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Stable Transformation of Medicago truncatula cv. Jemalong for Gene Analysis Using Agrobacterium tumefaciens

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Legume Genomics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1069))

Abstract

Medicago truncatula is a model legume that has all the genomic resources to be suitable as a model for functional genomics. Transformation to produce transgenic plants is part of this toolkit, enabling a spectrum of approaches to study gene function: including knockdown, overexpression, reporter genes for gene expression, and proteins tagged with fluorescent proteins such as GFP. A special genetic line is necessary for transformation and Jemalong 2HA derived from cv. Jemalong is used in the methods described. Leaf explants can be used for the transformation of the embryonic stem cells to produce the transgenic somatic embryos for regeneration. An auxin and a cytokinin are the key hormone requirements for regeneration by somatic embryogenesis but other hormones such as abscisic acid can be used to augment the system. As the explants used in this system are from leaves, rather than immature embryos or meristematic tissues often used in other species, it is a quite straightforward system. Agrobacterium tumefaciens containing a binary vector suitable for the particular objectives is used to deliver the transgene of interest.

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References

  1. Barker DG, Bianchi S, Blondon F, Datteé Y, Duc G, Essad S et al (1990) Medicago truncatula, a model plant for studying the molecular genetics of the Rhizobium-legume symbiosis. Plant Mol Biol Rep 8:40–49

    Article  CAS  Google Scholar 

  2. Cook DR (1999) Medicago truncatula—a model in the making! Curr Opin Plant Biol 2:301–304

    Article  PubMed  CAS  Google Scholar 

  3. Rose RJ (2008) Medicago truncatula as a model for understanding plant interactions with other organisms, plant development and stress biology: past, present and future. Funct Plant Biol 35:253–264

    Article  Google Scholar 

  4. Young ND, Debellé F, Oldroyd GED, Geurts R, Cannon SB, Udvardi MK et al (2011) The Medicago genome provides insight into the evolution of rhizobial symbioses. Nature 480:520–524

    Article  PubMed  CAS  Google Scholar 

  5. Tesfaye M, Silverstein KAT, Bucciarelli B, Samac DA, Vance CP (2006) The Affymetrix Medicago GeneChip® array is applicable for transcript analysis of alfalfa (Medicago sativa). Funct Plant Biol 33:783–788

    Article  CAS  Google Scholar 

  6. Benedito VA, Torres-Jerez I, Murray JD, Andriankaja A, Allen S, Kakar K et al (2008) A gene expression atlas of the model legume Medicago truncatula. Plant J 55:504–513

    Article  PubMed  CAS  Google Scholar 

  7. Cheng X, Wen J, Tadege M, Ratet P, Mysore KS (2011) Reverse genetics in Medicago truncatula using Tnt1 insertion mutants. In Plant reverse genetics: methods and protocols (ed. A. Pereira). Methods Mol Biol 678:179–190

    Article  PubMed  CAS  Google Scholar 

  8. Watson BS, Asirvatham VS, Wang L, Sumner LW (2003) Mapping the proteome of barrel medic (Medicago truncatula). Plant Physiol 131:1104–1123

    Article  PubMed  Google Scholar 

  9. Gallardo K, Le Signor C, Vandekerckhove J, Thompson RD, Burstin J (2003) Proteomics of Medicago truncatula seed development establishes the time frame of diverse metabolic processes related to reserve accumulation. Plant Physiol 133:664–682

    Article  PubMed  CAS  Google Scholar 

  10. Imin N, de Jong F, Mathesius U, van Noorden G, Saeed NA, Wang X-D, Rose RJ, Rolfe BG (2004) Proteome reference maps of Medicago truncatula embryogenic cell cultures generated from single protoplasts. Proteomics 4: 1883–1896

    Article  PubMed  CAS  Google Scholar 

  11. Imin N, Nizamidin M, Daniher D, Nolan KE, Rose RJ, Rolfe BG (2005) Proteomic analysis of somatic embryogenesis in Medicago truncatula. Explant cultures grown under 6-benzylaminopurine and 1-naphthaleneacetic acid treatments. Plant Physiol 137:1250–1260

    Article  PubMed  CAS  Google Scholar 

  12. Szittya G, Moxon S, Santos DM, Jing R, Fevereiro MPS, Moulton V, Dalmay T (2008) High- throughput sequencing of Medicago truncatula short RNAs identifies eight new miRNA families. BMC Genomics 9:593

    Article  PubMed  Google Scholar 

  13. Lelandais-Briére C, Naya L, Sallet E, Calenge F, Frugier F, Hartmann C, Gouzy J, Crespi M (2009) Genome-wide Medicago truncatula small RNA analysis revealed novel microRNAs and isoforms differentially regulated in roots and nodules. Plant Cell 21:2780–2796

    Article  PubMed  Google Scholar 

  14. Chen L, Wang T, Zhao M, Zhang W (2012) Ethylene-responsive miRNAs in roots of Medicago truncatula identified by high-throughput sequencing at whole genome level. Plant Sci 184:14–19

