Regeneration of triploid citrus germplasm by the crossing between tetraploid Huyou and Cocktail grapefruit

[Objective] Seed number has a crucial effect on the fruit quality for the fresh-fruit market and processing production in citrus. Triploid citrus can produce seedless fruits, and as polyploids, it usually shows the superiority of vigorous growth, enlarged fruits and enhanced stress resistance. In citrus breeding, the 4x × 2x interploidy cross is an effective way to obtain triploids and to select new seedless species. Cocktail grapefruit (Citrus paradisi ‘Cocktail’) has excellent fruit taste with high total soluble sugar and low titratable acid contents but has the defect of abundant seeds and poor tolerance to cold. So reducing the seed number to enhance the economic value has raised the awareness among citrus breeders. Changshan Huyou (Citrus aurantium ‘Changshanhuyou’) is a native citrus species in Zhejiang province and exhibits rich flavor and excellent cold resistance, and its seed is a mixed type of single and multiple embryos. A tetraploid Changshan Huyou was identified and bear seedy fruits for several years. Therefore, we attempted to produce triploid hybrids by interploidy hybridization between the tetraploid Changshan Huyou and the diploid grapefruit. [Methods] The 4x × 2x interploidy cross was conducted using a tetraploid Changshan Huyou (4x) as the female parent and the diploid Cocktail grapefruit (2x) as the pollen parent in this study. Pollen viability was determined by the result of Alexander staining and germination rate in vitro before artificial pollination. In the florescence stage of Cocktail grapefruit, the pollen was collected using a 2 mL centrifuge tube and stored in dry environment at 4 ℃ for a short period. Seeds were extracted from the ripened fruit of hybrids and divided into developed seeds of normal size, developed seeds of small size (1/3-1/6 of normal size) and undeveloped seeds. The normal size and small size seeds were sowed on Murashige and Tucker (MT) culture medium, and undeveloped seeds were sowed on MT+1.0 mg·L-1 GA3 culture medium. The seedlings were transplanted after the plant growing 4-5 functional leaves. The ploidy of progenies was then measured by flow cytometry using young leaves. After DNA extraction, the genetic origin of the offspring was analyzed using 4 Simple Sequence Repeats (SSR) markers, which could display different profiles in Changshan Huyou and Cocktail grapefruit. The morphological differences were compared using one-year-old seedlings from offsprings and parents. [Results] The pollen grains of Cocktail grapefruit had a staining activity of 89.40% and in vitro germination rate of 30.02%, indicating that the pollen of Cocktail grapefruit had satisfactory vitality for further pollination. As a result, a total of 40 flowers of the tetraploid Changshan Huyou were used in pollination, and 19 hybrid fruits were harvested in November with a fruit-setting rate of 47.5%. A total of 56 developed seeds (average 3.0 seeds per fruit), 117 small developed seeds (6.2 seeds per fruit), and 57 undeveloped seeds (3.0 seeds per fruit) were obtained, and 49, 69 and 4 seedlings were germinated in vitro with an average germination rate as 87.50%, 58.97% and 7.02% for these seeds, respectively. Ploidy analysis showed that 39 (31.96%) and 83 progenies were proven triploids and tetraploids, respectively. Among the offsprings of each group, a total of 2, 35 and 2 triploid seedlings were identified from the developed seeds, small developed seeds and undeveloped seeds, respectively. The majority (89.74%) of triploids were originated from the small developed seeds. The hybrid nature of all the 39 triploids and 1 out of 22 tetraploids randomly determined were confirmed by the SSR marker of F14, P72, MEST86 and CAG01, and were indicated as the characteristic stripes of both Changshan Huyou and Cocktail grapefruit. The remaining 21 tetraploids were confirmed to be derived from selfing or nucellus embryos due to the presence of the characteristic stripe of the female parent. The leaf shape index of male parents, female parents, triploid F1 seedlings (3x), and a tetraploid F1 seedling (4x) were 1.73, 2.00, 1.83 and 1.65, respectively. The triploid and tetraploid offsprings obtained from hybridization were closer to the tetraploid female parent in terms of leaf shape index, without statistical difference from the female parent but with significant difference (p<0.05) from the male parent. Leaf shape index of 3x hybrids was between 4x and 2x parents, however, the leaves of the 4x hybrid were wider and rounder than 4x female parent. The leaves of 4x hybrid offspring were significantly wider than the leaves of 3x hybrids. [Conclusion] The tetraploid Changshan Huyou was used as the maternal parent and hybridized with diploid pollen of Cocktail grapefruit in this study. After culture in vitro, ploidy identification and SSR molecular marker analysis, a total of thirty-nine heterozygous triploid offsprings were obtained, which mainly germinated from small developed seeds harvested from hybrid fruits in 4x × 2x interploidy cross, providing an effective strategy to ensure the seedless germplasms in citrus breeding by ploidy hybridization.