解凯东^,1, 彭珺¹, 袁东亚¹, 强瑞瑞¹, 谢善鹏¹, 周锐¹, 夏强明¹, 伍小萌¹, 柯甫志², 刘高平³, GROSSER Jude W⁴, 郭文武^,11华中农业大学园艺林学学院/园艺植物生物学教育部重点实验室,中国武汉 430070
²浙江省农业科学院柑桔研究所,中国浙江台州 318020
³浙江省台州市黄岩区果树技术推广总站,中国浙江台州 318020
⁴美国佛罗里达大学柑橘研究与教育中心,美国佛罗里达 33850

Production of Citrus Triploids Based on Interploidy Crossing with Bendizao and Man Tangerines as Female Parents

XIE KaiDong^,1, PENG Jun¹, YUAN DongYa¹, QIANG RuiRui¹, XIE ShanPeng¹, ZHOU Rui¹, XIA QiangMing¹, WU XiaoMeng¹, KE FuZhi², LIU GaoPing³, GROSSER Jude W⁴, GUO WenWu^,11College of Horticulture & Forestry Sciences, Huazhong Agricultural University/Key Laboratory of Horticultural Plant Biology (Ministry of Education), Wuhan 430070, China
²Citrus Research Institute, Zhejiang Academy of Agricultural Sciences, Taizhou 318020, Zhejiang, China
³Huangyan Fruit Tree Technology Promotion General Station, Taizhou 318020, Zhejiang, China
⁴Citrus Research and Education Center, IFAS, University of Florida, Lake Alfred FL 33850, USA

通讯作者: 郭文武,E-mail： guoww@mail.hzau.edu.cn

责任编辑: 赵伶俐
收稿日期:2020-09-1接受日期:2020-10-14网络出版日期:2020-12-01

基金资助:

国家自然科学基金国际合作重点项目.31820103011 湖北省科技支撑计划.2020BBA036 广东省科技计划.2018B020202009 中央高校基本科研业务费专项资金.2662019QD048 中央高校基本科研业务费专项资金.2662018PY007

Received:2020-09-1Accepted:2020-10-14Online:2020-12-01
作者简介 About authors
解凯东,Tel：027-87287393;E-mail： xiekaidong@mail.hzau.edu.cn。














摘要
【目的】基于二倍体与四倍体倍性杂交策略创制柑橘三倍体新种质。【方法】以二倍体为母本,四倍体为父本进行人工授粉,授粉后85 d采摘幼果并对未成熟种子实施幼胚离体挽救培养;再生植株后,用流式细胞仪和根尖染色体计数法对再生植株进行倍性鉴定;并用SSR标记对三倍体后代进行分子鉴定。【结果】以本地早橘和槾橘为母本,8个异源四倍体体细胞杂种和1个双二倍体为父本,配置9个倍性杂交组合,共授粉2 749朵花,坐果489个,平均坐果率17.8%;培养幼嫩种子2 239粒,经幼胚离体挽救培养,共再生植株260株;用流式细胞仪和根尖染色体计数检测再生植株倍性,获得三倍体141株;SSR分子鉴定表明从槾橘×NS组合随机选取的50株三倍体后代全部为双亲的有性后代;三倍体后代在温室生长一年后,采用嫁接（枳砧）将这些三倍体定植于田间。【结论】这些三倍体新种质为本地早橘和槾橘无核新品种培育奠定了宝贵的材料基础。
关键词： 柑橘;无核育种;三倍体;幼胚离体挽救培养;流式细胞仪

Abstract
【Objective】The aim of this study was to create the citrus triploids based on diploid and tetraploid ploidy cross strategy. 【Method】The artificial pollination was conducted with diploid as female parent and tetraploid as male parent. Young fruits were sampled at 85 d after pollination (DAP) and immature seeds were extracted and subjected to in vitro embryo rescue. Following plantlet regeneration from the embryos, their ploidy level was determined by flow cytometry and root-tip chromosome counting, as well as the genetic origin determined using simple sequence repeat (SSR) markers. 【Result】A total of nine interploidy crosses were carried out by using Bendizao tangerine and Man tangerine as female parents and eight allotetraploid somatic hybrids and one doubled diploids as male parents. From all crosses, 2 749 flowers were pollinated and 489 fruits were set, with an average fruit setting rate of 17.8%. By conducting in vitro immature embryo rescue, totally 260 plants were regenerated from 2 239 seeds cultured. By determining their ploidy level using flow cytometry and root-tip chromosome counting, 141 seedlings were proven to be triploids. SSR analysis showed that all of 50 randomly selected triploid plants from Man tangerine × NS were the hybrids of their both parents. After one year growing in greenhouse, all triploids were grafted onto Poncirus trifoliata in the field to accelerate flowering. 【Conclusion】These triploid citrus plants obtained herein provided elite materials for potential seedless variety selection.
Keywords：Citrus;seedless breeding;polyploid;embryo rescue;flow cytometry


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解凯东, 彭珺, 袁东亚, 强瑞瑞, 谢善鹏, 周锐, 夏强明, 伍小萌, 柯甫志, 刘高平, GROSSER Jude W, 郭文武. 以本地早橘和槾橘为母本倍性杂交创制柑橘三倍体[J]. 中国农业科学, 2020, 53(23): 4961-4968 doi:10.3864/j.issn.0578-1752.2020.23.020
XIE KaiDong, PENG Jun, YUAN DongYa, QIANG RuiRui, XIE ShanPeng, ZHOU Rui, XIA QiangMing, WU XiaoMeng, KE FuZhi, LIU GaoPing, GROSSER Jude W, GUO WenWu. Production of Citrus Triploids Based on Interploidy Crossing with Bendizao and Man Tangerines as Female Parents[J]. Scientia Agricultura Sinica, 2020, 53(23): 4961-4968 doi:10.3864/j.issn.0578-1752.2020.23.020


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0 引言

【研究意义】柑橘是世界第一大水果,也是我国南方栽培面积最广、经济地位最重要的果树,在乡村振兴和精准扶贫方面发挥着重要作用^[1]。我国柑橘主要供应鲜果市场,无核果实由于食用方便、品质佳,愈发受消费者青睐^[2,3]。虽然我国柑橘地方品种繁多,但多数有核,难以满足当今市场对无核优质果实的消费需求。因此,果实无核化是解决我国特色柑橘无核品种匮乏和提升地方品种商品价值的重要手段。【前人研究进展】本地早橘和槾橘均原产我国,本地早橘为早熟品种,味甜少酸,汁多化渣;槾橘是一个品质较优的晚熟鲜食品种。两者在浙江均有百年以上栽培历史,曾是当地大力发展的主栽品种。但由于其种子较多和长期无性繁殖引起的种性衰退,两者果实品质严重下降。特别是国外一些无核品种的引进和种植,对本地早橘和槾橘的冲击较大,导致其栽培面积大范围缩减^[4]。三倍体雌雄配子败育,是天然的不育类型,且其不育性状不受环境影响,是培育无核柑橘的重要手段^[5]。柑橘中,二倍体为母本,与四倍体为父本的倍性杂交是培育三倍体最经典的策略^[6]。基于该策略,美国、意大利、西班牙、日本和中国等国的科研工作者培育出了大量柑橘三倍体,并成功选育出一批表现优良的无核新品种^[2-3,7-12]。【本研究切入点】近年来,柑橘细胞工程育种技术（包括体细胞杂交和同源四倍体快速发掘技术）飞速发展,新获得了大批柑橘异源四倍体体细胞杂种和同源四倍体^[7,13-15],为柑橘倍性杂交创制无核三倍体提供了优良的育种亲本。异源四倍体体细胞杂种包含两个融合亲本的全部遗传物质,综合了其双亲优良性状,如体细胞杂种‘Succari甜橙+Page橘柚’果实风味佳,少核且早熟^[16],以其为父本倍性杂交创制的三倍体后代可能兼具3个亲本的优良性状,遗传变异丰富,有利于筛选果实品质性状优良且无核的柑橘新品种。【拟解决的关键问题】本研究以浙江黄岩地方品种本地早橘和槾橘为母本,与8个柑橘异源四倍体体细胞杂种和1个双二倍体为父本倍性杂交,结合幼胚离体挽救培养和流式细胞仪倍性分析,创制一批具有丰富遗传变异的三倍体植株,为我国柑橘无核新品种培育奠定宝贵的材料基础。

