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An Efficient Plant Regeneration, Detection and Identification of Secondary Metabolites from Propagate Plants of Peperomia Pellucida (L.) for Mass Cultivatione

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An Efficient Plant Regeneration, Detection and Identification of Secondary Metabolites from Propagate Plants of Peperomia Pellucida (L.) for Mass Cultivatione ISSN 2226-3063 e-ISSN 2227-9555 Modern Phytomorphology 15: 6–13, 2020 RESEARCH ARTICLE An efficient plant regeneration, detection and identification of secondary metabolites from propagate plants of Peperomia pellucida (L.) for mass cultivatione Abdul Bakrudeen Ali Ahmed1,2,3 , Teoh Lydia1 , Muneeb Muhamed Musthafa4 , Rosna Mat Taha1 , Faiz MMT Marikar 5* 1Institute of Biological Sciences, University of Malaya, 50603 Kuala Lumpur, Malaysia 2Department of Biochemistry and Biotechnology, Center for Research and Development (CRD), PRIST University, Vallam, Thanjavur - 613403, Tamil Nadu, India 3Department of Applied Sciences, Ton Duc Thang University, Ho Chi Minh City, Vietnam 4Department of Biosystems Technology, South Eastern University of Sri Lanka, University Park, Oluvil, 32360, Sri Lanka 5Department of Health Science and Education, The Open University of Sri Lanka, 10350, Sri Lanka; *[email protected] Received: 21.12.2020 | Accepted: 12.01.2021 | Published: 20.01.2021 Abstract Several species in the genus Peperomia Ruiz and Pav have giant chloroplasts in the palisade parenchyma of their leaves. Because of this unique feature, Peperomia pellucida L. has become a valuable model plant for studying plastid biology. However, the use of Peperomia for molecular studies has been limited by the lack of efficient regeneration and transformation protocols. This study describes an effective protocol for in vitro shoot induction, plant regeneration and rooting from internode of explant of Peperomia pellucida using Murashige and Skoog (MS) medium supplemented with different plant growth regulators. The multiple shoots were induced from internodes cultured on MS medium containing Kinetin (KN) (0.5 mg L-1) alone which induced six shoots per internodal explant. In combination Benzyladenine (BA) (1.5 mg L-1) and KN (1.5 mg L-1) induced 10 shoots per explant, while, length of shoots were significantly increased in BA (1.0 mg L-1) and KN (3.0 mg L-1) (0.8 cm per explant); KN (0.5 mg L-1) (0.7 cm per explant) respectively. Adventitious root induction was achieved on MS media with different concentrations of Indole-3-Butricacide (IBA) and Indole-3-Acetic Acid (IAA). The successful root induction was observed in MS medium supplemented with IBA and shown 100% responses while plantlets survival was 90% at greenhouse garden soil. Rooted plantlets were successfully acclimatized to pot containing soil, sand and farm manure (1:1:1) and established in tissue culture room. In vitro methanol and ethanol extracts gave the highest amount of Apiol and Phytol content in GC-MS analysis. This protocol could be very useful for mass cultivation of Peperomia pellucida for industrial exploitation with selected and standardized plants productiont. Keywords: Regeneration, root, shoot, metabolites, micropropagation, growth regulator. Introduction the leaves are broadly-ovate. The flowers are numerous, scattered on the spike and without petals but with only Genus Peperomia is one of the largest genera of basal stamens and an ovary. It also has fruits which are minute, angiosperms, comprising about 1500-1700 species and seeds produce mustard-like odour when crushed belongs to the family Piperaceae (Ilyas et al. 2014). (Majumder 2012). Now this plant is pantropical in Peperomia pellucida is a common fleshy tropical annual distribution and is abundantly available in Malaysia. This fibrous shallow-rooted herb, which grows as succulent plant has been reported from Bangladesh, Philippines, and erect plant (Majumder 2012). This plant grows in Thailand, Vietnam, Indonesia, Spain and Japan (Khan moist habitat (Ooi et al. 2012) and can reach up to 15- et al. 2010; Mutee et al. 2010). 45 cm long. The stem part is fleshy, round, delicate and glabrous (Majumder 2012) whereas the seeds are tiny dot P. pellucida served as valuable components of food, like and attached to several fruiting spikes. The leaves are medicines and has played a significant role in improving arranged alternately on the stalks for about 1-2 cm long. the quality of human life. Majumder et al., (2011) and It has shiny heart-shaped fleshy leaves and the blades of Sheikh et al., (2013) reported that the leaves and stems of © The Author(s) 2020. Published by Andriy Novikov, State Natural History Museum NAS of Ukraine on behalf of Modern Phytomorphology. This is an open access article under the Creative Commons BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/) freely available on https://phytomorphology.org/. 