Improved regeneration and Agrobacterium-mediated transformation of wild strawberry (Fragaria vesca L.)

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Virginia Tech

The Rosaceae contains many important commercially grown fruit crops. No comprehensive genomics platform is currently under development for fruit crops, giving functional genomics studies with wild strawberry (Fragaria vesca L.) the potential of identifying genes important in fruit crops. Fragaria vesca has a small genome size compared to the cultivated strawberry, Fragaria à ananassa Duch. (164 vs. 600 Mbp per 1C nucleus). This feature, in addition to a short life cycle (12-16 weeks) and small plant size make F. vesca a good candidate for a model plant for genetic and molecular studies. The specific objective of this work was to develop an efficient high-throughput Agrobacterium-mediated transformation protocol to generate an insertional mutant population to support the justification of F. vesca as a model organism for rosaceous crops. The transformation techniques described by Alsheikh et al. (2002) and Oosumi et al. (2005) were modified and applied to a range of germplasm obtained from the USDA National Germplasm Repository. We found that the modifications made to the Alsheikh protocol were unsuccessful when applied to our germplasm. With the Oosumi et al. (2005) protocol, transformation efficiencies ranging from 11 to 100% were obtained for two accessions when explants were exposed to varying durations on TDZ containing medium during shoot regeneration. The transformation efficiency was given as the mean number of GFP+ plants obtained per primary explant cultured. Multiplex PCR, for amplification of the hptII and GFP genes, was performed on a random sample of GFP+ plants to verify insertion of the T-DNA. The statistical power of our experiment was insufficient to detect treatment effect but based on our findings the transformation efficiencies were high enough to justify PI 551572 for use in the high throughput transformations that are required to generate a population of insertional mutants large enough for gene discovery in F. vesca.

Fragaria vesca, Agrobacterium, TDZ, multiplex PCR, regeneration, plant transformation