1 材料与方法

1.1 材料

2014—2018年,以柑橘单胚性品种—槾橘（Citrus tardiferax Hort. ex Tan）和单、多胚混合型品种—本地早橘（C. succosa Hort. ex Tanaka）为母本,异源四倍体体细胞杂种NS（C. reticulata Blanco × C. paradisi Macf. + C. sinensis L. Osbeck）^[7]、SP（C. sinensis L. Osbeck + C. reticulata Blanco × C. paradisi Macf.）^[7]、MD（C. reticulata Blanco × C. sinensis L. Osbeck + C. reticulata Blanco）^[16]、SM（C. sinensis L. Osbeck + C. reticulata Blanco × C. sinensis L. Osbeck）^[7]、PL（C. reticulata Blanco × C. paradisi Macf. + C. reticulata Blanco）^[16]、PM（C. reticulata Blanco × C. paradisi Macf. + C. reticulata Blanco × C. sinensis L. Osbeck）^[16]、VP（C. sinensis L. Osbeck + C. reticulata Blanco × C. paradisi Macf.）^[17]、PCS（C. reticulata Blanco × C. paradisi Macf. + C. clementina Hort. ex Tanaka × C. unshiu）^[16]和Succari甜橙（C. sinensis L. Osbeck）双二倍体（4X Suc）为父本,配置9个倍性杂交组合。所有四倍体花粉均采自华中农业大学柑橘研究所种质资源圃。本地早橘授粉地点为华中农业大学柑橘研究所种质资源圃;槾橘授粉地点为浙江省农业科学院柑桔研究所试验基地。

1.2 花粉制备及人工授粉

柑橘初花期,采摘四倍体父本即将开放的花带回实验室,用镊子将花药剥下后铺于有滤纸的培养皿,并将其置于烘箱28℃烘1—2 d,待花粉完全干燥后,将其收集至离心管后置于-20℃冰箱保存备用。人工授粉在柑橘盛花期前进行,选取生长健壮、花量大的枝条进行标记,将已开花和小花蕾全部去掉,枝条上仅保留待授粉的花;用镊子人工去雄后,用小毛笔蘸取少量父本花粉均匀涂于待授粉花的柱头上。若授粉后8 h内下雨,需重新授粉一次。

1.3 幼胚离体挽救培养

幼胚离体挽救培养参考XIE等^[12]的方法。采摘授粉后85 d的幼果带回实验室,无菌条件下将幼果浸泡于75%酒精消毒15 min,之后再置于酒精灯上燃烧消毒,酒精燃烧完后将果实剖开剥取出未成熟种子。为提高种子萌发率,将其种皮从合点端切开并向珠孔端轻轻撕至幼胚暴露,再将处理后的种子接种至萌发培养基（MT培养基+1 mg?L^-1 GA₃）。光照培养室培养1个月后,将形成的胚状体置于生芽培养基（MT + 0.5 mg?L^-1 BA + 0.5 mg?L^-1 KT + 0.1 mg?L^-1 NAA）中增殖生芽;待其长出2—3片叶后将茎切下,置于生根培养基（1/2 MT + 0.1 mg?L^-1 IBA + 0.5 mg?L^-1 NAA + 0.5 g?L^-1活性炭）中诱导生根。培养条件：温度：（25±1）℃;湿度：70%左右;光照强度：40 μmol?m^-2?s^-1;光照时间：16 h/天。

1.4 再生植株倍性分析

流式细胞仪（Sysmex,Japan）倍性分析参照解凯东等^[2]的方法。以二倍体母本叶片为对照,取待测样品0.5 cm²大小的叶片于塑料皿中,加入200 μL细胞裂解液（Precise-P,Sysmex）,并用刀片将其充分切碎;加入800 μL DAPI染色液（Precise-P,Sysmex）后,用33 μm微孔滤膜将样品过滤到2.5 mL试管并上机检测。样品倍性分析图像由Flomax软件（Sysmex,Japan）自动生成。

根尖染色体计数参考LAN等^[18]的方法。取1.5 mm左右生长旺盛的根尖,用饱和对二氯苯20℃预处理2—4 h;0.075 mol?L^-1 KCl低渗处理30 min后,用新鲜的卡诺固定液（无水乙醇:冰乙酸 = 3:1）室温下固定24 h,最后置于70%乙醇中4℃保存备用。制片前,将根尖用磷酸缓冲液清洗干净,并置于2%纤维素酶 + 2%果胶酶（1:1）混合酶液中,37℃下处理1 h左右;酶解结束后,吸取1—2个根尖于干净的载玻片上,滴1滴1%卡宝品红染液染色1 min后盖上盖玻片,用铅笔均匀敲打盖玻片直至材料分散均匀,最后用Olympus DP70显微镜镜检并拍照。

1.5 植株移栽及嫁接

移栽前将植株置于试管中室温下炼苗3—5 d,之后将其移入小塑料杯,待其成活后再移入大营养钵并置于温室。苗期需加强管理,保证幼苗的正常生长。植株在温室生长1年后,次年8月下旬取其木质化的枝条为接穗,以枳为砧木将其嫁接于田间（枳砧提前定植）。

1.6 SSR分子鉴定

基因组DNA提取参照CHENG等^[19]的方法,DNA浓度和质量用NanoDrop 1000紫外分光光度计（Thermo Scientific,USA）检测。DNA原液用TE缓冲液稀释至约50 ng?μL^-1。SSR引物（表1）由上海生工生物工程股份有限公司合成。SSR分析方法具体如下：PCR体系为10 μL：2×PCR反应mix（Vazyme）5 μL,正反向引物各0.1 μL,DNA模板1 μL,灭菌超纯水3.8 μL。PCR反应在ProFlex PCR仪（ABI,USA）上进行,扩增程序：94℃预变性5 min;94℃变性60 s,55℃退火30 s,72℃延伸60 s,32个循环;72℃延伸7 min;最后12℃保存。扩增完成后,PCR产物在恒定电压95 V条件下,用2.5% Metaphor琼脂糖凝胶（Lonza,USA）电泳约1 h后用凝胶成像系统（BIO-RAD,USA）拍照。

Table 1
表1
表13对SSR引物序列
Table 1Sequences of three pairs of SSR markers

引物名称 Marker name	正向序列 Forward sequence	反向序列 Reverse sequence	来源 Reference
Mest88	CATTAAAATATCCGTGCCGC	GAGCAAGTGCGTTGTTGTGT	[13]
Csin0191	GACTTGAACGGTTGCAATGA	TGATGTCCCAAAAGTGTGGA	[20]
Csin0504	CCCTTATTCAGACGCCAAAA	AGTCCGCCCAGTTACAACAT	[20]

