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Rapid plant regeneration, analysis of genetic fidelity and camptothecin content of micropropagated plants of Ophiorrhiza mungos Linn. — a potent anticancer Plant

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Abstract

An efficient protocol has been established for rapid multiplication of Ophiorrhiza mungos Linn. (Rubiaceae), a potent anticancer plant. Axillary and terminal buds of these in vitro-raised seedlings formed the primary source of explants for direct organogenesis. Explants were inoculated onto MS medium supplemented with different concentrations and combinations of 6-benzylaminopurine (BA) and kinetin (KN). The best morphogenic response was observed on MS media supplemented with 0.25 mg L−1 BA and 0.25 mg L−1 KN, which exhibited the highest regeneration frequency (84%), the maximum number of shoots/explants (63.1 ± 1.35) and shoot length (2.8 ± 1.15) within 4 weeks. Fortification of 1.0 mg L−1 GA3 enhanced the shoot elongation by 2.33 fold in 91% of shoot cluster cultures within 3 weeks. A high percent frequency of rooting (92.13%) was achieved within 15 days of shoot implantation on ½ strength MS media fortified with 100 mg L−1 activated charcoal. The rooted plantlets were successfully acclimatized with 95% survival rate. Randomly amplified polymorphic DNA (RAPD) analysis confirmed that all the regenerated plants were genetically identical to their mother plant, suggesting an absence of detectable genetic variation in the regenerated plantlets. High performance liquid chromatography (HPLC) was done to further confirm the existence of qualitative and quantitative differences in the major secondary metabolite (camptothecin) between the mother plant and in vitro-propagated plants. The present results evidently showed comparable chemical profiles. Thus, the present protocol can be used for clonal mass propagation of true-to-type elite O. mungos plants.

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Abbreviations

BA:

Benzyl-6-adenine

KN:

kinetin

MS:

Murashige and Skoog

NAA:

α-Naphthalene acetic acid

IAA:

Indole-3-acetic acid

GA3 :

Gibberellic acid

CTAB:

Cetyltrimethyl ammonium bromide

PCR:

Plolymerase chain re-action

HPLC:

High performance liquid chromatography

References

  • Agnihotri RK, Mishra J, Nandi SK. 2009. Improved in vitro shoot multiplication and rooting of Dendrocalamus hamiltonii Nees et Arn. Ex Munro: production of genetically uniform plants and field evaluation. Acta Physiol. Plant. 31: 961–967

    CAS  Google Scholar 

  • Ahmad N, Wali SA, Anis M. 2008. In vitro production of true-to-type plants of Vitex negundo L. from nodal explants. J. Hort. Sci. Biotech. 83(3): 313–317

    CAS  Google Scholar 

  • Bairu MW, Fennell CW, van Staden J. 2006. The effect of plant growth regulators on somaclonal variation in Cavendish banana (Musa AAA cv. ‘Zelig’). Sci. Hort. 108: 347–351

    Article  CAS  Google Scholar 

  • Beegum AS, Martin KP, Zhang CL, Nishitha IK, Ligimol, Slater A, Madhusoodanan PV. 2007. Organogenesis from leaf and internode explants of Ophiorrhiza prostrata, an anticancer drug (camptothecin) producing plant. Electronic Journal of Biotechnology 10(1): 114–123

    Article  Google Scholar 

  • Bodley AL, Cumming JN, Shapiro TA. 1998. Effects of camptothecin, a topoisomerase I inhibitor, on Plasmodium falciparum. Biochem. Pharmacol. 55: 709–711

    Article  CAS  PubMed  Google Scholar 

  • Chalageri G, Babu UV. 2012. In vitro plant regeneration via petiole callus of Viola patrinii and genetic fidelity assessment using RAPD markers. Turk. J. Bot. 36: 358–368

    CAS  Google Scholar 

  • Chaudhari KN, Ghosh S, Jha S. 2004. The root: A potential source of competent cells for high frequency regeneration in Tylophora indica. Plant Cell Rep. 22: 731–740

    Article  Google Scholar 

  • Chaudhuri RK, Pal A, Jha TB. 2007. Production of genetically uniform plants from nodal explants of Swertia chirata Buch.-Ham. Ex Wall.-an endangered medicinal herb. in vitro Cell. Dev. Biol.-Plant 43: 467–472

    Article  CAS  Google Scholar 

  • De Paiva Neto, Vespasiano Borges, Da Mota Tiago Ribeiro, Otoni Wagner Campos. 2003. Direct organogenesis from hypocotyl-derived explants of annatto (Bixa orellana). Plant Cell Tiss. Org. Cult. 75(2): 159–167

    Article  Google Scholar 

  • Figueiredo SFL. 2001. Micropropagation of Rollinia mucosa (Jacq.) Baill. In vitro Cell Dev. Biol.- Plant 37: 471–475

    Article  Google Scholar 

  • Gutiérrez IEM, Nepomuceno CF, Ledo CAS, Santana JRF. 2011. Micropropagation and acclimatization of Bauhinia cheilantha (an important medicinal plant). Afr. J. Biotechnol. 10(8): 1353–1358

