In vitro Callus Induction of Catunaregam spinosa Using Leaves as Explant
Journal of Pharmaceutical Research International,
Page 67-76
DOI:
10.9734/jpri/2022/v34i43A36313
Abstract
Aims: C. spinosa (Family Rubiaceae) is a valuable medicinal since years ago. A protocol was developed including best sterilization and best medium for callus induction of C. spinosa using leaf discs as explants.
Methodology: Sterilization protocol optimized using different concentrations of Carbendazim® (0.2,
0.3%) and Clorox (10, 15%) exposing to different time intervals (10, 15 min). Percentage survival and contaminations were calculated. Best medium was optimized using different concentrations of 6-Benzyl Amino Purine (BAP) and Naphthalene Acetic Acid (NAA) (1.0-6.0 mg L-1). Growth regulators free Murashige and Skoog (MS) medium was used as control. Completely randomized design was followed with ten replicates in each concentration. Days taken to initiate calli, morphological characteristics and mean dry weights of calli were evaluated after 3 months of incubation.
Results: Leaf discs sterilization with 0.3% Carbendazim for 10 min, 10 % Clorox for 10 min and 70% ethanol for 30 sec followed by two washings in sterile distilled water was found to be best sterilization protocol. It recorded lowest percentage contamination (13.34%) and highest percentage survival (86.66%) after 8 weeks. No observable changes were found in calli grown in growth regulators free MS medium. Calli growth and morphologies were significantly affected by the type of growth regulator and their concentrations in MS medium. Color of the calli varied from white opaque to yellow brown to green and the texture from foamy, loose, and friable to compact. Best medium with 1.0 mg L-1 BAP and 3.0 mg L-1 NAA produced green friable calli with 0.0969±0.01 g mean dry weight after 3 months. Some traits were found to be depended on synergetic effect of growth regulators and genotypic characteristics of explant.
Conclusion: The study provides a better sterilization protocol and medium for in vitro calli induction and growth of C. spinosa.
Keywords:
- Catunaregam spinosa
- tissue culture
- sterilization
- plant growth regulators
- callus
How to Cite
Lawrence, P. K., Senarath, W. T. P. S. K. and Munasinghe, M. L. A. M. S. (2022) “In vitro Callus Induction of Catunaregam spinosa Using Leaves as Explant”, Journal of Pharmaceutical Research International, 34(43A), pp. 67-76. doi: 10.9734/jpri/2022/v34i43A36313.
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Jain CS, Pancholi B and Jain R. In-vitro callus propagation and secondary metabolite quantification in Sericostoma pauciflorum. Iranian Journal of Pharmaceutical Research. 2012; 11(4):1103–1109.
Ahmad S and Spoor W. Effects of NAA and BAP on callus culture and plant regeneration in Curly Kale (Brassica oleraces L.). Pakistan Journal of Biological Sciences. 1999;2(1):109-112.
Lee Y, Lee DE, Lee HS, Kim SK, Lee WS, Kim SW and Kimet MW. Influence of auxins, cytokinins, and nitrogen on production of rutin from callus and adventitious roots of the white mulberry tree (Morus alba L.). Plant Cell, Tissue and Organ Culture (PCTOC). 2010;105(1): 9-19.
Begum F, Islam KMD, Paul RN, Mehedi M and Rani S. In vitro propagation of emetic nut Randia dumetorum (Lamb.). Indian Journal of Experimental Biology. 2003;41:1479-1481.
Sjahril R, Haring F, Riadi M, Rahim M, Khan R, Amir A and & Ar T. Performance of NAA, 2iP, BAP and TDZ on callus multiplication, shoots initiation and growth for efficient plant rege- neration system in Chrysanthemum (Chrysanthemum morifolium Ramat.). International Journal of Agriculture System. 2916;4(1):52-61.
Murashige T and Skoog F. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Plant Physiology. 1962;15:473–497.
Mostafiz SB and Wagiran A. Efficient callus induction and regeneration in selected Indica rice. Agronomy. 2018;8(5). DOI:10.3390/agronomy8050077.