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2 结果

2.1 授粉、胚抢救及植株再生

以二倍体槾橘和本地早橘为母本,8个异源四倍体体细胞杂种和1个双二倍体为父本,配置了9个倍性杂交组合（表2）。其中,以槾橘为母本,NS为父本配置了1个组合;以本地早橘为母本,7个异源四倍体（MD、SM、SP、PL、PM、VP、PCS）及双二倍体4X Suc为父本,配置8个杂交组合。由表2可知,9个杂交组合共授粉2 749朵花,坐果489个,平均坐果率17.8%。其中以本地早橘为母本的组合坐果率较低,介于10.3%—26.4%;而槾橘×NS坐果率较高,为42.9%。9个组合共离体培养未成熟种子2 239粒,其中以槾橘为母本培养种子267粒,以本地早橘为母本培养种子1 972粒。经幼胚离体挽救培养,9个组合共再生植株260株,平均植株再生率11.6%;其中,以本地早橘为母本的8个组合再生植株181株,植株再生率介于3.2%—37.1%;槾橘×NS组合再生植株79株,植株再生率29.6%。幼胚离体挽救培养、植株再生及田间嫁接过程见图1。

Table 2
表2
表2以本地早橘和槾橘为母本的倍性杂交组合及植株再生
Table 2Embryo rescue, plant regeneration and ploidy determination in the 2x × 4x interploidy crosses

杂交组合 Cross	授粉花数（朵） No. flowers	坐果数（个） No. fruits	坐果率（%） Fruit setting rate	种子数（粒） No. seeds	再生植株数（株） No. plants	三倍体（株） No. triploids
本地早橘×MD Bendizao tangerine × MD	294	66	22.4	364	14	4
本地早橘×SM Bendizao tangerine × SM	356	94	26.4	554	20	12
本地早橘×SP Bendizao tangerine × SP	357	69	19.3	376	12	6
本地早橘×PL Bendizao tangerine × PL	228	32	14.0	85	16	7
本地早橘×PM Bendizao tangerine × PM	221	38	17.2	91	9	5
本地早橘×VP Bendizao tangerine × VP	396	58	14.6	151	47	11
本地早橘× 4X Suc Bendizao tangerine × 4X Suc	363	54	14.9	246	24	7
本地早橘×PCS Bendizao tangerine × PCS	464	48	10.3	105	39	10
槾橘×NS Man tangerine × NS	70	30	42.9	267	79	79
总计 Total	2749	489	17.8	2239	260	141

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图1

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图1槾橘×NS组合幼胚离体挽救培养、植株再生及田间嫁接

a：授粉后85 d的种子（下）及对照种子（上）;b：接种于萌发培养基的种子;c：种子萌发形成胚状体;d：生芽培养;e：生根培养;f：植株炼苗;g：移入小塑料杯的三倍体;h：移入温室的三倍体群体;i：嫁接至田间的三倍体植株（枳砧）
Fig. 1Embryo rescue, plant regeneration and grafting of triploid plants from the cross of Man tangerine × NS

a: Seeds of ‘Man tangerine × NS’ obtained at 85 DAP (lower) and open-pollinated seeds (upper); b: Seeds cultured in medium for germination; c: Germinated embryos on the medium; d: Embryonoids transferred on the medium for shoot induction; e: A shoot transferred to the medium for root induction; f: Plantlet acclimation; g: A triploid plantlet in plastic pot; h: Triploid population in greenhouse; i: Grafted triploids in the field

2.2 再生植株倍性分析

用流式细胞仪对所有再生植株进行倍性分析（图2）,表明以单胚性槾橘为母本再生的79棵植株,经检测全部为三倍体（表2）,三倍体获得率100%。以单、多胚混合型本地早橘为母本的8个杂交组合再生的181棵植株,经检测三倍体有62株（表2）,三倍体获得率34.3%。此外,从‘槾橘×NS’组合随机挑选3株三倍体用于根尖染色体计数分析,表明三倍体染色体均为27条,验证了流式细胞仪倍性检测的准确性。上述多倍体在温室生长一年后,已全部嫁接于田间。

图2

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图2槾橘×NS组合再生植株的倍性鉴定

a：流式细胞仪倍性鉴定（PK1=50,二倍体;PK2=75,三倍体）;b、c：根尖染色体计数（b：二倍体,2n=2x=18;c：三倍体,2n=3x=27）,标尺=5 μm
Fig. 2Ploidy determination of regenerated plants from Man tangerine × NS

a: Ploidy analysis of regenerated plants and diploid control by flow cytometry (PK1=50, diploid; PK2=75, triploid); b-c: Shoot-tip chromosome counting (b: diploid, 2n=2x=18; c: triploid, 2n=3x=27), Bar=5 μm

2.3 三倍体后代的分子鉴定

用3对SSR引物对‘槾橘×NS’组合中随机选取的50株三倍体后代进行分子鉴定,显示50株三倍体后代均含有父母本特异条带,表明所有检测的三倍体后代均为双亲的有性后代（图3）。

图3

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图3槾橘×NS组合再生三倍体后代的SSR分子鉴定

引物Mest88的扩增谱图;M：槾橘;F：NS;1—50：三倍体后代
Fig. 3The SSR profile of 50 triploid plants obtained from Man tangerine × NS

SSR profile of primer Mest88; M: Man tangerine; F: NS; 1-50: The triploid progenies

3 讨论

柑橘是我国南方果树主导产业之一,栽培历史悠久,资源丰富,地方良种多;但我国目前规模化栽培的80余个柑橘品种,约一半引自国外^[21]。特别是一些优质的无核品种,如温州蜜柑、纽荷尔脐橙、伦晚脐橙、红美人等,在我国柑橘产区大面积引种栽培,导致一些地方特色品种,如本地早橘和槾橘的栽培面积逐年缩减。果实有核和品质衰退可能是导致这些品种在市场上面临淘汰的根本原因。柑橘长期无性繁殖容易感染并积累病毒,导致果实品质降低^[22],难以满足消费者对品质提升的需求。与有核果实相比,无核果实食用方便,倍受消费者青睐。因此,采用合理的育种手段,实现果实无核和其他品质性状的综合改良（或提纯复壮）是有效提升这些地方品种市场竞争力的重要保障。

与二倍体果树相比,三倍体由于细胞核内染色体增加了一套,其形态和生理往往会有一些新的变化,通常表现为器官巨大、育性降低、新陈代谢旺盛和对环境适应性增强等。如三倍体梨新品种‘华幸’^[23]、‘华香酥’^[24]均表现为果大、品质优和抗黑心病等特点;三倍体枇杷‘华玉无核1号’果实无核,可溶性固形物含量高,丰产性好^[25]。而柑橘三倍体除表现果实无核外,部分三倍体优系在抗逆性^[26,27]、成熟期调控^[28,29]和功能性成分代谢^[30,31]及抗氧化活性^[32]等方面也表现出优异的应用价值。就柑橘而言,由于多数品种单性结实能力强,不用担心由于三倍体育性降低而导致坐果难的问题;而柑橘等果树多数以无性繁殖为主,通过培育三倍体实现无核和其他优良性状结合,一旦改良成功,即可长期持续利用。如西班牙为解决柑橘果实有核和市场周年供应等问题,采用倍性杂交培育三倍体的策略,成功培育出‘Sofar’‘Garbí’‘Alborea’和‘Albir’等几个成熟期不同的三倍体新品种且大面积栽培,填补了西班牙柑橘鲜果市场1—3月份的空白^[28-29,33]。美国科学家针对柚和葡萄柚含有呋喃香豆素的问题,以柚为母本,与葡萄柚同源四倍体（父本）倍性杂交,成功培育出一个呋喃香豆素含量很低,无核、果实风味好且无苦味的三倍体葡萄柚新品种,在美国已商业化种植^[34]。因此,培育三倍体在柑橘遗传改良中具有巨大的应用潜力。

