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Role of activated charcoal and amino acids in developing an efficient regeneration system for foxtail millet (Setaria italica (L.) Beauv.) using leaf base segments

Abstract

An efficacious, reproducible direct in vitro regeneration system has been developed from leaf base segments (LBs) of six high yielding genotypes of foxtail millet (Setaria italica (L.) Beauv.). LBs excised from 4-day-old seedling were inoculated on Murashige and Skoog (MS) medium supplemented with different types and concentrations of cytokinins. The shoots induced per explant significantly increased with the supplementation of BAP to auxin containing medium. The results showed that a maximum shoot induction, 58.8% was obtained on MS medium incorporated with 8.9 µM BAP and 2.7 µM NAA in ‘CO5’ genotype. Further, the highest frequency of multiple shoots was produced on MS(I) medium containing 8.9 µM BAP, 2.7 µM NAA, 700 mg L−1 proline, 0.5 mg L−1 cysteine, 2.0 mg L−1 glycine and 150 mg L−1 arginine. MS(I) medium additionally fortified with 5.0 g L−1 activated charcoal (AC) was found to achieve the best precocious plant regeneration. Elongated shoots were rooted on half-strength MS medium amended with 2.9 µM IAA and achieved maximum root number (8.7) within 10 days. Rooted plantlets were acclimated in soil with 92% survival rate. Molecular marker analysis of in vitro regenerated and field grown plants revealed no somaclonal variations. Briefly, amino acids and activated charcoal could significantly enhance the foxtail millet direct multiple shoot proliferation and plant regeneration. Here we report, a short-term, genotype independent, direct plant regeneration protocol for future genetic transformation studies in foxtail millet genotypes.

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Acknowledgements

Author P. Rathinapriya is grateful to UGC BSR-SRF (25-1/2013-14 (BSR)/7-326/2011 dt 30.05.2014) the University Grants Commission, New Delhi, India for financial support in the form of fellowship. We thank the Department of Small Millets, Millet Research Station, Tamil Nadu Agricultural University and Main Agricultural Research Station, University of Agricultural Sciences, Dharwad, India for providing seed material used in this study. Also the authors gratefully acknowledge the use of Bioinformatics Infrastructure Facility, Alagappa University funded by Department of Biotechnology, Ministry of Science and Technology, Government of India: Grant No. (BT/BI/25/015/2012) for providing the computational facility and the authors thankfully acknowledge DST-FIST (Grant No. SR/FST/LSI-639/2015(C)), UGC-SAP (Grant No. F.5-1/2018/DRS-II(SAP-II)) and DST-PURSE (Grant No. SR/PURSE Phase 2/38 (G)) for providing instrumentation facilities. The first author gratefully expresses gratitude Prof. Rana P. Singh, Editor-in-Chief, and all the reviewers for their valuable suggestions to improve this manuscript.

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PR and MR conceived and designed research. PR carried out the experiments. SP performed the molecular marker analysis. ASR and RR analyzed data. PR prepared the manuscript, LS and MR proofread the manuscript. All authors read and approved the manuscript for publication.

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Correspondence to Manikandan Ramesh.

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Rathinapriya, P., Satish, L., Rameshkumar, R. et al. Role of activated charcoal and amino acids in developing an efficient regeneration system for foxtail millet (Setaria italica (L.) Beauv.) using leaf base segments. Physiol Mol Biol Plants 25, 533–548 (2019). https://doi.org/10.1007/s12298-018-0619-z

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Keywords

  • Additives
  • Direct multiple shoots
  • Molecular markers
  • Plant growth regulators
  • Plant regeneration