An efficient Agrobacterium-mediated genetic transformation method for foxtail millet (Setaria italica L.)


Key message

A simple and robust Agrobacterium-mediated gene expression system in the C4 panicoid model crop, foxtail millet has been developed with up to 27 % transformation efficiency.


Foxtail millet (Setaria italica L.) is a model crop to study C4 photosynthesis, abiotic stress tolerance, and bioenergy traits. Advances in molecular genetics and genomics had identified several potential genes in this crop that would serve as candidates for imparting climate-resilient traits in related millets, cereals, and biofuel crops. However, the lack of an efficient genetic transformation system has been impeding the functional characterization of these genes in foxtail millet per se. Given this, an easy and efficient regeneration and transformation protocol was optimized using mature seeds as a choicest explant. The suitability of secondary embryogenic calli over primary calli is underlined due to their high competence. The use of perfect combinations of plant growth regulators together with the ionic strength of organic and inorganics salts was found to influence regeneration and genetic transformation. We studied and optimized various crucial factors that affect the genetic transformation of foxtail millet calli using Agrobacterium tumefaciens-mediated approach. Secondary embryogenic calli and LBA44404 strain were found to be the best targets for transformation. The use of high sucrose and glucose, together with freshly prepared tobacco leaves extract, Silwet L-77 and acetosyringone, improved the efficiency of the genetic transformation of foxtail millet. Moreover, the use of an in vitro regeneration system with 84% callusing efficiency and 70–74% regeneration frequency led to a high recovery of transformants. Altogether, the present study reports a highly efficient (~ 27%) transformation system in foxtail millet that will expedite forward and reverse genetic studies in this important crop.

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  1. Anju C, Rabindran R, Velazhahan R, Ravikesavan R (2016) Callusing and regeneration in Finger Millet (Eleusine coracana (L.) Gaertn.). Res J Agric Sci 7:324–329

  2. Bennetzen JL, Schmutz J, Wang H, Percifield R, Hawkins J, Pontaroli AC et al (2012) Reference genome sequence of the model plant Setaria. Nat Biotechnol 30:555–561

  3. 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

  4. Ceasar SA, Baker A, Ignacimuthu S (2017) Functional characterization of the PHT1 family transporters of foxtail millet with development of a novel Agrobacterium-mediated transformation procedure. Sci Rep 7:14064

  5. Doust AN, Kellogg EA, Devos KM, Bennetzen JL (2009) Foxtail millet: a sequence-driven grass model system. Plant Physiol 149:2003–2005

  6. Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh tissue. Focus 12:13–15

  7. Enríquez-Obregón G (1999) Agrobacterium-mediated Japonica rice transformation: a procedure assisted by an antinecrotic treatment. Plant Cell Tissue Organ Cult 59:159–168

  8. Fursova O, Pogorelko G, Zabotina OA (2012) An efficient method for transient gene expression in monocots applied to modify the Brachypodium distachyon cell wall. Ann Bot 110:47–56

  9. Gamborg OL (1970) The effects of amino acids and ammonium on the growth of plant cells in suspension culture. Plant Physiol 45:372–375

  10. Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907

  11. Jeoung JM, Krishnaveni S, Muthukrishnan S, Trick HN, Liang GH (2002) Optimization of sorghum transformation parameters using genes for green fluorescent protein and beta-glucuronidase as visual markers. Hereditas 137:20–28

  12. Khanna HK, Daggard GE (2003) Agrobacterium tumefaciens-mediated transformation of wheat using a superbinary vector and a polyamine-supplemented regeneration medium. Plant Cell Rep 21:429–436

  13. Li P, Brutnell TP (2011) Setaria viridis and Setaria italica, model genetic systems for the Panicoid grasses. J Exp Bot 62:3031–3037

  14. Li C, Yue J, Wu X, Xu C, Yu J (2014) An ABA-responsive DRE-binding protein gene from Setaria italica, SiARDP, the target gene of SiAREB, plays a critical role under drought stress. J Exp Bot 65:5415–5427

  15. Li H, Li K, Guo Y et al (2018) A transient transformation system for gene characterization in upland cotton. Plant Methods 14:50

  16. Liu Y, Yu J, Zhao Q, Zhu D, Ao G (2005) Genetic transformation of millet (Setaria italica) by Agrobacterium-mediated. Chin J Agric Biotechnol 13:32–37

  17. Liu Y, Feng X, Xu Y, Yu J, Ao G, Peng Z, Zhao Q (2009) Overexpression of millet ZIP-like gene (SiPf40) affects lateral bud outgrowth in tobacco and millet. Plant Physiol Biochem 47:1051–1060