    Google Scholar 

  • Howell SH, Lall S, Che P. 2003. Cytokinins and shoot development. Trends Plant Sci. 8: 453–459

    Article  CAS  PubMed  Google Scholar 

  • Jaimsha RVK. 2012. Study on the production of camptothecin and its derivatives from selected Ophiorrhiza species and its in vitro culture. Mahatma Ghandhi University, Kerala, Doctor of Philosophy

    Google Scholar 

  • Johnson M, Yasmin N, Sonali D, Rajasekarapandian M. 2007. The role of cytokinin and auxin in organogenesis of Passiflora mollissima and evaluation of biochemical changes using isozyme. Eth. J. Sci. Technol. 4: 27–36

    Google Scholar 

  • Jose B, Satheesh KK. 2004. In vitro mass multiplication of Ophiorrhiza mungos Linn. Indian J. Exp. Biol. 42: 639–642

    PubMed  Google Scholar 

  • Kumaraswamy M, Anuradha M. 2010. Micropropagation of Pogostemon cablin Benth. through direct regeneration for production of true-to-type plants. Plant Tissue Cult. Biotechnol. 20(1): 81–89

    Article  Google Scholar 

  • Larkin PJ, Scowcroft WR. 1981. Somaclonal variation–a novel source of variability from cell cultures for plant improvement. Theor. Appl. Genet. 60: 197–214

    Article  CAS  PubMed  Google Scholar 

  • Lorence A, Nessler CL. 2004. Camptothecin, over four decades of surprising findings. Phytochemistry 65(20): 2735–2749

    Article  CAS  PubMed  Google Scholar 

  • Mallon R, Oubina JR, Gonzalez ML. 2010. In vitro propagation of the endangered plant Centaurea ultreiae: assessment of genetic stability by cytological studies, flow cytometry and RAPD analysis. Plant Cell Tiss. Org. Cult. 101: 31–39

    Article  Google Scholar 

  • Murashige T, Skoog F. 1962. A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol. Plant. 15: 473–497

    Article  CAS  Google Scholar 

  • Nandhakumar R, Vishwanathan H, Suresh T, Mohan PS. 2002. Antibacterial activity of Mappia foetida leaves and stem. Fitoterapia 73: 734–736

    Article  CAS  Google Scholar 

  • Namdeo AG, Sharma A, Mahadik KR. 2008. Some observations on Nothapodytes foetida: An overview. Pharmacogn Rev. 2(3): 110–115

    CAS  Google Scholar 

  • Namdeo AG, Priya T, Bhosale BB. 2012. Micropropagation and production of camptothecin form in vitro plants of Ophiorrhiza mungos. Asian Pac J Trop Biomed. 2(2): S662–S666

    Article  Google Scholar 

  • Nikam TD, Ghane SG, Nehul JN, Barmukh RB. 2009. Induction of morphogenic callus and multiple shoot regeneration in Momordica cymbalaria Fenzl. Indian J. Biotechnol. 8: 442–447

    CAS  Google Scholar 

  • Paul A, Thapa G, Basu A, Mazumdar P, Kalita MC, Sahoo L. 2010. Rapid plant regeneration, analysis of genetic fidelity and essential aromatic oil content of micropropagated plants of Patchouli, Pogostemon cablin (Blanco) Benth.— An industrially important aromatic plant. Ind. Crops Prod. 32(3): 366–374

    Article  CAS  Google Scholar 

  • Pawar PKNarkhede CS, Teli BA, Bhalsing NP, Pawar SR, Maheshwari VL. 2002. A technique for rapid propagation of Solanum surrattense Burm. F. Indian J. Biotechnol. 1: 201–204

    Google Scholar 

  • Purkayastha J, Sugla T, Paul A, Solleti S, Sahoo L. 2008. Rapid in vitro multiplication and plant regeneration from nodal explants of Andrographis paniculata: a valuable medicinal plant. in vitro Cell. Dev. Biol.- Plant 44: 442–447

    Article  CAS  Google Scholar 

  • Raja DH, Arokiasamy DI. 2008. In vitro propagation of Mentha viridis L. from nodal and shoot tip explants. Plant Tissue Cult. Biotech. 18: 1–6

    Google Scholar 

  • Rathore MS, Shekhawat NS. 2011. Micropropagation of Maerua oblongifolia: A rare ornamental from semi arid regions of Rajasthan, India. J. Dev. Biol. Tissue Eng. 3(8): 92–98

    CAS  Google Scholar 

  • Redinbo MR, Styewart L, Kuhn P, Champoux JJ, Hol WGJ. 1998. Crystal structures of human topoisomerase I in covalent and mono covalent complexes with DNA. Science 279: 1504–1513

    Article  CAS  PubMed  Google Scholar 

  • Roja G, Heble MR. 1994. The quinoline alkaloids camptothecin and 9-methoxycamptothecin from tissue cultures and mature trees of Nothapodytes nimmoniana. Phytochemistry 36: 65–66

