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

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Abstract

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.

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Acknowledgments

We thank Sriya Pathirana and Andrew Mullan for tissue culture media preparation and Ian King for maintaining the plants in the greenhouse. We also thank Nick Albert for supplying the binary vector pNWA37, and Mary Christey and Murray Boase for critical reading of the manuscript. The first author is grateful to Prof. Vernieri Paolo and Prof. Serra Giovanni for a scholarship granted by University of Pisa (Italy) and Scuola Superiore Sant’Anna (Italy) for a year and half of work at Plant and Food Research, New Zealand.

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The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. The New Zealand Institute for Plant and Food Research Ltd, Private Bag 11600, Palmerston North, New Zealand

    Alice Trivellini, Donald A. Hunter & Ranjith Pathirana

  2. Department of Agriculture, Food and Environment, Università degli Studi di Pisa, viale delle Piagge 23, 56124, Pisa, Italy

    Alice Trivellini

  3. Department of Agricultural and Environmental Sciences, Università degli Sudi di Milano, Via Celoria 2, 20133, Milan, Italy

    Antonio Ferrante

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  1. Alice Trivellini
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Correspondence to Ranjith Pathirana.

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Trivellini, A., Ferrante, A., Hunter, D.A. et al. Agrobacterium tumefaciens-mediated transformation of axillary bud callus of Hibiscus rosa-sinensis L. ‘Ruby’ and regeneration of transgenic plants. Plant Cell Tiss Organ Cult 121, 681–692 (2015). https://doi.org/10.1007/s11240-015-0738-y

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  • DOI: https://doi.org/10.1007/s11240-015-0738-y

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