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An efficient in vitro system for somatic embryogenesis and podophyllotoxin production in Podophyllum hexandrum Royle

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Abstract

Podophyllum hexandrum Royle known as Indian mayapple is an important medicinal plant found only in higher altitudes (2,700 to 4,200 m) of the Himalayas. The highly valued anticancer drug Podophyllotoxin is obtained from the roots of this plant. Due to over exploitation, this endemic plant species is on the verge of extinction. In vitro culture for efficient regeneration and the production of podophyllotoxin is an important research priority for this plant. Hence, in the present study, an efficient plant regeneration system for mass multiplication through somatic embryogenesis was developed. We have screened P. hexandrum seeds collected from three different regions in the Himalayas to find their regenerative potentials. These variants showed variation in germination percentage as well as somatic embryogenic frequency. The seeds collected from the Milam area of Pithoragarh district showed better germination response (99.3 %) on Murashige and Skoog (MS) medium fortified with Gibberellic acid (GA3 [5 mg/l]) and higher direct somatic embryogenic frequency (89.6 %). Maximum production of embryogenic callus (1.2 g fresh weight [FW]) was obtained when cotyledons containing the direct somatic embryo clusters were cultured in MS medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D [1.5 mg/l]) after 4 week of culture in complete darkness. In the present investigation, somatic embryogenesis was accomplished either by direct organogenesis or callus mediated pathways. The latter method resulted in a higher frequency of somatic embryo induction in hormone-free MS medium yielding 47.7 embryos/50 mg of embryogenic callus and subsequent germination in MS medium supplemented with GA3 (5 mg/l). Seventy-nine percent of embryos attained complete maturity and germinated into normal plants with well-developed roots. Systematic histological analysis revealed the origin of somatic embryo and their ontogenesis. The higher level of podophyllotoxin (1.8 mg/g dry weight [DW]) was recorded in germinated somatic embryos when compared to field grown plants. The present system can be widely used for mass propagation, transgenic recovery, and podophyllotoxin production for commercial utilization.

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Abbreviations

2,4-D:

2,4-dichlorophenoxy acetic acid

ABA:

Abscisic acid

DSE:

Direct somatic embryo

GA3 :

Gibberellic acid

HPLC:

High performance liquid chromatography

IAA:

Indole-3-acetic acid

IBA:

Indole-3-butyric acid

MS:

Murashige and Skoog medium

NAA:

Naphthaleneacetic acid

PGR:

Plant growth regulators

PTOX:

Podophyllotoxin

RT:

Retention time

References

  • Ammirato PV (1974) The effects of abscisic acid on the development of somatic embryos from cells of caraway (Carum carvi L.). Bot Gaz 135:328–337

    Article  CAS  Google Scholar 

  • Anbazhagan VR, Ahn CH, Harada E, Kim YS, Choi YE (2008) Podophyllotoxin production via cell and adventitious root cultures of Podophyllum peltatum. In Vitro Cell Dev Biol Plant 44:494–501

    Article  CAS  Google Scholar 

  • Arumugam N, Bhojwani SS (1990) Somatic embryogenesis in tissue cultures of Podophyllum hexandrum. Can J Bot 68:487–491

    Article  Google Scholar 

  • Badhwar RL, Sharma BK (1963) A note on the germination of Podophyllum seeds. Indian For 89:445–447

    Google Scholar 

  • Balzon TA, Luis ZG, Scherwinski-Pereira JE (2013) New approaches to improve the efficiency of somatic embryogenesis in oil palm (Elaeis guineensis Jacq.) from mature zygotic embryos. In Vitro Cell Dev Biol Plant 49:41–50

    Article  CAS  Google Scholar 

  • Bedir E, Khan I, Moraes RM (2001) Bioprospecting for podophyllotoxin. In: Janik J, Whipkey A (eds) Trends in new crops and new uses. ASHS, Alexandria, pp 545–549

    Google Scholar 

  • Berlin GP, Miksche JP (1976) Botanical microtechnique and cytochemistry, 3rd edn. Iowa State University Press, Ames, Iowa, USA

