Production of transgenic strawberries by temporary immersion bioreactor system and verification by TAIL-PCR

Background: Strawberry (Fragaria x ananassa) is an economically important soft fruit crop with polyploid genome which complicates the breeding of new cultivars. For certain traits, genetic engineering offers a potential alternative to traditional breeding. However, many strawberry varieties are quite recalcitrant for Agrobacterium-mediated transformation, and a method allowing easy handling of large amounts of starting material is needed. Also the genotyping of putative transformants is challenging since the isolation of DNA for Southern analysis is difficult due to the high amount of phenolic compounds and polysaccharides that complicate efficient extraction of digestable DNA. There is thus a need to apply a screening method that is sensitive and unambiguous in identifying the different transformation events. Results: Hygromycin-resistant strawberries were developed in temporary immersion bioreactors by Agrobacterium-mediated gene transfer. Putative transformants were screened by TAIL-PCR to verify T-DNA integration and to distinguish between the individual transformation events. Several different types of border sequence arrangements were detected. Conclusion: This study demonstrates that temporary immersion bioreactor system suits well for the regeneration of transgenic strawberry plants as a labour-efficient technique. Small amount of DNA required by TAIL-PCR is easily recovered even from a small transformant, which allows rapid verification of T-DNA integration and detection of separate gene transfer events. These techniques combined clearly facilitate the generation of transgenic strawberries but should be applicable to other plants as well.