Protocol: genetic transformation of the fern Ceratopteris richardii through microparticle bombardment

Plant Methods

Jane A Langdale¹  Ester H Rabbinowitsch¹  Andrew R G Plackett¹ 

[1] Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1 3RB, UK

关键词: Microparticle;    Protocol;    Transgenic;    Transformation;    Ceratopteris;    Fern;   

Others  :  1218675
DOI  :  10.1186/s13007-015-0080-8

 received in 2015-05-22, accepted in 2015-06-24,  发布年份 2015

【 摘 要 】

Background

The inability to genetically transform any fern species has been a major technical barrier to unlocking fern biology. Initial attempts to overcome this limitation were based on transient transformation approaches or achieved very low efficiencies. A highly efficient method of stable transformation was recently reported using the fern Ceratopteris richardii, in which particle bombardment of callus tissue achieved transformation efficiencies of up to 72%. As such, this transformation method represents a highly desirable research tool for groups wishing to undertake fern genetic analysis.

Results

We detail an updated and optimized protocol for transformation of C. richardii by particle bombardment, including all necessary ancillary protocols for successful growth and propagation of this species in a laboratory environment. The C. richardii lifecycle comprises separate, free-living gametophyte and sporophyte stages. Callus is induced from the sporophyte apex through growth on cytokinin-containing tissue culture medium and can be maintained indefinitely by sub-culturing. Transgene DNA is introduced into callus cells through particle bombardment, and stable genomic integration events are selected by regeneration and growth of T ₀sporophytes for a period of 8 weeks on medium containing antibiotics. Selection of T ₁transgenic progeny is accomplished through screening T ₁gametophytes for antibiotic resistance. In many cases sexual reproduction and development of transgenic embryos requires growth and fertilization of gametophytes in the absence of antibiotics, followed by a separate screen for antibiotic resistance in the resultant sporophyte generation.

Conclusions

Genetic transformation of C. richardii using this protocol was found to be robust under a broad range of bombardment and recovery conditions. The successful expansion of the selection toolkit to include a second antibiotic for resistance screening (G-418) and different resistance marker promoters increases the scope of transformations possible using this technique and offers the prospect of more complex analysis, for example the creation of lines carrying more than one transgene. The introduction of a robust and practicable transformation technique is a very important milestone in the field of fern biology, and its successful implementation in C. richardii paves the way for adoption of this species as the first fern genetic model.

【 授权许可】

   
2015 Plackett et al.

【 图 表 】

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