Abstract
Many citrus fruits have polyembryonic traits, and their seeds contain many nucellar embryos along with a single zygotic embryo, affecting the crossbreeding process. Generally, nucellar embryos are considered to have more vigorous growth than zygotic embryos. Therefore, the in vitro method using an embryo rescue culture is often chosen to obtain zygotic embryo-derived individuals. Nevertheless, hybrids can be obtained with a certain probability from the seeds sown in the soil. The in-soil method, which sows seeds in the soil, has distinct advantages over the in vitro method, including lower cost and simpler technology. However, the efficiency of obtaining hybrids from these methods has not been compared in detail. The current study evaluates the effectiveness of these methods for obtaining hybrids using polyembryonic Satsuma mandarin as the female parent. The number of mature embryos per seed using the in-soil method was less than one-third of that produced using the in vitro method. Although the in vitro method produced more hybrids than the in-soil method, the ratio of the hybrids to the resulting population was significantly higher in the in-soil method. Thus, the in-soil method was more efficient and practical than the in vitro method for selecting hybrids from polyembryonic Satsuma mandarin seeds. The observations of the individuals obtained using the in-soil method suggest that zygotic embryos were not poorer in growth than nucellar embryos when using our selected parental combinations.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Data availability
Not applicable.
Reference
Aleza P, Juárez J, Cuenca J, Ollitrault P, Navarro L (2012) Extensive citrus triploid hybrid production by 2x × 4x sexual hybridizations and parent-effect on the length of the juvenile phase. Plant Cell Rep 31:1723–1735. https://doi.org/10.1007/s00299-012-1286-0
Cameron JW, Soost RK (1980) Mono and poly embryony among tetraploid Citrus hybrids. Hortic Sci 15:730–731
Carimi F, de Pasquale F, Puglia AM (1998) In vitro rescue of zygotic embryos of sour orange, Citrus aurantium L., and their detection based on RFLP analysis. Plant Breed 117:261–266. https://doi.org/10.1111/j.1439-0523.1998.tb01936.x
Caruso M, Distefano G, Pietro PD, La Malfa S, Russo G, Gentile A, Recupero GR (2014) High-resolution melting analysis for early identification of citrus hybrids: a reliable tool to overcome the limitations of morphological markers and assist rootstock breeding. Sci Hortic 180:199206. https://doi.org/10.1016/j.scienta.2014.10.024
Delwart EL, Shpaer EG, Louwagie J, McCutchan FE, Grez M, Rübsamen-Waigmann H, Mullins JI (1993) Genetic relationships determined by a DNA heteroduplex mobility assay: analysis of HIV-1 env genes. Science 262:1257–1261. https://doi.org/10.1126/science.8235655,Pubmed:8235655
Deng XX, Yi HL, Li F, Guo WW (1996) Triploid plants regenerated from crossing diploid pummelo and tangerine with allotetraploid somatic hybrid of Citrus. Proc Intl Soc Citricult 1:189–192
Esen A, Soost RK (1971) Unexpected triploids in citrus: their origin, identification and possible use. J Hered 62:329–333. https://doi.org/10.1093/oxfordjournals.jhered.a108186
Esen A, Soost RK (1973) Seed development in citrus with special reference to 2x X 4x crosses. Am J Bot 60:448–462. https://doi.org/10.1002/j.1537-2197.1973.tb05945.x
Esen A, Soost RK (1977) Adventive embryogenesis in citrus and its relation to pollination and fertilization. Am J Bot 64:607–614. https://doi.org/10.2307/2441710
