Plant Cell, Tissue and Organ Culture (PCTOC)

, Volume 127, Issue 1, pp 85–94 | Cite as

Capability of the plant-associated bacterium, Ensifer adhaerens strain OV14, to genetically transform its original host Brassica napus

  • Dheeraj Singh Rathore
  • Fiona Doohan
  • Ewen MullinsEmail author
Original Article


Land plants exist in intimate associations with complex microbial communities across the phyllosphere, endosphere, and rhizosphere, with the latter inhabited by microbes that establish relationships with their host extending from parasitism to mutualism. For example, the rhizospheric Agrobacterium tumefaciens is pathogenic across a broad host range while its related rhizobia Sinorhizobium meliloti is an important symbiont of plants. Of interest, both species have a recorded capacity to genetically transform plant species with variable success. In this regard they have been recently joined by the rhizospheric non-pathogenic bacterium Ensifer adhaerens OV14, which has demonstrated an ability to genetically transform both dicots (Arabidopsis thaliana, Nicotiana tabaccum, and Solanum tuberosum) and monocot (Oryza sativa). The goal of this study was to investigate the potential of E. adhaerens strain OV14 to genetically transform Brassica napus, the host species from which it was isolated. By tailoring current A. tumefaciens-based protocols to suit the growth parameters of E. adhaerens strain OV14, here we report the successful transformation of the commercial B. napus cultivar Delight. The results indicated that co-cultivating 5 day old cotyledonary petiole explants with E. adhaerens strain OV14 (OD600nm = 0.8) for 5 days in the presence of 200 µM acetosyringone delivered transgenic plants of morphological equivalence to the original treated cv. Delight. A transformation frequency of 4.0 ± 0.2 % was attained based on stable integration patterns recorded for T1 individuals, which indicated transgene integrations of 1–3 copies/line. Segregation analysis based on the inheritance of the nptII transgene in the T2 generation showed Mendelian and non-Mendelian segregation patterns for the designated kanamycin resistance phenotype. To conclude, this practical study highlights the expanding host range of Ensifer-mediated transformation by confirming the ability of the symbiont Ensifer adhaerens OV14 to genetically engineer its original host.


Ensifer adhaerens OV14 EMT Oilseed rape Regeneration Transformation 









2,4-Dichlorophenoxyacetic acid


Gibberellic acid




Morpholinoethane sulfonic acid


Murashige and Skoog salt mixture


Naphthaline acetic acid


Teagasc-tryptone yeast extract medium


GUS positive



This research was supported by Teagasc Walsh Fellowship Scheme which funded D. S. Rathore.

Authors’ contribution

DSR performed all transformation experiments and molecular work, collected data, interpreted the results and drafted the manuscript. EM and FD supervised the oilseed rape transformation project and assisted in drafting the manuscript. All authors read and approved the final manuscript.

Compliance with ethical standards

Conflict of interest

E. Mullins and F. Doohan are authors of patent application PCT/EP2010/070681 which details the use of an isolated Ensifer adhaerens strain OV14 deposited under NCIMB Accession Number 41777 as a gene delivery system in the genetic transformation of plant material. Our manuscript has in no way been affected by this fact, nor has our participation in the work influenced in any manner the analysis of the generated datasets and/or the conclusions drawn.

Supplementary material

11240_2016_1032_MOESM1_ESM.docx (2 mb)
Supplementary material 1 (DOCX 2054 kb)


