Submitted to: Book Chapter
Publication Type: Book / Chapter
Publication Acceptance Date: 3/4/2010
Publication Date: N/A
Citation: N/A Interpretive Summary: These protocols describe methods for examining seed borne microbes associated with woody plants. In vitro propagation is used to eliminate interference from external microbes. Cryosectioning and light microscopy are used to detect microbes associated with micropropagated plant tissues. Amplification and sequencing of ribosomal DNA fragments is used to identify uncultured microbes associated with plant tissues. These methods are useful for examining seed borne microbial communities that may influence plant adaptation and phenotypes.
Technical Abstract: Micropropagated systems provide controlled habitats in which complex plant-endophyte interactions can be studied with minimal interference from superficial microbes. The series of protocols described herein can be used to examine seed borne endophyte consortia associated with woody plant species. Methods are described for establishing in vitro lines of the woody shrub, Atriplex canescens, detecting microbial endophytes in tissue cryosections of micropropagated plants, and amplifying, cloning, and sequencing internal transcribed spacer regions of endophyte ribosomal DNA genes.