Korean Journal of Agricultural Science (Korean J. Agric. Sci.; KJOAS)
Indexed in KCI (Korea Citation Index), Open Access, Peer Reviewed.
pISSN 2466-2402
eISSN 2466-2410

The role of cytogenetic tools in orchid breeding



Sevilleno SS, Cabahug-Braza RA, An HR, Lim KB, Hwang YJ. The role of cytogenetic tools in orchid breeding. Korean Journal of Agricultural Science 50:235-248.

Korean Journal of Agricultural Science (Korean J. Agric. Sci.) 2023 June, Volume 50, Issue 2, pages 50:235-248. https://doi.org/10.7744/kjoas.20230015

Received on 18 January 2023, Revised on 30 March 2023, Accepted on 20 April 2023, Published on 30 June 2023.

The role of cytogenetic tools in orchid breeding

Samantha Sevilleno Sevilleno1, Raisa Aone Cabahug-Braza2, Hye Ryun An3, Ki‑Byung Lim4, Yoon-Jung Hwang1,2,*

1Department of Convergence Science, Sahmyook University, Seoul 01795, Korea

2Plant Genetics and Breeding Institute, Sahmyook University, Seoul 01795, Korea

3Floriculture Research Division, National Institute of Horticultural & Herbal Science, Rural Development Administration, Wanju 55365, Korea

4Department of Horticulture Science, Kyungpook National University, Daegu 41566, Korea

*Corresponding author: hyj@syu.ac.kr


Orchidaceae species account for one-tenth of all angiosperms including more than 30,000 species having significant ecological, evolutionary, and economic importance. Despite Orchidaceae being one of the largest families among flowering plants, crucial cytogenetic information for studying species diversification, inferring phylogenetic relationships, and designing efficient breeding strategies is lacking, except for 10% or less of orchid species cases involving mostly chromosome number or karyotype analysis. Also, only approximately 1.5% of the identified orchid species from less than a hundred genera have genome size data that provide crucial information for breeders and molecular geneticists. Various molecular cytogenetic techniques, such as fluorescence in situ hybridization (FISH) and genomic in situ hybridization (GISH), have been developed for determining ploidy levels, analyzing karyotypes, and evaluating hybridity, in several ornamental crops including orchids. The estimation of genome size and the determination of nuclear DNA content using flow cytometry have also been employed in some Orchidaceae subfamilies. These different techniques have played an important role in supplementing beneficial knowledge for effective plant breeding programs and other related plant research. This review focused on orchid breeding summarizes the status of current cytogenetic tools in terms of background, advancements, different techniques, significant findings, and research challenges. Principal roles and applications of cytogenetics in orchid breeding as well as different ploidy level determination methods crucial for breeding are also discussed.


breeding, chromosome, cytogenetics, FISH (fluorescence in situ hybridization), orchids


Beck SL, Visser G, Dunlop RW. 2005. A comparison of direct (flow cytometry) and indirect (stomatal guard cell lengths and chloroplast numbers) techniques as a measure of ploidy in black wattle, Acacia mearnsii (de Wild). South African Journal of Botany 71:354-358.

Begum R, Alam SS, Menzel G, Schmidt T. 2009. Comparative molecular cytogenetics of major repetitive sequence families of three Dendrobium species (Orchidaceae) from Bangladesh. Annals of Botany 104:863-872.

Bishop R. 2010. Applications of fluorescence in situ hybridization (FISH) in detecting genetic aberrations of medical significance. Bioscience Horizons 3:85-95.

Bolanos-Villegas P, Chin SW, Chen FC. 2008. Meiotic chromosome behavior and capsule setting in Doritaenopsis hybrids. Journal of the American Society for Horticultural Science 133:107-116.

Borowsky RL. 2001. Estimating nucleotide diversity from random amplified polymorphic DNA and amplified fragment length polymorphism data. Molecular Phylogenetics and Evolution 18:143-148.

Brammer SP, Poersch LB, de Oliveira AR, Vasconcelos S, Brasileiro-Vidal AC. 2009. Genomic in situ hybridization in Triticeae: A methodological approach. Embrapa Trigo-Comunicado Técnico (INFOTECA-E) 270:15. (in Portuguese)

Cabral JS, Felix LP, Guerra M. 2006. Heterochromatin diversity and its co-localization with 5S and 45S rDNA sites in chromosomes of four Maxillaria species (Orchidaceae). Genetics and Molecular Biology 29:659-664.

Cai J, Liu X, Vanneste K, Proost S, Tsai WC, Liu KW, Chen LJ, He Y, Xu Q, Bian C, et al. 2015. The genome sequence of the orchid Phalaenopsis equestris. Nature Genetics 47:65-72.

Chan SW. 2010. Chromosome engineering: Power tools for plant genetics. Trends in Biotechnology 28:605-610.

