Mutagenesis in olive (Olea europaeaL.) calli caused by sodium azide and detection of mutants using ISSR and RAPD markers

Summary In vitro callus formation was established in the olive (Olea europaea L.) cultivar ‘Kronaki’ and in vitro mutagenesis of calli was carried out using five different concentrations (1.0 – 5.0 mM) of sodium azide (NaN3). Randomly amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers were used to detect genetic polymorphism among the olive mutants produced. Six RAPD and seven ISSR primers were used to amplify DNA segments from the genomes of untreated and NaN3-treated calli. The optimum medium for callus formation was 0.5× Murashige and Skoog (MS) medium, supplemented with 2 mg l–1 αnaphthaleneacetic acid and 0.5 mg l–1 2-isopentenyl-adenine. A total 60 RAPD amplicons and 60 ISSR fragments were detected. The sizes of the PCR amplicons varied from 200 – 3,000 bp in length. Fifteen of the 60 RAPD (40%) and ten of the 60 ISSR (16.6%) fragments were polymorphic. The results showed that 4.0 mM and 5.0 mM NaN3 were the most effective doses for mutagenesis. Based on RAPD, ISSR, and combined RAPD and ISSR marker analysis, it was shown that all NaN3 treatments caused detectable levels of mutation. The use of NaN3 for in vitro mutagenesis in olive calli resulted in genetic dissimilarity values of 0.14 – 0.21, and represents a useful way to induce mutants in olive which could be exploited in the future for trait improvement and in olive breeding programmes.