Adventitious shoot formation from Red Delicious apple cotyledonsin vitro

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Abstract
Embryo-free cotyledons of cv Red Delicious apple produced callus and adventitious shoots when they were cultured on a unique medium consisting of Nitsch and Nitsch (1969) salts supplemented with micronutrient solution, 6-benzylamino purine (4.0 mg l-1), several vitamins, as well as malt extract (400 mg l-1), myo-inositol (300 mg l-1). coconut milk (40 mg h-1) and glucose (25 g l-1). When the explanted cotyledons were placed in the dark for four days and then moved to lighted conditions, they formed a hard green callus which, differentiated into shoots within three weeks. The progress of shoot formation was delayed when the cotyledons were placed directly under lighted conditions. Seeds taken from slightly immature, freshly harvested apples formed shoots very quickly. The young shoots rooted readily on a medium which was free of coconut milk and malt extract but supplemented with 0.2 mg l-1 indoleacetic acid. These rooted shoots could be easily transplanted to the greenhouse. Cotyledons grown on a medium supplemented with 4 mg l-1 2,4-D developed a hard callus which failed to differentiate shoots even with BA treatment.