Advertisement

Journal of Crop Science and Biotechnology

, Volume 13, Issue 1, pp 13–19 | Cite as

High frequency direct plant regeneration, micropropagation and Shikonin induction in Arnebia hispidissima

  • Minakshi Pal
  • Ashok ChaudhuryEmail author
Research Article

Abstract

The data presented herein reports a rapid and efficient method for direct plant regeneration at high frequency without intervening callus formation from shoot tip (93%) and nodal segment (60%) cultured on MS media supplemented with 0.5 mg l−1 KIN, 0.25 mg l−1 BAP, 0.1 mg l−1 IAA and 100 mg l−1 CH. Conversely, leaf and internodal explants were poorly responsive. Adventitious shoot buds arose not only from the cut ends but all along the surface of the explants leading to the formation of clusters with multiple shoots. Multiple shoots upon transfer to MS media supplemented with 2.0 mg l−1 IBA induced efficient rooting (80%). In vitro flowering was observed when tissue culture-raised plantlets were maintained for extended period in culture. Shikonin was induced in roots of regenerated plants which often exudates in the culture medium was quantified spectrophotometerically by recording absorbance at 620 nm and estimated to be 0.50 mg g−1 fresh weight of tissue at the end of the 50 days of culture. The regenerated plants were successfully acclimatized, hardened, and transferred to soil in green house for micropropagation. The protocol developed here will be very useful for the supply of Arnebia hispidissima all year as a raw product necessary for obtaining Shikonin for the cosmetic, dyeing, food, and pharmaceutical industries.