    Google Scholar 

  • Bhatt A, Rawal RS, Dhar U (2005) Germination improvement in Swertia angustifolia: a high value medicinal plant of Himalaya. Curr Sci 89(6):1008–1011

    Google Scholar 

  • Brown DC, Thorpe TA (1995) Crop improvement through tissue culture. World J Microbiol Biotechnol 11:409–415

    Article  CAS  PubMed  Google Scholar 

  • Bush EJ, Jones DW (1995) Asymmetric total synthesis of (−)-podophyllotoxin. J Chem Soc Perkin Trans 1:151–155

    Google Scholar 

  • Chandra B, Palni LMS, Nandi SK (2006) Propagation and conservation of Picrorhiza kurrooa Royle ex Benth.: an endangered Himalayan medicinal herb of high commercial value. Biodivers Conserv 15:2325–2338

    Article  Google Scholar 

  • Choi YE, Kim JW, Yoon ES (1999) High frequency of plant production via somatic embryogenesis from callus or cell suspension cultures in Eleutherococcus senticosus. Ann Bot 83:309–314

    Article  CAS  Google Scholar 

  • Chugh A, Khurana PJ (2002) Gene expression during somatic embryogensis—recent advances. Curr Science 83:715–730

    Google Scholar 

  • De Kroon H, Whigham DF, Watson MA (1991) Developmental ecology of mayapple: effects of rhizome severing, fertilization and timing of shoot senescence. Funct Ecol 5:360–368

    Article  Google Scholar 

  • Eudes F, Acharya S, Laroche A, Selinger LB, Cheng KJ (2003) A novel method to induce direct somatic embryogenesis, secondary embryogenesis and regeneration of fertile green cereal plants. Plant Cell Tiss Org Cult 73:147–157

    Article  CAS  Google Scholar 

  • Feher A, Pasternak T, Dudits D (2003) Transition of somatic plant cells to an embryogenic state. Plant Cell Tiss Org Cult 74:201–228

    Article  CAS  Google Scholar 

  • Fehr A (2006) Why somatic plant cells start to form embryos? In Mujid A, Samaj J, eds., Somatic Embryogenesis., Plant Cell Monographs,Vol.2, Robinson DG, series ed., Springer-Verlag, Berlin Heidelberg, Germany, pp. 85–101

  • Fischer-Iglesias C, Sundberg B, Neuhaus G, Jones AM (2001) Auxin distribution and transport during embryonic pattern formation in wheat. Plant J 26:115–129

    Article  CAS  PubMed  Google Scholar 

  • Fitch MM (1993) High frequency somatic embryogenesis and plant regeneration from papaya hypocotyls callus. Plant Cell Tiss Org Cult 32(2):205–212

    Article  CAS  Google Scholar 

  • Gaj MD (2001) Direct somatic embryogenesis as a rapid and efficient system for in vitro regeneration of Arabidopsis thaliana (L.) Heynh. Plant Cell Tiss Org Cult 64:39–46

    Article  Google Scholar 

  • Jiménez VM (2005) Involvement of plant hormones and plant growth regulators on in vitro somatic embryogenesis. Plant Growth Regul 47:91–110

    Article  Google Scholar 

  • Kanchanapoom K, Domyoas P (1999) The origin and development of embryoids in oil palm (Elaeis guineensis Jacq.) embryo culture. Sci Asia 25:193–200

    Article  Google Scholar 

  • Kim SW, Oh SC, Liu JR (2003) Control of direct and indirect somatic embryogenesis by exogenous growth regulators in immature zygotic embryo cultures of rose. Plant Cell Tissue Org Cult 74(1):61–66

    Article  CAS  Google Scholar 

  • Kim YS, Lim S, Choi YE, Anbazhagan VR (2007) High frequency plant regeneration via somatic embryogenesis in Podophyllum peltatum L., an important medicinal plant for source of anticancer drug. Curr Sci 92:662–666

    CAS  Google Scholar 

  • Krishnaraj S, Vasil IK (1995) Somatic embryogenesis in herbaceous monocots. In: Thorpe TA (ed) In vitro embryogenesis in plants. Kluwer, Dordrecht, pp 417–471

