Phosphomannose-isomerase as a selectable marker for transgenic plum (Prunus domestica L.)

Abstract

A mannose selection system was adapted for Agrobacterium-mediated transformation of plum (Prunus domestica L.) hypocotyl explants and the recovery of transgenic plants. Adventitious regeneration from non-transformed hypocotyl sections was inhibited when 3 mg/l mannose, combined with 10 mg/l sucrose, was added to the medium. Mature seed hypocotyl slices from the cultivar ‘Claudia Verde’ were infected with A. tumefaciens AGL1, carrying the pNOVgus vector, and placed onto different selective media with mannose. A low mannose selection (1.5 g/l, regeneration below the inhibitory concentration) applied for 16 weeks led to the regeneration of escapes. However, when mannose at 1.5 g/l or at 3 g/l (the regeneration-inhibiting concentration) was applied for 6 weeks from the beginning of the experiments and, after that, was increased to 5 g/l, several independent transgenic lines were obtained. The transformation events were monitored by detection of the GUS enzymatic activity at different stages of the process. Nevertheless, stable integration of transgenes into the genome of the plum plants was confirmed by PCR and Southern blot analysis. The transformed shoots were rooted on a medium supplemented with 10 g/l sucrose and 4 g/l mannose. The transformation procedure described here, using the pmi/mannose system for selection of transgenic plum plants, represents an alternative for the production of transgenic plum plants under conditions that are safe regarding human health and the environment, and would permit the insertion of more transgene/s in a pre-existing transgenic line.