  1. Abbadi A, Leckband G (2011) Rapeseed breeding for oil content, quality, and sustainability. Eur J Lipid Sci Technol 113:1198–1206. doi: 10.1002/ejlt.201100063 CrossRefGoogle Scholar
  2. Altenbach S, Kuo C-C, Staraci L, Pearson K, Wainwright C, Georgescu A, Townsend J (1992) Accumulation of a Brazil nut albumin in seeds of transgenic canola results in enhanced levels of seed protein methionine. Plant Mol Biol 18:235–245. doi: 10.1007/BF00034952 CrossRefPubMedGoogle Scholar
  3. Banta L, Montenegro M (2008) Agrobacterium and plant biotechnology. In: Tzfira T, Citovsky V (eds) Agrobacterium: from biology to biotechnology. Springer, New York, pp 73–147. doi: 10.1007/978-0-387-72290-0_3
  4. Bhalla PL, Singh MB (2008) Agrobacterium-mediated transformation of Brassica napus and Brassica oleracea. Nat Protocols 3:181–189CrossRefPubMedGoogle Scholar
  5. Boszoradova E, Matusikova I, Jopcik M, Libantova J, Poloniova Z, Berenyi M, Moravcikova J (2011) Agrobacterium-mediated genetic transformation of economically important oilseed rape cultivars. Plant Cell Tissue Organ Culture 107:317–323. doi: 10.1007/s11240-011-9982-y CrossRefGoogle Scholar
  6. Botterman J, D’Halluin K, de Block M, de Greef W, Leemans J (1991) Engineering glufosinate resistance and evaluation under field conditions. In: Caseley J, Cussans G, Atkin R (eds) Herbicide resistance in weeds and crops. Butterworth Heinemann, Oxford, pp 355–365CrossRefGoogle Scholar
  7. Boulter M, Croy E, Simpson P, Shields R, Croy R, Shirsat A (1990) Transformation of Brassica napus L. (oilseed rape) using Agrobacterium tumefaciens and Agrobacterium rhizogenes—a comparison. Plant Sci 70:91–99CrossRefGoogle Scholar
  8. Broothaerts W et al (2005) Gene transfer to plants by diverse species of bacteria. Nature 433:629–633CrossRefPubMedGoogle Scholar
  9. Cannell M, Doherty A, Lazzeri P, Barcelo P (1999) A population of wheat and tritordeum transformants showing a high degree of marker gene stability and heritability. Theor Appl Genet 99:772–784CrossRefGoogle Scholar
  10. Cardoza V, Stewart CN Jr (2004) Brassica biotechnology: progress in cellular and molecular biology. In Vitro Cell Dev Biol Plant 40:542–551. doi: 10.1079/IVP2004568 CrossRefGoogle Scholar
  11. Choffnes D, Philip R, Vodkin L (2001) A transgenic locus in soybean exhibits a high level of recombination. In Vitro Cell Dev Biol Plant 37:756–762CrossRefGoogle Scholar
  12. Damgaard O, Jensen L, Rasmussen O (1997) Agrobacterium tumefaciens-mediated transformation of Brassica napus winter cultivars. Transgenic Res 6:279–288. doi: 10.1023/A:1018458628218 CrossRefGoogle Scholar
  13. De Cleene M (1985) The susceptibility of monocotyledons to Agrobacterium tumefaciens. J Phytopathol 113:81–89CrossRefGoogle Scholar
  14. De Cleene M, De Jozef L (1976) The host range of crown gall. Bot Rev 42:389–466CrossRefGoogle Scholar
  15. Doyle J (1991) DNA protocols for plants. In: Hewitt G, Johnston AB, Young JP (eds) Molecular techniques in taxonomy, vol 57. NATO ASI series. Springer, Berlin, pp 283–293. doi: 10.1007/978-3-642-83962-7_18
  16. Friedt W, Snowdon R (2010) Oilseed rape. In: Vollmann J, Rajcan I (eds) Oil crops, vol 4. Handbook of plant breeding. Springer, New York, pp 91–126. doi: 10.1007/978-0-387-77594-4_4
  17. Fry J, Barnason A, Horsch RB (1987) Transformation of Brassica napus with Agrobacterium tumefaciens based vectors. Plant Cell Rep 6:321–325CrossRefPubMedGoogle Scholar
  18. Fukuoka H, Ogawa T, Matsuoka M, Ohkawa Y, Yano H (1998) Direct gene delivery into isolated microspores of rapeseed (Brassica napus L.) and the production of fertile transgenic plants. Plant Cell Rep 17:323–328CrossRefGoogle Scholar
  19. Guerche P, Charbonnier M, Jouanin L, Tourneur C, Paszkowski J, Pelletier G (1987) Direct gene transfer by electroporation in Brassica napus. Plant Sci 52:111–116CrossRefGoogle Scholar
  20. Harloff HJ et al (2012) A mutation screening platform for rapeseed (Brassica napus L.) and the detection of sinapine biosynthesis mutants. Theor Appl Genet 124:957–969. doi: 10.1007/s00122-011-1760-z CrossRefPubMedGoogle Scholar
