Scientia Agricultura Sinica ›› 2020, Vol. 53 ›› Issue (23): 4961-4968.doi: 10.3864/j.issn.0578-1752.2020.23.020

• RESEARCH NOTES • Previous Articles    

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

XIE KaiDong1(),PENG Jun1,YUAN DongYa1,QIANG RuiRui1,XIE ShanPeng1,ZHOU Rui1,XIA QiangMing1,WU XiaoMeng1,KE FuZhi2,LIU GaoPing3,GROSSER Jude W4,GUO WenWu1()   

  1. 1College 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
  • Received:2020-09-01 Accepted:2020-10-14 Online:2020-12-01 Published:2020-12-09
  • Contact: WenWu GUO E-mail:xiekaidong@mail.hzau.edu.cn;guoww@mail.hzau.edu.cn

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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.

Key words: Citrus, seedless breeding, polyploid, embryo rescue, flow cytometry

Table 1

Sequences 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]

Table 2

Embryo 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

Fig. 1

Embryo 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"

Fig. 2

Ploidy 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"

Fig. 3

The 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"

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