Ferrante SP, Lucretti S, De Patrizio A, Abbate L, Tusa N, Scarano M (2010) Assessment of the origin of new citrus tetraploid hybrids (2n = 4x) by means of SSR markers and PCR based dosage effects. Euphytica 173:223–233. https://doi.org/10.1007/s10681-009-0093-3
Frost HB, Soost RK (1968) Seed reproduction. Development of gametes and embryos. In: Reuther W, Batchelor LD, Webber HJ (eds) The citrus industry, vol 2. University of California Press, Berkeley, pp 290–324
Furusato K, Ohta Y, Ishibashi K (1957) Studies on polyembryony in citrus. Seiken Ziho 8:40–48
Horiuchi S, Yuda E, Nakagawa S, Morimoto J, Gato Y (1990) Shapes of seed including well-developed zygotic embryo in polyembryonic citrus species/cultivars. J Jpn Soc Hortic Sci 59:225–235. https://doi.org/10.2503/jjshs.59.225 (in Japanese with English abstract)
Kaneyoshi J, Kanou T, Kuwata Y, Hirao A, Nakatani S, Kobayashi S (1997) Breeding of triploid citrus cultivars I: Production of triploids from Satsuma mandarin (Citrus unshiu Marc.) × Tetraploid Ponkan Mandarin (Citrus reticulata Blanco) crosses. Engei Gakkai zasshi 66:9–14. https://doi.org/10.2503/jjshs.66.9 (in Japanese with English abstract)
Khan IA, Roose ML (1988) Frequency and characteristics of nucellar and zygotic seedlings in three cultivars of trifoliate orange. J Am Soc Hortic Sci 113:105–110. https://doi.org/10.21273/JASHS.113.1.105
Kitamura M, Sakaki H, Sakanishi M, Fujita K, Kitazono K, Fukunaga Y, Mitsuta M, Isobe A (2014) Characteristic of early citrus cultivar ‘Kumamoto EC10.’ Kumamoto Prefectural Agric Res Cent 21:42–46 (in Japanese with English abstract)
Koltunow AM, Soltys K, Nito N, McClure S (1995) Anther, ovule, seed, and nucellar embryo development in Citrus sinensis cv. Valencia Can J Bot 73:1567–1582. https://doi.org/10.1139/b95-170
Lichten MJ, Fox MS (1983) Detection of non-homology-containing heteroduplex molecules. Nucl Acids Res 11:3959–3971. https://doi.org/10.1093/nar/11.12.3959,Pubmed:6223275
Mihara T, Kitamura M, Sakanishi M, Kitazono K (2018) New citrus cultivar ‘Kumamoto EC12’ can be shipped in December, with high quality. Research Kumamoto Prefectural Agric Res Cent 26:19–24 (in Japanese with English abstract)
Minamikawa MF, Nonaka K, Kaminuma E, Kajiya-Kanegae H, Onogi A, Goto S, Yoshioka T, Imai A, Hamada H, Hayashi T, Matsumoto S, Katayose Y, Toyoda A, Fujiyama A, Nakamura Y, Shimizu T, Iwata H (2017) Genome-wide association study and genomic prediction in citrus: potential of genomics-assisted breeding for fruit quality traits. Sci Rep 7:4721. https://doi.org/10.1038/s41598-017-05100-x
Moore GA, Castle WS (1988) Morphological and isozymic analysis of open-pollinated Citrus rootstock populations. J Hered 79:59–63. https://doi.org/10.1093/oxfordjournals.jhered.a110448
Navarro L, Aleza P, Cuenca J, Juárez J, Pina JA, Ortega C, Navarro A, Ortega V (2015) The mandarin triploid breeding program in Spain. Acta Horticult 1605:389–395. https://doi.org/10.17660/ActaHortic.2015.1065.48
Nishiura M, Iwashita T (1964) Studies on the citrus breeding. II: Number of seedlings per seed and number of gametic and nucellar seedlings from Satsuma mandarin by cross-pollination. Bull Hort Res Sta Ser B 3:1–9 (in Japanese with English abstract)
Noda T, Daiou K, Mihara T, Nagano Y (2020) Development of indel markers for the selection of Satsuma mandarin (Citrus unshiu Marc.) hybrids that can be used for low-cost genotyping with agarose gels. Euphytica 216:115. https://doi.org/10.1007/s10681-020-02654-2