7 | Ahmed et al. P. pellucida may be eaten as vegetable. The fresh plant has Materials and Methods the crispness celery and carrot stick as in salads. This plant Plant collection and identification is used for treating abdominal pain, boils, colic fatigue, abscesses, acne, convulsions, fever, gout, headache, renal Peperomia pellucida were collected from a population disorders, to lower blood cholesterol level, conjunctivitis, growing at the Institute of Biological Science garden of University Malaya and from various part of University skin diseases, breast cancer and rheumatic joint pain Malaya (Fig. 1a). The voucher specimens were verified (Khan et al. 2010; Ooi et al. 2012). To cure haemorrhage, (No. KLU47794) (Fig. 1b) at the University of Malaya the whole plant was crushed and mixed with water to Herbarium (KLU), Kuala Lumpur, Malaysia with the form a mixture; then heated and taken orally (Majumder voucher of specimen. et al. 2011). The aerial parts are extracted and used as dressing for wounds curing and their roots are used to Surface sterilization treat fevers. P. pellucida also has a popular emollient, cough The plants were washed under running tap water for suppressant and diuretic in Guyana and the Amazon as 30 min. and aerial parts of the plants were soaked and well as effective in the treatment of proteinuria. Aerial washed with three (3) drops of teepol and rinsed with parts of P. pellucida demonstrated dose-dependent sterile distilled water for 5 times. Then washed with analgesic and anti-inflammatory actions by interfering 50% sodium hypochlorite (v/v) for 2 min. and followed with prostaglandin synthesis (Mutee et al. 2010). by washing in a sterile 0.1% mercury chloride (w/v) for 1 min and rinsing with sterile distilled water for 5 times. Since, plants are used in modern medicine as Finally, explants were treated with an antifungal, 0.1% sources of direct therapeutic agents of many diseases. carbendazime (w/v) diluted in 70% alcohol (v/v) for 30 sec More complex semisynthetic chemical compounds, and rinsed with sterile distilled water for 5 times. used as a raw base material (Borokini and Omotayo Shoot regeneration and rooting 2012). Khan et al. (2010) revealed, the presence of flavonoids, apiols, phytosterols, substituted The surface sterilized leaves and roots explants styrenes, secolignans, tetrahydrofuranlignans, highly were removed from the shoots explants (1-1.5 cm) of metaoxylateddihydronaphthalenone, arylpropanoiids, P. pellucida. The internodal explants were cultured on MS sesamin, isoswertisin, xanthone glycoside and medium (Murashige and Skoog medium) supplemented peperomins A, B, C and E. The flavonoids and phytosterols with Cytokinins, BA (Benzyl Adenine) (0.5-2.0 mg L-1); -1 such as acacetin, apigenin, arylpropanoids (e.g. apiols), KN (Kinetin) (0.5-3.0 mg L ); Auxin, IAA (Indole Acetic -1 isovetexin, pellucidatincampesterol and stigmasterol, Acid) (0.5-2.0 mg L ); IBA (Indole Butyric Acid) (0.5 -1 substituted styrenes and dimeric ArC compound or -2.0 mg L ) respectively. The pH of the media was adjusted 2 -1 pellucidin A have been isolated from P. pellucida (Majumder to 5.7-5.8 and Carbendazime (10 mg L ) was added into the 2011). Whereas the compound of phytosterolscampesterol medium in order to prevent the fungal contamination. Acacetin, apiigenin, isovitexin and pellucidatin and Finally, the prepared medium was autoclaved at 121°C for 20 min. After autoclaved, the medium was dispensed stigmasterol (Majumder 2011). Since this plant has more in a culture tubes in laminar flow. The photoperiod of pharmacological application, there is a requirement to light was maintained for 16 h/8 h light/darkness. The establish an effective and efficient propagation protocol culture room usually maintained at 25 ± 2°C. Humidity for production of bioactive compounds and sustainable of the culture room also must be controlled at 75–90% conservation to the pharmaceutical industries. Plant and uniform forced-air ventilation. The healthy multiple tissue culture offers a viable alternative tool, widely used shoots formed were transferred into rooting medium for conservation, commercial propagation and bioactive supplemented with IBA (0.5-2.0 mg L-1) and IAA (0.5-2.0 compound production. This in vitro technique is very mg L-1) concentration. useful in ensuring sustainable optimization sources Acclimatization of plant derived natural products. In addition, this propagation method enhances optimal extraction of All plantlets were taken out carefully from the culture secondary metabolites by use of elicitors and applicable containers after 6 weeks and washed to remove the for large scale products. The main aims of the current agar. The plantlets were transferred to plastic pots (5 cm study are to establish highly efficient regeneration diameter) containing soil, sand and farm manure (1:1:1) -2 system for this species and to determine the secondary and maintained at 25± 2°C, 16 h day length (35-50 M Fm -2 metabolites content in dry in vivo leaves. S ) and at 75-80% relative humidity. The grown plantlets Modern Phytomorphology 15, 2020 An efficient plant regeneration, detection and identification of secondary metabolites | 8 from propagate plants of Peperomia pellucida (L.) for mass cultivation were transplanted into clean pots (10 cm diameter) propagation method was successfully accomplished in containing natural soil, kept under shade conditions for this technique for P. pellucida. The P. pellucida internodal 2 weeks and finally moved to the University of Malaya explants were used for mass propagation and showed green house.
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