柑橘3个单胚性品种四倍体高效活体诱导

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

摘要/Abstract

摘要：

以沃柑、香水柠檬和菊花芯柚等3个单胚性品种实生幼苗为材料，创制四倍体新种质。将实生苗上胚轴切断后置于黑暗条件保湿培养，9 d时切口处产生愈伤组织，在光照条件下采用0.025%秋水仙素溶液浸润切口处愈伤组织2 h，诱导其再生不定芽；待再生植株长出3 ~ 4片真叶后，利用流式细胞术和根尖染色体计数进行倍性鉴定，单/多胚InDel分子标记进行胚性鉴定。分别获得沃柑、香水柠檬、菊花芯柚单胚性四倍体8、21、9株，平均四倍体诱导率为16.2%；染色体加倍后的再生植株叶片变厚、气孔变大，叶形指数、油胞密度和气孔密度降低。本研究结果为以单胚四倍体为母本的倍性杂交且无需幼胚离体挽救培养创制无核三倍体新种质提供了核心种源。

关键词: 柑橘, 单胚, 四倍体, 秋水仙素, 种质创新

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

卢鑫, 任杰, 段耀园, 解凯东, 肖公傲, 夏泽文, 王蓉, 伍小萌, 郭文武. 柑橘3个单胚性品种四倍体高效活体诱导[J]. 园艺学报, 2025, 52(9): 2291-2299.

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.

图/表 8

表1 InDel引物序列

Table 1 Sequences of InDel primers

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

图1 秋水仙素处理沃柑上胚轴诱导四倍体流程 A：成熟果实；B：单胚种子催芽；C：实生幼苗；D：实生幼苗切断上胚轴；E、F：黑暗条件诱导上胚轴伤口形成愈伤组织；G、H：秋水仙素处理上胚轴愈伤组织；I：上胚轴愈伤组织再生不定芽；J：流式细胞仪倍性鉴定；K：再生植株移栽

Fig. 1 Process of tetraploid induction of Orah mandarin epicotyls by colchicine treatment A：Mature fruits：B：Mono-embryonic seed germination；C：Orah seedlings；D：Cutting off the epicotyls of seedlings；E-F：Inducing callus tissue on the wounds of epicotyls under dark；G-H：Treatment of callus tissue with colchicine；I：Regeneration of adventitious buds from callus tissue on the epicotyls；J：Ploidy identification by flow cytometry；K：Regenerated plants are transplanted

表2 秋水仙素处理柑橘3个单胚性品种上胚轴的四倍体诱导效果

Table 2 Effect of tetraploid induction from three mono-embryonic citrus cultivars by treating epicotyls with colchicine solution

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

图2 柑橘3个品种四倍体再生植株单/多胚InDel分子标记鉴定

Fig. 2 InDel molecular marker identification of mono/poly embryony in regenerated tetraploid plants of three citrus cultivars

表3 一年苗龄四倍体柑橘植株倍性鉴定

Table 3 Ploidy re-identification of regenerated citrus tetraploid plants

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

图3 流式细胞术（A）与根尖染色体计数（B）鉴定再生植株倍性

Fig. 3 Ploidy identification by flow cytometry（A）and root tip chromosome counting（B）in regenerated tetraploid plants

图4 柑橘3个品种四倍体与二倍体的叶形、油胞和气孔

Fig. 4 Leaf morphology，oil gland and stomata in tetraploid and diploid control plants of the three citrus cultivars

表4 柑橘3个品种四倍体与二倍体叶形、油胞和气孔比较

Table 4 Comparison of leaf morphology，oil gland and stomata in tetraploid and diploid control plants of the three citrus cultivars

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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