本研究基于2x×4x倍性杂交策略,以本地早橘和槾橘为母本培育三倍体,是实现这两个品种无核遗传改良的有效方法。近20年来,虽然国内外在柑橘三倍体无核育种方面取得了一定进展,但针对本地早橘和槾橘有核性状创制三倍体的报道较少;除美国外,国外多数国家倍性杂交主要以同源四倍体为父本^[8,9,10]。本研究选用的四倍体父本大多数为2个优良二倍体品种经原生质体融合再生创制的异源四倍体体细胞杂种,与同源四倍体相比,异源四倍体体细胞杂种综合了双亲的遗传物质,以其为父本与本地早橘和槾橘倍性杂交获得的三倍体后代不仅果实无核,而且可能兼具3个优良二倍体亲本的遗传物质,遗传变异丰富,有利于培育无核且其他性状（如高糖、极早熟、易剥皮等）优良的柑橘新品种。

前期研究发现,以本地早橘为母本倍性杂交,其坐果率和植株再生率均较低^[3],但以其为母本创制的三倍体后代果实不仅无核,且部分株系果实表现出极易剥皮或与亲本相比成熟期提前等优点（武汉地区9月中旬果实已转色,数据未发表）,表明本地早橘在培育柑橘极早熟和易剥皮品种方面是一个优良的二倍体育种亲本。因此,为获得一定数量的具有丰富遗传变异的本地早橘三倍体后代,本研究以其为母本配置了8个杂交组合,这8个组合坐果率（10.3%—26.4%）、植株再生率（3.19%—37.14%）确实较低,与笔者前期结果一致,可能与本地早橘品种特性有关。与本地早橘相比,以槾橘为母本不仅坐果率高,且易再生三倍体;但槾橘在本研究中是第一次用作亲本创制三倍体,其三倍体后代果实性状表现如何正在评价中;若田间反馈信息表明其为较好的育种亲本,未来将以其为亲本配置更多倍性杂交组合,用于柑橘无核三倍体新品种的培育。

4 结论

基于柑橘幼胚离体挽救培养和流式细胞仪快速倍性检测技术,从9个倍性杂交组合共再生三倍体141株,分子鉴定结果表明所有检测的三倍体后代均为有性杂种,为本地早橘和槾橘无核遗传改良奠定了宝贵的材料基础。

（责任编辑 赵伶俐）

参考文献 View Option 原文顺序
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被引期刊影响因子

[1] 郭文武, 叶俊丽, 邓秀新 . 新中国果树科学研究70年-柑橘
果树学报, 2019,36(10):1264-1272.
[本文引用: 1]

GUO W W, YE J L, DENG X X . Fruit scientific research in New China in the past 70 years: Citrus
Journal of Fruit Science, 2019,36(10):1264-1272. (in Chinese)
[本文引用: 1]

[2] 解凯东, 王惠芹, 王晓培, 梁武军, 谢宗周, 伊华林, 邓秀新, Grosser J W, 郭文武 . 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
中国农业科学, 2013,46(21):4550-4557.
DOI:10.3864/j.issn.0578-1752.2013.21.018URL [本文引用: 3]
【Objective】 The objective of this experiment is to produce citrus triploid hybrids by interploid crossing between elite monoembryonic diploid varieties as female parents and allotetraploid somatic hybrids. 【Method】 Pollinations were carried out between the selected male and female parents. Fruits were collected at 70-100 d after pollination and immature seeds were cultured in vitro. Ploidy level of the plantlets was determined by flow cytometry and chromosome counting. The genetic origin of triploids and tetraploids was analyzed by SSR markers.【Result】 In successive four years from 2009 to 2012, 14 ploidy crosses using eight diploid cultivars as seed parents and four allotetraploid somatic hybrids as pollen parents, were carried out with a total of 3 347 flowers pollinated. With 678 fruits harvested, an average fruit set ratio of 20.26% was obtained. As a result, 1 022 plants were recovered from 12 357 seeds cultured in vitro, which derived from 505 fruits. By flow cytometry analysis and chromosome counting, a total of 755 triploids and 19 tetraploids were verified. The results of SSR markers showed that all the triploids and tetraploids from the cross of Huanong red pummelo × NH were of hybrid origin. 【Conclusion】 The triploids obtained from these crosses are valuable materials for the selection of new seedless citrus varieties. The tetraploid hybrids are also of great value as potential parents for citrus triploid breeding.
XIE K D, WANG H Q, WANG X P, LIANG W J, XIE Z Z, YI H L, DENG X X, GROSSER J W, GUO W W . Extensive citrus triploid breeding by crossing monoembryonic diploid females with allotetraploid male parents
Scientia Agricultura Sinica, 2013,46(21):4550-4557. (in Chinese)
DOI:10.3864/j.issn.0578-1752.2013.21.018URL [本文引用: 3]
【Objective】 The objective of this experiment is to produce citrus triploid hybrids by interploid crossing between elite monoembryonic diploid varieties as female parents and allotetraploid somatic hybrids. 【Method】 Pollinations were carried out between the selected male and female parents. Fruits were collected at 70-100 d after pollination and immature seeds were cultured in vitro. Ploidy level of the plantlets was determined by flow cytometry and chromosome counting. The genetic origin of triploids and tetraploids was analyzed by SSR markers.【Result】 In successive four years from 2009 to 2012, 14 ploidy crosses using eight diploid cultivars as seed parents and four allotetraploid somatic hybrids as pollen parents, were carried out with a total of 3 347 flowers pollinated. With 678 fruits harvested, an average fruit set ratio of 20.26% was obtained. As a result, 1 022 plants were recovered from 12 357 seeds cultured in vitro, which derived from 505 fruits. By flow cytometry analysis and chromosome counting, a total of 755 triploids and 19 tetraploids were verified. The results of SSR markers showed that all the triploids and tetraploids from the cross of Huanong red pummelo × NH were of hybrid origin. 【Conclusion】 The triploids obtained from these crosses are valuable materials for the selection of new seedless citrus varieties. The tetraploid hybrids are also of great value as potential parents for citrus triploid breeding.