Noda T, Daiou K, Mihara T, Nagano Y (2021) Potential application of simple easy-to-use insertion-deletion (Indel) markers in citrus cultivar identification. Breed Sci 71:601–608. https://doi.org/10.1270/jsbbs.21021
Okudai N, Oiyama I, Takahara T (1981) Studies on the improvement of zygotic seedling yield of the polyebnryonic citrus. I: Differences in embryo number per seed and zygotic seedling yield among its varieties and strains. Bull Fruit Tree Res Stn Ser D 3:9–21 (in Japanese with English abstract)
Omura M, Shimada T (2016) Citrus breeding, genetics and genomics in Japan. Breed Sci 66:3–17. https://doi.org/10.1270/jsbbs.66.3,Pubmed:27069387
Ohta Y, Furusato K (1957) Embryo culture in citrus. Seiken Zilho Rpt Kihara Inst Biol Res 23:49–54
Ota S, Hisano Y, Muraki M, Hoshijima K, Dahlem TJ, Grunwald DJ, Okada Y, Kawahara A (2013) Efficient identification of TALEN-mediated genome modifications using heteroduplex mobility assays. Genes Cells 18:450–458. https://doi.org/10.1111/gtc.12050,Pubmed:23573916
Pan Z, Guan R, Zhu S, Deng X (2009) Proteomic analysis of somatic embryogenesis in Valencia sweet orange (Citrus sinensis Osbeck). Plant Cell Rep 28:281–289. https://doi.org/10.1007/s00299-008-0633-7
Pérez-Tornero O, Porras I (2008) Assessment of polyembryony in lemon: rescue and in vitro culture of immature embryos. Plant Cell Tiss Organ Cult 93:173–180. https://doi.org/10.1007/s11240-008-9358-0
Shen X, Gmitter FG, Grosser JW (2011) Immature embryo rescue and culture. Methods Mol Biol 710:75–92. https://doi.org/10.1007/978-1-61737-988-8_7,Pubmed:21207263
Shikano E, Hara T, Tachikawa T, Tanaka Y (1982) On the characters of the citrus variety ‘Beauty maple tangor.’ Bull Shizuoka Citrus Exp Sta 18:11–16 (in Japanese with English abstract)
Shimada T, Endo T, Fujii H, Nakano M, Sugiyama A, Daido G, Ohta S, Yoshioka T, Omura M (2018) MITE insertion-dependent expression of CitRKD1 with a RWP-RK domain regulates somatic embryogenesis in citrus nucellar tissues. BMC Plant Biol 18:166. https://doi.org/10.1186/s12870-018-1369-3
Shigematsu Y, Kita K, Yakushiji H, Ishikawa K, Inoue H, Nakataet H (2008) The new citrus cultivar ‘Kanpei.’ Bull Ehime Fruit Tree Exp Stn 22:1–4 (in Japanese with English abstract)
Singh J, Dhaliwal HS, Thakur A, Sidhu GS, Chhuneja P, Gmitter FG (2020) Optimizing recovery of hybrid embryos from interspecific citrus crosses of polyembryonic rough lemon (Citrus jambhiri lush). Agronomy 10:1940. https://doi.org/10.3390/agronomy10121940
Soares-Filho WdS, Lee LM, da Cunha Sobinho AP (1995) Influence of pollinators on polyembryony in Citrus. Acta Hortic 403:256–265
Soost RK, Roose ML (1996) Citrus. In: Janick J, Moore JN (eds) Fruit breeding, vol 1. Tree and Tropical Fruits. Wiley, New York, pp 257–323
Spiegel-Roy P, Bardi A, Shani A (1977) Peroxidase isozymes as a tool for early separation of nucellar and zygotic citrus seedlings. Fla-U S A. Second Meeting of the International Society of Citriculture 2:619–624
Takayanagi R, Miura Y, Kitanura T, Hidaka T (1991) Breeding of new citrus cultivar by embryo culture (I) Research bulletin of the Kanagawa prefectural agricultural research center 133: 75–81. (in Japanese with English abstract)
Tan ML, Song JK, Deng XX (2007) Production of two mandarin × trifoliate orange hybrid populations via embryo rescue with verification by SSR analysis. Euphytica 157:155–160. https://doi.org/10.1007/s10681-007-9407-5
