Production and molecular identification of potentially seedless germplasms derived from Wenling-gaocheng, a citrus local cultivar in Zhejiang province

Abstract: 【Objective】Seedlessness is an important trait for citrus fresh fruits because consumers do not like fruits with seeds. Wenling-gaocheng, a natural hybrid of pommelo and orange, is an excellent local cultivar in Zhejiang Province because of its unique flavor and rich nutritional value. However, it failed to expand the market outside Taizhou city due to the abundance of seeds in the fruits. The fruits of the triploid plants are generally seedless. There are several ways to produce citrus triploid plants.Among them, using diploid varieties as female parents and allotetraploid as the male parents is the most efficient way. The triploid plants produced by interploidy hybridization with allotetraploid as the male parent generally possess abundant genetic variations, which is conducive to breed new cultivars with good fruit traits. Production of diploid progenies using cytoplasmic male sterile (CMS) cultivar Huayou No. 2 as the female parent crossed with the seedy diploids is another useful way to breed seedless citrus germplasm because the CMS trait in citrus is female inherited, and the CMS trait can be inherited to all progenies. Therefore, the above two strategies were employed in this study to produce seedless progenies with the lineage of Wenling-gaocheng, to produce promising materials for selecting seedless cultivars with the lineage of Wenling-gaocheng.【Methods】Two crossing strategies were employed in this study. The first one was to produce triploid progenies using Wenling- gaocheng as female parent and two alloteraploids as male parents. The pollination of the two interploidy crosses was conducted at the full-bloom stage and the immature fruits were collected on the 90th days after pollination (DAP). The immature seeds were removed from the fruits and cultured in vitro in the germination medium. When the seedlings regenerated, their ploidy level was determined by the flow cytometry. Another strategy was to produce diploid progenies with CMS traits using Huayou No. 2 as the female parent and the Wenling- gaocheng as the male parent. The pollination of this cross was conducted at the full- bloom stage and the fruits were collected at the mature stage. After the seeds were extracted from the fruits and the episperms were peeled, they were placed in a controlled chamber to accelerate germination. The seeds were sown in pots after germination in a plant growth chamber. After the seedlings grew with three or more leaves, their ploidy level was determined by the flow cytometry. At last, the genetic origin of all seedlings from the three crosses were determined by SSR markers.【Results】In the two interploidy crosses, a total of 145 flowers were pollinated and 74 fruits were set, with an average fruit setting rate of 51.03%. 1520 immature seeds were in vitro cultured via embryo rescue and 553 seedlings were obtained. Among them, 239 plantlets were regenerated from the cross of Wenling-gaocheng × NS and 314 plantlets were originated from the cross of Wenling- gaocheng × BDZNS. After determining their ploidy level, a total of 45 triploids and 8 tetraploid seedlings were screened out. Three SSR markers were used to determine their genetic origin. All the analyzed triploids were the hybrids of their parents and all the tetraploids had the same bands with Wenling- gaocheng, indicating that all the tetraploids might be derived from the nucellar doubling of Wenling- gaocheng. For the cross of Huayou No. 2 × Wenling-gaocheng, a total of 69 flowers were pollinated. From the 20 fruits, 312 mature seeds were obtained. After sowing and germination, 203 seedlings were obtained, all of them were identified as diploid by flow cytometry. Four polymorphic SSR markers were selected to determine their genetic origin of 29 randomly selected progenies and it showed that all the diploid progenies were parental hybrids. 【Conclusion】The diploid hybrids derived from the cross of Huayou No. 2 × Wenling-gaocheng and the triploid hybrids derived from the two interploidy crosses could be used for seedless breeding of new varieties with Wenling-gaocheng genetic background. The autotetraploid progenies of Wenling-gaocheng also would be valuable for crossing with diploid Wenling-gaocheng to produce autotriploids.