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Orangemint leaf disks were infected with three strains of Agrobacterium tumefaciens: A281, a hyper-virulent strain containing plasmid pTiBo542; C58, a strain containing nopaline Ti plasmid pTiC58; and A136, a derivative of C58 lacking the nopaline Ti plasmid. After a 24 or 48 hr cocultivation, leaf disks were placed on a medium containing MS salts and vitamins, 2% sucrose, and 200 μgm l-1 cefotaxime. Callus formed only on those leaf disks infected with A281. Five callus lines (R-12, -71, -73, -81, -83) were putatively transformed based on succinopine production. Definitive transformations were confirmed via DNA slot blot analysis. All callus lines assayed (R-11. -12, -41, -73, -83) hybridized to a 7.7 KB fragment from the T-DNA region.
Particle bombardment has been shown to be useful for genetic manipulation of many plants; however, a critical component for successful transformation is the ability of transformed cells to regenerate plants. This study describes factors that affect the regeneration efficiency of apple leaf explants following particle bombardment. Basal leaf segments of micropropagated `Royal Gala' apple were treated with 1μm gold particles (0.5 μg/10μl), accelerated at either 4.5, 6.2, 7.6, 9.3 or 13.8 MPa, and cultured on N6 salts + l0μM TDZ regeneration medium for 5, 10 or 20 days in the dark. Both microprojectile-treated and control explants exhibited 85-100% regeneration. However, only 30-60% of the explants bombarded at 7.6, 9.3 and 13.8 MPa had more than 10 regenerants and 6-10% had more than 20 regenerants, whereas for control explants and those bombarded at 4.5 and 6.2 MPa, 70-90% had more than 10 regenerants and 30-50% had more than 20.