Rubus, Microscopic, Unresolved, Vegetative parts, Wild crop

INTRODUCTION: The genus Rubus Linnaeus (1753: 492) ¹ is one of the largest genera in the family Rosaceae comprises of about 1500 species ². Rubus consist of high potent plant species, which has been considered as good germplasm for breeding programme ³, and also documented as folk medicine in various traditional medicinal system ⁴. Due to the significance of nutritional quality and antioxidant capacity inedible fruit, Rubus species have been utilized for healthy diet ^{5, 6}. The fruit of many berry species are fleshier and are consumed in fresh, dried, frozen and product form ^{7, 8}, and have high vitamins, high antioxidant capacity, simple sugars and essential mineral contents ^{9, 10}.

However, the genus has been deemed as taxonomically disputed plants and is complicated by hybridization, polyploidy, agamospermy, and lack of a universal species concept ^{11, 12}. Rubus indicus is commonly known as Indian Raspberry and distributed mainly in India, Sri Lanka, and Thailand. In India, the species has occurred in southern Western Ghats ^{13, 14, 15}. The name was first proposed by Thunberg in 1813 ¹⁶ based on his collection, gathered it from Srilanka.

The species comes under the subgenus Malachobatus, and its ploidy level represents 56 ¹⁷. R. indicus is very attractive and can easily distinguish in naturally by its pretty vine-like leaves and laxly arranged inflorescence. The fruit has a red colour, very attractive and is edible ¹⁵. Presently, the species has been occurred as “unresolved” status ², due to the absence of necessary data except for some notes in the local flora. Anatomical structure of a plant is most likely to provide evidence concerning the inter-relationships of larger groups such as families. Though anatomical characters cannot be treated as the base of classification, it can be regarded rather as an extension and supplement to the external morphological characters. Anatomy sometimes proves very helpful for individual identification. Microscopic methods are of great value in establishing the identity of herbarium specimens which are not accompanied by flowers and fruits ¹⁸.

The aim of the present study was to rectify the status of R. indicus from the unresolved state. Focused on this aspect, a detailed anatomical study has been carried out, and the description was prepared based on live samples collected from the southern Western Ghats. Here, the sections were sliced out from the vegetative regions such as root, stem, and leaf. Alternatively, the study is expected to provide prior information, which would be useful in species identification of this unresolved species.

MATERIALS AND METHODS:

Plant Material and Vegetative Parts: The live samples of R. indicus Fig. 1 were collected from Gavi forest, Kerala, India, during January 2018. The confirmation of the specimen was done by the perusal of available literature and protologue and pressed in Centre for Medicinal Plant Research (CMPR) herbarium (Voucher no. 11784). The vegetative parts (root, stem, and leaf) of the specimen were preserved out well in cooling condition for anatomical work.

FIG. 1: REPRESENTS THE STUDY MATERIAL- RUBUS INDICUS

Anatomical Study: Hand/microtome sections of the root, stem, and leaves were taken and stained with diluted aqueous safranin, washed thoroughly and mounted in 40% glycerin and observed under the microscope. Anatomical microphotographs were transferred using the computer controlled microscopic system and camera. Images are examined thoroughly and compared the anatomical characteristics.

Histochemical Analysis: The depositions inside plant cells such as starch grains, oil globules, and lignin are located using proper stains. For detecting the presence of starch, the section is treated with iodine solution so that the starch grains if present will turn blue. If the section is treated with Sudan Red S, the oil globules will be colored orange-red. Lignified cells are detected by treating with phloroglucinol followed by Conc. HCl which will turn lignified cells into dark pink or cherry red color.

RESULTS AND DISCUSSION: Microscopic characterization of the root, stem, and leaves of Rubus indicus can be summarised as follows

Root: TS of the root is circular in outline with irregular margin, and well-developed cork, cortical and stellar regions. Pith is almost absent and occupied by the xylem tissues. Cork is composed of tangentially elongated cells, which is divided into three zones namely phellum phellogen and phelloderm. Cork is 8-10 layers wide, and cells contain reddish-brown pigmentation. Oil globules are also seen in cork cell. Cortical cells are loosely arranged parenchymatous cells. Depositions of rosette and cluster crystals of calcium oxalate can be seen in cortical cells. Normally cortical cell layers vary from 10 - 20 in numbers depending on the maturity of the plant. Pericyclic fibers are seen either in groups or in the single in the outer cortex region. Phloem made up of compactly arranged condensed cells and is interrupted by the medullary rays extended up to the cortical region. Xylem contains tracheids, vessels, and parenchyma. Vessels are of varying sizes and can be single or in groups, and the shape varies from oval to polygonal. Medullary rays are uni to multiseriate often filled with starch grains at places. Pith is occupied by lignified cells Table 1 and Fig. 2 A-C.