    Article  CAS  Google Scholar 

  • Romanelli SP, Perego G, Pratesi N, Carenini M, Tortoreto Zunino F. 1998. In vitro and in vivo interaction between cisplatin and topotecan in ovarian carcinoma systems. Cancer themother. Pharmacol. 41: 385–390

    Article  CAS  Google Scholar 

  • Saito K, Sudo H, Yamazaki M, Nakamura MK, Kitajima M, Takayama H, Aimi N. 2001. Feasible production of camptothecin by hairy root culture of Ophiorrhiza pumila. Plant Cell Rep. 20: 267–271

    Article  CAS  Google Scholar 

  • Stajner N, Bohanec B, Jakπe M. 2002. In vitro propagation of Asparagus maritimus–A rare Mediterranean saltresistant species. Plant Cell Tiss. Org. Cult. 70(3): 269–274

    Article  CAS  Google Scholar 

  • Sudipta KM, Kumara Swamy M, Balasubramanya S, Anuradha M. 2011. Cost effective approach for in vitro propagation of (Leptadenia reticulata Wight & Arn.) — a threatened plant of medicinal importance. Journal of Phytology 3: 72–79

    Google Scholar 

  • Sugla T, Purkayastha J, Singh SK, Solleti SK, Sahoo L. 2007. Micropropagation of Pongamia pinnata through enhanced axillary branching. in vitro Cell Dev. Biol.-Plant 43: 409–414

    Article  CAS  Google Scholar 

  • Sun Y, Zhao Y, Wang X, Qiao G, Chen G, Yang Y, Zhou J, Jin L, Zhuo R. 2009. Adventitious bud regeneration from leaf explants of Platanus occidentalis L. and genetic stability assessment. Acta Physiol. Plant 31: 33–41

    Article  Google Scholar 

  • Swamy MK, Balasubramanya S, Anuradha M. 2010. In vitro multiplication of Pogostemon cablin Benth. through direct regeneration. Afr. J. Biotechnol. 9(14): 2069–2075

    Google Scholar 

  • Swamy MK, Mohanty SK, Anuradha M. 2014. The effect of plant growth regulators and natural supplements on in vitro propagation of Pogostemon cablin Benth. J. Crop Sci. Biotech. 17(2): 71–78

    Article  Google Scholar 

  • Swaroopa G, Subhash K, Ghansham D. 2011. An improved plant regeneration system for high frequency multiplication of Rubia cordifolia L.: A rare medicinal plant. Asian J. Biotechnol. 3(4): 397–405

    Article  Google Scholar 

  • Thomas TD. 2008. The role of activated charcoal in plant tissue culture. Biotechnol. Adv. 26: 618–631

    Article  CAS  PubMed  Google Scholar 

  • Vineesh VR, Fijesh PV, Jelly Louis C, Jaimsha VK, Padikkala J. 2007. In vitro production of camptothecin (an anticancer drug) from mutant albino plants of Ophiorrhiza rugosa var. decumbens. Curr. Sci. 92: 1219

    Google Scholar 

  • Wall ME, Wani MC, Cook CE, Palmer KH, McPhail AT, Sim GA. 1966. Plant antitumor agents. I. Isolation and structure of camptothecin, a novel alkaloidal leukemia and tumor inhibitor from Camptotheca acuminata. J. Am. Chem. Soc. 88: 3888–3890

    Article  CAS  Google Scholar 

  • Watase I, Sudo H, Yamazaki M, Saito K. 2004. Regeneration of transformed Ophiorrhiza pumila plants producing camptothecin. Plant Biotech. 21: 337–342

    Article  CAS  Google Scholar 

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Authors and Affiliations

  1. Department of Biotechnology, Acharya Nagarjuna University, Guntur, 522510, India

    Pradeep S. Kaushik & Maniyam Anuradha

  2. Rishi Foundation, #234, 10th C main, 1st Block, Jayanagar, Bangalore, 560011, India

    Subbanarasiman Balasubramanya & Maniyam Anuradha

  3. Padmashree Institute of Management and Sciences, Kengeri, Bangalore, 560060, India

    Mallappa Kumara Swamy

  4. Department of Crop Science, Faculty of Agriculture, Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia

    Mallappa Kumara Swamy

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  1. Pradeep S. Kaushik
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  2. Mallappa Kumara Swamy
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  3. Subbanarasiman Balasubramanya
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  4. Maniyam Anuradha
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Correspondence to Mallappa Kumara Swamy.

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Kaushik, P.S., Swamy, M.K., Balasubramanya, S. et al. Rapid plant regeneration, analysis of genetic fidelity and camptothecin content of micropropagated plants of Ophiorrhiza mungos Linn. — a potent anticancer Plant. J. Crop Sci. Biotechnol. 18, 1–8 (2015). https://doi.org/10.1007/s12892-014-0001-9

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  • DOI: https://doi.org/10.1007/s12892-014-0001-9

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