Characterization of allotetraploids derived from protoplast fusion between navel orange and kumquat

In this study, we aimed to produce a citrus fruit with no seed and with edible peel. Therefore, the seedless Yoshida navel oranges (Citrus sinensis ‘Yoshida navel’) and Jangsil kumquat (Fortunella japonica) with edible peel were used as the starting materials. Since two varieties of different genera and different flowering periods would be difficult to crossbreed, protoplast fusion was conducted using the polyethylene glycol method. Yoshida navel orange and Jangsil kumquat were used as an embryogenic callus line and a mesophyll line, respectively. The regenerated plants were analyzed by flow cytometry to identify tetraploids, which were further evaluated by polymerase chain reaction, using simple sequence repeat markers specific for the nuclear and cytoplasmic organellar DNAs of the two parents, to confirm allotetraploids and cybrids. Sixteen allotetraploids were finally produced and characterized phenotypically for leaf morphology and fruit quality. All allotetraploids contained mitochondria originating from Yoshida navel orange; 12 contained chloroplasts derived from navel orange whereas four contained chloroplasts of kumquat origin. We investigated both plant and fruit characteristics of allotetraploids derived from the protoplast fusion of navel orange and kumquat. The sizes of the allotetraploid leaves were intermediate between those of both parents. However, in contrast to the spindle-shaped leaves of both parents, those of the allotetraploids were obovate. Petiole wings were absent in the allotetraploids, a known kumquat trait. Similar to navel oranges, flowering time was mid-May, and pollen was sterile, whereas fruit size, external shape, soluble solids content, and acidity were all intermediate between the two parents. Peel thickness and the number of segments were similar to those of kumquat, and flesh weight was similar to that of navel orange. Collectively, these results indicated that the intergeneric allotetraploids derived from navel orange and kumquat inherited favorable traits of both parents and could be produced and selected despite a lack of extensive parental variation. This is one of the first reports showing fruiting results of protoplast-fused citrus plants. The study reported characteristics of the fruits from both the original plants as well as from the protoplast-fused plant. Detailed characteristics of the allotetraploid fruit produced by protoplast fusion would be very helpful for future polyploid studies.