Energy Efficient Micropropagation of Dendrobium cruentum Rchb. f.: Chemical Sterilization, Net House Culture, Sucrose Optimization, and Acclimatization Substrate Selection
Article Sidebar
Main Article Content
Abstract
Dendrobium cruentum Rchb. f., a threatened native orchid of southern Thailand, faces extinction from habitat loss and over-collection; however, conventional micropropagation protocols remain economically inaccessible for most conservation programs due to high energy costs. This study developed an energy-efficient micropropagation protocol by evaluating sterilization methods, culture environments, sucrose concentrations, and acclimatization substrates. Six sterilization treatments were assessed: conventional autoclaving (positive control), four concentrations of Haiter® bleach (NaOCl; 0.5, 1.0, 1.5, and 2.0 mL/L), and a non-sterilized negative control. Seedlings were cultured under controlled-room (25 ± 2°C, 16-h photoperiod) or net house (ambient temperature, natural light, 70% shade) conditions with sucrose at 0, 20, or 40 g/L on half-strength MS medium. Acclimatization performance was evaluated using sphagnum moss, chopped coconut husk, and charcoal substrates. Chemical sterilization with 2.0 mL/L Haiter® achieved an acceptably low contamination rate of 10.00% versus 6.67% for autoclaving, while eliminating autoclave capital (USD 2,000–10,000), maintenance (USD 200–500/year), and energy costs (2–3 kWh/cycle), reducing sterilization expenditure by 60–70%. Net house culture combined with 40 g/L sucrose produced superior plantlets with 2.63 shoots, 14.33 roots, 3.60 cm height, and 1.14 g fresh weight, representing numerically more shoots (21%, p > 0.05), significantly more roots (68%, p ≤ 0.01), greater height (41%), and higher fresh weight (75.4%) than controlled-room culture, while substantially reducing operational electricity costs associated with climate-controlled culture rooms. Charcoal substrate achieved the highest acclimatization survival rate (85%). This integrated, low-cost protocol offers a practical, cost-effective tool for conservation and commercial production of this threatened native orchid.
Article Details
This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
References
Sangdum, S.; Thammasiri, K.; Chuenboonngarm, N.; Sopalun, K. Micropropagation of Dendrobium cruentum Rchb. f., a Rare Thai Orchid Species. Acta Hortic. 2017, 1167, 69–74. https://doi.org/10.17660/ActaHortic.2017.1167.10
Samala, S.; Thipwong, T. Influences of Organic Additives on Asymbiotic Seed Germination of Dendrobium cruentum Rchb. f. for in Vitro Micropropagation. Trends Sci. 2023, 20(3), 11. https://doi.org/10.48048/tis.2023.4181
CITES. Appendix I, II and III. Convention on International Trade in Endangered Species of Wild Fauna and Flora; CITES Secretariat: Geneva, Switzerland, 2023. https://cites.org/eng/app/index.php (accessed May 2, 2026).
IUCN. The IUCN Red List of Threatened Species, Version 2024-1; IUCN: Gland, Switzerland, 2024. https://www.iucnredlist.org (accessed May 2, 2026).
Keawsaard, Y.; Pentem, P.; Rotduang, P.; Weerapong, M.; Ramasoot, S. Induction of Mutation in Toothbrush Orchids Using Ethylmethane Sulphonate (EMS) and Detection of Genetic Variation by SSR (Simple Sequence Repeat) Marker. ASEAN J. Sci. Technol. Rep. 2025, 28(1), e255125. https://doi.org/10.55164/ajstr.v28i1.255125
Utami, E. S. W.; Hariyanto, S. Organic Compounds: Contents and Their Role in Improving Seed Germination and Protocorm Development in Orchids. Int. J. Agron. 2020, 2020, 2795108. https://doi.org/10.1155/2020/2795108
Avíla-Díaz, I.; Oyama, K.; Gómez-Alonso, C.; Salgado-Garciglia, R. In Vitro Propagation of the Endangered Orchid Laelia speciosa. Plant Cell Tissue Organ Cult. 2009, 99, 335–343. https://doi.org/10.1007/s11240-009-9609-8
Deb, C. R.; Pongener, A. Asymbiotic Seed Germination and In Vitro Seedling Development of Cymbidium aloifolium (L.) Sw.: A Multipurpose Orchid. J. Plant Biochem. Biotechnol. 2011, 20, 90–95. https://doi.org/10.1007/s13562-010-0031-4
