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

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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.

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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

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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.

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Authors and Affiliations

  1. Department of Botany, UGC Centre for Advanced Study, The University of Burdwan, Burdwan, 713104, West Bengal, India

    Sayanti Kundu, Avishek Dey & Abhijit Bandyopadhyay

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  1. Sayanti Kundu
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  2. Avishek Dey
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  3. Abhijit Bandyopadhyay
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Correspondence to Abhijit Bandyopadhyay.

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Communicated by A. Chandra.

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Kundu, S., Dey, A. & Bandyopadhyay, A. Stimulation of in vitro morphogenesis, antioxidant activity and over expression of kaurenoic acid 13-hydroxylase gene in Stevia rebaudiana Bertoni by chlorocholine chloride. Acta Physiol Plant 36, 2683–2693 (2014). https://doi.org/10.1007/s11738-014-1639-0

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