Integrated assessment of seed desiccation, storage, and viability of Grammatophyllum speciosum for ex-situ conservation
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Abstract. Puspitaningtyas DM, Diantina S, Yuniati R, Handini E, Aprilianti P, Lestari R. 2026. Integrated assessment of seed desiccation, storage, and viability of Grammatophyllum speciosum for ex-situ conservation. Biodiversitas 27 (3): d270323. https://doi.org/10.13057/biodiv/d270323. Orchid seed banking is recognized as an efficient ex-situ conservation strategy. However, standardized protocols for seed drying, storage, and viability assessment are still lacking for many tropical species. This study evaluated the effects of desiccation duration, frozen storage at -20°C, and tetrazolium chloride (TTC) staining to develop a practical conservation protocol for Grammatophyllum speciosum. Fresh seeds were dried over silica gel for 3-9 days and germinated asymbiotically on several basal media. Seed viability after one year of storage was assessed using eight TTC pre-treatments that combined NaOCl scarification, sucrose pre-conditioning, and two TTC concentrations. Germination was significantly influenced by drying duration, and a significant interaction between medium and drying period was detected (ANOVA, p<0.01). The highest germination (74.82%±6.01) was obtained after three days of drying on Vacin & Went (VW) medium, whereas prolonged drying (6-9 days) reduced germination to 55-65%. After one year of storage at -20°C, seeds retained high viability (71.87-72.98%), with no significant decline compared to fresh seeds, indicating tolerance to both desiccation and subzero conditions. TTC-based viability assessment revealed significant differences among pre-treatments (ANOVA, p<0.001). Combined NaOCl scarification and sucrose pre-conditioning improved staining clarity, producing the highest viability values (up to 73.57%±5.16). No significant difference was observed between 0.1% and 1% TTC concentrations, demonstrating that the lower concentration was sufficient for reliable viability estimation. TTC-based viability closely matched germination results, indicating the consistency of direct and indirect assessment methods. Overall, short-term desiccation (3 days), storage at -20°C, and optimized TTC pre-treatment provide a scalable and resource-efficient strategy for orchid seed banking. This protocol supports the long-term preservation of genetic diversity and contributes to conservation management of threatened epiphytic orchids.
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References
Bae K-H, Ko MS, Lee MH, Kim NY, Song JM, Song G. 2013. Effects of NaOCl treatment on in vitro germination of seeds of a rare endemic plant, Oreorchis coreana Finet. J Plant Biotechnol 40: 43-48. https://doi.org/10.5010/JPB.2013.40.1.043.
Custódio CC, Marks TR, Pritchard HW, Hosomi ST, Machado-Neto NB. 2016. Improved tetrazolium viability testing in orchid seeds with a thick carapace (Dactylorhiza fuchsii) or dark seed coat (Vanda curvifolia). Seed Sci Technol 44 (1): 1-12. https://doi.org/10.15258/sst.2016.44.1.17.
Diantina S, Kartikaningrum S, McCormick AC, Millner J, McGill C, Pritchard HW, Nadarajan J. 2020. Comparative in vitro seed germination and seedling development in tropical and temperate epiphytic and temperate terrestrial orchids. Plant Cell Tissue Organ Cult 143: 619-633. https://doi.org/10.1007/s11240-020-01947-7.
Dowling N, Jusaitis M. 2012. Asymbiotic in vitro germination and seed quality assessment of Australian terrestrial orchids. Aust J Bot 60: 592-601. https://doi.org/10.1071/BT12133.
Ellis RH, Hong TD, Roberts EH. 1989. A comparison of the low-moisture-content limit to the logarithmic relation between seed moisture and longevity in twelve species. Ann Bot 63 (6): 601-611. https://doi.org/10.1093/oxfordjournals.aob.a087788.
França-Neto JB, Krzyzanowski FC. 2019. Tetrazolium: An important test for physiological seed quality evaluation. J Seed Sci 41 (3): 359-366. https://doi.org/10.1590/2317-1545v41n3223104.
Gnasekaran P, Poobathy R, Mahmood M, Samian MR, Subramaniam S. 2012. Effects of complex organic additives on improving the growth of PLBs of Vanda Kasem’s Delight. Aust J Crop Sci 6 (8): 1245-1248.
Hay FR, Rezaei S, Wolkis D, McGill C. 2023. Determination and control of seed moisture. Seed Sci Technol 51 (2): 267-285. https://doi.org/10.15258/sst.2023.51.2.11.
Hosomi ST, Santos RB, Custódio CC, Seaton PT, Marks TR, Machado-Neto NB. 2011. Pre-conditioning Cattleya seeds to improve the efficacy of the tetrazolium test for viability. Seed Sci Technol 39: 178-189. https://doi.org/10.15258/sst.2011.39.1.15.
