依据须根形态学特征精准筛选柑橘四倍体

doi:10.16178/j.issn.0528-9017.20240725

摘要/Abstract

摘要：

柑橘四倍体资源可为三倍体杂交亲本和砧木遗传改良提供新种质。本研究建立了一套精准发掘柑橘四倍体的简易方法,将二倍体柑橘种子密播于蛭石基质中发芽成苗,待幼苗须根充分发育后肉眼观察,须根明显短粗的植株为柑橘四倍体。采用该方法,分别从枸头橙、常山胡柚、鸡尾葡萄柚、枳橙、枳壳、本地早的616、354、476、536、648和289株实生苗中,分别发掘出疑似四倍体8、6、8、7、4和1株,经流式细胞术倍性鉴定确认均为四倍体,6个品种的形态学筛选准确率均达到100%。上述6个品种的四倍体群体检出率分别为1.30%、1.69%、1.68%、1.30%、0.62%和0.35%。本方法四倍体群体检出率高、易操作,实现了100%的筛选准确率,在无倍性鉴定仪器条件下仍可完成四倍体选育工作。

关键词: 柑橘, 须根形态, 四倍体, 精准发掘

Abstract:

Citrus tetraploid resources can provide new germplasm for triploid hybrid parents and stock genetic improvement. In this study, a simple method was established to accurately explore citrus tetraploid, which involves densely sowing diploid citrus seeds in a vermiculite substrate to germinate and form seedlings. After the seedlings have fully developed their fibrous roots, the plants with significantly short and thick fibrous roots are observed with the naked eye as tetraploid citrus. By using this method, we screened 8, 6, 8, 7, 4 and 1 putative tetraploids respectively from 616, 354, 476, 536, 648 and 289 seedlings of Citrus aurantium cv. Goutoucheng, Citrus aurantium cv. Changshanhuyou, Citrus paradisi Macf., Citrus sinensis×Poncirus trifoliata, Poncirus trifoliata Raf., Citrus reticulata Blanco cv. Succosa. All the tetraploids were confirmed by flow cytometry, and the screening accuracy rate of morphological characteristics reached 100%. The tetraploid population detection rates of the above six varieties were 1.30%, 1.69%, 1.68%, 1.30%, 0.62% and 0.35%, respectively. This method has a high detection rate and is easy to operate, which achieves 100% screening accuracy and completes the work of tetraploid breeding without the condition of ploidy identification instrument.

Key words: citrus, fibrous root morphology, tetraploid, accurate exploration

中图分类号:

S666.1

王平, 柯甫志. 依据须根形态学特征精准筛选柑橘四倍体[J]. 浙江农业科学, 2026, 67(1): 62-66.

WANG Ping, KE Fuzhi. Accurate exploration of citrus tetraploid based on morphological characteristics of seedling fibrous roots[J]. Journal of Zhejiang Agricultural Sciences, 2026, 67(1): 62-66.

图/表 4

图1 枸头橙四倍体和二倍体幼苗须根形态学差异比较 A为四倍体,B为二倍体。

Fig.1 Morphological difference of fibrous roots between tetraploid and diploid seedlings of Citrus aurantium cv. Goutoucheng

图2 二倍体和四倍体幼苗嫩根组织的流式细胞仪出峰图左图为二倍体,右图为四倍体。图中横坐标表示DNA含量;纵坐标表示所检测的细胞数量。

Fig.2 Peak plots of root tissue of diploid and tetraploid seedling by flow cytometry

表1 枸头橙四倍体与二倍体幼苗的形态学指标

Table 1 Morphological indexes between tetraploid and diploid seedlings of Citrus aurantium cv. Goutoucheng

幼苗类型	叶片数	株高/cm	叶长/cm	叶宽/cm	叶形指数	主根粗/cm	主根长/ cm	主根尖粗/cm	须根数	第一须根粗/cm	第一须根长/cm	第一须根尖粗/cm	最长须根长/cm
二倍体	5.33±0.58 a	5.60±0.35 a	3.17±0.06 a	1.33±0.12 a	2.39±0.24 a	0.17±0.01 a	14.10±1.15 a	0.05±0.02 a	22.33±2.08 a	0.04±0.01 b	6.33±0.65 a	0.04±0.01 b	10.97±1.79 a
四倍体	3.67±0.58 b	4.67±0.15 b	2.50±0.10 b	1.43±0.15 a	1.75±0.11 b	0.19±0.01 a	8.17±0.76 b	0.09±0.02 a	18.00±2.00 a	0.07±0.02 a	3.23±1.25 b	0.07±0.01 a	4.77±1.12 b

表2 不同柑橘品种四倍体形态学筛选准确率和群体检出率

Table 2 Screening accuracy and population detection rate of tetraploid morphological of different citrus varieties

品种	种子数	成苗数	疑似数	四倍体株数	准确率/%	群体检出率/%
枸头橙	555	616	8	8	100	1.30
常山胡柚	321	354	6	6	100	1.69
鸡尾葡萄柚	420	476	8	8	100	1.68
枳橙	514	536	7	7	100	1.30
枳壳	609	648	4	4	100	0.62
本地早	245	289	1	1	100	0.35

