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

• Published in: Plant physiology (Bethesda) Vol. 165; no. 1; pp. 3 - 14
• Main Authors: Plackett, Andrew R.G., Huang, Liandong, Sanders, Heather L., Langdale, Jane A.
• Format: Journal Article
• Language: English
• Published: United States American Society of Plant Biologists 01.05.2014
• Subjects: antibiotics; Auxins; Biolistics - methods; Breakthrough Technologies; Callus; Ceratopteris richardii; Cinnamates - pharmacology; Crosses, Genetic; Cytokinins - pharmacology; Ferns; Ferns - drug effects; Ferns - genetics; ferns and fern allies; Gametophytes; Gene Expression - drug effects; genetically modified organisms; Germ Cells, Plant - drug effects; Germ Cells, Plant - physiology; Glucuronidase - metabolism; Hygromycin B - analogs & derivatives; Hygromycin B - pharmacology; Inheritance Patterns - genetics; Laboratory staining techniques; Plant cells; Plants; Plants, Genetically Modified; Regeneration - drug effects; Spores; Sporophytes; Transformation, Genetic - drug effects; Transgenes
• ISSN: 0032-0889; 1532-2548; 1532-2548
• DOI: 10.1104/pp.113.231357

Ferns represent the most closely related extant lineage to seed plants. The aquatic fern Ceratopteris richardii has been subject 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 microparticle bombardment. Indeterminate callus was generated and maintained from the sporophytes of both species using cytokinin treatment. In proof-of-principle experiments, a 35S::β-glucuronidase (GUS) expression cassette was introduced into callus cells via tungsten microparticles, and stable transformants were selected via a linked hygromycin 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.