Hosomi ST, Custódio CC, Seaton PT, Marks TR, Machado-Neto NB. 2012. Improved assessment of viability and germination of Cattleya (Orchidaceae) seeds following storage. In Vitro Cell Dev Biol Plant 48: 127-136. https://doi.org/10.1007/s11627-011-9404-1.
Hosomi ST, de Souza TB, Custódio CC, Machado-Neto NB. 2017. Refining the tetrazolium test for evaluation of Cattleya labiata and C. tigrina seeds viability. Aust J Crop Sci 11 (10): 1320-1326. https://doi.org/10.21475/ajcs.17.11.10.pne606.
Kauth PJ, Vendrame WA, Kane ME. 2006. In vitro seed culture and seedling development of Calopogon tuberosus. Plant Cell Tissue Organ Cult 85: 91-102. https://doi.org/10.1007/s11240-005-9055-1.
Kauth PJ, Dutra D, Johnson TR, Stewart SL, Kane ME, Vendrame WA. 2008. Techniques and applications of in vitro orchid seed germination. In: Teixeira da Silva JA (eds). Floriculture, Ornamental and Plant Biotechnology: Advances and Topical Issues. Global Science Books, UK.
Machado-Neto NB, Custódio CC. 2005. Orchid conservation through seed banking: Ins and outs. Selbyana 26 (1-2): 229-235.
Magrini S, De Vitis M. 2016. Impacts of scarification methods and nitrogen source on seeds germination in the genus Limodorum. Plant Biosyst 151: 419-428. https://doi.org/10.1080/11263504.2016.1179698.
Mercado SAS, Manrique JDG. 2015. Determining viability of orchid seeds using tetrazolium and carmín índigo tests. Rev Cienc 19: 59-69.
Mercado SAS, Delgado EAB, Caleño JDQ. 2019. Pre-treatment effect on the tetrazolium test on Epidendrum barbaricum Hágsater & Dodson seeds. Acta Agron 68 (4): 306-311. https://doi.org/10.15446/acag.v68n4.79619.
Mercado SAS, Caleño JDQ, Rozo LYM. 2020a. Improvement of the methodology of the tetrazolium test using different pretreatments in seeds of the genus Epidendrum (Orchidaceae). J Seed Sci 42: e202042013. https://doi.org/10.1590/2317-1545v42231028.
Mercado SAS, Caleño JDQ, Suárez JPR. 2020b. Optimization of the tetrazolium test in three species of orchids of the Andean forest. Aust J Crop Sci 14: 822-829. https://doi.org/10.21475/ajcs.20.14.05.p2276.
Merritt DJ, Hay FR, Swarts ND, Sommerville KD, Dixon KW. 2014. Ex-situ conservation and cryopreservation of orchid germplasm. Intl J Plant Sci 175: 46-58. https://doi.org/10.1086/673370.
Park HB, An J, Bae K-H, Hong SH, Park HJ, Kim S, Lee CW, Lee B-D, Baek JH, Kim NY, Hwang JE. 2023. Asymbiotic seed germination and in vitro seedling development of the endangered orchid species Cypripedium guttatum. Plants 12: 3788. https://doi.org/10.3390/plants12223788.
Popova E, Kim HH, Saxena PK, Engelmann F, Pritchard HW. 2016. Frozen beauty: The cryobiotechnology of orchid diversity. Biotechnol Adv 34: 380-403. https://doi.org/10.1016/j.biotechadv.2016.01.001.
POWO [Plants of the World Online]. 2025. Grammatophyllum speciosum Blume. https://powo.science.kew.org/taxon/urn:lsid:ipni.org:names: 636310-1. [10 January 2025]
Pradhan S, Regmi T, Parmar G, Pant B. 2013. Effect of different media on in vitro seed germination and seedling development of Cymbidium aloifolium (L.) Sw. Nepal J Sci Technol 14 (1): 51-56. https://doi.org/10.3126/njst.v14i1.8878.
Pradhan N, Fan X, Martini F, Chen H, Liu H, Gao J, Goodale UM. 2022. Seed viability testing for research and conservation of epiphytic and terrestrial orchids. Bot Stud 63 (1): 1-14. https://doi.org/10.1186/s40529-022-00333-0.
Pritchard HW, Seaton PT. 1993. Orchid seed storage: Historical perspective, current status, and future prospects for long-term conservation. Selbyana 14 (2): 89-104.
Puspitaningtyas DM, Handini E. 2014. Orchid seeds storage of Coelogyne spp. for ex situ conservation. Bul Kebun Raya 17 (2): 101-111. https://publikasikr.lipi.go.id/index.php/buletin/article/view/83. [Indonesian]
Puspitaningtyas DM, Handini E. 2020. Ex situ conservation of Cymbidium finlaysonianum by seed storage. Biodiversitas 21 (8): 3519-3524. https://doi.org/10.13057/biodiv/d210813.
