Successful Indirect Regeneration of Arnebia pulchra (Roemer and Schultes) as Medicinal Plant


1 Department of Plant Biotechnology, National Institute for Genetic Engineering and Biotechnology, Tehran, Iran.

2 Agriculture and Natural Resources Department, Islamic Azad University, Karaj Branch.

3 Pharmaceutical Sciences Research Center, Tehran University of Medical Sciences, Tehran, Iran


Arnebia pulchra (Willd. ex Roem. & Schult.) Edm. is an endangered, perennial member of Boraginaceae family. Spectrophotometric and LC-Mass analysis confirmed the existence of shikalkin derivatives in its root skin and polyphenolic compounds in the inner part of its root. Studies on the regeneration of A. pulchra from its callus resulted in successful callus induction in the root explants excised from the young plantlets on MS medium amended with 2,4-D (1 mg.l-1) and kinetin (0.5 mg.l-1) at 25°C in darkness. The highest frequency of shooting (15 shoots per callus) was achieved for the calli sub-cultured on MS medium supplemented with 2,4-D (0.1 mg.l-1) and kinetin (2 mg.l-1) in 2 weeks. The concentration and type of auxin were found critical for successful rooting. The best rooting conditions with a frequency rate of 80% were obtained for the regenerates which were sub-cultured on half-strength MS medium supplemented with IBA (1 mg.l-1), NAA (1 mg.l-1) and kinetin (0.2 mg.l-1). The regeneration method developed in this work provides a basis for germplasm conservation of A. pulchra.


1. Chandra SK, Rawal RS, Sanjay G, Balwant R. Arnebia nandadeviensis (Boraginaceae), a new species from India. J American Sci. 2009;5:105-106.

2. Papageorgiou VP, Assimopoulou AN, Couladouros EA, Hepworth D, Nicolaou KC. The chemistry and biology of alkannin, shikonin and related naphthazarin natural products. Angew Chem Int Ed. 1999;38:270-300.

3. Andújar I, Ríos JL, Giner RM, Recio MC. Pharmacological Properties of Shikonin. Planta Med. 2013;79:1685-1697.

4. Minakshi P, Chaudhury A. High frequency direct plant regeneration, Micropropagation and shikonin induction in Arnebia hispidissima. J Crop Sci Biotech. 2010;13:13-20.

5. Malik S, Bhushan S, Sharma M, Singh Ahuja P. Biotechnological approaches to the production of shikonins: a critical review with recent updates. Crit Rev Biotechnol. 2014;Online:1-14.

6. Karuppusamy S. A review on trends in production of secondary metabolites from higher plants by in vitro tissue, organ and cell cultures. J Med Plants Res. 2009;3:1222-1239.

7. Liu T, Ma C, Yang L, Wang W, Sui X, Zhao C, Zu Y. Optimization of Shikonin Homogenate Extraction from Arnebia euchroma Using Response Surface Methodology. Molecules. 2013;18:466-481.

8. Petersen M, Abdullah Y, Benner J, Eberle D, Gehlen K, Hücherig S, Janiak V, Hee K, Kim, Sander M, Weitzel C, Wolters S. Evolution of rosmarinic acid biosynthesis. Phytochem. 2009;70:1663-1679.

9. Haghbeen K, Pourmolaei S, Mareftjo MJ, Mousavi A, Akbari Noghabi K, Hosseini Shirazi F, Meshkat A. Detailed investigations on the solid cell culture and antimicrobial activities of the Iranian Arnebia euchroma .J Biomed Biotechnol. 2011;Article ID 165852, 8 pages.

10. Davydenkov VN, Patudin AV, Papov YG, Rabinovich SA. Cell culture of Arnebia euchroma (Royle) Jonst. novel source of shikonin production. Khim Farm Zh. 1991;1:53-55.

11. Singh B, Sharma RA. Anti-inflammatory and antimicrobial activity of Shikonin derivatives from Arnebia hispidissima (Lehm.) DC. Phytopharmaco. 2012;3:68-81.