    Chapter  Google Scholar 

  • Majumder A, Jha S (2009) Biotechnological approaches for the production of potential anticancer leads podophyllotoxin and paclitaxel: an overview. J Bio Sci 1:46–69

    Google Scholar 

  • Merkle SA, Sotak RJ, Wiecko AT, Sommer HE (1990) Maturation and conversion of Liriodendron tulipifera somatic embryos. In Vitro Cell Dev Biol 26:1086–1093

    Article  Google Scholar 

  • Mikuła A, Tykarska T, Zielińska M, Kurać M, Rybczyński J (2004) Ultrastructural changes in zygotic embryos of Gentiana punctata L. during callus formation and somatic embryogenesis. Acta Biol Cracov Ser Bot 46:109–120

    Google Scholar 

  • Moraes-Cerdeira RM, Dayan FE, Bedir E, Barrett H, Burandt C Jr, Canel C (2002) The lignans of Podophyllum. In: Rahman AU (ed) Studies in natural product chemistry, vol 26. Elsevier, New York, pp 149–182

    Google Scholar 

  • Nadeem M, Palni LMS, Purohit AN, Pandey H, Nandi SK (2000) Propagation and conservation of Podophyllum hexandrum Royle: an important medicinal herb. Biol Conserv 92:121–129

    Article  Google Scholar 

  • Nishiwaki M, Fujino K, Koda Y, Masuda K, Kikuta Y (2000) Somatic embryogenesis induced by the simple application of abscisic acid to carrot (Daucus carota L.) seedlings in culture. Planta 211:756–759

    Article  CAS  PubMed  Google Scholar 

  • Orozco-Segovia A, Batis AI, Rojas-Aréchiga M, Mendoza A (2003) Seed biology of palms: a review. Palms 47:79–94

    Google Scholar 

  • Paek KY, Chakrabarty D, Hahn EJ (2005) Application of bioreactor systems for large scale production of horticultural and medicinal plants. In: Hvoslef-Eide AK, Preil W (eds) Liquid culture systems for in vitro plant propagation. Springer, The Netherlands, pp 95–116

    Chapter  Google Scholar 

  • Pandey H, Nandi SK, Kumar A, Palni UT, Palni LMS (2007) Podophyllotoxin content in Podophyllum hexandrum Royle plants of known age of seed origin and grown at a lower altitude. Acta Physiol Plant 29:121–126

    CAS  Google Scholar 

  • Pasternak T, Prinsen E, Ayaydin F, Miskolczi P, Potters G, Asard H, Van Onckelen H, Dudits D, Fehér A (2002) The role of auxin, pH and stress in the activation of embryogenic cell division in leaf protoplast-derived cells of alfalfa (Medicago sativa L.). Plant Physiol 129:1807–1819

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Sadowska A, Wiweger M, Lata B, Obidoska G (1997) In vitro propagation of Podophyllum peltatum L. by the cultures of embrya and divided embrya. Biol Plant 39:331–336

    Article  Google Scholar 

  • Sajc L, Grubisic D, Vunjak-Novakovic G (2000) Bioreactors for plant engineering: an outlook for further research. Biochem Eng J 4:89–99

    Article  Google Scholar 

  • Sakakibara H, Takei K, Hirose N (2006) Interactions between nitrogen and cytokinin in the regulation of metabolism and development. Trends Plant Sci 11(9):440–448

    Article  CAS  PubMed  Google Scholar 

  • Samant SS, Dhar U, Palni LMS (1998) Medicinal plants of Himalaya: diversity distribution and potential values. Himvikas, Gyanodaya Prakashan, Nainital

    Google Scholar 

  • Scherwinski-Pereira JE, Guedes RS, Silva RA, Fermino PCP, Luis ZG, Freitas EO (2012) Somatic embryogenesis and plant regeneration in açaí palm (Euterpe oleracea). Plant Cell Tiss Org Cult 109:501–508