[3] 解凯东, 王晓培, 王惠芹, 梁武军, 谢宗周, 郭大勇, 伊华林, 邓秀新, Grosser J W, 郭文武 . 以柑橘多胚性二倍体母本倍性杂交培育三倍体
园艺学报, 2014,41(4):613-620.
URL [本文引用: 3]
In the four successive years from 2009 to 2012，to obtain triploid citrus hybrids，eight elite polyembryonic diploid female parents were crossed respectively with five allotetraploid somatic hybrids and one autotetraploid. A total of 4 442 flowers were pollinated in 15 crosses，from which 1 484 fruits were set，with an average fruit setting rate as 33.4%. As much as 12 578 immature seeds from 1 075 young fruits were cultured in vitro. After shooting and rooting induction，2 832 plants were regenerated，in which 401 and 121 plants proved to be triploids and tetraploids respectively by flow cytometry analysis. Up to 349 triploids and 98 tetraploids survived in the greenhouse after transplantation into greenhouse. The Simple sequence repeat（SSR）markers analysis showed that all of the 89 triploids derived from the cross of W. Murcott × NH were hybrids of both parents.
XIE K D, WANG X P, WANG H Q, LIANG W J, XIE Z Z, GUO D Y, YI H L, DENG X X, GROSSER J W, GUO W W . High efficient and extensive production of triploid citrus plants by crossing polyembryonic diploids with tetraploids
Acta Horticulturae Sinica, 2014,41(4):613-620. (in Chinese)
URL [本文引用: 3]
In the four successive years from 2009 to 2012，to obtain triploid citrus hybrids，eight elite polyembryonic diploid female parents were crossed respectively with five allotetraploid somatic hybrids and one autotetraploid. A total of 4 442 flowers were pollinated in 15 crosses，from which 1 484 fruits were set，with an average fruit setting rate as 33.4%. As much as 12 578 immature seeds from 1 075 young fruits were cultured in vitro. After shooting and rooting induction，2 832 plants were regenerated，in which 401 and 121 plants proved to be triploids and tetraploids respectively by flow cytometry analysis. Up to 349 triploids and 98 tetraploids survived in the greenhouse after transplantation into greenhouse. The Simple sequence repeat（SSR）markers analysis showed that all of the 89 triploids derived from the cross of W. Murcott × NH were hybrids of both parents.

[4] 柏德玟, 姚莹, 童琦珏 . 浙江省柑橘产业提升对策的探讨
浙江农业科学, 2019,60(8):1431-1434.
[本文引用: 1]

BAI D W, YAO Y, TONG Q J . Discussion of the countermeasures for citrus industrial promotion in Zhejiang province
Journal of Zhejiang Agricultural Science, 2019,60(8):1431-1434. (in Chinese)
[本文引用: 1]

[5] NAVARRO L, ALEZA P, CUENCA J, JUAREZ J, PINA J A, ORTEGA C, NAVARRO A, ORTEGA V . The mandarin triploid breeding program in Spain
Acta Horticulturae. 2015,1065:389-395.
[本文引用: 1]

[6] OLLITRAULT P, DAMBIER D, LURO F, FROELICHER Y . Ploidy manipulation for breeding seedless triploid citrus
Plant Breed Reviews, 2008,30:323-352.
[本文引用: 1]

[7] GROSSER J W, GMITTER F G . Protoplast fusion for production of tetraploids and triploids: Applications for scion and rootstock breeding in citrus
Plant Cell Tissue Organ Culture, 2011,104:343-357.
DOI:10.1007/s11240-010-9823-4URL [本文引用: 5]

[8] FATTA DEL BOSCO S, SIRAGUSA M, ABBATE L, LUCRETTI S, TUSA N . Production and characterization of new triploid seedless progenies for mandarin improvement
Scientia Horticulturae, 2007,114:258-262.
DOI:10.1016/j.scienta.2007.06.014URL [本文引用: 1]

[9] ALEZA P, JUAREZ J, CUENCA J, OLLITRAULT P, NAVARRO L . Extensive citrus triploid hybrid production by 2x × 4x sexual hybridizations and parent-effect on the length of the juvenile phase
Plant Cell Reports, 2012,31:1723-1735.
DOI:10.1007/s00299-012-1286-0URL [本文引用: 1]
The citrus fresh market demands the production of seedless citrus fruits, as seedy fruits are not accepted by consumers. The recovery of triploid plants has proven to be the most promising approach to achieve this goal, since triploids have very low fertility, are generally seedless and do not induce seeds in other cultivars by cross pollination. Triploid plants can be recovered by 2x x 4x sexual hybridization. In this work, we present an effective methodology to recover triploid plants from 2x x 4x hybridizations based on in vitro embryo rescue, ploidy level analysis by flow cytometry and genetic origin of triploid plants. The pollen viability of diploid and tetraploid citrus genotypes was analyzed by comparing the pollen germination rate in vitro. The pollen viability of tetraploid (doubled-diploid) genotypes is generally reduced but sufficient for successful pollination. Triploid embryos were identified in normal and undeveloped seeds that did not germinate under greenhouse conditions. The influence of parents and environmental conditions on obtaining triploid plants was analyzed and a strong interaction was noted between the parents and environmental conditions. The parental effect on the length of the juvenile phase was also demonstrated through observations of a large number of progeny over the last 15 years. The juvenile phase length of the triploid hybrids obtained with 'Fortune' mandarin as female parent and tetraploid 'Orlando' tangelo as male parent was shorter than the juvenile phase obtained with a clementine as female parent and tetraploids of 'Nova', 'W. Leaf' and 'Pineapple' male parents.
Key message Effective methodology to recover citrus triploid plants from 2x x 4x sexual hybridizations and the parental effect on the length of the juvenile phase.

[10] RECUPERO G R, RUSSO G, RECUPERO S . New promising citrus triploid hybrids selected from crosses between monoembryonic diploid female and tetraploid male parents
HortScience, 2005,40:516-520.
DOI:10.21273/HORTSCI.40.3.516URL [本文引用: 1]

[11] NAKANO M, SHIMIZU T, SUGAWA S, KANEYOSHI J, FUJII H, KITA M, YOSHIOKA T, KITAJIMA A . Determining the parental combinations of the triploid acid citrus cultivars ‘Yellow Bell’ and ‘Tahiti lime’ using DNA marker analyses
Scientia Horticulturae, 2019,246:893-897.
DOI:10.1016/j.scienta.2018.11.071URL

[12] XIE K D, YUAN D Y, WANG W, XIA Q M, WU X M, CHEN C W, CHEN C L, GROSSER J W, GUO W W . Citrus triploid recovery based on 2x × 4x crosses via an optimized embryo rescue approach
Scientia Horticulturae, 2019,252:104-109.
DOI:10.1016/j.scienta.2019.03.038URL [本文引用: 2]

[13] ALEZA P, FROELICHER Y, SCHWARZ S, AGUSTI M, HERNANDEZ M, JUAREZ J, LURO F, MORILLON R, NAVARRO L, OLLITRAULT P . Tetraploidization events by chromosome doubling of nucellar cells are frequent in apomictic citrus and are dependent on genotype and environment
Annals of Botany, 2011,108:37-50.
DOI:10.1093/aob/mcr099URL [本文引用: 1]
Background and Aims Polyploidy is a major component of plant evolution. The citrus gene pool is essentially diploid but tetraploid plants are frequently encountered in seedlings of diploid apomictic genotypes. The main objectives of the present study were to establish the origin of these tetraploid plants and to ascertain the importance of genotypic and environmental factors on tetraploid formation.
Methods Tetraploid seedlings from 30 diploid apomictic genotypes were selected by flow cytometry and geno-typed with 24 single sequence repeat (SSR) markers to analyse their genetic origin. Embryo rescue was used to grow all embryos contained in polyembryonic seeds of 'Tardivo di Ciaculli' mandarin, followed by characterization of the plantlets obtained by flow cytometry and SSR markers to accurately establish the rate of tetraploidization events and their potential tissue location. Inter-annual variations in tetraploid seedling rates were analysed for seven genotypes. Variation in tetraploid plantlet rates was analysed between different seedlings of the same genotype ('Carrizo' citrange; Citrus sinensis x Poncirus trifoliata) from seeds collected in different tropical, subtropical and Mediterranean countries.
Key Results Tetraploid plants were obtained for all the studied diploid genotypes, except for four mandarins. All tetraploid plants were identical to their diploid maternal line for SSR markers and were not cytochimeric. Significant genotypic and environmental effects were observed, as well as negative correlation between mean temperature during the flowering period and tetraploidy seedling rates. The higher frequencies (20 %) of tetraploids were observed for citranges cultivated in the Mediterranean area.
Conclusions Tetraploidization by chromosome doubling of nucellar cells are frequent events in apomictic citrus, and are affected by both genotypic and environmental factors. Colder conditions in marginal climatic areas appear to favour the expression of tetraploidization. Tetraploid genotypes arising from chromosome doubling of apomictic citrus are extensively being used as parents in breeding programmes to develop seedless triploid cultivars and have potential direct use as new rootstocks.

