US EPA

1936070 

Journal Article 

Stable transformation of ferns using spores as targets: Pteris vittata and Ceratopteris thalictroides 

Muthukumar, B; Joyce, BL; Elless, MP; Stewart, CN, Jr 

2013 

1 

Plant Physiology
ISSN: 0032-0889
EISSN: 1532-2548 

163 

2 

648-658 

English 

23933990 

10.1104/pp.113.224675 

WOS:000325554100019 

Ferns (Pteridophyta) are very important members of the plant kingdom that lag behind other taxa with regards to our understanding of their genetics, genomics, and molecular biology. We report here the first instance of stable transformation of fern with recovery of transgenic sporophytes. Spores of the arsenic hyperaccumulating fern Pteris vittata (Chinese brake fern) and tetraploid Ceratopteris thalictroides (C-fern 'Express') were stably transformed by Agrobacterium tumefaciens with constructs containing the P. vittata actin promoter driving a GUSPlus reporter gene. Reporter gene expression assays were performed on multiple tissues and growth stages of gametophytes and sporophytes. Southern blot analysis confirmed stable transgene integration in recovered sporophytes and also confirmed that no plasmid from Agrobacterium was present in the sporophyte tissues. We recovered seven independent transformants of P. vittata and four independent C. thalictroides transgenics. Inheritance analyses using GUS histochemical staining revealed that the GUS transgene was stably expressed in second (T2) generation C. thalictroides sporophytic tissues. In an independent experiment, the gusA gene that was driven by the 2x CaMV 35S promoter was bombarded into P. vittata spores using biolistics in which putatively stable transgenic gametophytes were recovered. Transformation procedures required no tissue culture or selectable marker genes. However, we did attempt to use hygromycin selection, which was ineffective to recover transgenic ferns. This simple stable transformation method should help facilitate functional genomics studies in ferns.