Agrobacterium tumefaciens-mediated transformation of axillary bud callus of Hibiscus rosa-sinensis L. ‘Ruby’ and regeneration of transgenic plants

Hibiscus rosa-sinensis L. is a popular ornamental species valued for its large brightly coloured ephemeral flowers and has a range of health-promoting properties. The value of H. rosa-sinensis could be improved even further if there were ways to prolong the display life of its short-lived flowers, and to improve its frost tolerance. Development of an efficient plant transformation and regeneration procedure that allows introduction of genes into the plant will greatly facilitate this. Here we outline a transformation and regeneration procedure that is the first to produce transformed H. rosa-sinensis plants successfully. We first optimised callus induction and shoot regeneration efficiency. The highest shoot regeneration frequency of 66.7 % was achieved in the cultivar ‘Ruby’ when callus induced from axillary buds using a basal medium supplemented with 2.22 µM benzylaminopurine and 2.47 µM β-naphthoxyacetic acid was cultured on shoot regeneration medium. The frequency of shoot regeneration from callus was lower in ‘Ben James’ and absent in ‘Bright Light’, indicating genotypic differences. When axillary bud-derived callus of ‘Ruby’ was co-cultured with Agrobacterium tumefaciens harbouring a β-glucuronidase (GUS) reporter plasmid, 49 % of calli produced shoots on selection media. All tested plantlets were confirmed as transformed based on the presence of the GUS transgene in the genomic DNA and GUS activity measurements. Roots were induced on transgenic plantlets using half-strength basal medium supplemented with 2.85 µM indole-3-acetic acid. This simple protocol can be used to improve the ornamental, agronomic and health-promoting traits of H. rosa-sinensis hitherto recalcitrant to A. tumefaciens-mediated transformation.