High-efficiency stable transformation of the model fern species Ceratopteris richardii via microparticle bombardment.

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::ß-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.

Conserved transport mechanisms but distinct auxin responses govern shoot patterning in Selaginella kraussiana.

To provide a comparative framework to understand the evolution of auxin regulation in vascular plants, the effect of perturbed auxin homeostasis was examined in the lycophyte Selaginella kraussiana. Polar auxin transport was measured by tracing tritiated IAA in excised shoots. Shoots were cultured in the presence of auxin efflux inhibitors and exogenous auxin, and developmental abnormalities were documented. Auxin transport in Selaginella shoots is exclusively basipetal, as in angiosperms. Perturbed auxin transport results in the loss of meristem maintenance and abnormal shoot architecture. Dichotomous root branching in Selaginella appears to be regulated by an antagonistic relationship between auxin and cytokinin. The results suggest that basipetal polar auxin transport occurred in the common ancestor of lycophytes and euphyllophytes. Although the mechanisms of auxin transport appear to be conserved across all vascular plants, distinct auxin responses govern shoot growth and development in lycophytes and euphyllophytes.

Sector analysis and predictive modelling reveal iterative shoot-like development in fern fronds.

Plants colonized the terrestrial environment over 450 million years ago. Since then, shoot architecture has evolved in response to changing environmental conditions. Our current understanding of the innovations that altered shoot morphology is underpinned by developmental studies in a number of plant groups. However, the least is known about mechanisms that operate in ferns--a key group for understanding the evolution of plant development. Using a novel combination of sector analysis, conditional probability modelling methods and histology, we show that shoots, fronds ('leaves') and pinnae ('leaflets') of the fern Nephrolepis exaltata all develop from single apical initial cells. Shoot initials cleave on three faces to produce a pool of cells from which individual frond apical initials are sequentially specified. Frond initials then cleave in two planes to produce a series of lateral merophyte initials that each contributes a unit of three pinnae to half of the mediolateral frond axis. Notably, this iterative pattern in both shoots and fronds is similar to the developmental process that operates in shoots of other plant groups. Pinnae initials first cleave in two planes to generate lateral marginal initials. The apical and marginal initials then divide in three planes to coordinately generate the determinate pinna. These findings impact both on our understanding of fundamental plant developmental processes and on our perspective of how shoot systems evolved.