Using GFP to select for transgenic sweet orange [Citrus sinensis (L.) Oseck] derived from electroporated protoplasts

Genetic information of plant cytoplasmic DNA presents a great interest for phylogenic studies. Recently, in Citrus, a new interest for cytoplasmic genome analysis has resulted from the impediment of somatic hybridization. Indeed this technique allows new patterns of nuclear, chloroplastic, and mitochondrial genomes that must be characterized. Cytoplasmic genome analysis among Citrus and related genera was generally done by Southern blots. This method is powerful but not applicable to in vitro plants because it requires too much fresh material. A few years ago we published a preliminary work that demonstrated the successful amplification of Citrus cytoplasmic DNA by using mitochondrial and chloroplastic universal primers and the possibility in some cases to display polymorphism by combining these amplifications with an enzymatic restriction. In the present work, the usefulness of such CAPS analysis, for cytoplasmic genome differentiation at the infra-generic level (Citrus) and intergeneric level inside of the Aurantioideae sub family, was studied on 44 genotypes from 31 species. With the combinations of primers and enzymes tested, the CAPS Technique appears more powerful for chloroplast than for mitochondria to display polymorphisms, even at the infra-generic level. Phylogenic inference are made from a cluster analysis of chloroplastic data obtained with 4 primers coupled each with 2 to 4 restriction enzymes. An example of application for somatic hybrid cytoplasmic genome characterization is presented.