Chen WH, Kao YL, Tang CY, Tsai CC, Lin TY. 2013. Estimating nuclear DNA content within 50 species of the genus Phalaenopsis Blume (Orchidaceae). Scientia Horticulturae 161:70-75.

Chen WH, Tang CY, Kao YL. 2010. Polyploidy and variety improvement of Phalaenopsis orchids. Acta Horticulture 878:133-138.

Choi SC, Chung YS, Kim C. 2016. Perspectives on the genomics research of important crops in the tribe Andropogoneae: Focusing on the Saccharum complex. Korean Journal of Agricultural Science 43:1-13.

Chugh S, Guha S, Rao IU. 2009. Micropropagation of orchids: A review on the potential of different explants. Scientia Horticulturae 122:507-520.

Daviña JR, Grabiele M, Cerutti JC, Hojsgaard DH, Almada RD, Insaurralde IS, Honfi AI. 2009. Chromosome studies in Orchidaceae from Argentina. Genetics and Molecular Biology 32:811-821.

D’Emerico S, Galasso I, Pignone D, Scrugli A. 2001. Localization of rDNA loci by fluorescent in situ hybridization in some wild orchids from Italy (Orchidaceae). Caryologia 54:31-36.

De Chandra L, Pathak P, Rao AN, Rajeevan PK. 2019. Breeding approaches for improved genotypes. p. 300. In Commercial Orchids. De Gruyter Open Ltd., Berlin, Germany.

Devi J, Ko JM, Seo BB. 2005. FISH and GISH: Modern cytogenetic techniques. Indian Journal of Biotechnology 4:307-315.

Dhooghe E, Van Laere K, Eeckhaut T, Leus L, Van Huylenbroeck J. 2011. Mitotic chromosome doubling of plant tissues in vitro. Plant Cell, Tissue and Organ Culture (PCTOC) 104:359-373.

Dias VM, Manelli-Oliveira R, Machado-Santelli GM. 2005. Using fluorescence for improvement of the quantitative analysis of micronucleus in cell culture. Mutation Research/Genetic Toxicology and Environmental Mutagenesis 565:173-179.

Doležel J, Bartoš J. 2005. Plant DNA flow cytometry and estimation of nuclear genome size. Annals of Botany 95:99-110.

Dutrillaux AM, Lemonnier-Darcemont M, Darcemont C, Krpač V, Fouchet P, Dutrillaux B. 2009. Origin of the complex karyotype of the polyploid parthenogenetic grasshopper Saga pedo (Orthoptera: Tettigoniidae). European Journal of Entomology 106:477-483.

Germanà MA. 2012. Use of irradiated pollen to induce parthenogenesis and haploid production in fruit crops. Plant mutation breeding and biotechnology. pp. 411-421. CABI, Wallingford, UK.

He P, Li L, Cheng L, Wang H, Chang Y. 2018. Variation in ploidy level and morphological traits in the progeny of the triploid apple variety Jonagold. Czech Journal of Genetics and Plant Breeding 54:135-142.

Heslop-Harrison JS. 2000. Comparative genome organization in plants: From sequence and markers to chromatin and chromosomes. Plant Cell 12:617-635.

Hsiao YY, Pan ZJ, Hsu CC, Yang YP, Hsu YC, Chuang YC, Shih HH, Chen WH, Tsai WC, Chen HH. 2011. Research on orchid biology and biotechnology. Plant Cell Physiol 52:1467-1486.

Hsu CC, Chung YL, Chen TC, Lee YL, Kuo YT, Tsai WC, Hsiao YY, Chen YW, Wu WL, Chen HH. 2011. An overview of the Phalaenopsis orchid genome through BAC end sequence analysis. BMC Plant Biology 11:3.

Hwang YJ, Cabahug RA, Mancia FH, Lim KB. 2019. Molecular cytogenetics and its application to major flowering ornamental crops. Horticulture, Environment, and Biotechnology 61:1-9.

Hwang YJ, Kim HH, Kim JB, Lim KB. 2011. Karyotype analysis of Lilium tigrinum by FISH. Horticulture, Environment, and Biotechnology 52:292-297.

Jauhar PP. 2006. Modern biotechnology as an integral supplement to conventional plant breeding: The prospects and challenges. Crop Science 46:1841-1859.

Jiang J, Gill BS. 2006. Current status and the future of fluorescence in situ hybridization (FISH) in plant genome research. Genome 49:1057-1068.

Kao YY, Lin CC, Huang CH, Li YH. 2007. The cytogenetics of Phalaenopsis orchids. In Orchid Biotechnology edited by Chen WH, Chen HH. pp. 115-134. World Scientific Publishing Co., Ltd., Singapore, Singapore.

