Clonal micropropagation of Coelogyne mooreana Sander ex Rolfe

Coelogyne mooreana Sander ex Rolfe, a valuable, rare, and endemic orchid species of the Vietnamese flora, is characterized by high ornamental qualities. This orchid is highly ornamental and often referred to as the “queen” of the genus Coelogyne due to its large, white, fragrant flowers adorned with black spots and a central yellow stripe. Therefore, this study aimed to develop a protocol for clonal micropropagation by modifying the nutrient medium with organic additives and plant growth regulators. Immature seeds collected from unopened fruits six months after artificial pollination were used as starting material. This work involved several stages: asymbiotic seed germination, protocorm development, seedling formation, and rooting. The highest seed germination rate (93%) was achieved on Gamborg (B5) nutrient medium supplemented with 0.5 mg/L 6-BAP. Maximum adventitious shoot formation was observed on Murashige and Skoog (½ MS) nutrient medium with organic additives but without plant growth regulators (5.64 and 4.56 shoots, respectively). Favorable results for protocorm development and seedling formation were obtained on ½ MS medium supplemented with 0.5 mg/L 6-BAP and coconut water (100 mL/L). At the rooting stage, the most effective medium was ½ MS supplemented with 0.5 mg/L IBA and 100 mL/L coconut water, which promoted the development of a strong root system, with a rooting percentage of 96.7%, 5.05 ± 0.36 roots per explant, a root length of 2.43 ± 0.05 cm, and a plant length of 5.95 ± 0.24 cm. Seedlings planted in a 1:1:1 ratio of bark, perlite, and peat exhibited high viability when adapted to ex vitro conditions. For the first time, a complete protocol for clonal micropropagation of C. mooreana encompassing all stages was developed. These results provide recommendations for the conservation and mass propagation of this rare and valuable orchid species.

1. Khasim S.M., Hegde S.N., González-Arnao M.T., Thammasiri K. Orchid Biology: Recent Trends & Challenges. Singapore, Singapore: Springer, 2020:547. https://doi.org/10.1007/978-981-32-9456-1

2. Ai Y.Y., Liu Q., Hu H.X., Shen T. et al. Terrestrial and Epiphytic Orchids Exhibit Different Diversity and Distribution Patterns Along an Elevation Gradient of Mt. Victoria, Myanmar. Global Ecology and Conservation. 2023;42: e02408. https://doi.org/10.1016/j.gecco.2023.e02408

3. Korovkin O.A., Cheryatova Yu.S. Botany: a textbook. Moscow, Russia: KnoRus, 2024:464. EDN: CBVVAR (In Russ.)

4. Cheryatova Yu.S. Evolutionary-ecological adaptations and the biosphere role of plants. Biosfernoe khozyaystvo: teoriya i praktika. 2022;(10):45-49. (In Russ.)

5. Djordjević V., Tsiftsis S. The Role of Ecological Factors in Distribution and Abundance of Terrestrial Orchids. Brno, Czech Republic: Springer International Publ., 2022;71. https://doi.org/10.1007/978-3-030-11257-8_4-1

6. Shefferson R.P., Jacquemyn H., Kull T., Hutchings M.J. The Demography of Terrestrial Orchids: Life History, Population Dynamics and Conservation. Botanical Journal of the Linnean Society. 2020;192(2):315-332. https://doi.org/10.1093/botlinnean/boz084

7. Clayton D. The Genus Coelogyne: A Synopsis. Borneo, Republic of Indonesia: Natural History Publications; Kew, United Kingdom: The Royal Botanic Gardens, 2002:305.

8. Chase M.W., Gravendeel B., Sulistyo B.P., Wati R.K. Expansion of the Orchid Genus Coelogyne (Arethuseae; Epidendroideae) to Include Bracisepalum, Bulleyia, Chelonistele, Dendrochilum, Dickasonia, Entomophobia, Geesinkorchis, Gynoglottis, Ischnogyne, Nabaluia, Neogyna, Otochilus, Panisea and Pholidota. Phytotaxa. 2021:94-134. https://doi.org/10.11646/phytotaxa.510.2.1

9. Yeung E.C. The Orchid Embryo – “ An Embryonic protocorm”. Botany. 2022;100(9):691-706. https://doi.org/10.1139/cjb-2022-0017

10. 10 . Kolomeitseva G.L., Koval V.A., Ryabchenko A.S., Babosha A.V. Megasporogenesis and Megagametogenesis in Coelogyne speciosa subsp. Fimbriata (J.J.Sm.) Gravendeel (Orchidaceae Juss.). Int. J. Plant Biol. 2023;14:190-198. https://doi.org/10.3390/ijpb14010016

11. Kolomeytseva G.L., Tsavkelova E.A., Gusev E.M., Malina N.E. On the symbiosis of some orchids and the active strain of Bacillus pumilus bacteria in in vitro culture. Bulletin of the Central Botanical Garden. 2002;183:117-126. (In Russ.)

12. Shirokov A.I., Kolomeytseva G.L., Burov A.V., Kameneva E.V. Cultivation of Orchids of European Russia: a monograph. Nizhny Novgorod, Russia, 2005:64. (In Russ.)

