Biological Systems: Theory and Innovation

Microclonal propagation of the orchid Vanda (Orchidaceae Vanda)

Oksana Klyachenko, Oleksandr Subin, Olha Oliinyk, Diana Shyshkina
Download article
Abstract

Biotechnological methods of propagation of Vanda orchids represent an effective approach for obtaining large quantities of genetically uniform and disease-free plant material. This study aimed to investigate the specific features of microclonal propagation of Vanda orchids. To obtain planting material of different Vanda genotypes, namely ‘Vanda coerulea’ and ‘Vanda sanderiana’, direct and indirect morphogenesis, as well as in vitro rhizogenesis followed by adaptation to in vivo conditions, were applied. A stepwise method for obtaining aseptic material was developed, involving treatment with 70% ethyl alcohol for 1 minute followed by the application of the main sterilising agent, 2.5% NaClO, for 5-10 minutes. This protocol made it possible to obtain 70%-80% sterile and viable explants while reducing the level of fungal and bacterial contamination. The results of direct and indirect morphogenesis, callus formation, and rhizogenesis in in vitro cultures of Vanda explants are presented. No significant differences in callus formation were observed between the studied cultivars. The frequency of callusogenesis for both genotypes reached 100%, indicating a high capacity for callus tissue formation. The best results in growth and shoot formation were achieved on Murashige and Skoog (MS) nutrient medium supplemented with 0.5 mg/L 6-benzylaminopurine (6-BAP). For rooting, the most effective medium was MS with half‑strength macro- and microelements supplemented with 0.5 mg/L α-naphthaleneacetic acid (NAA). This medium is recommended for the induction of rhizogenesis in regenerated plants of different Vanda cultivars. For the adaptation of regenerated plants, a substrate composed of sphagnum moss, peat, crushed beech leaves, and pine bark in a ratio of 1:1:1:1 was used. The survival rate of ‘Vanda coerulea’ plants under in vivo conditions reached 87.5%, whereas under the same conditions, the survival rate of ‘Vanda sanderiana’ plants was 83%. The obtained results may serve as a basis for the development of propagation protocols for Vanda orchids, ensuring a high level of control at all stages of cultivation. The implementation of such protocols is important for breeding programmes, the conservation of rare cultivars, and the improvement of plant material quality for commercial floriculture