Tusa N, Fatta DF, Nardi L, Lucretti S (1996) Obtaining triploid plants by crossing Citrus Lemon cv ‘Femminello’ 2n × 4N allotetraploid somatic hybrids. Proc Int Soc Citricult 1:133–136
Ueno I, Iwamasa M, Nishiura M (1967) Embryo number of various varieties of Citrus and its relatives. Bull Hort Res Sta Jpn 7:11–22
Viloria Z, Grosser JW (2005) Acid citrus fruit improvement via interploid hybridization using allotetraploid somatic hybrid and autotetraploid breeding parents. J Amer Soc Hort Sci 130(3):392–402. https://doi.org/10.21273/JASHS.130.3.392
Wakana A, Uemoto S (1988) Adventive embryogenesis in Citrus (Rutaceae). II Post-Fertilization Development Am J Bot 75:1033–1047. https://doi.org/10.1002/J.1537-2197.1988.TB08810.X
Woo JK, Park YC, Lee JW, Yun SH, Kim M, Park S, Lee Y, Song KJ, Kim HB (2019) Evaluation of polyembryony for genetic resources and efficacy of simple sequence repeat markers for the identification of nucellar and zygotic embryo-derived individuals in citrus. Appl Biol Chem 62:30. https://doi.org/10.1186/s13765-019-0437-1
Xie KD, Yuan DY, Wang W, Xia QM, Wu XM, Chen CW, Chen CL, Grosser JW, Guo WW (2019) Citrus triploid recovery based on 2x × 4x crosses via an optimized embryo rescue approach. Sci Hortic 252:104–109. https://doi.org/10.1016/j.scienta.2019.03.038
Xu Q, Chen L, Ruan X, Chen D, Zhu A, Chen C, Bertrand D (2013) The draft genome of sweet orange (Citrus sinensis). Nature Genet 45:59–66
Yildiz E, Kaplankiran M, Demİrkeser TH, Uzun A, Toplu C (2013) Identification of zygotic and nucellar individuals produced from several Citrus crosses using SSRs markers. Not Bot Hort Agrobot Cluj 41:478–484. https://doi.org/10.15835/nbha4129037
Zhu S, Wu B, Ma Y, Chen J, Zhong G (2013) Obtaining citrus hybrids by in vitro culture of embryos from mature seeds and early identification of hybrid seedlings by allele-specific PCR. Sci Hortic 161:300–305. https://doi.org/10.1016/j.scienta.2013.07.003
Acknowledgements
The authors would like to thank Naomi Matsuda and Keiko Otaguro for their technical assistance. We would like to thank Editage (www.editage.com) for the English editing services.
Funding
This work was partly supported by a Grant-in-Aid for Scientific Research (18K05623) from the Japan Society for the Promotion of Science to Yukio Nagano.
Author information
Authors and Affiliations
Contributions
TN, KD, TM, HM, and YN conceived and designed the experiments. TM prepared the citrus genetic resources. TN and KD performed the experiments. The manuscript was drafted by TN and edited by YN, and was subsequently reviewed by all authors.
Corresponding author
Ethics declarations
Ethics approval and consent to participate
On behalf of all authors, the corresponding author states that the experimental research on plants were performed in accordance with relevant institutional, national, and international guidelines and legislation.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Additional information
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Below is the link to the electronic supplementary material.
Rights and permissions
Springer Nature or its licensor holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Noda, T., Daiou, K., Mihara, T. et al. Efficient method for generating citrus hybrids with polyembryonic Satsuma mandarin as the female parent. Mol Breeding 42, 51 (2022). https://doi.org/10.1007/s11032-022-01324-6
Received:
Accepted:
Published:
DOI: https://doi.org/10.1007/s11032-022-01324-6