13. Orlova N.D., Molkanova O.I., Konovalova T.Yu., Yatsenko I.O. Features of Decaisnea fargesii Franch. regeneration in in vitro culture. Tauride Bulletin of Agrarian Science. 2024;(1(37)):125-135. (In Russ.) https://doi.org/10.5281/zenodo.10930671

14. Tatarenko I.V. Mycorrhiza of Orchidaceae of Primorsky Krai. Botanicheskii Zhurnal. 1995;80(8):64-72. (In Russ.)

15. Bolshakova E.V., Emelyanova I.S., Lukatkin A.S., Mokshin E.V. Influence of nutrient media on the morphogenesis of Vanda hybrid and Anoectochilus roxburghii. Mezhdunarodnaya nauchno-prakticheskaya konferentsiya ‘Nauka segodnya: opyt, traditsii, innovatsii’. July 26, 2017. Vologda, Russia: Marker, 2017:8-10. (In Russ.)

16. Velmyaykin I.N., Mokshin E.V., Lukatkin A.S. Effect of growth regulators on in vitro shoot growth and propagation of Cymbidium hybridum Hort. Vestnik Nizhegorodskogo universiteta imeni N.I. Lobachevskogo. 2013;(3-1):133-137. (In Russ.)

17. Hussien M., Konovalova T.Yu., Molkanova O.I. Clonal micropropagation of representatives of the genus Dactylorhiza Neck. ex Nevski. AgroEcoInfo. 2021;(4):20. (In Russ.) https://doi.org/10.51419/20214417

18. Hussien M., Molkanova O.I., Raeva-Bogoslovskaya E.N., Makarov S.S. Plant Growth Regulators and Organic Additives on the Proliferation of Protocorm-like Bodies and Plantlet Regeneration of Cattleya gaskelliana (N.E.Br.) B.S. Williams. Agricultural Sciences (Ciência e Agrotecnologia). 2024;48: e018223. https://doi.org/10.1590/1413-7054202448018223

19. Hussien M., Molkanova O.I., Orlova E.E., Koval V.A. Improving rooting of Cattleya gaskelliana (N.E.Br.) B.S. Williams (Orchidaceae Juss.) under in vitro conditions. Proceedings of the Russian Academy of Sciences. Biological Series. 2025;(1):35-43. https://doi.org/10.31857/S1026347025010045 (In Russ.)

20. Makarov S.S., Kalashnikova E.A., Kuznetsova I.B., Kirakosyan R.N. Growing forest berry plants in in vitro conditions. Laboratory practical training: a textbook. Karavaevo, Russia: Kostroma State Agricultural Academy, 2019:48. (In Russ.)

21. Makarov S.S., Antonov A.M., Kulikova E.I. Biotechnology in horticulture. growing fruit and rare berry plants in in vitro culture. Laboratory practical training: a textbook. St. Petersburg, Russia: Lan’, 2023:128. (In Russ.)

22. Utami E.S.W., Hariyanto S. Organic Compounds: Contents and Their Role in Improving Seed Germination and Protocorm Development in Orchids. International Journal of Agronomy. 2020;2020(1):2795108. https://doi.org/10.1155/2020/2795108

23. Huh Y.S., Lee J.K., Nam S.Y., Paek K.Y. et al. Improvement of Asymbiotic Seed Germination and Seedling Development of Cypripedium macranthos Sw. with Organic Additives. Journal of Plant Biotechnology. 2016;43(1):138-145. https://doi.org/10.5010/JPB.2016.43.1.138

24. Salsabila S.N., Fatimah K., Noorhazira S., Halimatun T.S.T.A.B. et al. Effect of Coconut Water and Peptone in Micropropagation of Phalaenopsis amabilis (L.) Blume Orchid. In: IOP Conference Series: Earth and Environmental Science. 2022;1102(1):012002. https://doi.org/10.1088/1755-1315/1102/1/012002

25. Dewir Y.H., El-Mahrouk M.E., Murthy H.N., Paek K.Y. Micropropagation of Cattleya: Improved In Vitro Rooting and Acclimatization. Horticulture, Environment, and Biotechnology. 2015;56:89-93. https://doi.org/10.1007/s13580-015-0108-z

26. Hussien M., Molkanova O., Mitrofanova I.V. Micropropagation of Trichopilia suavis Lindl. & Paxton. Ornamental Horticulture. 2023;29:365-374. https://doi.org/10.1590/2447-536X.v29i3.2653

27. Nabieva A.Y. Asymbiotic Seed Germination and In Vitro Seedling Development of Orchis militaris, an Endangered Orchid in Siberia. Journal of Genetic Engineering and Biotechnology. 2021;19(1):122. https://doi.org/10.1186/s43141-021-00223-1

28. Jagiełło-Kubiec K., Nowakowska K., Łukaszewska A.J., Pacholczak A. Acclimation to Ex Vitro Conditions in Ninebark. Agronomy. 2021;11(4):612. https://doi.org/10.3390/agronomy11040612

29. Sanghamitra M., Babu J.D., Bhagavan B.V.K., et al. Standardization of Different Potting Media on Physiological Growth, Yield and Vase Life of Dendrobium Orchid cv. Sonia 17 under Shade Net Conditions in High Altitude Tribal Zone of Andhra Pradesh. Journal of Pharmacognosy and Phytochemistry. 2019;8(5):128-132. https://krishikosh.egranth.ac.in/handle/1/5810181497