Liu J, Feng H, Ma Y, Zhang L, Han H and Huang X. Effects of different plant hormones on callus induction and plant regeneration of miniature roses (Rosa hybrida L.). Horticulture International Journal. 2018;2(4):201-206.
Yaseen M, Ahmad T, Sablok G, Standardi A and Hafiz IA. Review: role of carbon sources for in vitro plant growth and development. Molecular Biology Reports. 2012;40(4):2837-2849.
Rana MS, Ramírez J, Gutiérrez-Alejandre A, Ancheyta J, Cedeño L, Maity SK. Support effects in CoMo hydrodesulfurization catalysts prepared with EDTA as a chelating agent. Journal of Catalysis. 2007;246(1): 100-8.
Seyyedyousefi SR, Kaviani B, Dehkaei NP and Salehzadeh A. Callus induction in Alstroemeria using NAA and BAP. European Journal of Experimental Biology. 2013;3(5):137-140.
Yildiz M, Fatih OS., Cansu TK and Tuna E. The effect of sodium hypochlorite solutions on the viability and in vitro regeneration capacity of the tissue, The Natural Products Journal. 2012; 2(4).
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Irene WM, Alumiro HL, Asava KK, Agwanda CO and Anami SE. Effects of genotype and plant growth regulators on callus induction in leaf cultures of Coffea arabica L. F1 Hybrid. Journal of Plant Biochemistry & Physiology. 2019;7(2): 236.
Ikeuchi M, Sugimoto K and Iwase A. Plant callus: Mechanisms of induction and repression. The Plant Cell. 2013; 25:3159– 3173.
Neibaur I, Gallo M and Altpeter F. The effect of auxin type and cytokinin concentration on callus induction and plant regeneration frequency from immature inflorescence segments of Seashore Paspalum (Paspalum vaginatum Swartz). In vitro Cellular & Developmental Biology. Plant. 2008;44(6):480–486.
Available:http://www.jstor.org/stable/20461 750.
DOI: 10.3389/fphar.2016.00292.
Khan H. Medicinal plants need biological screening: a future treasure as therapeutic agents. Biology and Medicine. 2014b;6: e110.
DOI: 10.4172/0974-8369.1000e110.
Elekofehinti OO, Iwaloye O, Olawale F and Ariyo EO. Saponins in cancer treatment: Current progress and future prospects. Pathophysiology. 2021;28(2):250- 272.
DOI:10.3390/pathophysiology28020017.
Ejelonu OC, Elekofehinti OO and Adanlawo IG. Tithonia diversifolia saponin- blood lipid interaction and its influence on immune system of normal wistar rats. Biomedicine & Pharmacotherapy. 2017; 87:589-595.
DOI:10.1016/j.biopha.2017.01.017.ISSN 1950-6007. PMID 28086134.
Kim JJJ, Xiao H, Tan Y, Wang ZZ, Seale JP and Qu X. The Effects and mechanism of saponins of Panax notoginseng on glucose metabolism in 3T3-L1 cells. The American Journal of Chinese Medicine. 2009;37(6):1179–1189.
DOI:10.1142/S0192415X09007582. ISSN 0192-415X. PMID 19938225.
Jangwan JS and Singh R. In vitro cytotoxic activity of triterpene isolated from bark of Randia dumentorum Lamk. Journal of Current Chemical and Pharmaceutical Sciences. 2014;4(1):1-9.
Ragib A, Hossain T, Hossain J and Jakaria J. Antioxidant potential and cytotoxicity of Randia dumetorum Lam. leaf extract. Journal of Pharmacognosy and Phytotherapy. 2017;9(9):138-145.
Jawale CS. Piscicidal plants of India. Trends in Fisheries Reserach. 2018;7(2): 2319–4758.
Dominic R and Ramanujam SN. Traditional knowledge and ethnobotanical uses of piscicidal plants of Nagaland, North east India. Indian Journal of Natural Products and Resources. 2012;3(4):582-588.
Negi KS, Kanwal KS. Plants used as fish toxins in Garhwal region of Uttarakhand Himalaya. Indian Journal of Traditional Knowledge. 2009;8(4);535-538.