  21. Hasan M, Seyis F, Badani AG, Pons-Kühnemann J, Friedt W, Lühs W, Snowdon RJ (2006) Analysis of genetic diversity in the Brassica napus L. gene pool using SSR markers. Genet Resour Crop Evol 53:793–802. doi: 10.1007/s10722-004-5541-2 CrossRefGoogle Scholar
  22. Hong HP, Gerster JL, Datla RSS, Albani D, Scoles G, Keller W, Robert LS (1997) The promoter of a Brassica napus polygalacturonase gene directs pollen expression of β-glucuronidase in transgenic Brassica plants. Plant Cell Rep 16:373–378. doi: 10.1007/BF01146776 Google Scholar
  23. Hooykaas P, Schilperoort R (1992) Agrobacterium and plant genetic engineering. In: Schilperoort R, Dure L (eds) 10 years plant molecular biology. Springer, Amsterdam, pp 15–38. doi: 10.1007/978-94-011-2656-4_2
  24. Hooykaas P, Klapwijk P, Nuti M, Schilperoort R, Rorsch A (1977) Transfer of the Agrobacterium tumefaciens Ti plasmid to avirulent Agrobacteria and to Rhizobium ex planta. J Gen Microbiol 98(477–474):484Google Scholar
  25. Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907PubMedPubMedCentralGoogle Scholar
  26. Jefferson RA et al (2006) Freedom to co-operate: transbacter as a biological open source (BIOS) tool for gene transfer. 8th international congress of plant molecular biology abstracts. Plant Mol Biol Rep 24:141–160CrossRefGoogle Scholar
  27. Jones KM, Kobayashi H, Davies BW, Taga ME, Walker GC (2007) How rhizobial symbionts invade plants: the Sinorhizobium–Medicago model. Nat Rev Microbiol 5:619–633. doi: 10.1038/nrmicro1705 CrossRefPubMedPubMedCentralGoogle Scholar
  28. Khan MR, Rashid H, Ansar M, Chaudry Z (2003) High frequency shoot regeneration and Agrobacterium-mediated DNA transfer in Canola (Brassica napus). Plant Cell Tissue Organ Cult 75:223–231. doi: 10.1023/A:1025869809904 CrossRefGoogle Scholar
  29. Mashayekhi M, Shakib AM, Ahmad-Raji M, Ghasemi Bezdi K (2008) Gene transformation potential of commercial canola (Brassica napus L.) cultivars using cotyledon and hypocotyl explants. Afr J Biotechnol 7:4459–4463Google Scholar
  30. Mei J, Fu Y, Qian L, Xu X, Li J, Qian W (2011) Effectively widening the gene pool of oilseed rape (Brassica napus L.) by using Chinese B. rapa in a ‘virtual allopolyploid’ approach. Plant Breed 130:333–337. doi: 10.1111/j.1439-0523.2011.01850.x CrossRefGoogle Scholar
  31. Moloney MM, Walker JM, Sharma KK (1989) High efficiency transformation of Brassica napus using Agrobacterium vectors. Plant Cell Rep 8:238–242. doi: 10.1007/bf00778542 CrossRefPubMedGoogle Scholar
  32. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  33. Nagaharu U (1935) Genome analysis in Brassica with special reference to the experimental formation of B. napus and peculiar mode of fertilization. Jpn J Bot 7:389–452Google Scholar
  34. Neuhaus G, Spangenberg G, Scheid OM, Schweiger H-G (1987) Transgenic rapeseed plants obtained by the microinjection of DNA into microspore-derived embryoids. Theor Appl Genet 75:30–36CrossRefGoogle Scholar
  35. Ondřej M, Kocábek T, Rakouský S, Wiesnerová D (1999) Segregation of T-DNA inserts in the offspring of Arabidopsis thaliana after Agrobacterium transformation. Biol Plant 42:185–195CrossRefGoogle Scholar
  36. Ormeño-Orrillo E et al (2015) Taxonomy of Rhizobia and Agrobacteria from the Rhizobiaceae family in light of genomics. Syst Appl Microbiol. doi: 10.1016/j.syapm.2014.12.002 Google Scholar
  37. Pawlowski WP, Torbert KA, Rines HW, Somers DA (1998) Irregular patterns of transgene silencing in allohexaploid oat. Plant Mol Biol 38:597–607CrossRefPubMedGoogle Scholar
  38. Ponstein AS, Bade JB, Verwoerd TC, Molendijk L, Storms J, Beudeker RF, Pen J (2002) Stable expression of Phytase (phyA) in canola (Brassica napus) seeds: towards a commercial product. Mol Breed 10:31–44CrossRefGoogle Scholar
  39. Poulsen GB (1996) Genetic transformation of Brassica. Plant Breed 115:209–225. doi: 10.1111/j.1439-0523.1996.tb00907.x CrossRefGoogle Scholar
