A Regeneration and Agrobacterium-mediated Transformation System for Genetically Diverse Chrysanthemum Cultivars

Abstract

An efficient, high-frequency regeneration and Agrobacterium -mediated transformation system was developed allowing the genetic engineering of three chrysanthemum ( Dendranthema grandiflora Tzvelev) cultivars: the formerly recalcitrant and economically important cut-flower mum `Polaris' and two potted mums, `Hekla' and `Iridon'. The regeneration protocol used leaf explants on a sequence of media with four hormone regimes. Explants were first cultured on an embryogenesis-type medium containing a high concentration of 2,4-D, which promoted callus formation. Shoot primordia were induced by culture on medium lacking 2,4-D, followed by shoot elongation on a high-cytokinin plus gibberellic acid medium. Finally, elongated shoots were rooted on a low-auxin rooting medium. Transformed plants of the three cultivars were obtained following co-culture of leaf explants with A. tumefaciens strain EHA 105 harboring the plasmid pBI121 containing genes for neomycin phosphotransferase II (NPTII) and β-glucuronidase (GUS). Stable transformation of the three cultivars was verified via GUS assays and Southern analysis.