Efficient in vivo Induction of Tetraploids from Three Mono-Embryonic Citrus Cultivars

doi:10.16420/j.issn.0513-353x.2024-0974

Acta Horticulturae Sinica ›› 2025, Vol. 52 ›› Issue (9): 2291-2299.doi: 10.16420/j.issn.0513-353x.2024-0974

• Genetic & Breeding·Germplasm Resources·Molecular Biology • Next Articles

Efficient in vivo Induction of Tetraploids from Three Mono-Embryonic Citrus Cultivars

LU Xin¹, REN Jie¹, DUAN Yaoyuan¹, XIE Kaidong¹, XIAO Gong’ao¹, XIA Zewen¹, WANG Rong¹^,², WU Xiaomeng¹, GUO Wenwu¹^,^**()

¹ National Key Laboratory for Germplasm Innovation & Utilization of Horticultural Crops， Huazhong Agricultural University， Wuhan 430070， China
² College of Landscape & Horticulture， Yunnan Agricultural University， Kunming 650201， China

Received:2025-01-02 Revised:2025-03-03 Online:2025-09-25 Published:2025-09-24
Contact: GUO Wenwu

Abstract

Abstract:

Mono-embryonic tetraploid plants were produced from the seedlings of three mono-embryonic citrus cultivars（Orah mandarin，Xiangshui lemon and Juhuaxinyou pummelo）. After cutting off the epicotyls of seedlings，callus tissue was induced under dark and humid conditions for 9 d，then the callus tissue was treated with 0.025% colchicine solution for 2 h，and adventitious buds were induced to regenerate on the epicotyls under light. After growing to 3-4 real leaves，the regenerated plants were identified for ploidy and mono-embryony by using flow cytometry and InDel molecular marker analysis. The results showed that 8，21，and 9 mono-embryonic tetraploids plants of Orah mandarin，Xiangshui lemon，and Juhuaxinyou pummelo were obtained，with an average induction rate of 16.2%. The regenerated plants exhibited stable ploidy，with increased leaf thickness and enlarged stomata after chromosome doubling，while leaf shape index，density of oil gland and stomata decreased. This study produced valuable core mono-embryonic tetraploid germplasm resources as seed parents for seedless triploid breeding without immature embryo rescue via tissue culture in citrus.

Key words: citrus, mono-embryonic, tetraploid, colchicine, germplasm innovation

LU Xin, REN Jie, DUAN Yaoyuan, XIE Kaidong, XIAO Gong’ao, XIA Zewen, WANG Rong, WU Xiaomeng, GUO Wenwu. Efficient in vivo Induction of Tetraploids from Three Mono-Embryonic Citrus Cultivars[J]. Acta Horticulturae Sinica, 2025, 52(9): 2291-2299.

Figures/Tables 8

References 28

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引物名称Primer name	引物序列（5′-3′）Primer sequence	参考文献Reference
mite_p1	F：GTACCGAATTACCMCCCATAA；R：GTAGGATTTGGGTTATTGATG	Wang et al.,2017
mite_p2	F：TCTGGTTCATTGAGAATCCGCG；R：ATCATGTGGGTCATGGTAC	Wang et al.,2017

引物名称Primer name	引物序列（5′-3′）Primer sequence	参考文献Reference
mite_p1	F：GTACCGAATTACCMCCCATAA；R：GTAGGATTTGGGTTATTGATG	Wang et al.,2017
mite_p2	F：TCTGGTTCATTGAGAATCCGCG；R：ATCATGTGGGTCATGGTAC	Wang et al.,2017

品种 Cultivar	诱导上胚轴数 Number of induced epicotyls	再生植株数 Number of regeneration	四倍体数 Number of tetraploids	混倍体数 Number of mixoploids	四倍体诱导/% Tetraploid induction rate	混倍体发生率/% Mixoploid incidence
沃柑Orah mandarin	115	55	18	8	32.7	14.5
香水柠檬Xiangshui lemon	271	233	32	16	13.7	6.9
菊花芯柚Juhuaxinyou pummelo	105	77	9	3	11.7	3.9
总计/平均Sum/Average	491	365	59	27	16.2	7.4

品种 Cultivar	诱导上胚轴数 Number of induced epicotyls	再生植株数 Number of regeneration	四倍体数 Number of tetraploids	混倍体数 Number of mixoploids	四倍体诱导/% Tetraploid induction rate	混倍体发生率/% Mixoploid incidence
沃柑Orah mandarin	115	55	18	8	32.7	14.5
香水柠檬Xiangshui lemon	271	233	32	16	13.7	6.9
菊花芯柚Juhuaxinyou pummelo	105	77	9	3	11.7	3.9
总计/平均Sum/Average	491	365	59	27	16.2	7.4

品种 Cultivar	初测4x数/株 Number of tetraploids identified in the first time	一年苗龄植株二次检测/株 Secondary ploidy identification			4x纯合率/% Tetraploid ratio
品种 Cultivar	初测4x数/株 Number of tetraploids identified in the first time	4x	2x	混倍体Mixoploid	4x纯合率/% Tetraploid ratio
沃柑Orah mandarin	8	8	0	0	100.0
香水柠檬Xiangshui lemon	32	24	3	5	75.0
菊花芯柚Juhuaxinyou pummelo	9	9	0	0	100.0
总计/平均 Sum/Average	49	41	9	7	83.7

Efficient in vivo Induction of Tetraploids from Three Mono-Embryonic Citrus Cultivars

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Figures/Tables 8

References 28

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品种 Cultivar	倍性 Ploidy	叶片厚度/mm Leaf thickness	叶形指数 Leaf shape index	油胞密度/ （个 · cm^-2） Oil gland density	气孔密度/ （个 · mm^-2） Stomatal density	气孔长轴长度/μm Long axis length of stomatal	气孔短轴长度/μm Short axis length of stomata
沃柑Orah mandarin	2x	0.27 ± 0.03	2.49 ± 0.18	102.1 ± 16.4	278.1 ± 23.0	22.0 ± 1.6	18.8 ± 1.0
沃柑Orah mandarin	4x	0.35 ± 0.02^**	2.28 ± 0.31	43.3 ± 16.0^**	165.2 ± 23.9^**	31.4 ± 2.3^**	26.0 ± 2.2^**
香水柠檬Xiangshui lemon	2x	0.27 ± 0.03	2.11 ± 0.20	98.9 ± 26.4	377.1 ± 69.6	21.4 ± 1.8	17.7 ± 1.8
香水柠檬Xiangshui lemon	4x	0.33 ± 0.02^**	1.81 ± 0.14^**	65.2 ± 14.3^**	275.7 ± 51.5^**	28.5 ± 2.1^**	22.9 ± 1.6^**
菊花芯柚Juhuaxinyou pummelo	2x	0.33 ± 0.03	2.17 ± 0.22	97.7 ± 20.5	333.6 ± 37.5	24.8 ± 1.6	18.7 ± 1.1
菊花芯柚Juhuaxinyou pummelo	4x	0.37 ± 0.05^*	1.81 ± 0.20^**	33.0 ± 14.2^**	224.8 ± 33.5^**	30.3 ± 2.7^**	22.6 ± 2.0^**

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