Establishment of an efficient and reproducible in vitro protocol for callogenesis of Silybum marianum

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Research Paper 01/01/2019
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Establishment of an efficient and reproducible in vitro protocol for callogenesis of Silybum marianum

Zara Jabeen, Khalil Ur Rahman, Muhammad Anjum Zia, Nazish Jhan
Int. J. Biosci.14( 1), 402-410, January 2019.
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Silybum marianum (milk thistle) is belongs to astereaceae family and a wild herb in Pakistan. Silymarin is an isomeric mixture of flavonoids (silybin, silydianin, silibinin and silychristin) and a flavonolignans (taxifolin) extracted from seeds of milk thistle, and used against liver diseases. Due to problems in traditional cultivation and extraction of its bioactive compound (silymarin), different strategies have been employed in order to fulfil the scarce demand of Silybum marianum. In vitro micropropagation method has great potential to produce medicinal plants in large amount in less duration of time. So in vitro propagation protocol was established for this medicinal crop by using explants from 21 days old-seedlings induced in vitro from seeds. For efficient callogenesis, seeds were surface sterilized and cultured on hormone free MS0 medium. After induction of seedlings, leaf, stem and hypocotyl were excised from the mother plants and used as explant for callus induction. Six different plant growth hormones (PGR) were checked for their callogenesis ability from three explants. It was obvious from the results that hypocotyl explant induce maximum callus induction frequency (100%) within minimum time i.e (11±2.87) days when cultured on MS+1mg/l 2,4-D+1mg/l IBA+1mg/l IAA (CIM-5) medium as compared to other medium. Hypocotyl explant was considered best explant for the highest production of callus as compared to leaf and stem explants of S. marianum.


Abbasi B, Khan M, Mahmood T, Ahmad M, Chaudhary M, Khan M. 2010. Shoot regeneration and free-radical scavenging activity in Silybum marianum L. Plant Journal of Cell Tissue and Organ Culture 101, 371–376.

Ahmad M, Khan MA, Hasan A, Zafar M, Sultana S. 2008. Chemotaxonomic standardization of herbal drugs milk thistle and globe thistle. Asian Journal of Chemistry 20, 4443–4459.

Arif M, Rauf S, Din AU, Rauf M, Afrasiab H. 2014. High frequency plant regeneration from leaf derived callus of Dianthus caryophyllus L. American Journal of Plant Sciences 5, 2454-2463.

Basiglio CL, Pozzi EJS, Mottino AD, Roma MG. 2009. Differential effects of silymarin and its active component silibinin on plasma membrane stability and hepatocellular lysis. Chemico-biological interactions 179, 297-303.

Bekheet SA, Taha HS, Ahmed M, Gabr M. 2017. Protocol for in vitro morphogenesis and hairy root cultures of Milk thistle (Silybum marianum L. Gaertn). Journal of Applied Science Research 9, 860-866.

Cimino C, Cavalli SV, Spina F, Natalucci C, Priolo N. 2006. Callus culture for biomass production of milk thistle as a potential source of milk clotting peptidases. Electronic Journal of Biotechnology 9(3).

Eari S, Aghdasi M, Ahmadzadeh E, Mianabadi M. 2017. Influence of Plant Growth Regulators on Callus Induction, Silymarin Production and Antioxidant Activity in Milk Thistle (Silybum marianum L. Gaertn.) under Tissue Culture Medium. Journal of Medicinal Plants and By-products 1, 59-69.

Elfahmi. 2006. Phytochemical and biosynthetic studies of Lignans with afocus on Indonesian medicinal plants. Ph. D. Dissertation, University of Groningen, Indonisa.

Engelberth AS, Carrier DJ, Clausen EC. 2008. Separation of silymarins from milk thistle (Silybum marianum L.) extracted with pressurized hot water using fast centrifugal partition chromatography. Journal of Liquid Chromatography 31, 3001-3011.

Gopi C, Vatsala TM. 2006. In vitro studies on effects of plant growth regulators on callus and suspension culture biomass yield from Gymnemasylvestre. African Journal of Biotechnology 12, 1215-1219.

Gupta O, Sing S, Bani S, Sharma N, Malhotra S, Gupta B, Banerjee S, Handa S. 2000. Anti-inflammatory and anti-arthritic activities of  silymarin acting through inhibition of 5-lipoxygenase. Phytomedicine 7, 21-24.

Hammouda FM, Ismail SI, Hassan NM, Zaki AK, Kamel A, Rimpler H. 1993. Evaluation of the silymarin content in Silybum marianum (L.) Gaertn. cultivated under different agricultural conditions. Phytotherapy Research, 90–91.

