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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Agarwal T, Gupta AK, Patel AK, Shekhawat NS (2015) Micropropagation and validation of genetic homogeneity of Alhagi maurorum using SCoT, ISSR and RAPD markers. Plant Cell Tissue Organ Cult 120:313–323
Al Khateeb W, Bahar E, Lahham J, Schroeder D, Hussein E (2013) Regeneration and assessment of genetic fidelity of the endangered tree Moringa peregrina (Forsk.) Fiori using Inter Simple Sequence Repeat (ISSR). Physiol Mol Biol Plants 19:157–164
Al-Turki TA, Basahi MA (2015) Assessment of ISSR based molecular genetic diversity of Hassawi rice in Saudi Arabia. Saudi J Biol Sci 22:591–599
Arockiasamy S, Ignacimuthu S (2007) Regeneration of transgenic plants from two indica rice (Oryza sativa L.) cultivars using shoot apex explants. Plant Cell Rep 26:1745–1753
Asad S, Arshad M, Mansoor S, Zafar Y (2009) Effect of various amino acids on shoot regeneration of sugarcane (Sacchrum officinarum L.). Afr J Biotechnol 8:1214–1218
Aydin M, Pour HA, Haliloglu K, Tosun M (2016) Effect of polyamines on somatic embryogenesis via mature embryo in wheat. Turk J Biol 40:1178–1184
Burner DM, Grisham MP (1995) Induction and stability of phenotypic variation in sugarcane as affected by propagation procedure. Crop Sci 35:875–880
Buter B, Pescitelli SM, Berger K, Schmid JE, Stamp P (1993) Autoclaved and filter sterilized liquid media in maize anther culture: significance of activated charcoal. Plant Cell Rep 13:79–82
Ceasar SA, Ignacimuthu S (2008) Efficient somatic embryogenesis and plant regeneration from shoot apex explants of different Indian genotypes of finger millet (Eleusine coracana (L.) Gaertn.). Vitro Cell Dev Biol Plant 44:427–435
Ceasar SA, Ignacimuthu S (2011) Agrobacterium-mediated transformation of finger millet (Eleusine coracana (L.) Gaertn.) using shoot apex explants. Plant Cell Rep 30:1759–1770
Ceasar SA, Hodge A, Baker A, Baldwin SA (2014) Phosphate concentration and Arbuscular mycorrhizall colonisation influence the growth, yield and expression of twelve PHT1 family phosphate transporters in foxtail millet (Setaria italica). PLoS ONE 9:e108459. https://doi.org/10.1371/journal.pone.0108459
Choi HW, Lemaux PG, Cho M (2000) High frequency of cytogenetic aberration in transgenic oat (Avena sativa L.) plants. Plant Sci 156:85–94
Chutipaijit S, Sutjaritvorakul T (2018) Application of activated charcoal and nanocarbon to callus induction and plant regeneration in aromatic rice (Oryza sativa L.). Chem Spec Bioavailab 30:1–8
Devi P, Zhong H, Sticklen MB (2000) In vitro morphogenesis of pearl millet (Pennisetum glaucum. (L.) R.Br.): efficient production of multiple shoots and inflorescences from shoot apices. Plant Cell Rep 19:546–550
Dey M, Bakshi S, Galiba G, Sahoo L, Panda SK (2012) Development of a genotype independent and transformation amenable regeneration system from shoot apex in rice (Oryza sativa spp. Indica) using TDZ. 3 Biotech 2:233–240
Dey M, Panda SK, Sahoo L (2015) Establishment of an efficient regeneration system amenable to Agrobacterium mediated transformation of two elite indica rice varieties of north east India. Int J Appl Sci Biotechnol 3:680–686
Dharishini MP, Moorthy MK, Balasubramanian K (2015) Effects of plant growth regulators and activated charcoal on regeneration and plantlet development in Neer Brahmi (Bacopa monnieri). J Acad Ind Res 4:69–74
Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh tissue. Focus 12:13–15
Dumas E, Monteuuis O (1995) In vitro rooting of micropropagated shoots from juvenile and mature Pinus pinaster explants: influence of activated charcoal. Plant Cell Tissue Organ Cult 40:231–519
FAOSTAT (2005) Agriculture, Rome. http://faostat.fao/org/
Gless C, Lorz H, Jahne-Gartner A (1998) Transgenic oat plants obtained at high efficiency by microprojectile bombardment of leaf base segments. J Plant Physiol 152:151–157
Grewal D, Gill R, Gosal SS (2006) Role of cysteine in enhancing androgenesis and regeneration of indica rice (Oryza sativa L.). Plant Growth Regul 49:43–47
Gupta PK, Roy JK (2002) Molecular markers in crop improvement: present status and future needs in India. Plant Cell Tissue Organ Cult 70:229–234
Gupta R, Verma K, Joshi DC, Yadav D, Singh M (2010) Assessment of genetic relatedness among three varieties of finger millet with variable seed coat color using RAPD and ISSR markers. Genet Eng Biotechnol J 2:1–9
Huang XQ, Wei ZM (2004) High-frequency plant regeneration through callus initiation from mature embryos of maize (Zea mays L.). Plant Cell Rep 22:793–800
Hutchinson MJ, Onamu R, Obukosia S (2004) Effect of thidiazuron, benzylaminopurine and naphthalene acetic acid on in vitro propagation of tuberose (Polianthes tuberosa L.) from shoot tip explants. J Agri Sci Technol 6:48–59
Karakas FP, Sahin G, Turker A (2016) Enhancement of direct shoot regeneration and determination of bioactive secondary metabolites in leaves of Galega officinalis L. Turk J Biol 40:1311–1319
Karthikeyan A, Pandian STK, Ramesh M (2009) High frequency plant regeneration from embryogenic callus of a popular indica rice (Oryza sativa L.). Physiol Mol Biol Plants 15:371–375
Kaur P, Kothari SL (2004) In vitro culture of kodo millet: influence of 2,4-d and picloram in combination with kinetin on callus initiation and regeneration. Plant Cell Tissue Organ Cult 77:73–79
Kim SK, Sohn EY, Lee IJ (2009) Starch properties of native foxtail millet, Setaria italic Beauv. J Crop Sci Biotechnol 12:59–62
Kunitake H, Nakashima T, Mori K, Tanaka M, Mii M (1995) Plant regeneration from mesophyll protoplasts of lisianthus (Eustoma grandiflorum) by adding activated charcoal into protoplast culture medium. Plant Cell Tissue Organ Cult 43:59–65
Lambe P, Mutambel HSN, Deltour R, Dinant M (1999) Somatic embryogenesis in pearl millet (Pennisetum glaucum): strategies to reduce genotype limitation and to maintain longterm totipotency. Plant Cell Tissue Organ Cult 55:23–29
Lata C, Gupta S, Prasad M (2013) Foxtail millet: a model crop for genetic and genomic studies in bioenergy grasses. Crit Rev Biotechnol 33:328–343
Manchanda P, Gosal SS (2012) Effect of activated charcoal, carbon sources and gelling agents on direct somatic embryogenesis and regeneration in sugarcane via leaf roll segments. Sugar Tech 14:168–173
Manoharan M, Dahleen L (2002) Genetic transformation of the commercial barley (Hordeum vulgare L.) cultivar Conlon by article bombardment of callus. Plant Cell Rep 21:76–80
Martins M, Sarmento D, Oliveira MM (2004) Genetic stability of micropropagated almond plantlets, as assessed by RAPD and ISSR markers. Plant Cell Rep 23:492–496
Matsuda Y, Bushnell WR, Somers DA, Rines HW (1998) Rapid embryogenesis and plant regeneration of target tissue derived from juvenile shoots for transformation of oat by microprojectile bombardment. Plant Biotechnol 15:139–143
