Use of petal explants for successful transformation of Dendranthema × grandiflorum Kitamura 'Orlando' mediated by Agrobacterium tumefaciens

Abstract

An Agrobacterium-mediated genetic transformation system for chrysanthemum ‘Orlando’ using petal explants was developed. After decontamination, petals were divided into two parts; terminal (position 1) and middle (position 2) parts, and were pre-cultured for two days on Murashige and Skoog (MS) medium containing 1.0 mg·L^-1 indole acetic acid (IAA), 1.0 mg·L^-1 6-benzylaminopurine (BA), and 0.1 mg·L^-1 kinetin (SIM, shoot induction medium). Then, the explants were transformed by co-cultivation with Agrobacterium tumefaciens strain LBA-4404 harboring a binary vector pCAMBIA 2301 carrying the reporter gene β-glucuronidase (GUS), and the marker gene neomycin phosphotransferase (NPT II). The highest frequency of transgenic shoot induction was obtained when the explants were co-cultivated with A. tumefaciens for two days. The formation of transgenic shoots varied with the position of explant. The highest regeneration frequency (13.3) was obtained when position 2 explants were cultured on the shoot induction medium supplemented with 7.5 mg·L^-1 kanamycin and 250 mg·L^-1 cefotaxime. Putative transgenic plants were obtained after rooting on the MS medium supplemented with 20 mg·L^-1 kanamycin and 250 mg·L^-1 cefotaxime. Histochemical GUS assay and polymerase chain reaction analysis of transgenic plants confirmed the presence of the GUS gene. Thus, successful transformation of chrysanthemum 'Orlando' petal explants mediated by A. tumefaciens was confirmed.

Keywords: Chrysanthemum, co-cultivation, GUS, kanamycin, NPTII, petal explants