Innovation of Citrus Triploids by Ploidy Hybridization Between Tetraploid Huyou and Four Pomelo Cultivars

Pummelo (Citrus grandis) is seedy under pollination to maintain fruit set and fruit quality. The objective of this study is to develop novel triploid citrus germplasm by 4x×2x intreploid hybridization, thereby providing a germplasm foundation for the breeding of seedless pummelo varieties.

Allotriploids were obtained from four combinations of interploidy hybridization sharing a tetraploid Changshan Huyou (C. changshan-huyou) as their common female parent and having C. grandis Guanximiyou, C. grandis Zaoxiangyou, C. grandis Dongshizaoyou, and C. grandis Hongbaoshiyou as the male parent, respectively. Upon fruit maturity, seeds were harvested and categorized based on their sizes for inoculation on MT medium for tissue culture to obtain regenerated plants. Ploidy analysis of regenerated plants was conducted using flow cytometry, and genetic identification of triploid progenies was performed using SSR molecular markers. Additionally, systematic analysis was conducted on growth vigor, leaf morphology, and segregation patterns of monoembryony/polyembryony in triploid progenies.

A total of 97 flowers were pollinated across the four hybrid combinations, yielding 29 fruits with an average fruit set rate of 29.90%. Variation in fruit set rate was observed among different male parent combinations, and the combination using Hongbaoshiyou as the male parent exhibited the highest fruit set rate (57.14%), whereas the Dongshizaoyou combination showed the lowest (9.09%). A total of 526 seeds were harvested, from which 303 regenerated plants were produced through tissue culture, resulting in an average plant regeneration rate of 57.60%. A total of 66 triploid plants were revealed by analysis of flow cytometry. These triploids were regenerated from seeds of various sizes, with the highest incidence of triploids observed in small seeds (86.36%). Triploid progenies displayed rich genetic variation in traits such as plant height and leaf morphology. In some combinations, the height of progenies was significantly greater than the female parent. The wing leaf length-to-width ratio of triploid progenies from the tetraploid Huyou×Hongbaoshiyou combination was intermediate between both parents, while in other combinations, the wing leaf length-to-width ratio of the progenies showed no significant difference from that of the female parent. Genetic identification of the 66 triploid progenies using five pairs of SSR primers showed that all triploids amplified specific bands from both parents, confirming their hybrid origin. Specifically, primer Ma3-96 amplified five types of bands in progenies from the combination of the tetraploid Huyou×Guanximiyou, and the observed ratio of these types was in accordance with the theoretical segregation ratio (4:1:1:5:1) expected for a double diploid upon chi-square test. Molecular marker identification of monoembryony/polyembryony traits indicated that the female parent, tetraploid Huyou, was polyembryonic, while the four diploid male parents were all monoembryonic. Among the 66 triploid progenies, 56 exhibited polyembryony and 10 exhibited monoembryony. The segregation ratio of monoembryony to polyembryony (5:35) in the progenies from the combination of tetraploid Huyou×Guanximiyou conformed to the theoretical expectation (1:5) upon chi-square test, indicating that the inheritance of the monoembryony/polyembryony trait in this combination followed Mendelian segregation patterns.

A batch of novel allotriploid germplasm was successfully generated, providing valuable genetic resources for breeding of new seedless pummelo varieties.