Short communication. N-(2-chloro-4-pyridyl)-N-phenylurea(4-CPPU) enhances in vitro direct shoot organogenesis of Citrus aurantium L. epicotyl segments compared to other commonly used cytokinins

The effect of three concentrations of five different cytokinins, i.e. 6-benzylamino purine, 2-isopentyl adenine, kinetin (Kin), thidiazuron and N-(2-chloro-4-pyridyl)-N-phenylurea (4-CPPU), was evaluated on the in vitro direct shoot organogenesis of epicotyl explants of sour orange (Citrus aurantium L.). The basal medium used was that of Murashige and Tucker and epicotyl explants were incubated in medium supplemented with the prementioned cytokinins for 45 days. The addition of Kin and 4-CPPU in the medium enhanced the direct shoot organogenesis of sour orange epicotyl segments. The concentration of each of these two cytokinins which gave the best results, was combined with indole-3-acetic acid (IAA) or α-naphthalene acetic acid (α-NAA) at concentrations ranging from 0.01 mg L–1 to 0.2 mg L–1. The inclusion of IAA at 0.2 mg L–1 in the medium with 4-CPPU at 0.05 mg L–1 resulted in 100% successful direct shoot organogenesis, while the combination of Kin at 0.25 mg L–1 with IAA or α-NAA each at 0.01 mg L–1 presented equally high organogenesis percentages (91.7%). The incubation of the produced shoots, in medium supplemented with either indole-3-butyric acid or α-NAA resulted in high rooting percentages (up to 90%) and the rooted explants were successfully acclimatized under mist (85%). Although 4-CPPU has been used in in vitro culture of various species, this is the first report on its use in the direct shoot organogenesis of citrus species and could be of great value in citrus genetic transformation protocols using epicotyl segments, since this cytokinin resulted in the absolute organogenesis percentage.