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Acta Physiologiae Plantarum

, Volume 36, Issue 10, pp 2683–2693 | Cite as

Stimulation of in vitro morphogenesis, antioxidant activity and over expression of kaurenoic acid 13-hydroxylase gene in Stevia rebaudiana Bertoni by chlorocholine chloride

  • Sayanti Kundu
  • Avishek Dey
  • Abhijit BandyopadhyayEmail author
Original Paper

Abstract

Phytoconstituents from medicinal plants are considered as important source of raw materials of drugs for pharmaceutical industries. Biotechnology has become an inevitable approach in the area of research and development of medicinal plants for many decades. The present work has been carried out to ascertain the role of chlorocholine chloride (CCC) on in vitro morphogenesis, antioxidant activity and expression level of kaurenoic acid 13-hydroxylase (KA13H) gene in Stevia rebaudiana. To fulfill these purposes chlorocholine chloride was applied in the Murashige and Skoog (Physiol Plant 15(3):473–497, 1962) medium in combination with other plant growth regulators such as 1-naphthalene acetic acid, kinetin and thidiazuron. Chlorocholine chloride was found to contribute significant role on in vitro morphogenesis of S. rebaudiana as evidenced by the formation of embryogenic calli and increase in callusing and microshooting efficiency of explant, i.e., cotyledonary leaf. Moreover, antioxidant enzyme activity as well as ascorbic acid content of the calli and leaves was also stimulated after application of chlorocholine chloride. Q-PCR amplification using gene-specific primers revealed that CCC also promoted the expression level of KA13H gene in S. rebaudiana leaves. The overall study highlighted the promising role of chlorocholine chloride on regeneration efficiency of cotyledonary leaf, significant promotion in antioxidant potential and expression of KA13H gene in S. rebaudiana.

Keywords

Stevia rebaudiana Chlorocholine chloride In vitro morphogenesis Antioxidant activity Kaurenoic acid 13-hydroxylase 

Abbreviations

ANOVA

Analysis of variance

CCC

Chlorocholine chloride

Hr

Hour

Lb

Pound

MS

Murashige and Skoog

N

Normality of solution

NAA

1 Naphthalene acetic acid

NBT

Nitroblue tetrazolium chloride

nm

Nanometer

Q-PCR

Quantitative polymerase chain reaction

PVP

Polyvinyl pyrrolidone

RH

Relative humidity

rpm

Rotation per minute

TCA

Trichloro acetic acid

TDZ

Thidiazuron

UV

Ultraviolet

w/v

Weight by volume

v/v

Volume by volume

Notes

Acknowledgments

The authors are grateful to their supervisor for valuable and constructive suggestions. Authors also express their thankfulness to the Head, Department of Botany, University of Burdwan for providing laboratory and infrastructure facilities. Authors are also grateful to Dr. Anupam Basu, Associate Professor and Head of the Department, Department of Zoology, University of Burdwan for his assistance in Q-PCR analysis. Financial support in form of fellowship from Government of West Bengal, India is duly acknowledged.

