Particle Bombardment Transformation of Citrus

The objective of this study was to develop a particle bombardment transformation system for citrus. Plasmid DNA, containing the nptII selectable marker and the gfp reporter, was bombarded into immature citrange rootstock, cv. Carrizo (Citrus sinensis Osb. x Poncirus trifoliata L. Raf.) and transgenic shoots were regenerated. Although the number of independent stably transformed tissues/total number of explants, recorded by monitoring GFP fluorescence four weeks after bombardment was high at 18.4%, the regenerated transgenic shoots was low at 0.7%. Fluorescing GFP putative transgenic shoots were micro-grafted onto immature Carrizo rootstocks in vitro, confirmed by PCR amplification of nptII and gfp coding regions, followed by secondary grafting onto older rootstocks in the soil. Southern blot analysis using probe from nptII coding region indicated that all the fluorescing shoots were transgenic. The particle bombardment transformation system might be applied to transformation of other citrus cultivars with minimal modification on tissue culture conditions and be appropriate for transformation with minimal cassettes which is a potential method for transformation of clean DNA (free of pest sequence) into citrus genome.  This is the first report of biolistics and plant regeneration in citrus.