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Efficient in vitro plant regeneration from leaf-derived callus and genetic fidelity assessment of an endemic medicinal plant Ranunculus wallichianus Wight & Arnn by using RAPD and ISSR markers

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A Correction to this article was published on 09 August 2021

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Abstract

Ranunculus wallichianus is a medicinally important plant and an endemic species to the Western Ghats of South India. An efficient and reliable indirect regeneration protocol system for R. wallichianus was developed from leaf explants in the present investigation. Leaf explants were cultured on both full-strength and half-strength MS (Murashige & Skoog) medium supplemented with different concentrations (0.5 mg L− 1 to 3.0 mg L− 1) of 2,4-D and NAA. Among the different concentrations tested, the highest percentage of yellowish-green compact nodular callus formation was observed on a half-strength MS medium with 2.0 mg L− 1 of 2, 4-D. Then, the in vitro raised organogenic callus was cultured on a half-strength MS medium containing various concentrations (0.5 mg L− 1 to 3.0 mg L− 1) of BA, KIN, and TDZ with 0.5 mg L− 1 NAA and 10 % CW for in vitro shoot regeneration. The highest percentage of regeneration response (97 %) and a maximum number of shoots formation (11.1 ± 0.13 shoots/culture with 9.2 ± 0.21 cm mean shoot length) were obtained from MS medium containing 2.5 mg L− 1 BA with 0.5 mg L− 1 NAA and 10 % CW. The well elongated in vitro raised shoots were effectively rooted in a half-strength MS medium with 2.5 mg L− 1 IBA + 250 mg L− 1 activated charcoal. The well-rooted plantlets were successfully hardened and acclimatized with a survival rate of 94 %. Clonal fidelity of in vitro raised plantlets was assessed by using DNA-based RAPD and ISSR molecular markers. A total of 56 and 47 monomorphic bands were obtained from RAPD and ISSR markers respectively. This present in vitro propagation protocol system could be effective for the conservation of R. wallichianus with their genetic purity and its further investigations.

Key message

The present study develops a protocol for mass multiplication of Ranunuculus wallichianus through indirect organogenesis and evaluates the clonal fidelity of regenerants by using RAPD and ISSR markers.

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Fig. 1
Fig. 2
Fig. 3

Change history

Abbreviations

BA :

6-Benzyladenine

KIN :

Kinetin

TDZ :

Thidiazuron

CW :

Coconut Water

2,4-D :

2,4-dichlorophenoxyacetic acid

NAA:

1-Napthyl Acetic Acid

IAA:

Indole-3-Acetic Acid

IBA:

Indole-3-Butyric acid

PGR:

Plant Growth Regulator

DNA:

Deoxyribonucleic acid

RAPD:

Random Amplified polymorphic DNA

ISSR:

Inter Simple Sequence Repeats

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Acknowledgements

The Authors gratefully acknowledge the management of St. Joseph’s college, Tiruchirappalli, India for the laboratory facilities. The authors also acknowledge the DST-SERB, New Delhi for financial support.

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Authors and Affiliations

  1. Department of Botany, Gnanarethinam Centre for In vitro Conservation (GCIC), St. Joseph’s College, Affiliated to Bharathidasan University, Tiruchirappalli, Tamil Nadu, 620 002, India

    P. Srinivasan, H. David Raja & R. Tamilvanan

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  1. P. Srinivasan
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  2. H. David Raja
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  3. R. Tamilvanan
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Contributions

PS and RT perform the study and examine the experimental data. PS prepared the manuscript. HDR gave the draw of the experimental design and complete the final version of the manuscript.

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Correspondence to P. Srinivasan.

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Communicated by Mohammad Faisal.

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The original version of this article has been revised: The bottom three entries of the 4th column in Table 1 have been corrected.

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Srinivasan, P., Raja, H.D. & Tamilvanan, R. Efficient in vitro plant regeneration from leaf-derived callus and genetic fidelity assessment of an endemic medicinal plant Ranunculus wallichianus Wight & Arnn by using RAPD and ISSR markers. Plant Cell Tiss Organ Cult 147, 413–420 (2021). https://doi.org/10.1007/s11240-021-02134-y

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