  40. Radke SE, Andrews BM, Moloney MM, Crouch ML, Kridl JC, Knauf VC (1988) Transformation of Brassica napus L. using Agrobacterium tumefaciens: developmentally regulated expression of a reintroduced napin gene. Theor Appl Genet 75:685–694. doi: 10.1007/BF00265588 CrossRefGoogle Scholar
  41. Rathore DS, Lopez-Vernaza MA, Doohan F, Connell DO, Lloyd A, Mullins E (2015) Profiling antibiotic resistance and electrotransformation potential of Ensifer adhaerens OV14; a non-Agrobacterium species capable of efficient rates of plant transformation. FEMS Microbiol Lett 362. doi: 10.1093/femsle/fnv126
  42. Rogel MA, Hernandez-Lucas I, Kuykendall LD, Balkwill DL, Martinez-Romero E (2001) Nitrogen-fixing nodules with Ensifer adhaerens harboring Rhizobium tropici symbiotic plasmids. Appl Environ Microbiol 67:3264–3268. doi: 10.1128/AEM.67.7.3264-3268.2001 CrossRefPubMedPubMedCentralGoogle Scholar
  43. Rudder S, Doohan F, Creevey CJ, Wendt T, Mullins E (2014) Genome sequence of Ensifer adhaerens OV14 provides insights into its ability as a novel vector for the genetic transformation of plant genomes. BMC Genom 15:268–285. doi: 10.1186/1471-2164-15-268 CrossRefGoogle Scholar
  44. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning, vol 2. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
  45. Schierholt A, Rücker B, Becker HC (2001) Inheritance of high oleic acid mutations in winter oilseed rape (Brassica napus L.). Crop Sci. doi: 10.2135/cropsci2001.4151444x Google Scholar
  46. Snowdon R, Lühs W, Friedt W (2007) Oilseed rape. In: Kole C (ed) Oilseeds. Genome mapping and molecular breeding in plants, vol 2. Springer, Berlin, pp 55–114. doi: 10.1007/978-3-540-34388-2_2
  47. Tan S, Evans RR, Dahmer ML, Singh BK, Shaner DL (2005) Imidazolinone-tolerant crops: history, current status and future. Pest Manag Sci 61:246–257. doi: 10.1002/ps.993 CrossRefPubMedGoogle Scholar
  48. USDA (2015) United States Department of Agriculture, Foreign Agricultural Service report, Oilseeds: World Markets and Trade.
  49. Vigeolas H, Waldeck P, Zank T, Geigenberger P (2007) Increasing seed oil content in oil-seed rape (Brassica napus L.) by over-expression of a yeast glycerol-3-phosphate dehydrogenase under the control of a seed-specific promoter. Plant Biotechnol J 5:431–441. doi: 10.1111/j.1467-7652.2007.00252.x CrossRefPubMedGoogle Scholar
  50. Wendt T, Doohan F, Winckelmann D, Mullins E (2011) Gene transfer into Solanum tuberosum via Rhizobium spp. Transgenic Res 20:377–386. doi: 10.1007/s11248-010-9423-4 CrossRefPubMedGoogle Scholar
  51. Wendt T, Doohan F, Mullins E (2012) Production of Phytophthora infestans-resistant potato (Solanum tuberosum) utilising Ensifer adhaerens OV14. Transgenic Res 21:567–578. doi: 10.1007/s11248-011-9553-3 CrossRefPubMedGoogle Scholar
  52. Yin Z, Plader W, Malepszy S (2003) Transgene inheritance in plants. J Appl Genet 45:127–144Google Scholar
  53. Zhang Y, Singh MB, Swoboda I, Bhalla PL (2005) Agrobacterium-mediated transformation and generation of male sterile lines of Australian canola. Aust J Agric Res 56:353–361. doi: 10.1071/AR04175 CrossRefGoogle Scholar
  54. Zhou G et al (2013) Biodegradation of the neonicotinoid insecticide thiamethoxam by the nitrogen-fixing and plant-growth-promoting rhizobacterium Ensifer adhaerens strain TMX-23. Appl Microbiol Biotechnol 97:4065–4074. doi: 10.1007/s00253-012-4638-3 CrossRefPubMedGoogle Scholar
  55. Zuniga-Soto E, Mullins E, Dedicova B (2015) Ensifer-mediated transformation: an efficient non-Agrobacterium protocol for the genetic modification of rice. SpringerPlus 4:1–10CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2016

Authors and Affiliations

  1. 1.Department of Crop ScienceTeagasc Crops Research CentreCarlowIreland
  2. 2.UCD School of Biology and Environmental Sciences and UCD Earth InstituteUniversity College DublinDublinIreland

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