    Article  CAS  Google Scholar 

  • Sivanandhan G, Mariashibu TS, Arun M, Rajesh M, Kasthurirengan S, Selvaraj N, Ganapathi A (2011) The effect of polyamines on the efficiency of multiplication and rooting of Withania somnifera (L.) Dunal and content of some withanolides in obtained plants. Acta Physiol Plant 33:2279–2288

    CAS  Google Scholar 

  • Sivanandhan G, Arun M, Mayavan S, Rajesh M, Jeyaraj M, Kapil Dev G, Manickavasagam M, Selvaraj N, Ganapathi A (2012a) Optimization of elicitation conditions with methyl jasmonate and salicylic acid to improve the productivity of withanolides in the adventitious root culture of Withania somnifera (L.) Dunal. Appl Biochem Biotechnol 168:681–696

    Article  CAS  PubMed  Google Scholar 

  • Sivanandhan G, Arun M, Mayavan S, Rajesh M, Mariashibu TS, Manickavasagam M, Selvaraj N, Ganapathi A (2012b) Chitosan enhances withanolides production in adventitious root cultures of Withania somnifera (L.) Dunal. Ind Crop Prod 37:124–129

    Article  CAS  Google Scholar 

  • Sultan P, Shawl AS, Ramteke PW, Jan A, Chisti N, Jabeen N, Shabir S (2006) In vitro propagation for mass multiplication of Podophyllum hexandrum: a high value medicinal herb. Asian J Plant Sci 5:179–184

    Google Scholar 

  • van Uden W, Pras N, Visser JF, Malingre TM (1989) Detection and identification of podophyllotoxin produced by cell cultures derived from Podophyllum hexandrum Royle. Plant Cell Rep 8:165–168

    Article  PubMed  Google Scholar 

  • Vasil IK (2008) A short history of plant biotechnology. Phytochem Rev 7:387–394

    Google Scholar 

  • Williams EG, Maheswaran G (1986) Somatic embryogenesis: factors influencing coordinated behaviour of cells as an embryogenic group. Ann Bot 57:443–462

    Google Scholar 

Download references

Acknowledgments

The authors are thankful to the Life Science Research Board, Defence Research and Development Organisation (DRDO), Govt. of India for financial support (DLS/81/48222/LSRB–171 BTB/2008) used to carry out the present work. The corresponding author is thankful to the University Grants Commission (UGC), Govt. of India, for providing an emeritus fellowship under the BSR scheme. The first author is thankful to Prof. S.K. Nandi, G.B. Pant Institute of Himalayan Environment and Development, Almora, Uttarakhand, India, Prof. M.C. Nautiyal, H.N.B. Garhwal University, Garhwal Srinagar, Uttarakhand, India, and Dr. Lokho Puni IFS, Forest Research Institute, Dehradun, Uttaranchal for help with the collection of seeds at different locations. The first author is thankful to Dr. V. Nandhagopal, National College, Trichy, for his critical guidance in histology work. The authors are thankful to Dr. R. Boopathy, Head of the Department and Dr. S. Girija, Associate Professor, Department of Biotechnology, Bharathiar University, Coimbatore for providing HPLC facility.

Conflict of interest

The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. Department of Biotechnology and Genetic Engineering, School of Biotechnology, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India

    Manoharan Rajesh, Ganeshan Sivanandhan, Murugaraj Jeyaraj, Rajan Chackravarthy, Markandan Manickavasagam & Andy Ganapathi

  2. Temasek Life Sciences Laboratory Limited, 1 Research Link, National University of Singapore, Singapore, 117604, Singapore

    Rajan Chackravarthy

  3. Periyar E.V.R College, Tiruchirappalli, Tamil Nadu, 620023, India

    N. Selvaraj

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  1. Manoharan Rajesh
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  2. Ganeshan Sivanandhan
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  3. Murugaraj Jeyaraj
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  5. Markandan Manickavasagam
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  6. N. Selvaraj
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Correspondence to Andy Ganapathi.

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Rajesh, M., Sivanandhan, G., Jeyaraj, M. et al. An efficient in vitro system for somatic embryogenesis and podophyllotoxin production in Podophyllum hexandrum Royle. Protoplasma 251, 1231–1243 (2014). https://doi.org/10.1007/s00709-014-0632-1

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