[14] 梁武军, 解凯东, 郭大勇, 谢宗周, 伊华林, 郭文武 . 10个柑橘砧木类型同源四倍体的发掘与SSR鉴定
果树学报, 2014,31(1):1-6.


LIANG W J, XIE K D, GUO D Y, XIE Z Z, YI H L, GUO W W . Spontaneous generation and SSR molecular characterization of autotetraploids in ten citrus rootstocks
Journal of Fruit Science, 2014,31(1):1-6. (in Chinese)


[15] 梁武军, 解凯东, 郭大勇, 谢宗周, 徐强, 伊华林, 郭文武 . 柑橘10个品种实生后代多倍体的发掘及SSR鉴定
园艺学报, 2014,41(3):409-416.
URL [本文引用: 1]
Seeds were extracted from mature fruits and undeveloped seeds were cultured in vitro and
mature seeds were sowed in field. Seedlings with 2–3 true leaves after germination were analyzed by
flow cytometry to screen polyploids，and simple sequence repeat （SSR） markers were used to characterize
the genetic origin of these polyploids. The results showed that a total of 44 tetraploid seedlings were obtained from all studied diploid genotypes，i.e. 6，1，3，12，3，6，2，1 and 10 seedlings from Early Gold
sweet orange，Cutter Valencia，Tarocco blood orange，Sunburst mandarin，Ortanique tangor，Murcott
tangor，Huanong Bendizao tangerine，Zhuhongju tangerine and Flame grapefruit respectively. A total of 8
triploid seedlings were obtained from all studied diploid genotypes，i.e. 1，1，2，4 seedlings from Early
Gold sweet orange，Tarocco blood orange，Itabori sweet orange and Ortanique tangor respectively. The
genetic origin of polyploids obtaining by embryo rescue were determined by 13 SSR markers，and the
results showed that the regenerated triploid plants of Early Gold and Itabori sweet oranges were
allotriploid and that of Tarocco blood orange and Ortanique tangor maybe were autotriploid. The
regenerated tetraploid plants of Early Gold sweet orange and Ortanique tangor maybe were autotetraploid
and those of Tarocco blood orange were allotetraploid. SSR analysis validated that the band profiles of
tetraploids derived from other eight diploid cultivars were identical to their diploid maternal line. These
exploited natural polyploids hold great value for both citrus seedless breeding and basic research.
LIANG W J, XIE K D, GUO D Y, XIE Z Z, XU Q, YI H L, GUO W W . Spontaneous generation and SSR characterization of polyploids from ten citrus cultivars
Acta Horticulturae Sinica, 2014,41(3):409-416. (in Chinese)
URL [本文引用: 1]
Seeds were extracted from mature fruits and undeveloped seeds were cultured in vitro and
mature seeds were sowed in field. Seedlings with 2–3 true leaves after germination were analyzed by
flow cytometry to screen polyploids，and simple sequence repeat （SSR） markers were used to characterize
the genetic origin of these polyploids. The results showed that a total of 44 tetraploid seedlings were obtained from all studied diploid genotypes，i.e. 6，1，3，12，3，6，2，1 and 10 seedlings from Early Gold
sweet orange，Cutter Valencia，Tarocco blood orange，Sunburst mandarin，Ortanique tangor，Murcott
tangor，Huanong Bendizao tangerine，Zhuhongju tangerine and Flame grapefruit respectively. A total of 8
triploid seedlings were obtained from all studied diploid genotypes，i.e. 1，1，2，4 seedlings from Early
Gold sweet orange，Tarocco blood orange，Itabori sweet orange and Ortanique tangor respectively. The
genetic origin of polyploids obtaining by embryo rescue were determined by 13 SSR markers，and the
results showed that the regenerated triploid plants of Early Gold and Itabori sweet oranges were
allotriploid and that of Tarocco blood orange and Ortanique tangor maybe were autotriploid. The
regenerated tetraploid plants of Early Gold sweet orange and Ortanique tangor maybe were autotetraploid
and those of Tarocco blood orange were allotetraploid. SSR analysis validated that the band profiles of
tetraploids derived from other eight diploid cultivars were identical to their diploid maternal line. These
exploited natural polyploids hold great value for both citrus seedless breeding and basic research.

[16] GUO W W, PRASAD D, SERRANO P, GMITTER F G J R, GROSSER J W . Citrus somatic hybridization with potential for direct tetraploid scion cultivar development
Journal of Horticultural Science & Biotechnology, 2004,79(3):400-405.
[本文引用: 5]

[17] GROSSER J W, OLLITRAULT P, OLIVARES-FUSTER O . Somatic hybridization in citrus: An effective tool to facilitate variety improvement
In Vitro Cellular & Developmental Biology-Plant, 2000,36:434-449.
[本文引用: 1]

[18] LAN H, CHEN C L, MIAO Y, YU C X, GUO W W, XU Q, DENG X X . Fragile sites of ‘Valencia’ sweet orange (Citrus sinensis) chromosomes are related with active 45s rDNA
PLoS ONE, 2016,11(3):e0151512.
DOI:10.1371/journal.pone.0151512URLPMID:26977938 [本文引用: 1]
Citrus sinensis chromosomes present a morphological differentiation of bands after staining by the fluorochromes CMA and DAPI, but there is still little information on its chromosomal characteristics. In this study, the chromosomes in 'Valencia' C. sinensis were analyzed by fluorescence in situ hybridization (FISH) using telomere DNA and the 45S rDNA gene as probes combining CMA/DAPI staining, which showed that there were two fragile sites in sweet orange chromosomes co-localizing at distended 45S rDNA regions, one proximally locating on B-type chromosome and the other subterminally locating on D-type chromosome. While the chromosomal CMA banding and 45S rDNA FISH mapping in the doubled haploid line of 'Valencia' C. sinensis indicated six 45S rDNA regions, four were identified as fragile sites as doubled comparing its parental line, which confirmed the cytological heterozygosity and chromosomal heteromorphisms in sweet orange. Furthermore, Ag-NOR identified two distended 45S rDNA regions to be active nucleolar organizing regions (NORs) in diploid 'Valencia' C. sinensis. The occurrence of quadrivalent in meiosis of pollen mother cells (PMCs) in 'Valencia' sweet orange further confirmed it was a chromosomal reciprocal translocation line. We speculated this chromosome translocation was probably related to fragile sites. Our data provide insights into the chromosomal characteristics of the fragile sites in 'Valencia' sweet orange and are expected to facilitate the further investigation of the possible functions of fragile sites.