    Article  PubMed  CAS  Google Scholar 

  15. Mantiri FR, Kurdyukov S, Lohar DP, Sharapova N, Saeed NA, VandenBosch KA, Rose RJ (2008) The transcription factor MtSERF1 of the ERF subfamily identified by transcriptional profiling is required for somatic embryogenesis induced by auxin plus cytokinin in Medicago truncatula. Plant Physiol 146:1622–1636

    Article  PubMed  CAS  Google Scholar 

  16. Jayasena KW, Hajimorad MR, Law EG, Rehman A-U, Nolan KE, Zanker T, Rose RJ, Randles JW (2001) Resistance to Alfalfa mosaic virus in transgenic barrel medic lines containing the virus coat protein gene. Aust J Agric Res 52:67–72

    Article  Google Scholar 

  17. Nolan KE, Kurdyukov S, Rose RJ (2009) Expression of the SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE 1 (SERK1) gene is associated with developmental change in the life cycle of the model legume Medicago truncatula. J Exp Bot 60:1759–1771

    Article  PubMed  CAS  Google Scholar 

  18. Nolan KE, Rose RJ, Gorst JR (1989) Regeneration of Medicago truncatula from tissue culture: increased somatic embryogenesis using explants from regenerated plants. Plant Cell Rep 8:278–281

    Article  Google Scholar 

  19. Rose RJ, Nolan KE, Bicego L (1999) The development of the highly regenerable seed line Jemalong 2HA for transformation of Medicago truncatula—implications for regenerability via somatic embryogenesis. J Plant Physiol 155:788–791

    Article  CAS  Google Scholar 

  20. Hoffmann B, Trinh TH, Leung J, Kondorosi A, Kondorosi E (1997) A new Medicago truncatula line with superior in vitro regeneration, transformation, and symbiotic properties isolated through cell culture selection. Mol Plant-Microbe Interact 10:307–315

    Article  CAS  Google Scholar 

  21. Thomas MR, Rose RJ, Nolan KE (1992) Genetic transformation of Medicago truncatula using Agrobacterium with genetically modified Ri and disarmed Ti plasmids. Plant Cell Rep 11:113–117

    Article  CAS  Google Scholar 

  22. Wang JH, Rose RJ, Donaldson BI (1996) Agrobacterium-mediated transformation and expression of foreign genes in Medicago truncatula. Aust J Plant Physiol 23:265–270

    Article  Google Scholar 

  23. Chabaud M, Larsonneau C, Marmouget C, Huguet T (1996) Transformation of barrel medic (Medicago truncatula Gaertn.) by Agrobacterium tumefaciens and regeneration via somatic embryogenesis of transgenic plants with the MtENOD12 nodulin promoter fused to the gus reporter gene. Plant Cell Rep 15: 305–310

    Article  CAS  Google Scholar 

  24. Thomas MR, Johnson LB, White FF (1990) Selection of interspecific somatic hybrids of Medicago by using Agrobacterium transformed tissues. Plant Sci 69:189–198

    Article  Google Scholar 

  25. Nolan KE, Rose RJ (1998) Plant regeneration from cultured Medicago truncatula with particular reference to abscisic acid and light treatments. Aust J Bot 46:151–160

    Article  CAS  Google Scholar 

  26. Rose RJ, Nolan KE (1995) Regeneration of Medicago truncatula from protoplasts isolated from kanamycin-sensitive and kanamycin-resistant plants. Plant Cell Rep 14:349–353

    Article  CAS  Google Scholar 

  27. Chabaud M, de Carvalho-Niebel F, Barker DG (2003) Efficient transformation of Medicago truncatula cv. Jemalong using the hypervirulent Agrobacterium tumefaciens strain AGL1. Plant Cell Rep 22:46–51

    Article  PubMed  CAS  Google Scholar 

  28. Lazo GR, Stein PA, Ludwig RA (1991) A DNA transformation-competent Arabidopsis genomic library in Agrobacterium. Nat Biotechnol 9:963–967

    Article  CAS  Google Scholar 

  29. Hellens R, Mullineaux P, Klee H (2000) A guide to Agrobacterium binary Ti vectors. Trends Plant Sci 5:446–452

    Article  PubMed  CAS  Google Scholar 

  30. Curtis MD, Grossniklaus U (2003) A gateway cloning vector set for high-throughput functional analysis of genes in planta. Plant Physiol 133:462–469

    Article  PubMed  CAS  Google Scholar 

  31. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497

    Article  CAS  Google Scholar 

  32. Dalton CC, Iqbal K, Turner DA (1983) Iron phosphate precipitation in Murasgige and Skoog media. Physiol Plant 57:472–476

    Article  CAS  Google Scholar 

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Song, Y., Nolan, K.E., Rose, R.J. (2013). Stable Transformation of Medicago truncatula cv. Jemalong for Gene Analysis Using Agrobacterium tumefaciens . In: Rose, R. (eds) Legume Genomics. Methods in Molecular Biology, vol 1069. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-613-9_14

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  • DOI: https://doi.org/10.1007/978-1-62703-613-9_14

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-612-2

  • Online ISBN: 978-1-62703-613-9

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