Stem: TS of the stem is almost circular with wavy margins. TS show distinct epidermis followed by cortex, stele and pith regions. Epidermis is a single layer of elongated rectangular cells with thick cuticular covering and the limited number of elongated trichomes. Cortex is collenchymatous followed by chlorenchymatous hypodermis and parenchymatous cells. Some of the parenchymatous cells are also filled with chlorophyll are also seen. Rosette crystals of calcium oxalate are seen in cortical cells. Pericyclic fiber patches are present as a wavy ring above the phloem region. A thin-walled compactly arranged condensed phloem cells are found capped above the xylem. Calcium oxalate crystals are seen in this region also. Xylem consists of fibers, vessels, and parenchyma. The size of the vessel varies in each bundle. Each bundle consists of 3-4 big sized vessels and some small ones.

Medullary rays are multiseriate extends to phloem at places, making phloem discontinues band. Pith is a wide zone composed of thin-walled parenchymatous cells. The size and shape of the cells may vary and a smaller sized cell surrounded by 4 – 6 bigger cells gives flower-like arrangement. Calcium oxalate crystal deposition can also be noticed at places Table 1; Fig. 2 D-G.

 TABLE 1: MAJOR MICROSCOPIC CHARACTERS OF RUBUS INDICUS

Leaves: TS of the leaf passing through the midrib is of dorsi ventrally convex arrangement with lower portion is more elevated. TS show an arc of centrally located broad merestele and few rudimentary vascular strands above it. Both the epidermis is followed by collenchymatous tissue. At lamina portion upper epidermis followed by palisade cells and mesophyll cells and lower epidermis with numerous stomata at places. In detailed view, upper epidermis and lower epidermis bear few uni to multicellular trichomes. At the midrib portion, an arc of centrally located merestele with 25-30 rows of vertically running 7-8 xylem vessels is present. Vessels are smaller in size at ends. Above the arc rudimentary vascular strands are seen. Phloem cells filled with small sized crystals of calcium oxalate. The remaining ground tissue is parenchymatous and rosette crystals of calcium oxalate are scattered throughout the region. Lamina shows 1-3 rows of palisade cells. Upper epidermis followed by narrow mesophyll cells and lower epidermis Table 1; Fig. 2 H-J.

FIG. 2: MICROSCOPICAL FEATURES OF ROOT, STEM, AND LEAVES OF RUBUS INDICUS

Plate 1. Microscopy of Rubus indicus Thunb. A – C, TS of Root; D – G, TS of Stem; H – J, TS of leaves. chl, chlorenchyma; ck, cork; col, collenchyma; ct, cortex; epi, epidermis; lepi, lower epidermis; mr, medullary rays; perf, pericyclic fibres; ph, phloem; pi, pith; rcr, rosette crystals of calcium oxalate; uepi, upper epidermis; v, vessels; x, xylem

The anatomical features of plant parts have been preferred as resource material for taxonomic inferences in different groups of flowering plants ^{19, 20, 21, 22}. But in the case of Rubus species, the anatomy-based taxonomic discrimination was carried out by using seed, pollen and petal parts ^{23, 24, 25, 26}, even though, the information regarding the vegetative parts of Rubus species is still unknown.