  18. Liu N, Xie K, Jia Q, Zhao J, Chen T, Li H, Wei X, Diao X, Hong Y, Liu Y (2016) Foxtail mosaic virus-induced gene silencing in Monocot plants. Plant Physiol 171:1801–1807

  19. Matheka J, Tripathi JN, Merga I, Gebre E, Tripathi L (2019) A simple and rapid protocol for the genetic transformation of Ensete ventricosum. Plant Methods 15:130

  20. Mohanty A, Sarma NP, Tyagi AK (1999) Agrobacterium- mediated high frequency transformation of an elite indica rice variety Pusa Basmati 1 and transmission of the transgenes to R2 progeny. Plant Sci 147:127–137

  21. Murashighe T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–479

  22. Muthamilarasan M, Prasad M (2015) Advances in Setaria genomics for genetic improvement of cereals and bioenergy grasses. Theor Appl Genet 128:1–14

  23. Păcurar DI, Thordal-Christensen H, Nielsen KK, Lenk I (2008) A high-throughput Agrobacterium mediated transformation system for the grass model species Brachypodium distachyon L. Transgenic Res 17:965–975

  24. Pius J, George L, Eapen S, Rao PS (1994) Influence of genotype and phytohormones on somatic embryogenesis and plant regeneration in finger millet. Proc Indian Natl Sci Acad 60:53–56

  25. Qin FF, Zhao Q, Ao GM et al (2008) Co-suppression of Si401, a maize pollen specific Zm401 homologous gene, results in aberrant anther development in foxtail millet. Euphytica 163:103–111

  26. Rao AM, Kavi Kishor PB, Ananda Reddy L, Vaidvanath K (1988) Callus induction and high frequency plant regeneration in Italian millet (Setaria italica). Plant Cell Rep 7:557–559

  27. Reddy LA, Vaidyanath K (1990) Callus formation and regeneration in two induced mutants of foxtail millet (Setaria italica). J Genet Breed 44:133–138

  28. Saha P, Majumder P, Dutta I, Ray T, Roy SC, Das S (2006) Transgenic rice expressing Allium sativum leaf lectin with enhances resistance against sap-sucking insect pests. Planta 223:1329–1343

  29. Satish L, Rathinapriya P, Ceasar SA, Rency AS, Pandian S, Rameshkumar R et al (2015) 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(2):140–153

  30. Sood P, Prasad M (2017) Genetic transformation of setaria: a new perspective. In: Prasad M (ed) The foxtail millet genome, compendium of plant genomes. Springer, Berlin, pp 105–121

  31. Sood P, Singh RK, Manoj P (2019) Millets genetic engineering: the progress made and prospects for the future. Plant Cell Tissue Organ Cult 137:421–439

  32. Visarada KBRS, Sailaja M, Sarma NP (2002) Effect of callus induction media on morphology of embryogenic calli in rice genotypes. Biol Plant 45:495–502

  33. Wang M, Pan Y, 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

  34. Wang M, Li P, Li C, Pan Y, Jiang X, Zhu D, Zhao Q, Yu J (2014) SiLEA14, a novel atypical LEA protein, confers abiotic stress resistance in foxtail millet. BMC Plant Biol 14:290

  35. Zhang G, Liu X, Quan Z, Cheng S, Xu X, Pan S et al (2012) Genome sequence of foxtail millet (Setaria italica) provides insights into grass evolution and biofuel potential. Nat Biotechnol 30:549–554

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Authors research in the area of foxtail millet genomics is supported by Science and Engineering Research Board (SERB), Department of Science and Technology (DST), Govt. of India [Grant no. EMR/2015/000464]. PS acknowledges the Young Scientist Award from DST-SERB, Govt. of India [File No. YSS/2014/000870/LS]. RKS acknowledges the research fellowship received from the Council of Scientific and Industrial Research, Govt. of India. Dr. Muthamilarasan, DST INSPIRE Faculty, ICAR-NIPB, New Delhi is acknowledged for his critical inputs during this study. The authors are also thankful to DBT-eLibrary Consortium (DeLCON) for providing access to e-resources.

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MP conceived and designed research. PS conducted experiments and analysed data. PS and RKS wrote the manuscript. All authors read and approved the manuscript.

Correspondence to Manoj Prasad.

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Communicated by Amit Dhingra.

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Sood, P., Singh, R.K. & Prasad, M. An efficient Agrobacterium-mediated genetic transformation method for foxtail millet (Setaria italica L.). Plant Cell Rep (2020) doi:10.1007/s00299-019-02507-w

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  • Setaria italica
  • Mature seed-derived calli
  • Transformation
  • Regeneration
  • Agrobacterium tumefaciens