Scientia Agricultura Sinica ›› 2026, Vol. 59 ›› Issue (10): 2288-2298.doi: 10.3864/j.issn.0578-1752.2026.10.016

• RESEARCH NOTES • Previous Articles    

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

SHEN ShangJie1(), CHEN Hui1(), ZHOU HongJian2, HONG YaYan3, YE ZheXi2, HU ZhiYong2, WU JinJie2, WANG Gang4, ZHENG LiZhen5, ZHANG Lin6, ZHANG Min2(), ZHANG Chi1()   

  1. 1 School of Horticulture, Zhejiang A&F University/Key Laboratory of Quality and Safety Control for Subtropical Fruit and Vegetable, Ministry of Agriculture and Rural Affairs, Hangzhou 311300
    2 Zhejiang A&F University/State Key Laboratory of Forest Food Resources Development and Utilization, Hangzhou 311300
    3 The People’s Government of Hejia Town, Changshan 324200, Zhejiang
    4 Agriculture and Rural Bureau of Changshan County, Changshan 324200, Zhejiang
    5 Bureau of Agriculture and Rural Affairs of Kecheng District, Quzhou 324000, Zhejiang
    6 Zhejiang Agricultural Technology Extension Center, Hangzhou 310020
  • Received:2026-01-04 Accepted:2026-03-02 Online:2026-05-16 Published:2026-05-20
  • Contact: ZHANG Min, ZHANG Chi

Abstract:

【Objective】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.【Method】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.【Result】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.【Conclusion】A batch of novel allotriploid germplasm was successfully generated, providing valuable genetic resources for breeding of new seedless pummelo varieties.

Key words: Citrus grandis, Citrus changshan-huyou, allotriploid, flow cytometry, SSR markers

Table 1

Sequence of the primers"

引物名称 Primer name 正向序列 Forward sequence (5′-3′) 反向序列 Reverse sequence (5′-3′) 参考文献Reference
P72 GTGAGGCAAAACGGAAAGAG GGGCCCATACAACGTAGAAG [16]
Ma3-96 ATCATTTCAACAAGGGACGG TCGCCAATCAAACAACAAAA [17]
P8 TTGCCAAGAGATTAAACGAACA GACGAGAGGTCCAGAAATCG [18]
COSC4-9 GTCCTCTTTTGACGCCTCCT CCTTTGGGTTTGTTGGACGAC [18]
CAG01 AACACTCGCACCAAATCCTC TAAATGGCAACCCCAGCTTTG [19]

Table 2

The pollination and plant regeneration from the crosses used tetraploid Huyou as the female parent"

杂交组合
Cross
授粉花数
No. of flowers
坐果数
No. of fruits
坐果率
Fruit setting rate (%)
种子数
No. of seeds
再生植株
No. of plants
三倍体
No. of
triploids
四倍体‘胡柚’ב琯溪蜜柚’ Tetraploid Huyou×Guanximiyou 61 16 26.22 328 232 40
四倍体‘胡柚‘ב早香柚’ Tetraploid Huyou×Zaoxiangyou 11 4 36.36 112 33 14
四倍体‘胡柚’ב东试早柚’ Tetraploid Huyou×Dongshizaoyou 11 1 9.09 22 14 5
四倍体‘胡柚’ב红宝石柚’ Tetraploid Huyou×Hongbaoshiyou 14 8 57.14 64 24 7
总计Total 97 29 29.90 526 303 66

Fig. 1

Classification of seeds and plant regeneration of the tetraploid C. changshan-huyou×C. grandis Guanximiyou hybridization A: Seed classification; B: Sowing on the culture medium; C: Emergence of seedlings; D: Transplantation"

Fig. 2

Flow cytometric identification of ploidy in regenerated plants"

Table 3

Plant height and basal diameter of triploid offspring plants from four hybrid combinations"

品种
Cultivar
株高
Plant height
(cm)
地径
Ground diameter (mm)
株高/地径
Plant height/Ground diameter
四倍体‘胡柚’ב琯溪蜜柚’子代(n=40) Tetraploid Huyou × Guanximiyou 33.73±5.26a 5.97±0.73 5.65±0.62a
四倍体‘胡柚’ב早香柚’(n=14) Tetraploid Huyou × Zaoxiangyou 26.03±7.83b 5.75±0.35 4.49±1.12b
四倍体‘胡柚’ב东试早柚’(n=5) Tetraploid ‘Huyou × Dongshizaoyou 29.72±8.28ab 5.71±0.54 5.20±1.40ab
四倍体‘胡柚’ב红宝石柚’(n=7) Tetraploid Huyou × Hongbaoshiyou 33.61±7.94a 5.69±0.65 5.91±0.95a
四倍体‘胡柚’ Tetraploid Huyou 27.00±3.84b 5.71±0.66 5.08±0.68ab

Fig. 3

Comparison of leaf index and wing leaf length-to-width ratio of 4 hybrid combinations and their parents A-D: Leaf shape indices of different crosses; E-H: Length-to-width ratios of wing leaves for the corresponding crosses. HD: Tetraploid Huyou; GX: Guanximiyou; ZXY: Zaoxiangyou; HBS: Hongbaoshiyou; DSZ: Dongshizaoyou; F1(3X): Triploid hybrid progenies of the corresponding cross combinations. Different lowercase letters above the bars indicate significant differences among treatments (P<0.05). The same as below"

Fig. 4

Morphological characteristics of the hybrid progeny plants and leaves A: Progeny plants from the cross between tetraploid Huyou and diploid Guanximiyou; B: Comparison of leaf morphology between parents and progeny of each cross combination. F1-1-3: Progeny from tetraploid Huyou×Guanximiyou; F1-4-6: Progeny from tetraploid Huyou×Dongshizaoyou; F1-7-9: Progeny from tetraploid Huyou×Hongbaoshiyou; F1-10-12: Progeny from tetraploid Huyou×Zaoxiangyou"

Fig. 5

SSR molecular identification of four combination-derived regenerated triploid progeny A, B: Identification results using primer P72; C, D: Identification results using primer Ma3-96; E: Identification results using primer CAG01; F: Identification results using primer P8; G: Identification results using primer COSC4-9. H: Tetraploid Huyou; G: Guanximiyou; Z: Zaoxiangyou; D: Dongshizaoyou; B: Hongbaoshiyou; 1-40: Triploid progenies. The same as below"

Fig. 6

PCR profiles of hybrids randomly selected from the four cross combinations with the MITE marker M:2 kb DNA marker"

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