Micropropagation of thirteen Malus cultivars and rootstocks,and effect of antibiotics on proliferation

Several factors that affect in vitro establishment, proliferation, and rooting of thirteen Malus cultivars and rootstocks were studied. Apple shoot tips (1.5±0.5 cm in length) were established using ascorbic and citric acids as antioxidants. Four proliferation media containing 1.0 mg 1^–1 BA and different concentrations of IBA and GA₃ were tested. Proliferation rates varied depending on the genotype and medium used. The highest proliferation rate was obtained for a rootstock that produced 11.6±2.5 shoots (1.5±0.8 cm in length) per tube per month. Rooting was induced with IBA for all the genotypes tested. The optimal IBA concentration was cultivar dependent (between 0.1 and 1.0 mg 1^–1 IBA), and lower concentrations were necessary to induce rooting in liquid rather than in solid medium.The effects on shoot-tip proliferation of cefotaxime, carbenicillin and kanamycin, three antibiotics commonly used for transformation studies, were also evaluated. Cefotaxime at 200 mg 1^–1 stimulated shoot growth and development, but at 500 mg 1^–1 caused abnormal shoot morphology. Carbenicillin at 500 mg 1^–1, alone or in combination with cefotaxime at 200 mg 1^–1, inhibited proliferation and caused excessive enlargement of the basal leaves, inducing callus formation and release of phenolic compounds in the medium. Kanamycin at 50 mg 1^–1 was phytotoxic and caused shoot chlorosis and necrosis. Consideration of the toxicity of these antibiotics is critical when designing transformation schemes for selection and recovery of transgenic apple plants.Abbreviations BA benzyladenine - cef cefotaxime - crb carbenicillin - GA₃ gibberellic acid - IBA indole-3-butyric acid - Kan kanamycin - ms Murashige and Skoog [19] macro- and micro-nutrients - NAA naphthalene-acetic acid