Key words

Arnebia hispidissima callus direct plant regeneration in vitro shikonin 

Abbreviations

BAP

6-benzylaminopurine

CH

Casein hydrolysate

IAA

Indole acetic acid

IBA

Indole butyric acid

KIN

Kinetin

NAA

naphthalene acetic acid

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Arous S, Boussaïd M, Marrakchi M. 2001. Plant regeneration from zygotic embryo hypocotyls of Tunisian chili (Capsicum annuum L.). J. Appl. Hort. 3: 17–22Google Scholar
  2. Chaudhury A, Pal M. 2004. Guru Jambheshwar University of Science & Technology, Hisar, Haryana, India & Department of Biotechnology, Ministry of Science & Technology, Government of India, New Delhi, India. Method of Direct Regeneration and Shikonin Induction in Callus of Arnebia hispidissima. Patent Application # 2066/DEL/2004 dated 21-10-2004. Indian Patent. (Patent Pending)Google Scholar
  3. Chaudhury A, Pal M. 2005. Guru Jambheshwar University of Science & Technology, Hisar, Haryana, India & Department of Biotechnology, Ministry of Science & Technology, Government of India, New Delhi, India. Method of Direct Regeneration, Shikonin Induction in Callus and Agrobacterium-mediated genetic transformation of Arnebia hispidissima. Patent Application # 05256545 dated 21-10-2005. European Patent Granted Published 10th October, 2008Google Scholar
  4. Davydenkov VN, Patudin AV, Popov YuG, Rabinovich SA, Miroshnikov AI. 1991. Cultured Arnebia euchroma (Royle) Jonst.— cells a new source of Shikonin production. Khimiko Farmatsevticheskii Zhurnal 25(1): 53–55 (in Russian)Google Scholar
  5. Dong J, Ye H, Wu X, Li G, Wu Z, Chen J. 1993. Studies of sus pension culture and fermentable culture of Arnebia euchro ma. Acta Bot. Sin. 35: 57–61 (in Chinese)Google Scholar
  6. Fujita Y, Hara Y, Ogino T, Suga C. 1981a. Production of Shikonin derivatives by cell suspension cultures of Lithospermum erythrorhizon: I. Effects of nitrogen sources on the production of Shikonin derivatives. Plant Cell Rep. 1:59–60CrossRefGoogle Scholar
  7. Fujita Y, Hara Y, Suga C, Morimoto T. 1981b. Production of Shikonin derivatives by cell suspension cultures of Lithospermum erythrorhizon. II. A new medium for the pro duction of Shikonin derivatives. Plant Cell Rep. 1: 61–63CrossRefGoogle Scholar
  8. Fujita Y, Tabata M, Nishi A, Yamada Y. 1982. New medium and production of secondary compounds with the two stage culture method. In A Fujiwara, ed, Plant Tissue Culture, Maruzen, Tokyo, pp 399–400Google Scholar
  9. Fujita Y, Maeda Y, Suga C, Morimoto T. 1983. Production of Shikonin derivatives by cell suspension cultures of Lithospermum erythrorhizon. III. Comparison of Shikonin derivatives of cultured cells and Ko-shikon. Plant Cell Rep. 2: 192–193CrossRefGoogle Scholar
  10. Fukui H, Yoshikawa N, Tabata M. 1983. Induction of Shikonin formation by agar in Lithospermum erythrorhizon cell sus pension cultures. Phytochem. 22: 2451–2453CrossRefGoogle Scholar
  11. Ge F, Wang XD, Wang YC. 2004. Studies on highly efficient induction of callus from Arnebia euchroma and rapid prolif eration of callus. Chinese Pharma J. 39(10): 735–737Google Scholar
  12. Geetha N, Venkatachalam P, Prakash V, Lakshmi Sita G. 1998. High frequency induction of multiple shoots and plant regen eration from seedling explants of pigeonpea (Cajanus cajan L.)Google Scholar
  13. Hayashi M, Tsurumi S, Fujimura H. 1969. Pharmacological effect of Shikon. Folia Pharmacol. Jpn. 65:195–196.Google Scholar
  14. Khatoon S, Mehrotra S, Bajpai VK, Mehrotra BN. 1994. Ultramorphology of some Boraginaceous taxa used as Ratanjot. Feddes-Repertorium 105: 61–71Google Scholar
  15. Khatoon S, Mehrotra BN, Mehrotra S. 2003. Pharmacognistic evaluation of Ratanjot of Arnebia nobilis. Ref. Natl. Prod. Sci. 9: 286–290Google Scholar
  16. Kim DJ, Chang HN. 1990. Effect of growth hormone modifica tion on Shikonin production from Lithospermum erythrorhi zon cell culture with in situ extraction. Biotech. Lett. 12(4): 289–294CrossRefGoogle Scholar
  17. Manjkhola S, Dhar U, Joshi M. 2005. Organogenesis, embryo genesis and synthetic seed production in Arnebia euchroma-A critically endangered medicinal plant of the Himalaya. In vitro Cell. Dev. Biol-Plant 41: 244–248CrossRefGoogle Scholar