Moghaieb REA, El-Arabi NI, Momtaz OA, Youssef SS, Soliman MH (2010) Genetic transformation of mature embryos of bread (T. aestivum) and pasta (T. durum) wheat genotypes. GM Crops 1:87–93
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497
Nhut DT, Le BV, Fukai S, Tanaka M, Van KTT (2001) Effects of activated charcoal, explant size, explant position and sucrose concentration on plant and shoot regeneration of Lilium longiflorum via young stem culture. Plant Growth Regul 33:59–65
Osuna-Avila P, Nava-Cedillo A, Jofre-Garfias AE (1995) Plant regeneration from shoot apex explants of foxtail millet. Plant Cell Tissue Organ Cult 40:33–35
Ozawa K, Komamine A (1989) Establishment of a system of high-frequency embryogenesis from long-term cell suspension cultures of rice (Oryza sativa L.). Theor Appl Genet 77:205–211
Pan MJ, van Staden J (1998) The use of charcoal in in vitro culture—a review. Plant Growth Regul 26:155–163
Pan Y, Ma X, Liang H, Zhao Q, Zhu D, Yu J (2015) Spatial and temporal activity of the foxtail millet (Setaria italica) seed-specific promoter pF128. Planta 241:57–67
Panaud O (2006) Foxtail millet. In: Kole C (ed) Cereals and millet, vol 1. Springer, Berlin, pp 325–332
Park S, Sivanesan I (2015) Effect of plant growth regulators on axillary shoot multiplication from nodal explants of Ajuga multiflora Bunge. Propag Ornam Plants 15:42–44
Parmar SS, Sainger M, Chaudhary D, Jaiwal PK (2012) Plant regeneration from mature embryo of commercial Indian bread wheat (Triticum aestivum L.) cultivars. Physiol Mol Biol Plants 18:177–183
Rajendran HA, Muthusamy R, Stanislaus AC, Krishnaraj T, Kuppusamy S, Ignacimuthu S, Al-Dhabi NA (2016) Analysis of molecular variance and population structure in southern Indian finger millet genotypes using three different molecular markers. J Crop Sci Biotechnol 19:275–283
Ramakrishnan M, Ceasar SA, Duraipandiyan V, Ignacimuthu S (2014) Efficient plant regeneration from shoot apex explants of maize (Zea mays) and analysis of genetic fidelity of regenerated plants by ISSR markers. Plant Cell Tissue Organ Cult 119:183–196
Ramesh M, Murugiah V, Gupta AK (2009) Efficient in vitro plant regeneration via leaf base segments of indica rice (Oryza sativa L.). Indian J Exp Biol 47:68–74
Rao AM, Kishor PB, Reddy LA, Vaidyanath K (1988) Callus induction and high frequency plant regeneration in Italian millet (Setaria italica). Plant Cell Rep 7:557–559
Rao AM, Sree KP, Kishor PBK (1995) Enhanced plant regeneration in grain and sweet sorghum by asparagine, proline and cefotaxime. Plant Cell Rep 15:72–75
Rashid A (2003) Somatic embryogenesis from mesocotyl and leaf-base segments of Paspalum scrobiculatum L., a minor millet. Vitro Cell Dev Biol Plant 39:485–489
Reddy LA, Vaidyanath K (1990) Callus formation and regeneration in two induced mutants of foxtail millet (Setaria italica). J Genet Breed 44:133–138
Rency AS, Satish L, Pandian S, Rathinapriya P, Ramesh M (2017) In vitro propagation and genetic fidelity analysis of alginate-encapsulated Bacopa monnieri shoot tips using Gracilaria salicornia extracts. J Appl Phycol 29:481–494
Rout GR, Samantaray S, Das P (1998) The role of nickel on somatic embryogenesis in Setaria italica L. in vitro. Euphytica 101:319–324
