Recovery of citrus cybrid plants with diverse mitochondrial and chloroplastic genome combinations by protoplast fusion followed by in vitro shoot, root, or embryo micrografting

Somatic embryogenesis and plant regeneration are basic processes for the success of citrus somatic
hybridization via protoplast fusion. In many cases, few embryos develop normally and only a small number of plants are recovered. The development of methodologies able to increase the recovery of plants after protoplast fusion experiments it is an important requirement to improve the efficiency of the procedure. Here, plants were regenerated at high efficiency using in vitro micrografting of shoots, roots, and embryos recovered after different somatic hybridizations. Hybridizations were performed
using protoplasts isolated from Chios mandarin callus with protoplasts isolated from Clementine mandarin leaves and from Sanguinelli sweet orange callus. Recovered plants were analyzed with flow cytometry and nuclear simple sequence repeat (SSR), mitochondrial InDel, and chloroplast SSR markers to determine genomic structure. One tetraploid cybrid and numerous diploid cybrids were
recovered, and these exhibited a range of mitochondrial and chloroplastic genome combinations.