Use of Petal Explants for Successful Transformation of Chrysanthemum x Morifolium "Orlando" Mediated by Agrobacterium Tumefaciens

Chrysanthemum is among the most popular cut flower and potted plant crops in the floriculture industry. This situation has promoted studies on the development of an efficient gene transformation protocol in chrysanthemum. Previously it has been demonstrated that petal explants had higher regeneration efficiency than other explants for chrysanthemum ‘Orlando’, but use of petal explants for transformation has not been previously reported.  Therefore, development of a transformation protocol using petal explants would be beneficial for future work inserting genetic traits of interest in chrysanthemum. Petals were surface-disinfected in a 70% (v/v) ethanol solution for 30 seconds and in a 1.5% (v/v) sodium hypochlorite solution for 10 minutes, followed by three rinses with sterilized water. After sterilization, petals were divided into two parts, terminal (position 1) and middle parts (position 2), and were cultured on the MS medium supplemented with 1.0 mg·L^-1 IAA, 1.0 mg·L^-1 BA, and 0.1 mg·L^-1 kinetin with 3% (w/v) sucrose and 0.8% (w/v) agar (shoot induction medium, SIM). In order to examine the effect of concentration of kanamycin, two experiments were conducted. In experiment 1, explants were cultured on a SIM supplemented with kanamycin (0.0, 2.5, 5.0, 7.0, or 10.0 mg·L^-1) for shoot development, and in experiment 2, explants were cultured on the MS medium supplemented with kanamycin (0.0, 5.0, 10.0, 15.0, or 20.0 mg·L^-1) for root development. The explants were submerged in a 25 mL Agrobacterium tumefaciens suspension (O.D. 0.8), harboring the binary vector LBA4404, containing the GUS gene and were shaken for 30 min. Inoculated petal explants were transferred on a SIM and co-cultured for 1, 2, or 3 days in the dark. Selections in experiment 1 were made at 7.5 mg·L^-1 kanamycin due to explant sensitivity to kanamycin and its suitability to prevent escape formation (non-transgenic plants). Selections in experiment 2 were made at 20 mg·L^-1 kanamycin because regenerated shoots did not induce roots and turned brown in color. A two day co-culture with A. tumefaciens significantly increased the number of transgenic plants. In order to confirm the efficiency of this transformation system, expression of the transgenic plant was demonstrated by polymerase chain reaction (PCR) analysis and GUS histochemical assay. Successful transformation of chrysanthemum 'Orlando' petal explants mediated by A. tumefaciens was confirmed in this research.