Neuwinger HD. Plants used for poison fishing in tropical Africa. Toxicon. 2004; 44:417–430.
Sunita K, Kumar P, Khan SMA, Husain SA and Singh DK. Anthelminthic/larvicidal activity of some common medicinal plants. European Journal of Biological Research. 2017; 7(4):324-336.
Jyothirmayi B, Tejaswini L, Latha GA, Swathi C, Nishitha G and Unissa R. Anthelmintic evaluation of Randia dumetorum fruit extracts. Journal of Pharmacy Research. 2018;12(6):880- 883.
Satpute KL, Patil MJ, Bafna AR, Bodas K, Shafi S. In vitro antioxidant activity of fruits of Randia Dumetorum Lamk. Journal of Global Trends in Pharmaceutical Sciences. 2014; 5(4):2103-2107.
Saini H, Dwivedi J, Paliwal H, Kataria U and Sharma M. An Ethno-pharmacological evaluation of Catunaregam Spinosa (Thumb.) Tirveng for antioxidant activity. Journal of Drug Delivery and Therapeutics. 2019;9(4-s):280-284.
Kandimalla R, Singh SKYP, Saikia B, Kalita K, Dash S, Choudhury B and Kotoky J. Antioxidant and hepatoprotective potentiality of Randia dumetorum Lam. leaf and bark via inhibition of oxidative stress and inflammatory cytokines. Frontiers in Pharmacology. 2016;7.
Ghante MH, Bhusari KP, Duragkar NJ, Jain NS and Warokar AS. Bronchorelaxant, mast cell stabilizing, anti- inflammatory and antioxidant activity of Randia Dumetorum (Retz.) Lamk. extracts. Acta Poloniae Pharmaceutica - Drug Research. 2012;69(3):465-474.
Garg AK, Chouhan P, Ghuraiya S and Sharma BK. Madanaphala (Randia Dumetorum): A Pharmacological And Pharmacognostical Review. International Journal of Recent Scientific Research. 2019;10( 04):32061-32064.
Anoopkumar AN, Rebello S, Sudhikumar AV, Puthur S and Aneesh EM. A novel intervention on the inhibiting effects of Catunaregam spinosa induced free radical formation and DNA damage in Aedes aegypti (Diptera: Culicidae): a verdict for new perspectives on microorganism targeted vector control approach. International Journal of Tropical Insect Science. 2020;40:989–1002(2020).
Available: https://doi.org/10.1007/s42690- 020-00157-0
Ashton R. The incredible pomegranate. Plant and Fruit, ed. Baer B and Silverstein D. United States of America: ThirdMillennium Publishing. 2006;162.
Lakhera K, Kumar A, Rani A, Dixit R and Rana S. Plant tissue culture and its application. Bulletin of Pure & Applied Sciences- Botan, 2018;37b:32. DOI:10.5958/2320-3196.2018.00004.6.
Tomar UK, Dantu PK. Protoplast Culture and Somatic Hybridization, in Cellular and Biochemical Sciences, Tripathi G, editor. I.K. International House Pvt Ltd New Delhi. 2010:877-890.
Ibrahim IA, Sezgin M. Embryo culture and applications in biotechnology, in 3rd International Eurasian Conference on Biological and Chemical Sciences. Ankara, Turkey; 2020.
Hernández COR, García SET, Sandoval CO, Castillo FYR, Muro AL, Gonzalez FJA and Barrera ALG. Cell culture: History, Development and Prospect. International Journal of Current Research and Academic Review. 2014;2(12):188-200.
Varshney A, Anis M, Aref IM. Potential role of cytokinin–auxin synergism, antioxidant enzymes activities and appraisal of genetic stability in Dianthus caryophyllus L.—an important cut flower crop. In Vitro Cell Development Biology Plant. 2013;49:166– 174.
Available: https://doi.org/10.1007/s11627- 012-9474-8.
Dar SA, Nawchoo IA, Tyub S and Kamili AN. Effect of plant growth regulators on in vitro induction and maintenance of callus from leaf and root explants of Atropa acuminata Royal ex Lindl. Biotechnology Reports. 2021;32.