参考文献 19

[1]	GORA J S, RAM C, POONIA P K, et al. Polyploid rootstocks in citrus for mitigation of biotic and abiotic stresses: a review[J]. Journal of Agriculture and Ecology, 2022, 13(1): 1-19.
[2]	NARUKULLA V, LAHANE Y, UGE P, et al. Production of triploid seedless sweet orange [Citrus sinensis (L.) osbeck] cv. mosambi: a success story[J]. Agronomy, 2024, 14(4): 829.
[3]	王平, 罗君琴, 聂振朋, 等. 凯旋柑小种子三倍体选育初报[J]. 浙江柑橘, 2019, 36(4): 24-26.
	WANG P, LUO J Q, NIE Z P, et al. Preliminary report on triploid breeding of small seeds of Kaixuan citrus[J]. Zhejiang Ganju, 2019, 36(4): 24-26.
[4]	解凯东, 王惠芹, 王晓培, 等. 单胚性二倍体为母本与异源四倍体杂交大规模创制柑橘三倍体[J]. 中国农业科学, 2013, 46(21): 4550-4557.
	XIE K D, WANG H Q, WANG X P, et al. Extensive Citrus triploid breeding by crossing monoembryonic diploid females with allotetraploid male parents[J]. Scientia Agricultura Sinica, 2013, 46(21): 4550-4557.
[5]	GUERRA D, WITTMANN M T S, SCHWARZ S F, et al. Comparison between diploid and tetraploid citrus rootstocks: morphological characterization and growth evaluation[J]. Bragantia, 2014, 73(1): 1-7.
[6]	GROSSER J W, BARTHE G A, CASTLE B, et al. The development of improved tetraploid citrus rootstocks to facilitate advanced production systems and sustainable citriculture in Florida[J]. Acta Horticulturae, 2015(1065): 319-327.
[7]	DUTT M, MAHMOUD L M, CHAMUSCO K, et al. Utilization of somatic fusion techniques for the development of HLB tolerant breeding resources employing the Australian finger lime (Citrus australasica)[J]. PLoS One, 2021, 16(8): e0255842.
[8]	SALEH B, ALLARIO T, DAMBIER D, et al. Tetraploid citrus rootstocks are more tolerant to salt stress than diploid[J]. Comptes Rendus Biologies, 2008, 331(9): 703-710.
[9]	MAHMOUD L M, KILLINY N, HOLDEN P, et al. Physiological and biochemical evaluation of salt stress tolerance in a citrus tetraploid somatic hybrid[J]. Horticulturae, 2023, 9(11): 1215.
[10]	HUSSAIN S, SOHAIL H, NOOR I, et al. Physiological and biochemical determinants of drought tolerance in tetraploid vs diploid sour orange citrus rootstock[J]. The Journal of Horticultural Science and Biotechnology, 2023, 98(6): 772-785.
[11]	DUTT M, VASCONCELLOS M, SONG K J, et al. In vitro production of autotetraploid Ponkan mandarin (Citrus reticulata Blanco) using cell suspension cultures[J]. Euphytica, 2010, 173(2): 235-242.
[12]	YAMAMOTO M, ARITA S, KOZAI N. Simple methods for producing tetraploids in polyembryonic citrus[J]. Japan Agricultural Research Quarterly: JARQ, 2024, 58(2): 113-119.
[13]	REN J, LU X, DUAN Y Y, et al. In vivo tetraploid induction of mono-embryonic citrus genotypes by colchicine treatment[J]. Scientia Horticulturae, 2024, 338: 113701.
[14]	梁武军, 解凯东, 郭大勇, 等. 柑橘10个品种实生后代多倍体的发掘及SSR鉴定[J]. 园艺学报, 2014, 41(3): 409-416.
	LIANG W J, XIE K D, GUO D Y, et al. Spontaneous generation and SSR characterization of polyploids from ten citrus cultivars[J]. Acta Horticulturae Sinica, 2014, 41(3): 409-416.
[15]	周锐, 解凯东, 王伟, 等. 依据多倍体形态特征快速高效发掘柑橘四倍体[J]. 园艺学报, 2020, 47(12): 2451-2458.
	ZHOU R, XIE K D, WANG W, et al. Efficient identification of tetraploid plants from seedling populations of apomictic citrus genotypes based on morphological characteristics[J]. Acta Horticulturae Sinica, 2020, 47(12): 2451-2458.
[16]	ALEZA P, FROELICHER Y, SCHWARZ S, et al. Tetraploidization events by chromosome doubling of nucellar cells are frequent in apomictic citrus and are dependent on genotype and environment[J]. Annals of Botany, 2011, 108(1): 37-50.
[17]	陈昊, 谢善鹏, 解凯东, 等. 柑橘13个多胚品种同源四倍体高效发掘与分子鉴定[J]. 果树学报, 2023, 40(11): 2297-2306.
	CHEN H, XIE S P, XIE K D, et al. Efficient exploration and SSR identification of autotetraploids from the seedlings of thirteen apomictic citrus genotypes[J]. Journal of Fruit Science, 2023, 40(11): 2297-2306.
[18]	谢善鹏, 解凯东, 夏强明, 等. 柑橘6个地方品种资源四倍体高效发掘及分子鉴定[J]. 果树学报, 2022, 39(1): 1-9.
	XIE S P, XIE K D, XIA Q M, et al. Efficient exploration and SSR identification of 53 doubled diploid seedlings from six local citrus cultivars and germplasm resources[J]. Journal of Fruit Science, 2022, 39(1): 1-9.
[19]	王平, 聂振朋, 孙立方, 等. 柑橘实生幼苗倍性检测的最佳部位[J]. 浙江农业科学, 2020, 61(11): 2260-2261.
	WANG P, NIE Z P, SUN L F, et al. Confirmation of optimum organ of citrus seedling for ploidy identification[J]. Journal of Zhejiang Agricultural Sciences, 2020, 61(11): 2260-2261.