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

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Abstract

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.

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Abbreviations

BAP:

6-Benzylaminopurine

Cf:

Cefotaxime

cv:

Cultivar

2,4-D:

2,4-Dichlorophenoxyacetic acid

GA3 :

Gibberellic acid

GUS:

β-Glucuronidase

MES:

Morpholinoethane sulfonic acid

MS:

Murashige and Skoog salt mixture

NAA:

Naphthaline acetic acid

TTY:

Teagasc-tryptone yeast extract medium

GUS+:

GUS positive

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Acknowledgments

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.

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Authors and Affiliations

  1. Department of Crop Science, Teagasc Crops Research Centre, Oak Park, Carlow, Ireland

    Dheeraj Singh Rathore & Ewen Mullins

  2. UCD School of Biology and Environmental Sciences and UCD Earth Institute, University College Dublin, Belfield, Dublin, 4, Ireland

    Dheeraj Singh Rathore & Fiona Doohan

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  1. Dheeraj Singh Rathore
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  2. Fiona Doohan
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Correspondence to Ewen Mullins.

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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.

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Rathore, D.S., Doohan, F. & Mullins, E. Capability of the plant-associated bacterium, Ensifer adhaerens strain OV14, to genetically transform its original host Brassica napus . Plant Cell Tiss Organ Cult 127, 85–94 (2016). https://doi.org/10.1007/s11240-016-1032-3

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