Ramos MS, Bahena MS, Estrada AR, Antonio, AB, Sosa FC, Christen JG, Fernández, JJA, Arango, IP and Alvarez,L. Establishment and phytochemical analysis of a callus culture from Ageratina pichinchensis (Asteraceae) and its anti- inflammatory activity. Molecules. 2018;23:1258.
Jain CS, Pancholi B and Jain R. In-vitro callus propagation and secondary metabolite quantification in Sericostoma pauciflorum. Iranian Journal of Pharmaceutical Research. 2012; 11(4):1103–1109.
Ahmad S and Spoor W. Effects of NAA and BAP on callus culture and plant regeneration in Curly Kale (Brassica oleraces L.). Pakistan Journal of Biological Sciences. 1999;2(1):109-112.
Lee Y, Lee DE, Lee HS, Kim SK, Lee WS, Kim SW and Kimet MW. Influence of auxins, cytokinins, and nitrogen on production of rutin from callus and adventitious roots of the white mulberry tree (Morus alba L.). Plant Cell, Tissue and Organ Culture (PCTOC). 2010;105(1): 9-19.
Begum F, Islam KMD, Paul RN, Mehedi M and Rani S. In vitro propagation of emetic nut Randia dumetorum (Lamb.). Indian Journal of Experimental Biology. 2003;41:1479-1481.
Sjahril R, Haring F, Riadi M, Rahim M, Khan R, Amir A and & Ar T. Performance of NAA, 2iP, BAP and TDZ on callus multiplication, shoots initiation and growth for efficient plant rege- neration system in Chrysanthemum (Chrysanthemum morifolium Ramat.). International Journal of Agriculture System. 2916;4(1):52-61.
Murashige T and Skoog F. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Plant Physiology. 1962;15:473–497.
Mostafiz SB and Wagiran A. Efficient callus induction and regeneration in selected Indica rice. Agronomy. 2018;8(5). DOI:10.3390/agronomy8050077.
Liu J, Feng H, Ma Y, Zhang L, Han H and Huang X. Effects of different plant hormones on callus induction and plant regeneration of miniature roses (Rosa hybrida L.). Horticulture International Journal. 2018;2(4):201-206.
Yaseen M, Ahmad T, Sablok G, Standardi A and Hafiz IA. Review: role of carbon sources for in vitro plant growth and development. Molecular Biology Reports. 2012;40(4):2837-2849.
Rana MS, Ramírez J, Gutiérrez-Alejandre A, Ancheyta J, Cedeño L, Maity SK. Support effects in CoMo hydrodesulfurization catalysts prepared with EDTA as a chelating agent. Journal of Catalysis. 2007;246(1): 100-8.
Seyyedyousefi SR, Kaviani B, Dehkaei NP and Salehzadeh A. Callus induction in Alstroemeria using NAA and BAP. European Journal of Experimental Biology. 2013;3(5):137-140.
Yildiz M, Fatih OS., Cansu TK and Tuna E. The effect of sodium hypochlorite solutions on the viability and in vitro regeneration capacity of the tissue, The Natural Products Journal. 2012; 2(4).
Available:https://dx.doi.org/10.2174/22103 15511202040328.
Irene WM, Alumiro HL, Asava KK, Agwanda CO and Anami SE. Effects of genotype and plant growth regulators on callus induction in leaf cultures of Coffea arabica L. F1 Hybrid. Journal of Plant Biochemistry & Physiology. 2019;7(2): 236.
Ikeuchi M, Sugimoto K and Iwase A. Plant callus: Mechanisms of induction and repression. The Plant Cell. 2013; 25:3159– 3173.
Neibaur I, Gallo M and Altpeter F. The effect of auxin type and cytokinin concentration on callus induction and plant regeneration frequency from immature inflorescence segments of Seashore Paspalum (Paspalum vaginatum Swartz). In vitro Cellular & Developmental Biology. Plant. 2008;44(6):480–486.
Available:http://www.jstor.org/stable/20461 750.
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