References

  1. Agnihotri S, Singh RR, Chaturved HC (2001) In vitro high frequency regeneration of plantlets of Vigna mungo and their ex vitro growth. Indian J Exp Biol 39:916–920PubMedGoogle Scholar
  2. Apel K, Hirt H (2004) Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annual Rev Plant Biol 55:373–399CrossRefGoogle Scholar
  3. Asada K (1999) The water–water cycle in chloroplasts, scavenging of active oxygens and dissipation of excess photons. Annu Rev Plant Physiol Plant Mol Biol 50:601–639PubMedCrossRefGoogle Scholar
  4. Beauchamp, Fedovich BC (1976) Superoxide dismutase assay and an assay applicable to acrylamide gel. Anal Biochem 10:276–287Google Scholar
  5. Bettaieb T, Laribi B, Rouatbi N, Kouki K (2012) In vitro propagation of Stevia rebaudiana (Bert.): A non caloric sweetener and antidiabetic medicinal plant. Int J Med Arom Plants 2:333–339Google Scholar
  6. Blackmon WJ, Reynolds BD (1982) In vitro shoot regeneration of Hibiscus acetosella, muskmelon, watermelon, winged bean. Hort Sci 17:588–589Google Scholar
  7. Brandle JE, Telmer P (2007) Steviol glycoside biosynthesis. Phytochemistry 68:1855–1863PubMedCrossRefGoogle Scholar
  8. Dey A, Kundu S, Bandyopadhyay A, Bharracharjee A (2013a) Efficient micropropagation and chlorocholine chloride induced stevioside production of Stevia rebaudiana Bertoni. Comp Rend Biol 336:17–28CrossRefGoogle Scholar
  9. Dey A, Paul S, Kundu S, Bandyopadhyay A, Bhattacharjee A (2013b) Elevated antioxidant potential of chlorocholine chloride-treated in vitro grown Stevia rebaudiana Bertoni. Acta Physiol Plant 35:1775–1783CrossRefGoogle Scholar
  10. Felippe GM, Lucas NMC, Behar L, Oliveira MAC (1971) Observacoes a respeito de germinacao de Stevia rebaudiana. Bert Hoehnea 1:81–93Google Scholar
  11. Fu CX, Zhao DX, Huang Y (2005) Cellular aggregate size as the critical factor for flavonoid production by suspension cultures of Saussurea medusa. Biotechnol Lett 27:91–95PubMedCrossRefGoogle Scholar
  12. Ghanta S, Banerjee A, Poddar A, Chattopadhyay S (2007) Oxidative DNA damage preventive activity and antioxidant potential of Stevia rebaudiana Bertoni, a natural sweetener. J Agr Food Chem 55:10962–10967CrossRefGoogle Scholar
  13. Graebe JE (1987) Gibberellin biosynthesis and control. Ann Rev Plant Physiol 38:419–465CrossRefGoogle Scholar
  14. Hossain T et al (2010) In vitro bulb production in Hippeastrum (Hippeastrum hybridum). J Cent Eur Agric 11(4):469–474CrossRefGoogle Scholar
  15. Ievinsh G, Inguna G, Kruzmane D (2002) Effect of CCC and pH on shoot elongation in Sedum rubrotinctum R.T. Clausen. Plant Sci 163:647–651CrossRefGoogle Scholar
  16. Jeppensen PB, Gregerson S, Poulsen CR, Harmansen K (2002) Stevioside induces antihyperglycemic, insulinotropic and glucagonostatic effects in vivo: studies in the diabetic goto- kakizaki (gk) rats. Phytomedicine 9:9–14CrossRefGoogle Scholar
  17. Kar M, Mishra D (1976) Catalase, peroxidase, polyphenol oxidase activities during rice leaf senescence. Plant Physiol 57:315–319PubMedCrossRefPubMedCentralGoogle Scholar
  18. Kim KK, Sawa Y, Shibata H (1996) Hydroxylation of ent-kaurenoic acid to steviol in Stevia rebaudiana Bertoni: purification and partial characterization of the enzyme. Arch Biochem Biophys 332:223–230PubMedCrossRefGoogle Scholar
  19. Kozak D, Grodek J (2005) The consequent effect of growth retardants on the growth and development of Tibouchina urvilleana Cogn. shoots in vitro. Acta Sci Pol Hortorum Cultus 4(2):123–128Google Scholar
  20. Livak KJ, Schmittgen TD (2001) Analysis of realtive gene expression data using realtime quantitative PCR and the 2−ΔΔCT method. Methods 25:402–408Google Scholar
  21. Mendoza AB, Hattori K, Nishimura T, Fustuhara Y (1993) Histological and scanning electron microscopic observations on plant regeneration in mungbean (Vigna radiata (L.) Wilczek) culture in vitro. Plant Cell Tiss Org Cult 32:137–143CrossRefGoogle Scholar
  22. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol Plant 15(3):473–497CrossRefGoogle Scholar
  23. Nagy M, Tari I (1986) Ethylene production and IAA distribution in bean hypocotyls treated with CCC. Biochem Physiol Pflanzen 181:611–614CrossRefGoogle Scholar
  24. Omaye ST, Turnbull JD, Sauberlich HE (1979) Selected methods for the determination of ascorbic acid in animal cells, tissues and fluids. Method Enzymol 62:3–11CrossRefGoogle Scholar
  25. Ramachandra SR, Ravishankar GA (2002) Plant cell cultures, chemical factories of secondary metabolites. Biotechnol Adv 20:101–153CrossRefGoogle Scholar
  26. Rao MV, Paliyath G, Ormrod DP (1996) Ultraviolet-B-radiation and ozone-induced biochemical changes in the antioxidant enzymes of Arabidopsis thaliana. Plant Physiol 110:125–136PubMedCrossRefPubMedCentralGoogle Scholar
  27. Sakaguchi M, Kan T (1982) Japanese researches on Stevia rebaudiana (Bert.) Bertoni and stevioside. Ci Cult 34:235–248Google Scholar
  28. Shao HB, Chu LY, Zhao HL, Kang C (2008) Primary antioxidant free radical scavenging and redox signalling pathways in higher plant cells. Int J Biol Sci 4(1):8–14CrossRefPubMedCentralGoogle Scholar
  29. Shibata H, Sawa Y, Oka T, Sonoke S, Kim KK, Yoshioka M (1995) Steviol and steviol-glycoside—glucosyltransferase activities in Stevia rebaudiana Bertoni—purification and partial characterization. Arch Biochem Biophys 321:390–396PubMedCrossRefGoogle Scholar
  30. Sivaram L, Mukundan U (2003) In vitro culture studies on Stevia rebaudiana. In Vitro Cell Dev Biol Plant 39:520–523CrossRefGoogle Scholar
  31. Smirnoff N (1996) The function and metabolism of ascorbic acid in plant. Ann Bot 78:661–669CrossRefGoogle Scholar
  32. Snell FD, Snell CT (1971) Colorimetric methods of analysis. Van Nostrand Reinhold Co., New York, pp 7–145Google Scholar
  33. Starratt AN, Kirby CW, Pocs R, Brandle JE (2002) Rebaudioside F, a diterpene glycoside from Stevia rebaudiana. Phytochemistry 59:367–370Google Scholar
  34. Totté N, Charon L, Rohmer M, Compernolle F, Baboeuf I, Geuns JMC (2000) Biosynthesis of the diterpenoid steviol, an ent-kaurene derivative from Stevia rebaudiana Bertoni, via the methylerythritol phosphate pathway. Tetrahedron Lett 41:6407–6410CrossRefGoogle Scholar

Copyright information

© Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków 2014

Authors and Affiliations

  • Sayanti Kundu
    • 1
  • Avishek Dey
    • 1
  • Abhijit Bandyopadhyay
    • 1
    Email author
  1. 1.Department of Botany, UGC Centre for Advanced StudyThe University of BurdwanBurdwanIndia

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