The lifecycle of Ceratopteris richardii and its relation to microparticle-mediated transformation. The C. richardii lifecycle comprises two separate, free-living stages, the haploid gametophyte (a–d) and the diploid sporophyte (e–g). Dispersed as haploid spores (a), these germinate and develop rhizoids and a two-dimensional thallus (b), assuming either hermaphrodite (c) or male (d) characteristics by sexual maturity (approximately 9–11 days after germination). Fertilization of gametes results in the development of sporophyte embryos on the hermaphrodite thallus (e), which continue to develop independently of the gametophyte, producing first simple fronds (f) and then fronds of increasing size and complexity until the mature, spore-bearing stage is achieved (g). Genetic transformation of C. richardii is performed on the sporophyte (h–j). Young sporophytes are treated with cytokinin (CK) to induce callus tissue at the shoot apex (h) in place of new fronds (f) . This callus is isolated in tissue culture and transformed through particle bombardment (i) of plasmid DNA carrying the transgene of interest and a linked antibiotic resistance marker. Stable transgene integrations into the C. richardii genome are identified by screening for antibiotic resistance during sporophyte regeneration (j): antibiotic is added to the media, killing untransformed callus cells. Regenerated transformant shoots (designated T0) can then be grown to maturity and transgenic T1 spores (the progeny) harvested.