Ružić DV, Vujović TI (2008) The effects of cytokinin types and their concentration on in vitro multiplication of sweet cherry cv. Lapins (Prunus avium L.). Hortic Sci 35:12–21
Salvi ND, George L, Eapen S (2001) Plant regeneration from leaf base callus of turmeric and random amplified polymorphic DNA analysis of regenerated plants. Plant Cell Tissue Organ Cult 66:113–119
Satish L, Ceasar SA, Shilpha J, Rency AS, Rathinapriya P, Ramesh M (2015) Direct plant regeneration from in vitro-derived shoot apical meristems of finger millet (Eleusine coracana (L.) Gaertn.). Vitro Cell Dev Biol Plant 51:192–200
Satish L, Rathinapriya P, Ceasar SA, Rency AS, Pandian S, Rameshkumar R, Subramanian A, Ramesh M (2016a) Effects of cefotaxime, amino acids and carbon source on somatic embryogenesis and plant regeneration in four Indian genotypes of foxtail millet (Setaria italica L.). Vitro Cell Dev Biol Plant 52:140–153
Satish L, Rathinapriya P, Rency AS, Ceasar SA, Pandian S, Rameshkumar R, Ramesh M (2016b) Somatic embryogenesis and regeneration using Gracilaria edulis and Padina boergesenii seaweed liquid extracts and genetic fidelity in finger millet (Eleusine coracana). J Appl Phycol 28:2083–2098
Satish L, Shilpha J, Pandian S, Rency AS, Rathinapriya P, Ceasar SA, Largia MJV, Kumar AA, Ramesh M (2016c) Analysis of genetic variation in sorghum (Sorghum bicolor (L.) Moench) genotypes with various agronomical traits using SPAR methods. Gene 576:581–585
Sato K, Mukainari Y, Naito K, Fukunaga K (2013) Construction of a foxtail millet linkage map and mapping of spikelet-tipped bristles 1 (stb1) by using transposon display markers and simple sequence repeat markers with genome sequence information. Mol Breed 31:675–684
Sivakumar P, Law YS, Ho CL, Harikrishna JA (2010) High frequency plant regeneration from mature seed of elite, recalcitrant Malaysian indica rice (Oryza sativa L.) CV. MR 219. Acta Biol Hung 61:313–321
Sivanesan I, Kyoung KE, Kyoung KM, Young KE, Park SW (2015) Somatic embryogenesis and plant regeneration from zygotic embryo explants of onion. Hortic Bras 33:441–447
Thomas TD (2008) The role of activated charcoal in plant tissue culture. Biotechnol Adv 26:618–631
Vasanth K, Lakshmiprabha A, Jayabalan N (2006) Amino acids enhancing plant regeneration from cotyledon and embryonal axis of peanut (Arachis hypogaea L.). Indian J Crop Sci 1:79–83
Wang MZ, Pan YL, Li C, Liu C, Zhao Q, Ao GM, Yu JJ (2011) Culturing of immature inflorescences and Agrobacterium-mediated transformation of foxtail millet (Setaria italica). Afr J Biotechnol 10:16466–16479
Wernicke W, Brettell RIS (1982) Morphogenesis from cultured leaf tissue of sorghum bicolor culture initiation. Protoplasma 111:19–27
Yoshida S, Forno DA, Cock JH, Gomez KA (1976) Laboratory manual for physiological studies of rice. International Rice Research Institute, Manila
Yu H, Wang W, Wang Y, Hou B (2012) High frequency wheat regeneration from leaf tissue explants of regenerated plantlets. Adv Biosci Biotechnol 3:46–50
Zhong H, Srinivasan C, Sticklen MB (1992) In vitro morphogenesis of corn (Zea mays L.). I. Differentiation of multiple shoot clumps and somatic embryos from shoot tips. Planta 187:483–489
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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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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DOI: https://doi.org/10.1007/s12298-018-0619-z