The anatomical examinations of R. indicus has provided the information about the presence and nature of pericyclic fiber; size and shape of calcium oxalate crystals, presence or absence and a number of starch grains and oil globules, nature of epidermis and number of trichomes and arrangements of medullary rays. Data obtained in this study revealed the anatomical identity of current Rubus species, and they may be qualified as significant at the species level. According to Metcalfe and Chalk (1950) ¹⁸, the nature of pericyclic fiber is useful in species or genus level classification. Here, the microscopic stem image shows that the pericyclic fibers have occurred as a continuous ring with a wavy outline, but in root anatomy, it shows a group of fibre and seen in the outer cortex region. Various types of calcium oxalate crystals have been seen in cortex region of both stem and root and also in the ground tissue of leaves; hence the presence and shape of crystals and the occurrences of two or more type crystals might be useful for taxonomic classification ^{27, 28}. These crystals have been located in various tissue such as epidermis, phloem, xylem, pith and cortex ²⁹. The unique observation of flower-like cell arrangements in the pith region of the stem can be considered as a distinguishing feature. Uni, bi or multiseriate medullary rays extended towards phloem region may also be considered for good diagnostic characters for plant discrimination ³⁰. Starch grains found in plants have characteristic morphological features, physical and chemical properties, which may be used as diagnostic characters to identify the plant species ³¹.

CONCLUSION: Studies on the microscopic characters of the vegetative parts of R. indicus such as roots, stem, and leaves are carried out, which can be a supplementary data for the plant identification. Anatomical and histochemical characters of the abovementioned parts were done, and this is the first time report on the present species. Nature of epidermis and trichomes, nature of pericyclic fiber; the presence of calcium oxalate crystals, presence or absence of starch grains and oil globules, and arrangements of vessels and medullary rays, etc can be considered as diagnostic features of the species.

ACKNOWLEDGEMENT: Authors are thankful to Dr. Indira Balachandran, Director, Centre for Medicinal Plant Research (CMPR), Malappuram for the facilities given. We are also grateful to Dr. K. M. Prabhukumar Scientists, Plant Systematic and Genetic Resources Division, CMPR, for their support and help for species identification. Authors are grateful to the Department of Forest, Govt. of Kerala, for providing necessary assistance during the fieldwork.

CONFLICT OF INTEREST: The authors declare there is no conflict of interest.

REFERENCES:

1. Linnaeus C: Species Plantarum 1. Holmiae, Sweden 1753; 560.
2. The Plant List. “Version 1.1”. Royal Botanic Gardens Kew 2018. Available from: http://www.theplantlist.org/ (accessed 07 March 2018).
3. Wei-Lin L, Wen-Long W and Zhi-Dong Z: The utilization value and potential of Chinese bramble (Rubus L.). Acta Horticulturae 2002; 585: 133-138. DOI: 10.17660/ ActaHortic.2002.585.18
4. Hummer KE: Rubus pharmacology: antiquity to the present. HortScience 2010; 45: 1587-1591.
5. Ahmad M, Masood S, Sulthana S, Hadda TB, Bader M and Zafar M: Antioxidant and nutraceutical value of wild medicinal Rubus berries. Pakistan Journal of Pharmaceutical Science 2015; 28 (1): 241-47.
6. George BP, Parimelazhagan T and Chandran R: Evaluation of total phenolic content, antioxidant and analgesic potential of Rubus fairholmianus International Journal of Pharmacy and Pharmaceutical Sciences 2013; 5(3): 484-88.
7. Milivojevi´c J, Maksimovi´c V, Nikoli´c M, Bogdanovi´c J, Maleti´c R and Milatovi´c D: Chemical and antioxidant properties of cultivated and wild Fragaria and Rubus berries. Journal of Food Quality 2011; 34(1): 1-9.
8. Moreno-Medina BL, Casierra-Posada F and Cutler J: Phytochemical composition and potential use of Rubus species. Gesunde Pflanzen 2018: 70.
9. Maatta-Riihinen KR, Kamal-Eldin A and Torronen AR: Identification and quantification of phenolic compounds in berries of Fragaria and Rubus species (Family Rosaceae). Journal of Agricultural and Food Chemistry 2004; 52: 6178-87.
10. Moyer R, Hummer K, Finn C, Wrolstad R and Frei B: Anthocyanins, phenolics, and antioxidant capacity in diverse small fruits: Vaccinium, Rubus, and Ribes. Journal of Agricultural and Food Chemistry 2002; 50: 519-25.
11. Alice LA and Cambell CS: Phylogeny of Rubus (Rosaceae) based on nuclear ribosomal DNA Internal Transcribed Spacer region sequences 1999; 86(1): 81-97.
12. Wang Y, Chen Q, Chen T, Tang H, Liu L and Wang X: Phylogenetic insights into Chinese Rubus (Rosaceae) from multiple chloroplast and nuclear DNAs. Frontiers Plant Science 2016; 7: 968. doi: 10.3389/fpls.2016.00968
13. Gamble JS: Rubus, in Flora of the Presidency of Madras. London: Adlard & Son & West Newman, Ltd. Vol. II, 1919: 439-442.
14. Sasidharan N: Biodiversity documentation for Kerala. Part.6: Flowering Plants. Kerala Forest Research Institute, Peechi, Kerala 2004: 166-67.
15. Dassanayake MD  and Fosberg FR:  A revised handbook to the flora of Ceylon, Amerind Publishing Company, New Dehli, Vol. III, 1981: 339.
16. Thunberg CP: Dissertationem botanico-medicam de rubo. H.A.M.S. Upasaliae 1813: 5-7.
17. Thompson MM: Survey of chromosome numbers in Rubus (Rosaceae: Rosoideae). Annual Reports Missouri Botanical Garden 1997; 84: 128-64.
18. Metcalfe CR and Chalk L: Anatomy of the Dicotyledons. Clarendon Press, Oxford, Vol. II, 1950: 243-45.
19. Edeoga HO, Omosun G, Osuagwu, GGE and Emezue OO: Microscopic anatomy and histochemistry of stem and root of some Mimosa species. (Leguminosae- Mimosoideae). Asian Journal of Plant Sciences 2007; 6(4): 688-91.
20. Okoli BE: On the probable function and taxonomic value of calcium oxalate crystals in Cucurbitaceae. Feddes Repert 1988; 99: 139-42.
21. Edeoga HO and Okoli BE: Anatomy and systematics in the Costusafer, C. lucanusianus complex (Costaceae). Acta Phytotaxonomica et Geobotanica 1997; 48: 151-58.
22. RemaShree AB, Jayanthi A,Thushar KV, Ravindran PN, and Indira B: Anatomy of Coscinium fenestratum (Gaertn.) Colebr. A critically endangered medicinal plant from the Western Phytomorphology 2005; 55: 249-58.
23. Hedba RJ and Chinnappa CC: Studies on pollen morphology of Rosaceae. Acta botanica Gallica 1994; 141(2): 183-93.
24. Wada S and Reed BM: Morphological analysis of Rubus seed. Acta Horticulture 2008; 782: 67-74.
25. Fredes M, Munoz C, Prat L, Torres F, Saez P, Bustamante L, Paiva L and Pertuze R: Seed morphology and anatomy of Rubusgeoides Sm. Chilean Journal of Agricultural Research 2016; 76(3): 385-89.
26. Sharifnia F and Shakib SB: Epidermal petal patterns of 13 Iranian Rubus L. (Rosaceae) species. Annals of Biological Research 2012; 3(6): 2734-40.
27. Franceschi VR and Horner HT: Calcium oxalate crystals in plants. The Botanical Review 1980; 46(4): 361-27.
28. Herman PPJ, Robbertse PJ and Grobbelaar N: The morphology of the vegetative shoot apex and stem of some southern African Pavetta species. South African. Journal of Botany 1986; 52(3): 212-20.
29. Konyar ST, Ozturk N and Dane F: Occurrence, type and distribution of calcium oxalate crystals in leaves and stems of some species of poisonous plants. Botanical Studies 2014; 55(32): 1-9.
30. Gabr SK, Bakr RO, Elshishtawy HM, El-Fishawy AM and El-alfy TS: Botanical and genetic characters of Erythrina neillii cultivated in Egypt. Brazilian Journal of Pharmacognosy 2017; 27: 273-81.
31. Selvam ABD: Standardisation of descriptive terminology of starch granules with reference to the identification of row drugs. International Journal of Pharmaceutical Research and Development 2013; 5(7): 1-6.
    

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    How to cite this article:

    Narayanan B, Mana HC and Thara UB: Microscopic characterisation of root, stem and leaves of an unresolved species: Rubus indicus (Rosaceae). Int J Pharm Sci & Res 2019; 10(8): 3715-20. doi: 10.13040/IJPSR.0975-8232.10(8).3715-20.

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