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Effect of purified vermicast extract with plant growth regulators on indirect regeneration of Naregamia alata Wight & Arn from leaf explants and genetic fidelity analysis of regenerants

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Abstract

An efficient indirect organogenesis protocol for Naregamia alata was standardized in the present investigation. Purified form of vermicast extract (VCE) was used in combination with various plant growth regulators for callus induction, shoot regeneration, and root induction. Leaf explants were inoculated on MS medium with various concentrations (1.0–2.5 mg L−1) of 2, 4-D and NAA combined with 20%, 30% and 40% of purified VCE for callus induction. Among the various concentrations tested, 2.0 mg L−1 2, 4-D, and 20% VCE produced maximum callus induction (96.2%) with green nodular callus. Then, the calli were transferred to MS medium fortified with different concentrations (1.0–2.5 mg L−1) of TDZ and BA in combination with purified VCE (20%, 30% and 40%). The highest percentage of adventitious shoot regeneration (100%) was observed on MS medium supplemented with 1.5 mg L−1 TDZ and 30% purified VCE with respect to the number of shoots (20.7 ± 0.28 shoots/callus) and shoot length (11.4 ± 0.21 cm). The well-elongated shoots were rooted in MS medium with IBA and IAA (1.0–2.5 mg L−1) with purified VCE (20%, 30% and 40%). The maximum percentage of root formation (99%), number of roots (9.6 roots /shoot) and root length (5.9 cm) were observed in 2.0 mg L−1 IBA with 30% VCE combination. Healthy rooted plants were successfully acclimatized with a survival rate of 98% in field conditions. The genetic fidelity analysis of in vitro raised plants of N. alata was evaluated using RAPD and ISSR markers and they were found to be true genetic traits. The present work suggests the addition of purified VCE with the plant growth regulators would enhance the regeneration ability of N. alata from leaf explants resulting in regenerants with genetic stability.

Key message

The present study was designed for large scale production of genetically pure clones of N. alata via indirect organogenesis using purified Vermicast Extract and various concentrations of plant growth regulators from leaf explants.

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Abbreviations

PGR:

Plant growth regulator

VCE:

Vermicast extract

MS medium:

Murashige and Skoog medium

2, 4-D:

2, 4-Dichlorophenoxyacetic acid

NAA:

1-Napthyl acetic acid

TDZ:

Thidiazuron

BA:

6-Benzyladenine

IBA:

Indole-3-butyric acid

IAA:

Indole-3-acetic acid

PFD:

Photon flux density

CTAB:

Cetyltrimethylammonium bromide

PCR:

Polymerase chain reaction

dNTP:

Deoxynucleotide triphosphate

EDTA:

Ethylene diamine tetra acetic acid

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Acknowledgements

The authors thankfully acknowledge Rev. Fr. Dr. Peter SJ, Secretary and Rev. Fr. Dr. M. Arockiasamy Xavier SJ, Principal of St. Joseph’s college (Autonomous), Tiruchirappalli, India, for providing the necessary amenities.

Funding

The present study was funded by the management of St. Joseph’s College (Autonomous), Tiruchirappalli, India, through SJC Research Grant 2021.

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Authors and Affiliations

  1. Fr.Gnanarethinam Unit for In Vitro Conservation (GUIC), Department of Botany, St. Joseph’s College (Autonomous), Affiliated to Bharathidasan University, Tiruchirappalli, Tamil Nadu, 620 002, India

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

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  1. H. David Raja
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  2. P. Srinivasan
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  3. R. Tamilvanan
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HDR designed the study. HDR and PS conducted the experiments and writing an original manuscript. PS analyzed statistical data. RT collected the literature review.

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Correspondence to H. David Raja.

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Raja, H.D., Srinivasan, P. & Tamilvanan, R. Effect of purified vermicast extract with plant growth regulators on indirect regeneration of Naregamia alata Wight & Arn from leaf explants and genetic fidelity analysis of regenerants. Plant Cell Tiss Organ Cult 151, 23–34 (2022). https://doi.org/10.1007/s11240-022-02326-0

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