  18. Martin KP, Joseph D, Madassery J, Philip VJ. 2003. Direct shoot regeneration from lamina explants of two commercial cut flower cultivars of Anthurium andraenanum Horticulture. In Vitro Cell. Dev. Biol-Plant 39: 500–504CrossRefGoogle Scholar
  19. Mizukami H, Konoshima M, Tabata M. 1977. Effect of nutri tional factors on Shikonin derivative formation in Lithospermum callus culture. Phytochem. 16: 1183–1186CrossRefGoogle Scholar
  20. Pal M. 2005. In vitro culture and induction of Shikonin produc tion in Arnebia hispidissima. Ph. D. Thesis submitted to Department of Biotechnology, Guru Jambheshwar University of Science & Technology, Hisar, Haryana, IndiaGoogle Scholar
  21. Papageorgiou VP. 1978. Wound healing properties of naph thaquinones pigments from Alkanna tinctoria. Experimentia 34: 1499–1501CrossRefGoogle Scholar
  22. Papageorgiou VP. 1980. Naturally occurring Isohexenylnaphthazarin pigments: a new class of drugs. Planta Med. 38: 193–203CrossRefPubMedGoogle Scholar
  23. Papageorgiou VP, Assimopoulou AN, Couladouros EA, Hepworth D, Nicolaou KC. 1999. The chemistry and biology of alkannin, Shikonin, and related naphthazarin natural prod ucts. Angewandte Chemie Int. Ed. 38: 270–301CrossRefGoogle Scholar
  24. Pimenova ME, Tareeva NV. 1980. Shikonin content variability in the underground organs of macrotomia dyeing. Rastit Resur. 16: 82–86 (in Russian)Google Scholar
  25. Singh B, Sahu PM, Jain SC, Singh S. 2004. Estimation of Napthaquinones from Arnebia hispidissima (Lehm.) DC. In vivo and In vitro. I. Anti-inflammatory screening. Phytotherapy Res. 18: 154–159CrossRefGoogle Scholar
  26. Sokha V, Nikolaeva L, Pank F. 1996. The Shikonin products in plant tissue culture: Selection of cell lines with high produc tivity in Arnebia euchroma and Lithospermum erythrorhizon. Proc. International Symp. Breed. Res. Medicinal Aromatic Plants, Quedlinburg, Germany. 30(2): 322–326.Google Scholar
  27. Suzuki S, Fujino H, Yamazaki N, Tatsuo Y, Yoshizaki M. 1990. Propagation of Oenothera erythrosepala Borbas by shoot apex culture. Jpn. J. Breed. 40: 367–370.Google Scholar
  28. Terada A, Tanoue Y, Taniguchi H. 1990. Chemistry of Shikonin, ancient purple pigment and its derivatives. J. Syn. Org. Chem. Jpn. 48: 866–875 (in Japanese).Google Scholar
  29. Thengane SR, Deodhar SR, Bhosle SV, Rawal SK. 2006. Direct somatic embryogenesis and plant regeneration in Garcinia indica Choiss Current Sci. 91: 1074–1078.Google Scholar
  30. Thomas TD, Shankar S. 2008. Multiple shoot induction and cal lus regeneration in Sarcostemma brevistigma Wight & Arnott, a rare medicinal plant. Plant Biotechnol. Rep. 3: 67–74CrossRefGoogle Scholar
  31. Toshiro O. 2001. Massive growth of Boraginaceae plant by tis sue culture and massive production of Shikonin derivative thereby, Patent Japan JP2000004702Google Scholar
  32. Tsukada M, Fukui H, Habara C, Tabata M. 1983. Comparative studies on naphthoquinone derivatives in various crude drugs of “Zicao” (Shikon). Shoyakugaku Zasshi 37: 299–305Google Scholar
  33. Yadav R, Arora P, Kumar D, Katyal D, Dilbaghi N, Chaudhury A. 2009. High frequency direct plant regeneration from leaf, internode, and root segments of Eastern Cottonwood (Populus deltoides). Plant Biotechnol. Rep. 3: 175–182CrossRefGoogle Scholar
  34. Yazaki K, Tanaka S, Matsuoka H, Sato F. 1998. Stable transfor mation of Lithospermum erythrorhizon by Agrobacterium rhizogenes and Shikonin production of transformants. Plant Cell Rep. 18: 214–219CrossRefGoogle Scholar
  35. Yazaki K, Matsuoka H, Shimomura K, Bechthold A, Sato F. 2001. A novel dark inducible protein, LeDI-2 and its involve ment in root-specific secondary metabolism in Lithospermum erythrorhizon. Plant Physiol. 125: 1831–1841CrossRefPubMedGoogle Scholar
  36. Yu HJ, Oh SK, Oh MH, Choi DW, Kwon YM, Kim SG. 1997. Plant regeneration from callus cultures of Lithospermum ery throrhizon. Plant Cell Rep. 16(5): 261–266Google Scholar

Copyright information

© Korean Society of Crop Science and Springer Netherlands 2010

Authors and Affiliations

  1. 1.Department of Bio & Nano TechnologyGuru Jambheshwar University of Science & TechnologyHisarIndia

Personalised recommendations