Puspitaningtyas DM, Handini E. 2021. Seed germination evaluation of Phalaenopsis amabilis in various media for long-term conservation. Biodiversitas 22 (11): 5231-5238. https://doi.org/10.13057/biodiv/d221162.
Samala S, Te-chato S, Yenchon S, Thammasiri K. 2014. Protocorm-like body proliferation of Grammatophyllum speciosum through asymbiotic seed germination. ScienceAsia 40: 379-383. https://doi.org/10.2306/scienceasia1513-1874.2014.40.379.
Seaton PT, Pritchard HW. 2003. Orchid germplasm collection, storage and exchange. In: Dixon KW, Kell SP, Barrett RL, Cribb PJ (eds). Orchid Conservation. Natural History Publications, Kota Kinabalu, Sabah. 227-258.
Seaton PT, Pritchard HW. 2011. Orchid seed stores for sustainable use: A model for future seed-banking activities. Lankesteriana 11: 349-353. https://doi.org/10.15517/lank.v11i3.18290.
Seaton P, Kendon JP, Pritchard HW, Puspitaningtyas DM, Marks TR. 2013. Orchid conservation: The next ten years. Lankesteriana 13 (1-2): 93-101. https://doi.org/10.15517/lank.v0i0.11545.
Seaton PT, Hosomi ST, Custódio CC, Marks TR, Machado-Neto NB, Pritchard HW. 2018. Orchid seed and pollen: A toolkit for long-term storage, viability assessment and conservation. In: Lee Y-I, Yeung EC-T (eds). Orchid Propagation: From Laboratories to Greenhouses—Methods and Protocols. Springer Protocols Handbooks, New York. https://doi.org/10.1007/978-1-4939-7771-0_4.
Shekarriz P, Kafi M, Deilamy SD, Mirmasoumi M. 2014. Coconut water and peptone improve seed germination and protocorm-like body formation of hybrid Phalaenopsis. Agric Sci Dev 3 (10): 317-322.
Sopalun K, Thammasiri K, Ishikawa K. 2010. Micropropagation of the Thai orchid Grammatophyllum speciosum Blume. Plant Cell Tissue Organ Cult 101: 143-150. https://doi.org/10.1007/s11240-010-9671-2.
Srivastava D, Gayatri MC, Sarangi SK. 2015. In vitro seed germination and plant regeneration of an epiphytic orchid Aerides ringens (Lindl.) Fischer. Indian J Biotechnol 14: 574-580.
Stewart SL, Kane ME. 2006. Symbiotic seed germination of Habenaria macroceratitis (Orchidaceae), a rare Florida terrestrial orchid. Plant Cell Tissue Organ Cult 86: 159-167.
Swartz ND, Dixon KW. 2017. Conservation methods for terrestrial orchids. J Ross Publishing, Plantation, FL, USA.
Udomdee W, Wen P, Chin S, Chen F. 2014. Effect of storage temperature on seed viability and in vitro germination of nobile Dendrobium hybrids. Thai Agric Res J 32 (2): 201-217. https://doi.org/10.14456/thaidoa-agres.2014.9.
Utami ESW, Hariyanto S. 2020. Organic compounds: Contents and their role in improving seed germination and protocorm development in orchids. Intl J Agron 2020: 1-12. https://doi.org/10.1155/2020/2795108.
Vacin EF, Went FW. 1949. Some pH changes in nutrient solutions. Bot Gaz 110 (4): 605-613.
Vendrame WA, Carvalho VS, Dias JMM. 2007. In vitro germination and seedling development of cryopreserved Dendrobium hybrid mature seeds. Sci Hortic 114 (2): 188-193.
Vertucci CW, Roos EE. 1990. Theoretical basis of protocols for seed storage. Plant Physiol 94 (3): 1019-1023. https://doi.org/10.1104/pp.94.3.1019.
Walters C. 1998. Understanding the mechanisms and kinetics of seed aging. Seed Sci Res 8: 223-244. https://doi.org/10.1017/S096025850000413X.
Yong JWH, Ge L, Ng YF, Tan SNg. 2009. The chemical composition and biological properties of coconut (Cocos nucifera L.) water. Molecules 14 (12): 5144-5164. https://doi.org/10.3390/molecules14125144.
Zakiyah A, Setiari N, Nurchayati Y. 2025. The growth of Dendrobium spectabile (Blume) Miq. orchid plantlet in Vacin and Went (VW) medium with different concentrations of coconut water. Berita Biologi 24 (2): 333-343. https://doi.org/10.55981/berita_biologi.2025.222.
Zeng S, Wu K, da Silva JAT, Zhang J, Chen Z, Xia N, Duan J. 2012. Asymbiotic seed germination, seedling development and reintroduction of Paphiopedilum wardii Sumerh., an endangered terrestrial orchid. Sci Hortic 138: 198-209. https://doi.org/10.1016/j.scienta.2012.02.026.