Transient and stable transformation of Ceratopteris richardii gametophytes

Ferns, being vascular yet seedless, present unparalleled opportunities to investigate important questions regarding the evolution and development of land plants. Ceratopteris richardii, a diploid, homosporous fern has been advanced as a model fern system; however, the tenuous ability to transform the genome of this fern greatly limited its usefulness as a model organism. Here we report a simple and reliable Agrobacterium-mediated method for generating transient and stable transformants of mature C. richardii gametophytes.

Transformation success was achieved by enzyme treatment that partially digested the cell walls of mature gametophytes to facilitate Agrobacteria infection. Co-incubation of Agrobacteria with enzymatically treated gametophytes was sufficient to generate transient transformants at a frequency of nearly 90% under optimal conditions. Stable transformation was achieved at a rate of nearly 3% by regenerating entire gametophytes from single transformed cells from T0 gametophytes on selective media.

This transformation method will allow for the immediate observation of phenotypes in the haploid gametophytes of transformed plants, as well as the generation of stably transformed C. richardii lines for further analysis. Transformation capability will greatly facilitate gene functional studies in C. richardii, more fully realizing the potential of this model fern species. These protocols may be adapted to other plant species that are recalcitrant to Agrobacterium-mediated transformation.