Keywords

in vitro; callusogenesis; morphogenesis; rhizogenesis; in vivo adaptation

Suggested citation
Klyachenko, O., Subin, O., Oliinyk, O., & Shyshkina, D. (2025). Microclonal propagation of the orchid Vanda (Orchidaceae Vanda). Biological Systems: Theory and Innovation, 16(4), 40-56. https://doi.org/10.31548/biologiya/4.2025.40
References
  1. Baby, R., Valsala, P.A., & Manjesh, S. (2019a). Genetic fidelity evaluation of micropropagated plantlets of Vanda hybrid Dr. Anek. International Journal of Current Microbiology and Applied Sciences, 8(8), 2373-2381. doi: 10.20546/ijcmas.2019.808.276.
  2. Baby, R., Valsala, P.A., & Doddamani, M.B. (2019b). In vitro micropropagation protocol for Vanda hybrid ‘dr. Anek.’ International Journal of Current Microbiology and Applied Sciences, 8(4), 2073-2084. doi: 10.20546/ijcmas.2019.804.244.
  3. Bhattacharjee, B., & Islam, S.M.S. (2014a). Development of an efficient protocol for in vitro germination and enhancing protocorm-like body development in three indigenous orchid species in Bangladesh. Asian Pacific Journal of Molecular Biology and Biotechnology, 22(3), 209-218.
  4. Bhattacharjee, B., & Islam, S.M.S. (2014b). Effects of plant growth regulators on multiple shoot induction in Vanda tessellate (Roxb.) Hook. Ex G.Don an endangered medicinal plant. International Journal of Science and Nature, 5, 707-712.
  5. Chen, J.-T. (2018). In vitro organogenesis of a slipper orchid, paphiopedilum ‘Alma Gavaert’. Notulae Scientia Biologicae, 10(4), 607-613. doi: 10.15835/nsb10410368.
  6. Chugh, S., Guha, S., & Rao, I.U. (2009). Micropropagation of orchids: A review on the potential of different explants. Scientia Horticulturae, 122(4), 507-520. doi: 10.1016/j.scienta.2009.07.016.
  7. Convention on Biological Diversity. (1992, June). Retrieved from https://zakon.rada.gov.ua/laws/show/995_030#Text.
  8. Dar, S.A., Nawchoo, I.A., Tyub, S., & Kamili, A.N. (2021) Effect of plant growth regulators on in vitro induction and maintenance of callus from leaf and root explants of Atropa acuminata Royal ex. Lindl. Biotechnology Reports, 32, article number e00688. doi: 10.1016/j.btre.2021.e00688.
  9. Davis, B.J., Phillips, R.D., Wright, M., Linde, C.C., & Dixon, K.W. (2015). Continent-wide distribution in mycorrhizal fungi: Implications for the biogeography of specialized orchids. Annals of Botany, 116(3), 413-421. doi: 10.1093/aob/mcv084.
  10. Hardjo, P.H., Savitri, W.D., Artadana, I.B.M., Putra, S.E.D., Parac, E.P., & Jan, A. (2021). Callus-mediated somatic embryogenesis and plant regeneration in Vanda tricolor Lindl. var. Pallida. Jordan Journal of Biological Sciences, 14(05), 933-937. doi: 10.54319/jjbs/140508.
  11. Kaeppler, S.M., Kaeppler, H.F., & Rhee, Y. (2000). Epigenetic aspects of somaclonal variation in plants. Plant Molecular Biology, 43, 179-188. doi: 10.1023/a:1006423110134.
  12. Kolomiets, Yu.V., Kliachenko, O.L., & Subin, O.V. (2022). Biotechnology. Kyiv: Comprint.
  13. Kumar, K., & Rao, I.U. (2012). Morphophysiologicals problems in acclimatization of micropropagated plants in -ex vitro conditions- a reviews. Journal of Ornamental Plants, 2, 271-283.
  14. Kushnir, G.P., & Sarnatska, V.V. (2005). Microclonal propagation of plants. Kyiv: Naukova Dumka.
  15. Laily, A.N., Maharijaya, A., Wulandari, D., Puspitaningtyas, D.M., Ariate, S.R., Martin, A.F., & Hapsari, B.W. (2024). Optimizing micropropagation of Indonesian conserved orchid Vanda celebica using organic compunds and a temporary immersion system. Notulae Botanicae Horti Agrobotanici Cluj-Napoca, 52(4), article number 14065. doi: 10.15835/nbha52414065.
  16. Lang, N.T., & Hang, N.T. (2006). Using biotechnological approaches for Vanda orchid improvement. Omonrice, 14, 140-143.
  17. Lee, Y.-I., Hsu, S.-T., & Yeung, E.C. (2013). Orchid protocorm-like bodies are somatic embryos. American Journal of Botany, 100(11), 2121-2131. doi: 10.3732/ajb.1300193.
