High-Efficiency Stable Transformation of the Model Fern Species Ceratopteris richardii via Microparticle Bombardment    

Abstract

Ferns represent the most closely related extant lineage to seed plants. The aquatic fern Ceratopteris richardii has been subject to research for a considerable period of time, but analyses of the genetic programs underpinning developmental processes have been hampered by a large genome size, a lack of available mutants, and an inability to create stable transgenic lines. In this paper, we report a protocol for efficient stable genetic transformation of C. richardii and a closely related species Ceratopteris thalictroides using microparticle bombardment. Indeterminate callus was generated and maintained from the sporophytes of both species using cytokinin treatment. In proof-of-principle experiments, a 35S::b-glucuronidase (GUS) expression cassette was introduced into callus cells via tungsten microparticles, and stable transformants were selected via a linked hygromycin B resistance marker. The presence of the transgene in regenerated plants and in subsequent generations was validated using DNA-blot analysis, reverse transcription-polymerase chain reaction, and GUS staining. GUS staining patterns in most vegetative tissues corresponded with constitutive gene expression. The protocol described in this paper yields transformation efficiencies far greater than those previously published and represents a significant step toward the establishment of a tractable fern genetic model.

Bringing the multicellular fern meristem into focus

“…1f) as in the peripheral zone of seed plants (Frank et al, 2015). There have been several recent advances in knockout technologies in ferns (Muthukumar et al, 2013;Plackett et al, 2014;Bui et al, 2015) and transcriptome analyses (Frank et al, 2015). The firm recognition of the fern and lycophyte meristems as multicellular with cytohistochemical zonation is integral for the interpretation of broad comparative developmental analyses.…”

Field Guide to Plant Model Systems

“…Ferns are often polyploids with large genomes, and while no draft fern genome sequences are currently available (Wolf et al, 2015), transcriptome assemblies of Azolla filiculoides (an aquatic fern; genome of 740 Mb) and Equisetum giganteum (horsetail) have provided some insight into gene content (Vanneste et al, 2015; Brouwer et al, 2014). A derived fern, Ceratopteris richardii (11.26 Gb) has been developed to some extent as a model system with genetic approaches; e.g., investigations of sex determination mechanisms (Strain et al, 2001; Atallah and Banks, 2015) and transformation protocols have been established (Muthukumar et al, 2013; Plackett et al, 2014). …”

Can mosses serve as model organisms for forest research?

“…In plant sciences, several seed plant model species are established, whereas lycophytes and ferns are more problematic model organisms due to their low transformability or large genome size (Plackett et al 2014). Liverworts, mosses, and hornworts constitute the paraphyletic group of bryophytes and represent early diverging lineages of land plants with an intermediate position between vascular plants and algae (Kenrick and Crane 1997;Lang et al 2010).…”