Huseini HF, Larijani B, Heshmat R, Fakhrzadeh H, Radjabipour B, Toliat T, Raza M. 2006. The efficacy of Silybum marianum (L.) Gaertn. (silymarin) in the treatment of type II diabetes: a randomized, double‐blind, placebo-controlled, clinical trial. Phytotherapy Research 20, 1036-1039.

John SA, Koperuncholan M. 2012. Direct root regeneration and indirect organogenesis in Silybum marianum and preliminary phytochemical, antimicrobial studies of its callus. International Journal of Pharmacy 2, 392-400.

Liu SQ, Cai QG. 1990. Callus formation from protoplasts and plant regeneration from tissue culture of Silybum marianum gaertn. Journal Integral Plant Biology 32, 19-25.

Manaf H, Kawther H, Rabie AE, AbdFl-Aal MS. 2009. In Vitro Callus Formation and Plant Regeneration of Silybum marianum (L) Geartn. Agricultural Sciences 54, 283-289.

Nikolova MT, Ivancheva SV. 2005. Quantitative flavonoid variations of Artemisia vulgaris L. and Veronica chamaedrys L. in relation to altitude and polluted environment. Acta Biological Szegediensis. 49, 29–32.

Ozturk B, Kocaoglu EH, Durak ZE. 2015. Effects of aqueous extract from Silybum marianumon adenosine deaminase activity in cancerous and noncancerous human gastric and colon tissues. Pharmacognosy Magazine 11, 143-146.

Pourjabar A, Mohammadi SA, Ghahramanzadeh A, Salimi GH. 2012. Effect of Genotype, Explant Type and Growth Regulators on The Accumulation of Flavonoides of (Silybum marianum L.) in In vitro Culture Tissue. Agricultural and Biosystems Engineering 6, 514-516.

Rady MR, Matter MA, Ghareeb HA, Hanafy MS, Saker MM, Eid SA, Hammoda FM, Imbaby SI, Nazief NH. 2013. In vitro cultures of Silybum marianum and silymarin accumulation. Journal of Genetic Engineering Biotechnology 12, 75–79.

Rafieian-Kopaie M, Nasri H. 2012. Silymarin and diabetic nephropathy. Journal Renal Injection Prevention 1, 3-5.

Saller R, Meier R, Brignoli R. 2001. The use of silymarin in the treatment of liver diseases. Drugs 61, 2035–2063.

Sanchez-Sampedro MA, Fernández-Tarrago J, Corchete P. 2005. Yeast extract and methyl jasmonate-induced silymarin production in cell cultures of Silybum marianum (L.) Gaernt. Journal of Biotechnology 119, 60-69.

Shaarawy SM, Tohamy AA, Elgendy SM, Abd Elmageed ZY, Bahnasy A, Mohamed MS. 2009. Protective effects of garlic and silymarin on NDEA-induced rats hepatotoxicity. International Journal of Biological Sciences 5, 549-557.

Shaker E, Mahmoud H, Mnaa S. 2010. Silymarin, the antioxidant component and Silybum marianum extracts prevent liver damage. Food Chemical Toxicology 48, 803-806.

Shinwari ZK, Rehman H, Rabbani MA. 2014. Morphologicaltraits based genetic diversity in safflower (Carthamus tinctorius L.). Pakistan Journal of Botany 46, 1389-1395.

Soto C, Perez J, Garcia V, Uría E, Vadillo M, Raya L. 2010. Effect of silymarin on kidneys of rats suffering from alloxan-induced diabetes mellitus. Phytomedicine 17, 1090-1094.

Vanisree ML, Shu-Fung C, Hsin-Nalawade L, Yih-Lin SM, Tsay CS. 2004. Studies on the production of some important secondary metabolites from medicinal plants by plant tissue cultures. Botanical Bulletin of the Academia Sinica 45, 410-415.

Wallace S, Vaughn K, Stewart BW, Viswanathan T, Clausen E, Nagarajan S, Carrier DJ. 2008. Milk thistle extracts inhibit the oxidation of low-density lipoprotein (LDL) and subsequent scavenger receptor-dependent monocyte adhesion. Journal of Agricultural Food Chemistry. 56, 3966–3972.

Yu-wei LV, Wamg RJ, Yawei LV, Yang ZS, Wang YJ. 2017. In vitro propagation of Silybum marianum (L) Geartn. And genetic fidelity assessment of micropropagation plants. Pakistan Journal of Botany 49, 673-680.

Zia M, Mannan A, Chaudhary MF. 2007. Effect of growth regulators and amino acids on artemisinin production in the callus of Artemisia absinthium. Pakistan Journal of Botany 39, 799-58.