title: Wheat seed embryo excision enables the creation of axenic seedlings and Koch's postulates testing of putative bacterial endophytes.
abstract: Early establishment of endophytes can play a role in pathogen suppression and improve seedling development. One route for establishment of endophytes in seedlings is transmission of bacteria from the parent plant to the seedling via the seed. In wheat seeds, it is not clear whether this transmission route exists, and the identities and location of bacteria within wheat seeds are unknown. We identified bacteria in the wheat (Triticum aestivum) cv. Hereward seed environment using embryo excision to determine the location of the bacterial load. Axenic wheat seedlings obtained with this method were subsequently used to screen a putative endophyte bacterial isolate library for endophytic competency. This absence of bacteria recovered from seeds indicated low bacterial abundance and/or the presence of inhibitors. Diversity of readily culturable bacteria in seeds was low with 8 genera identified, dominated by Erwinia and Paenibacillus. We propose that anatomical restrictions in wheat limit embryo associated vertical transmission, and that bacterial load is carried in the seed coat, crease tissue and endosperm. This finding facilitates the creation of axenic wheat plants to test competency of putative endophytes and also provides a platform for endophyte competition, plant growth, and gene expression studies without an indigenous bacterial background.
authors.affiliation: Rothamsted Research, West Common, Harpenden, AL5 2JQ, UK.
authors.forename: Rebekah J
authors.initials: RJ
authors.lastname: Robinson
authors.affiliation: Rothamsted Research, West Common, Harpenden, AL5 2JQ, UK.
authors.forename: Bart A
authors.initials: BA
authors.lastname: Fraaije
authors.affiliation: Rothamsted Research, West Common, Harpenden, AL5 2JQ, UK.
authors.forename: Ian M
authors.initials: IM
authors.lastname: Clark
authors.affiliation: School of Biological Sciences, University of Reading, Whiteknights, RG6 6AH, UK.
authors.forename: Robert W
authors.initials: RW
authors.lastname: Jackson
authors.affiliation: Rothamsted Research, West Common, Harpenden, AL5 2JQ, UK.
authors.forename: Penny R
authors.initials: PR
authors.lastname: Hirsch
authors.affiliation: Rothamsted Research, West Common, Harpenden, AL5 2JQ, UK.
authors.forename: Tim H
authors.initials: TH
authors.lastname: Mauchline
mesh_terms: D001419:Bacteria / Q000302:isolation & purification / Q000378:metabolism*; D060026:Endophytes / Q000502:physiology*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D036226:Seedlings / Q000196:embryology* / Q000382:microbiology*; D012639:Seeds / Q000196:embryology* / Q000382:microbiology*; D014908:Triticum / Q000196:embryology* / Q000382:microbiology*
chemical_list: D012336:RNA, Ribosomal, 16S