Green fluorescent protein as a screenable marker to increase the efficiency of generating transgenic woody fruit plants

The green fluorescent protein (GFP) from Aequorea victoria has been introdu ced into three different citrus genotypes [Citrus aurantium L., C. aurantif olia (Christm.) Swing. and C. sinensis L. Osbeck x Poncirus trifoliata (L.) Raf.] which are considered recalcitrant to transformation, mainly due to l ow transformation frequencies and to the regeneration of escape shoots at h igh frequencies from the Agrobacterium-inoculated explants. High-level GFP expression was detected in transgenic cells, tissues and plants. Using GFP as a vital marker has allowed us to localize the sites of transgene express ion in specific cells, always occurring in callus tissue formed from the ca mbium of the cut ends of explants. Whereas green fluorescent shoots regener ated in all cases from this callus, most escapes regenerated directly from explants with almost no callus formation. Thus, development of callus from cambium is a prerequisite for citrus transformation. Furthermore, in vivo m onitoring of GFP expression permitted a rapid and easy discrimination of tr ansgenic and escape shoots. The selection of transgenic shoots could be eas ily favored by eliminating the escapes and/or by performing shoot-tip graft ing of the transgenic buds soon after their origin. GFP-expressing shoots h ave also been observed in citrus explants cocultivated with Agrobacterium b ut cultured in a medium without the selective agent kanamycin. This opens t he possibility to rescue the transgenic sectors and to regenerate transgeni c plants without using selectable marker genes conferring antibiotic or her bicide resistance, which is currently a topic of much discussion for the co mmercialization of transgenic plants.