[19] CHENG Y J, GUO W W, YI H L, PANG X M, DENG X X . An efficient protocol for genomic DNA extraction from Citrus species
Plant Molecular Biology Reporter, 2003,21:177.
DOI:10.1007/BF02774246URL [本文引用: 1]
We describe a simple and efficient method for genomic DNA extraction from woody fruit crops containing high polysaccharide levels. This method involves a modified CTAB or SDS procedure employing a purification step to remove polysaccharides by using water-saturated ether and 1.25 M NaCl. Precipitation with an equal volume of isopropanol caused a DNA pellet to form. After being washed with 70% ethyl alcohol, the pellet easily dissolved in TE buffer. Using this method, DNA was extracted from samples of more than 1000Citrus spp., including young leaves, old leaves, frosted old leaves, withered old leaves, and callus lines. The average yield of DNA ranged from 50–500 μg/g of sample. DNA was suitable for PCR and RFLP analyses and long-term storage. Recently, the procedure was used to isolate DNA from withered old leaves of more than 20 tropical and subtropical fruit crops.

[20] XU Q, CHEN L L, RUAN X A, CHEN D, ZHU A, CHEN C, BERTRAND D, JIAO W B, HAO B H, LYON M P, CHEN J, GAO S, XING F, LAN H, CHANG J W, GE X, LEI Y, HU Q, MIAO Y, WANG L, XIAO S, BISWAS M K, ZENG W, GUO F, YANG X, XU X W, CHENG Y J, XU J, LIU J H, LUO O J, TANG Z, GUO W W, KUANG H, ZHANG H Y, ROOSE M L, NAGARAJAN N, DENG X X, RUAN Y . The draft genome of sweet orange (Citrus sinensis)
Nature Genetics, 2013,45:59-66.
DOI:10.1038/ng.2472URL
Oranges are an important nutritional source for human health and have immense economic value. Here we present a comprehensive analysis of the draft genome of sweet orange (Citrus sinensis). The assembled sequence covers 87.3% of the estimated orange genome, which is relatively compact, as 20% is composed of repetitive elements. We predicted 29,445 protein-coding genes, half of which are in the heterozygous state. With additional sequencing of two more citrus species and comparative analyses of seven citrus genomes, we present evidence to suggest that sweet orange originated from a backcross hybrid between pummelo and mandarin. Focused analysis on genes involved in vitamin C metabolism showed that GalUR, encoding the rate-limiting enzyme of the galacturonate pathway, is significantly upregulated in orange fruit, and the recent expansion of this gene family may provide a genomic basis. This draft genome represents a valuable resource for understanding and improving many important citrus traits in the future.

[21] 邓秀新, 王力荣, 李绍华, 张绍铃, 张志宏, 从佩华, 易干军, 陈学森, 陈厚彬, 钟彩虹 . 果树育种40年回顾与展望
果树学报, 2019,36(4):514-520.
[本文引用: 1]

DENG X X, WANG L R, LI S H, ZHANG S L, ZHANG Z H, CONG P H, YI G J, CHEN X S, CHEN H B, ZHONG C H . Retrospection and prospect of fruit breeding for last four decades in China
Journal of Fruit Science, 2019,36(4):514-520. (in Chinese)
[本文引用: 1]

[22] 刘科宏, 周彦, 李中安 . 柑橘茎尖嫁接脱毒技术研究进展
园艺学报, 2016,43(9):1665-1674.
DOI:10.16420/j.issn.0513-353x.2016-0079URL [本文引用: 1]
Shoot-tip grafting（STG）is one of effective techniques for eliminating the diseases from infected citrus to obtain the virus-free budwoods. The core of STG is success and virus elimination rate. In this review，factors influenced success and virus elimination rate of grafted shoot tips was mainly summarized in order to provide some experiences and tricks for virus-free citrus producers.
LIU K H, ZHOU Y, LI Z A . Technical progress on shoot-tip grafting of citrus
Acta Horticulturae Sinica, 2016,43(9):1665-1674. (in Chinese)
DOI:10.16420/j.issn.0513-353x.2016-0079URL [本文引用: 1]
Shoot-tip grafting（STG）is one of effective techniques for eliminating the diseases from infected citrus to obtain the virus-free budwoods. The core of STG is success and virus elimination rate. In this review，factors influenced success and virus elimination rate of grafted shoot tips was mainly summarized in order to provide some experiences and tricks for virus-free citrus producers.

[23] 王斐, 方成泉, 姜淑苓, 林盛华, 欧春青, 李连文, 马力 . 大果优质三倍体梨新品种‘华幸’
园艺学报, 2014,41(11):2355-2356.
URL [本文引用: 1]
‘Huaxing’is a new triploid pear cultivar hybrid selected from‘Dayali’（tetraploid）בXuehua’（diploid）. The fruit is large and the average fruit weight is 295 g. The skin is green-yellow and smooth. The flesh is white and fine with less stone cells，and characterized by crispness and juiciness，sour-sweet with 11.5%–12.5% soluble solids content. It matures in late September in Xingcheng. ‘Huaxing’has various desirable characteristics including early bearing，high yield，enduring storage and high resisitance to scab.
WANG F, FANG C Q, JIANG S L, LIN S H, OU C Q, LI L W, MA L . A new triploid pear cultivar ‘Huaxing’
Acta Horticulturae Sinica, 2014,41(11):2355-2356. (in Chinese)
URL [本文引用: 1]
‘Huaxing’is a new triploid pear cultivar hybrid selected from‘Dayali’（tetraploid）בXuehua’（diploid）. The fruit is large and the average fruit weight is 295 g. The skin is green-yellow and smooth. The flesh is white and fine with less stone cells，and characterized by crispness and juiciness，sour-sweet with 11.5%–12.5% soluble solids content. It matures in late September in Xingcheng. ‘Huaxing’has various desirable characteristics including early bearing，high yield，enduring storage and high resisitance to scab.

[24] 张莹, 曹玉芬, 田路明, 董星光, 霍宏亮, 李树玲, 黄礼森, 赵德英, 齐丹, 徐家玉, 闫帅, 王立东 . 三倍体梨新品种‘华香酥’
园艺学报, 2020,47(5):1009-1010.
[本文引用: 1]

ZHANG Y, CAO Y F, TIAN L M, DONG X G, HUO H L, LI S L, HUANG L S, ZHAO D Y, QI D, XU J Y, YAN S, WANG L D . A new triploid pear cultivar ‘Huaxiangsu’
Acta Horticulturae Sinica, 2020,47(5):1009-1010. (in Chinese)
[本文引用: 1]

[25] 党江波, 郭启高, 向素琼, 何桥, 孙海燕, 吴頔, 景丹龙, 王淑明, 夏燕, 李晓林, 梁国鲁 . 大果无核枇杷新品种‘华玉无核1号’
园艺学报, 2019,46(S2):2765-2766.
[本文引用: 1]

DANG J B, GUO Q G, XIANG S Q, HE Q, SUN H Y, WU D, JING D L, WANG S M, XIA Y, LI X L, LIANG G L . A new seedless loquat cultivar ‘Huayu Wuhe 1’ with large fruit
Acta Horticulturae Sinica, 2019,46(S2):2765-2766. (in Chinese)
[本文引用: 1]