Kiihl PRP, Pereira ARA, Godoy SM de, Stenzel NMC, Risso-Pascotto C. 2011. Chromosome stickiness during meiotic behavior analysis of Passiflora serrato-digitata L. (Passifloraceae). Ciência Rural 41:1018-1023.

Kui L, Chen H, Zhang W, He S, Xiong Z, Zhang Y, Yan L, Zhong C, He F, Chen J, et al. 2017. Building a genetic manipulation toolbox for orchid biology: Identification of constitutive promoters and application of CRISPR/Cas9 in the orchid, Dendrobium officinale. Frontiers in Plant Science 7:2036.

Kumar M, Chaudhary V, Sharma VR, Sirohi U, Singh J. 2018. Advances in biochemical and molecular marker techniques and their applications in genetic studies of orchid: A review. International Journal of Chemical Studies 6:806-822.

Kuo YT, Hsu HL, Yeh CH, Chang SB. 2016. Application of a modified drop method for high-resolution pachytene chromosome spreads in two Phalaenopsis species. Molecular Cytogenetics 9:44.

Kurniawati J, Sugiyarto L, Yulianti E, Nurcahyo H, Mercuriani IS. 2019. Molecular identification of several orchid species based on OPA10 and OPA18 RAPD marker. International Journal of Chemical Studies 1397:12042.

Kus A, Kwasniewska J, Hasterok R. 2017. Brachypodium distachyon–a useful model in the qualification of mutagen-induced micronuclei using multicolor FISH. PLoS One 12:e0170618.

Kwasniewska J, Bara AW. 2022. Plant cytogenetics in the micronuclei investigation–the past, current status, and perspectives. International Journal of Molecular Sciences 23:1306.

Lan T, Albert VA. 2011. Dynamic distribution patterns of ribosomal DNA and chromosomal evolution in Paphiopedilum, a lady’s slipper orchid. BMC Plant Biology 11:126.

Lee BJ, Eo SH. 2016. Current trends in forest science research using microsatellite markers in Korean national journals. Korean Journal of Agricultural Science 43:221-231. [in Korean]

Lee YI, Chang FC, Chung MC. 2011. Chromosome pairing affinities in interspecific hybrids reflect phylogenetic distances among lady’s slipper orchids (Paphiopedilum). Annals of Botany 108:113-121.

Lee YI, Chung MC. 2008. Identification of genome relationships among Paphiopedilum species by genomic and fluorescent in situ hybridization. Acta Horticulturae 766:331-334.

Lee YI, Tseng YF, Lee YC, Chung MC. 2020. Chromosome constitution and nuclear DNA content of Phalaenopsis hybrids. Scientia Horticulturae 262:10908.

Lin S, Lee HC, Chen WH, Chen CC, Kao YY, Fu YM, Chen YH, Lin TY. 2001. Nuclear DNA contents of Phalaenopsis sp. and Doritis pulcherrima. Journal of the American Society for Horticultural Science 126:195-199.

Maluszynska J. 2003. Cytogenetic tests for ploidy level analyses–chromosome counting. In Doubled Haploid Production in Crop Plants edited by Maluszynski M, Kasha KJ, Forster BP, Szarejko I. pp. 391-395. Springer, Dordrecht, Netherlands.

Matsuba A, Fujii M, Lee SS, Suzuki G, Yamamoto M, Mukai Y. 2015. Molecular cytogenetic use of BAC clones in Neofinetia falcata and Rhynchostylis coelestis. Nucleus 58:207-210.

Moraes AP, Olmos SA, Ojeda ADI, De Barros F, Forni-Martins ER. 2016. Detecting mechanisms of karyotype evolution in Heterotaxis (Orchidaceae). PLoS One 11:e0165960.

Moscone EA, Samuel R, Schwarzacher T, Schweizer D, Pedrosa-Harand A. 2007. Complex rearrangements are involved in Cephalanthera (Orchidaceae) chromosome evolution. Chromosome Research 15:931-943.

Ochatt SJ. 2008. Flow cytometry in plant breeding. Journal of Quantitative Cell Science 73:581-598.

Pamarthi RK, Devadas R, Kumar R, Rai D, Kiran Babu P, Meitei AL, De LC, Chakrabarthy S, Barman D, Singh DR. 2019. PGR diversity and economic utilization of orchids. International Journal of Current Microbiology and Applied Science 8:1865-1887.

Perumal S, Waminal NE, Lee J, Lee J, Choi BS, Kim HH, Grandbastien MA, Yang TJ. 2017. Elucidating the major hidden genomic components of the A, C, and AC genomes and their influence on Brassica evolution. Science Reports 7:17986.

Raven PH. 1975. The bases of angiosperm phylogeny: Cytology. Annals of the Missouri Botanical Garden 1:724-764.

