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.

在这项研究中，我们旨在生产没有种子且可食用果皮的柑橘类水果。因此，无籽吉田脐橙（Citrus sinensis'Yoshida navel'）和长实金橘（Fortunella japonica））以可食用果皮为原料。由于不同属和不同开花期的两个品种将难以杂交，因此使用聚乙二醇方法进行了原生质体融合。吉田脐橙和Jangsil金桔分别用作胚性愈伤组织系和叶肉系。通过流式细胞仪分析再生的植物以鉴定四倍体，然后使用对两个亲本的核和细胞质细胞器DNA具有特异性的简单序列重复标记，通过聚合酶链反应进一步评估四倍体，以确认异源四倍体和cybrids。最终产生了十六种异源四倍体，并在表型上表征了叶片的形态和果实品质。所有的异源四倍体都含有源自吉田脐橙的线粒体。12个包含来自脐橙的叶绿体，而四个包含金橘来源的叶绿体。我们调查了脐橙和金橘原生质体融合产生的异源四倍体的植物和果实特性。异源四倍体叶片的大小在两个亲本之间。但是，与两个亲本的纺锤形叶子相反，同种四倍体的叶子是倒卵形的。异种四倍体（一种已知的金橘特征）中没有叶柄翼。与脐橙类似，开花时间为五月中旬，花粉不育，而果实大小，外形，可溶性固形物含量和酸度在两个亲本之间居中。果皮厚度和段数与金橘相似，果肉重量与脐橙相似。总的来说，这些结果表明，源自脐橙和金桔的属间异源四倍体具有遗传优势，并且尽管缺乏广泛的亲本变异，仍可以生产和选择。这是显示原生质体融合柑橘类植物结果的第一批报道之一。该研究报告了来自原始植物以及融合原生质体植物的果实的特征。由原生质体融合产生的异源四倍体果实的详细特征将对未来的多倍体研究非常有帮助。这是显示原生质体融合柑橘类植物结果的第一批报道之一。该研究报告了来自原始植物和融合原生质植物的果实的特征。由原生质体融合产生的异源四倍体果实的详细特征将对未来的多倍体研究非常有帮助。这是显示原生质体融合柑橘类植物结果的第一批报道之一。该研究报告了来自原始植物以及融合原生质体植物的果实的特征。由原生质体融合产生的异源四倍体果实的详细特征将对未来的多倍体研究非常有帮助。