  18. Likhanov, A.F., Overchenko, O.V., Kostenko, S.M. & Subin, O.V. (2017). Specificity of cytodifferentiation in calluses in vitro of Aesculus Hippocastanum form resistant to horse-chestnut leaf miner. Plant Physiology and Genetics, 49(6), 495-505. doi: 10.15407/frg2017.06.495.
  19. Liu, X.-f., Xiang, L.-l., Huang, Y., Li, Y.-j., & Li, F. (2020). Efficient propagation with in vitro seed germination of Vanda falcata. Journal of Zhejiang University-SCIENCE B, 21, 999-1004. doi: 10.1631/jzus.b2000505.
  20. Maharjan, S., Pradhan, S., Thapa, B.B., & Pant, B. (2019). In vitro propagation of endangered orchid, Vanda pumila Hook.f. through protocorms culture. American Journal of Plant Sciences, 10(07), 1220-1232. doi: 10.4236/ajps.2019.107087.
  21. Muharyati, Y., Setiyadi, M.W., Khatimah, A., & Ardiansyah, A. (2024). Propagation of the Vanda helvola orchid in vitro. Jurnal Pijar Mipa, 19(3), 488-492. doi: 10.29303/jpm.v19i3.6600.
  22. Murashige, T., & Skoog, F. (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum, 15, 473-497. doi: 10.1111/j.1399-3054.1962.tb08052.x.
  23. Nongdam, P., Beleski, D.G., Tikendra, L., Dey, A., Varte, V., EL Merzougui, S., Pereira, V.M., Barros, P.R., & Vendrame, W.A. (2023). Orchid micropropagation using conventional semi-solid and temporary immersion systems: A review. Plants, 12(5), article number 1136. doi: 10.3390/plants12051136.
  24. Nowakowska, K., Marciniak, P., & Pacholczak, A. (2022). A protocol for efficient micropropagation of rare orchid Vanda brunnea Rchb.f. South African Journal of Botany, 150, 233-239. doi: 10.1016/j.sajb.2022.07.023.
  25. Palama, T.L., Menard, P., Fock, I., Choi, Y.H., Bourdon, E., Govinden-Soulange, J., Bahut, M., Payet, B., Verpoorte, R., & Kodja, H. (2010). Shoot differentiation from protocorm callus cultures of Vanilla planifolia (Orchidaceae): Proteomic and metabolic responses at early stage. BMC Plant Biology, 10(1), article number 82. doi: 10.1186/1471-2229-10-82.
  26. Pathak, P., Kumari, A., Chandler, B.D., & Zettler, L.W. (2023). In vitro propagation and phytochemical analysis of Vanda cristata Wall. ex Lindl: An endangered medicinal orchid of biopharmaceutical importance. South African Journal of Botany, 153, 109-123. doi: 10.1016/j.sajb.2022.11.023.
  27. Podhaetskyi, A.A., Matskevych, V.V., & Podhaetskyi, A.A. (2018). Features of microclonal propagation plant species: Monography. Bila Tserkva: BNAU.
  28. Rahman, M., Hasan, M., Das, R., Hossain, M., & Rahman, M. (2009). In vitro micropropagation of orchid (Vanda tessellata L.) from shoot tip explant. Journal of Bio-Science, 17, 139-144. doi: 10.3329/jbs.v17i0.7122.
  29. Rasmussen, H.N., & Rasmussen, F.N. (2014). Seedling mycorrhiza: A discussion of origin and evolution in Orchidaceae. Botanical Journal of the Linnean Society, 175(3), 313-327. doi: 10.1111/boj.12170.
  30. Setiaji, A., Annisa, R.R., Santoso, A.D., Kinasih, A., & Riyadi, A.D. (2021). Review: Factors affecting mass propagation of Vanda orchid in vitro. Cell Biology and Development, 5(2). doi: 10.13057/cellbioldev/v050201.
  31. Shemediuk, N., & Skip, O. (2014) Application biotechnological methods for the biodiversity representative of the family Orchidaceae Juss. Scientific Bulletin of the S.Z. Gzhytskyi National University of Veterinary Medicine and Biotechnologies Lviv, 16(3), 345-357.
  32. Sheyko, O.A., & Musatenko, L.I. (2013). The induction callus formation in vitro from explants of rare and endangered orchid. Chornomorski Botanical Journal, 9(4), 595-604.
  33. Thomas, T.D. (2008). The role of activated charcoal in plant tissue culture. Biotechnology Advances, 26(6), 618-631. doi: 10.1016/j.biotechadv.2008.08.003.
  34. Tikendra, L., Singh, A.R., Vendrame, W.A., & Nongdam, P. (2025). In vitro propagation of endangered ‘Vanda coerulea’ Griff. ex Lindl.: Asymbiotic seed germination, genetic homogeneity assessment, and micro-morpho-anatomical analysis for effective conservation. Agronomy, 15(5), article number 1195. doi: 10.3390/agronomy15051195.