Capability of the plant-associated bacterium, Ensifer adhaerens strain OV14, to genetically transform its original host Brassica napus
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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.
KeywordsEnsifer adhaerens OV14 EMT Oilseed rape Regeneration Transformation
Morpholinoethane sulfonic acid
Murashige and Skoog salt mixture
Naphthaline acetic acid
Teagasc-tryptone yeast extract medium
This research was supported by Teagasc Walsh Fellowship Scheme which funded D. S. Rathore.
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.
- 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
- 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
- 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
- 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
- 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
- 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
- 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
- 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
- Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning, vol 2. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
- 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
- USDA (2015) United States Department of Agriculture, Foreign Agricultural Service report, Oilseeds: World Markets and Trade. http://apps.fas.usda.gov/psdonline/circulars/oilseeds.pdf
- 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
- Yin Z, Plader W, Malepszy S (2003) Transgene inheritance in plants. J Appl Genet 45:127–144Google Scholar