Russell G. 2004. Stomatal guard cell measurements using leaf prints. Certified Senior Advisor Journal 4:137-139.

Sakiroglu M, Kaya MM. 2012. Estimating genome size and confirming ploidy levels of wild tetraploid alfalfa accessions (Medicago sativa subsp. X varia) using flow cytometry. Turkish Journal of Field Crops 17:151-156.

Sattler MC, Carvalho CR, Clarindo WR. 2016. The polyploidy and its key role in plant breeding. Planta 243:281-296.

Semiarti E, Nopitasari S, Setiawati Y, Lawrie MD, Purwantoro A, Widada J, Ninomiya K, Asano Y, Matsumoto S, Yoshioka Y. 2020. Application of CRISPR/Cas9 genome editing system for molecular breeding of orchids. Indonesian Journal of Biotechnology 25:61-68.

Sharma SK, Mehra P, Kumari J, Kumar S, Kumaria S, Tandon P, Rao SR. 2012. Physical localization and probable transcriptional activity of 18S–5.8 S–26S rRNA gene loci in some Asiatic Cymbidiums (Orchidaceae) from north-east India. Genes 499:362-366.

Sharma SK, Mukai Y. 2015. Chromosome research in orchids: Current status and future prospects with special emphasis from molecular and epigenetic perspective. Nucleus (India) 58:173-184.

Silva GS, Souza MM. 2013. Genomic in situ hybridization in plants. Genetics and Molecular Research 12:2953-2965.

Singh RJ. 2016. Plant cytogenetics. CRC Press, London, UK.

Stace CA. 2000. Cytology and cytogenetics as a fundamental taxonomic resource for the 20th and 21st centuries. Taxonomy 49:451-477.

Tong CG, Wu FH, Yuan YH, Chen YR, Lin CS. 2020. High-efficiency CRISPR/Cas-based editing of Phalaenopsis orchid MADS genes. Plant Biotechnology Journal 18:889-891.

Trávníček P, Ponert J, Urfus T, Jersáková J, Vrána J, Hřibová E, Doležel J, Suda J. 2015. Challenges of flow-cytometric estimation of nuclear genome size in orchids, a plant group with both whole-genome and progressively partial endoreplication. Cytometry Part A 87:958-966.

Tsai WC, Dievart A, Hsu CC, Hsiao YY, Chiou SY, Huang H, Chen HH. 2017. Post genomics era for orchid research. Botanical Studies 58:1-22.

Vanstechelman I, Eeckhaut T, Van Huylenbroeck J, Van Labeke MC. 2010. Histogenic analysis of chemically induced mixoploids in Spathiphyllum wallisii. Euphytica 174:61-72.

Vilcherrez-Atoche JA, Iiyama CM, Cardoso JC. 2022. Polyploidization in orchids: From cellular changes to breeding applications. Plants 11:469.

Vu THT, Le TL, Nguyen TK, Tran DD, Tran HD. 2017. Review on molecular markers for identification of Orchids. Vietnam Journal of Science, Technology and Engineering 59:62-75.

Yan L, Wang X, Liu H, Tian Y, Lian J, Yang R, Hao S, Wang X, Yang S, Li Q, et al. 2015. The genome of Dendrobium officinale illuminates the biology of the important traditional Chinese orchid herb. Molecular Plant 8:922-934.

Yuan S, Liu Y, Fang Z, Yang L, Zhuang M, Zhang Y, Sun P. 2009. Relationship between the ploidy level of microspore-derived plants and the number of chloroplast in stomatal guard cells in Brassica oleracea. Scientia Agricultura Sinica 42:189-197.

Zhang GQ, Liu KW, Li Z, Lohaus R, Hsiao YY, Niu SC, Wang JY, Lin YC, Xu Q, Chen LJ, et al. 2017. The Apostasia genome and the evolution of orchids. Nature 549:379-383.

Zhang GQ, Xu Q, Bian C, Tsai WC, Yeh CM, Liu KW, Yoshida K, Zhang LS, Chang SB, Chen F, et al. 2016. The Dendrobium catenatum Lindl. genome sequence provides insights into polysaccharide synthase, floral development and adaptive evolution. Scientific Reports 6:19029.

Authors Information

Samantha S. Sevillen, https://orcid.org/0000-0001-9906-4992

Raisa Aone M. Cabahug, https://orcid.org/0000-0003-0863-4721

Hye Ryun An, https://orcid.org/0000-0002-3606-9195

Ki‑Byung Lim, https://orcid.org/0000-0003-2342-4052

Yoon-Jung Hwang, https://orcid.org/0000-0002-5984-8968

Conflicts of interest

No potential conflict of interest relevant to this article was reported.


This work was carried out with the support of the Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ01502405), Rural Development Administration, Republic of Korea.