AcGRF4-AcGIF1 chimeric protein improves the transformation and regeneration efficiency in kiwifruit

• An Agrobacterium rhizogenes -mediated transformation for Actinidia latifolia was developed. • AcGRF4-AcGIF1 chimeric protein improves the transformation and regeneration efficiency in Actinidia latifolia . • AcGRF4-AcGIF1 strategy also works in A. tumefaciens -mediated transformation of A. eriantha . Kiwifruit ( Actinidia spp.) is a woody plant producing delicious fruits with high values of nutrients. Some species in the genus Actinidia are non-transformable yet. In this study, we developed an Agrobacterium rhizogenes -mediated marker-free transformation method for Actinidia latifolia , which has not been previously transformed yet. Agrobacterium rhizogenes- mediated induction of hairy root is practicable in A. latifolia , but the regeneration efficiency of hairy root-induced callus is relatively low. The qRT-PCR assays indicated that the expression levels of GRF4 and GIF1 in A. latifolia (namely AlGRF4 and AlGIF1 ) were much lower than those of AcGRF4 and AcGIF1 in A. chinensis , suggesting that the low regeneration efficiency of A. latifolia might be caused by low expression levels of AlGRF4 and AlGIF1 . Therefore, we cloned AcGRF4 and AcGIF1 to create a fusion gene AcGRF4 - AcGIF1 . Overexpression of the fusion gene substantially boosted the hairy root induction rate by 77 % in A. latifolia . The regeneration efficiency was also increased by the fusion gene from 20 % to 60 %. Besides, the AcGRF4 - AcGIF1 system is also compatible with the previously-established Agrobacterium tumefaciens -mediated transformation of A. eriantha , whose regeneration efficiency was increased from 26 % to 40 %. Collectively, our results indicate that the AcGRF4-AcGIF1 system is a powerful strategy facilitating the genetic transformation of kiwifruit, especially for those non-transformable species.