12. Gamborg OL, Collins F, Hall RD. Basic Techniques-Cell and Tissue Culture of Model Species in Plant Tissue Culture Manual, Fundamentals and Applications (3rd eds), Kluwer Academic Publisher, Dordrecht, the Netherlands, 1991.

13. Haghbeen K, Mozaffarian V, Ghaffari F, Pourazeezi E, Saraji M. Lithospermum ocinale callus produces shikalkin. Biologia. 2006;20:393-399.

14. Kumar R, Sharma N, Malik S, Bhushan S, Sharma UK, Kumari D, Sinha AK, Sharma M, Singh Ahuja P. Cell suspension culture of Arnebia euchroma (Royle) Johnston-A potential source of naphthoquinone pigments. J Med Plants Res. 2011;5:6048-6054.

15. Davis PH. Flora of Turkey, Edinburg University Press. 1998;10:183.

16. Assimopoulou AN, Papageorgiou VP. Study on isohexenylnaphthazarins polymerization in alkaline media. Biomed Chromatogr. 2004;18:492-500.

17. Assimopoulou AN, Karapanagiotis I, Vasiliou A, Kokkini S, Papageorgiou VP. Analysis of alkannin derivatives from Alkannaspecies by high-performance liquid chromatography photodiodearray/mass spectrometry. Biomed Chromatogr. 2006;20:1359-1374.

18. He T, Jia JF. Breaking dormancy in seeds of Anisodus tanguticus: An endangered medicinal herb of high altitude in the Qinghai-Tibet Plateau. Seed Sci Technol. 2009;37:229-231.

19. Ganaie KA, Aslam S, Nawchoo IA. No Chilling Obligation for Germination in Seeds of Arnebia Benthamii: A Critically Endangered Alpine Medicinal Plant of North West Himalayas. Int J Biodivers Conserv. 2011;3:155-159.

20. Chaudhary A, Minakahi P. Method of direct regeneration, Shikonin induction in callus and Agrobacterium rhizogenes-mediated genetic transformation of Arnebia hispidissima. (European patent). 2006;EP1649743 (A1).

21. Gürel S, Gürel E, Kaya Z. Callus development and indirect shoot regeneration from seedling explants of sugar beet (Beta vulgaris L.) cultured in vitro. Turk J Bot. 2001;25:25-33.

22. Farhadi S, Haghbeen K, Marefatjo MJ, Ghiyami Hoor M, Shahbani Zahiri H, Rahimi K. Anionic peroxidase production by Arnebia euchroma callus. Biotech Appl Biochem. 2011;58:456-63

23. Alemi M, Sabouni F, Sanjarian F, Haghbeen K, Ansari S. Anti-inflammatory effect of seeds and callus of Nigella sativa L. extracts on mix glial cells with regard to their thymoquinone content. AAPS Pharm Sci Tech. 2013;14:160-167.

24. Mahipal S, Shekhawat NS. Micropropagation of Arnebia hispidissima (Lehm). DC. and production of alkannin from callus and cell suspension culture. Acta Physiol Plant. 2011;33:1445-1450.

25. Atak Ç,  Çelik Ö. Micropropagation of Anthurium Andraeanum from Leaf Explants. Pak J Bot. 2009;41:1155-1161.

26. Rai MK, Asthana P, Jaiswal VS, Jaiswal U. Biotechnological advances in guava (Psidium guajava L.): recent developments and prospects for further research. Trees Struct Funct. 2010;24:1-12.

27. Rathore MS, Shekhawat NS. Micropropagation of Pueraria tuberosa (Roxb. Ex Willd) and determination of puerarin content in different tissues. Plant Cell Tiss Organ Cult. 2009;99:327-334.

28. Rathore MS, Shekhawat NS. Ex vivo implications of phytohormones on various in vitro responses in Leptadenia reticulata (Retz.) an endangered plant. Environ Exp Bot. 2013;86:86-93.

29. Jiang B, Yang YG, Guo YM, Guo ZC, Chen YZ. Thidiazuron-induced in vitro shoot organogenesis of the medical plant Arnebia euchroma (Royle) johnst. In vitro Cell Devop Biol Plant. 2005;41:677-681.