[26] VILORIA Z, DROUILLARD D L, GRAHAM J H, GROSSER J W . Screening triploid hybrids of ‘Lakeland’ limequat for resistance to citrus canker
Plant Disease, 2004,88:1056-1060.
DOI:10.1094/PDIS.2004.88.10.1056URLPMID:30795244 [本文引用: 1]
Resistance of citrus genotypes to Xanthomonas axonopodis pv. citri, the cause of Asiatic citrus canker (ACC), was evaluated by injection infiltration of 10(3) and 10(4) CFU/ml through stomates on the abaxial surface of immature leaves. Citrus genotypes for screening comprised two autotetraploids and nine triploid hybrids of 'Lakeland' limequat (Citrus aurantifolia x Fortunella japonica) and their progenitors ('Lakeland' limequat, the autotetraploids 'Femminello' lemon (Citrus limon) and 'Giant Key' lime (C. aurantifolia), and the somatic hybrids 'Key' [also known as 'Mexican'] lime + 'Valencia' orange and 'Hamlin' orange + 'Femminello' lemon). 'Meiwa' kumquat (Fortunella crassifolia) and 'Pineapple' sweet orange (C. sinensis) were used as known resistant and susceptible standards, respectively. Lesion number per inoculation site and bacterial population per lesion were recorded 15 to 19 days after inoculation. The assay was performed four times during a spring-summer-fall period under greenhouse conditions. Canker lesions were consistently produced by stomatal inoculation with 10(4) but not 10(3) CFU/ml. Susceptible and resistant genotypes were separated based on lesion number per inoculation site and bacterial population per lesion. Spearman's rank correlation analysis for lesion numbers on 15 genotypes common to all four assays showed significant correlations among the genotype rankings. Genotype rankings were also significantly correlated between the two bacterial population assays. Lesion number per inoculation site is sufficient for assessment of resistance of citrus genotypes to ACC without the necessity of conducting bacterial population assays. 'Lakeland' limequat is a promising seed parent for breeding acid citrus fruit that is resistant to ACC.

[27] LOURKISTI R, FROELICHER Y, HERBETTE S, MORILLON R, TOMI F, GIBERNAU M, GIANNETTINI J, BERTI L, SANTINI J . Triploid citrus genotypes have a better tolerance to natural chilling conditions of photosynthetic capacities and specific leaf volatile organic compounds
Frontiers in Plant Science, 2020,11. doi: 10.3389/fpls.2020.00330.
DOI:10.3389/fpls.2020.572686URLPMID:33281842 [本文引用: 1]
Pathogen infections limit plant growth and productivity, thus contributing to crop losses. As the site of photosynthesis, the chloroplast is vital for plant productivity. This organelle, communicating with other cellular compartments challenged by infection (e.g., apoplast, mitochondria, and peroxisomes), is also a key battlefield in the plant-pathogen interaction. Here, we focus on the relation between reactive oxygen species (ROS)-redox signaling, photosynthesis which is governed by redox control, and biotic stress response. We also discuss the pathogen strategies to weaken the chloroplast-mediated defense responses and to promote pathogenesis. As in the next decades crop yield increase may depend on the improvement of photosynthetic efficiency, a comprehensive understanding of the integration between photosynthesis and plant immunity is required to meet the future food demand.

[28] ALEZA P, CUENCA J, JUAREZ J, PINA J A, NAVARRO L . ‘Garbí’ mandarin: A new late-maturing triploid hybrid
HortScience, 2010,45:139-141.
DOI:10.21273/HORTSCI.45.1.139URL [本文引用: 2]

[29] CUENCA J, ALEZA P, JUAREZ J, PINA J A, NAVARRO L . ‘Safor’ mandarin: A new citrus mid-late triploid hybrid
HortScience, 2010,45:977-980.
DOI:10.21273/HORTSCI.45.6.977URL [本文引用: 2]

[30] CHEN C X, CANCALON P, HAUN C, GMITTER F G . Characterization of furanocoumarin profile and inheritance toward selection of low furanocoumarin seedless grapefruit cultivars
Journal of the American Society for Horticultural Science, 2011,136:358-363.
URL [本文引用: 1]
Furanocoumarins are organic chemical components in grapefruit (Citrus puradisi) juice that have been shown to induce potentially deleterious drug interactions. In this study we measured seven furanocoumarins (FCs) [bergamottin, 6',7'-dihydroxybergamottin (6,7-DHB), paradisin C, bergaptol, isoimperatorin, 5',8'-dimethylallyloxypsoralen (5,8-DMP), and epoxybergamottin (EBM)] in fruit of three grapefruit cultivars [Foster (Fos), Low Acid Foster (LAF), and Hudson (Hod)], one pummelo (C. nuvcima) cultivar [Hirado Buntan (HBP)], 17 randomly selected hybrids from HBPx Hud, and 31 other triploid hybrids. Bergamotton, 6,7-DHB, and paradisin C were not detected or extremely low in HBP (0.00, 0.11, and 0.00 mg.L(-1)) and LAF (0.40, 3.83, and 0.00 mg.L(-1)) compared with Hud (13.03. 9.58, and 6.11 mg.L(-1)) and Fos (6.48, 14.38, and 6.11 mg.L(-1)). In these hybrids, 6,7-DHB, bergamottin, and paradisin C obviously cosegregated in an approximate rate of 1:1. The three FCs in eight hybrids were not detected or extremely low, like H BP, the maternal parent; those in the other nine were as high as or higher than Hud, the paternal parent. The same segregation tendency was also observed in these triploid hybrids. Based on all the cultivars and hybrids, strong correlations existed among 6,7-DHB, bergamottin, and paradisin C (coefficient up to 0.909). Such strong correlations may reflect their metabolic links in the bergamottin pathway. The 1:1 cosegregation and strong correlation among the three FCs suggested that the trait of FCs is likely controlled by one single enzymatic or regulatory gene in the pathway. The FC profiles and inheritance may lead to a genomic and breeding solution to the grapefruit FC drug interaction issue. Selection of FC-low or FC-free seedless grapefruit cultivars is underway.

[31] AHMED D, EVRARD J C, OLLITRAULT P, FROELICHER Y . The effect of cross direction and ploidy level on phenotypic variation of reciprocal diploid and triploid mandarin hybrids
Tree Genetics & Genomes, 2020,16:25.
DOI:10.1007/s11295-020-1417-7URL [本文引用: 1]

[32] SDIRI S, CUENCA J. NAVARRO P, SALVADOR A, BERMEJO A . New triploids late-maturing mandarins as a rich source of antioxidant compounds
European Food Research and Technology, 2020,246:225-237.
DOI:10.1007/s00217-019-03407-9URL [本文引用: 1]

[33] CUENCA J, ALEZA P, JUAREZ J, PINA J A, NAVARRO L . Two new IVIA triploid mandarin hybrids: ‘Alborea’ and ‘Albir’
Acta Horticulturae, 2015,1065:209-214.
[本文引用: 1]

[34] OLLOTRAULT P, GERMANA M A, FROELICHER Y, CUENCA J, ALEZA P, MORILLON R, GROSSER J W, GUO W W. Ploidy Manipulation for Citrus Breeding, Genetics, and Genomics//GENTILE A, LA MALFA S, DENG Z. The Citrus Genome. Switzerland: Springer Press, 2020: 75-105.
[本文引用: 1]