Overexpression of Vitis GRF4-GIF1 improves regeneration efficiency in diploid Fragaria vesca Hawaii 4

Plant breeding played a very important role in transforming strawberries from being a niche crop with a small geographical footprint into an economically important crop grown across the planet. But even modern marker assisted breeding takes a considerable amount of time, over multiple plant generations, to produce a plant with desirable traits. As a quicker alternative, plants with desirable traits can be raised through tissue culture by doing precise genetic manipulations. Overexpression of morphogenic regulators previously known for meristem development, the transcription factors Growth-Regulating Factors (GRFs) and the GRF-Interacting Factors (GIFs), provided an efficient strategy for easier regeneration and transformation in multiple crops. We present here a comprehensive protocol for the diploid strawberry Fragaria vesca Hawaii 4 (strawberry) regeneration and transformation under control condition as compared to ectopic expression of different GRF4-GIF1 chimeras from different plant species. We report that ectopic expression of Vitis vinifera VvGRF4-GIF1 provides significantly higher regeneration efficiency during re-transformation over wild-type plants. On the other hand, deregulated expression of miRNA resistant version of VvGRF4-GIF1 or Triticum aestivum (wheat) TaGRF4-GIF1 resulted in abnormalities. Transcriptomic analysis between the different chimeric GRF4-GIF1 lines indicate that differential expression of FvExpansin might be responsible for the observed pleiotropic effects. Similarly, cytokinin dehydrogenase/oxygenase and cytokinin responsive response regulators also showed differential expression indicating GRF4-GIF1 pathway playing important role in controlling cytokinin homeostasis. Our data indicate that ectopic expression of Vitis vinifera VvGRF4-GIF1 chimera can provide significant advantage over wild-type plants during strawberry regeneration without producing any pleiotropic effects seen for the miRNA resistant VvGRF4-GIF1 or TaGRF4-GIF1.