Ngampanya, B.; Homla-aor, W. Simple Media for Dendrobium Orchid Seed Germination and Protocorm Development. Acta Hortic. 2010, 878, 219–223. https://doi.org/10.17660/ActaHortic.2010.878.26
Thongpukdee, A.; Thepsithar, C.; Rojanawong, T. Optimum Conditions for Seed Germination of Phalaenopsis Silky Moon. Acta Hortic. 2010, 878, 237–242. https://doi.org/10.17660/ActaHortic.2010.878.29
Kodym, A.; Zapata-Arias, F. J. Low-Cost Alternatives for the Micropropagation of Banana. Plant Cell Tissue Organ Cult. 2001, 66, 67–71. https://doi.org/10.1023/A:1010661521438
Kadam, D. D.; Chhatre, A. A.; Lavale, S. A.; Shinde, N. A. Low-Cost Alternatives for Conventional Tissue Culture Media. Int. J. Curr. Microbiol. Appl. Sci. 2018, 7, 2523–2529. https://doi.org/10.20546/ijcmas.2018.704.288
Chen, C. Cost Analysis of Plant Micropropagation of Phalaenopsis. Plant Cell Tissue Organ Cult. 2016, 127, 751–766. https://doi.org/10.1007/s11240-016-1060-x
O’Callaghan, J.; Fitzpatrick, J.; Lalor, F.; Byrne, E. Investigating the Energy, Environmental, and Economic Challenges and Opportunities Associated with Steam Sterilisation Autoclaves. Chem. Prod. Process Model. 2023, 18(4), 671–689. https://doi.org/10.1515/cppm-2022-0053
Kumara, P. U.; Atapattu, A. G. K. K. M.; Herath, H. M. I.; Fonseka, D. L. C. K.; Aruna Kumara, K. K. I. U.; Marikar, F. M. M. T. Low-Cost Gelling Agent in Tissue Culture Technology for the Orchid Regeneration. Rom. J. Hortic. 2022, 3, 53–60. https://doi.org/10.51258/RJH.2022.07
Bouaaza, G.; Chetto, O.; Beniken, L.; Dziri, L.; Majji, M.; Benkirane, R.; Benyahia, H. Effect of Gelling Agent and Plant Growth Regulators on In Vitro Shooting and Rooting of Stevia rebaudiana Bertoni. Adv. Agric. 2024, 2024, 5131187. https://doi.org/10.1155/aia/5131187
Brondani, G. E.; Oliveira, L. S.; Bergonci, T.; Brondani, A. E.; França, F. A. M.; Silva, A. L. L.; Gonçalves, A. N. Chemical Sterilization of Culture Medium: A Low Cost Alternative to In Vitro Establishment of Plants. Sci. For. 2013, 41(98), 257–264.
Cardoso, J. C.; Imthurn, A. C. P. Easy and Efficient Chemical Sterilization of the Culture Medium for In Vitro Growth of Gerbera Using Chlorine Dioxide (ClO₂). Ornam. Hortic. 2018, 24(3), 218–224. https://doi.org/10.14295/oh.v24i3.1222
Cardoso, J. C.; Silva, J. A. T. Micropropagation of Gerbera Using Chlorine Dioxide (ClO₂) to Sterilize the Culture Medium. In Vitro Cell. Dev. Biol. Plant 2012, 48(3), 362–368. https://doi.org/10.1007/s11627-011-9418-8
Teixeira, S. L.; Ribeiro, J. M.; Teixeira, M. T. Influence of NaClO on Nutrient Medium Sterilization and on Pineapple (Ananas comosus cv. Smooth Cayenne) Behavior. Plant Cell Tissue Organ Cult. 2006, 86(3), 375–378. https://doi.org/10.1007/s11240-006-9121-3
Tiwari, A. K.; Tripathi, S.; Lal, M.; Mishra, S. Screening of Some Chemical Disinfectants for Media Sterilization during In Vitro Micropropagation of Sugarcane. Sugar Tech 2012, 14(4), 364–369. https://doi.org/10.1007/s12355-012-0178-5
Ferreira, L. S.; Oliveira, A. L. S.; Soares, F. P.; Costa, A. S. D.; Blank, A. F.; Arrigoni-Blank, M. D. F. Better Light Spectral Quality and Thermal Amplitude inside the Greenhouse Stimulate Growth and Improve Acclimatization of In Vitro-Grown Cattleya warneri T. Moore. In Vitro Cell. Dev. Biol. Plant 2021, 57, 567–574. https://doi.org/10.1007/s11627-021-10162-8
Kodym, A.; Zapata-Arias, F. J. Natural Light as an Alternative Light Source for the In Vitro Culture of Banana (Musa acuminata cv. ‘Grande Naine’). Plant Cell Tissue Organ Cult. 1998, 55, 141–145. https://doi.org/10.1023/A:1006119114107
Saranya, S.; Gayathri, M. K.; Krishna, R. R.; Bindumole, V. R.; Remakanthan, A. A Rapid and Cost Effective Hardening Method for Tissue Culture Derived Banana Plantlets. Plant Tissue Cult. Biotechnol. 2024, 34(1), 11–24. https://doi.org/10.3329/ptcb.v34i1.74335
Bhattacharyya, P.; Kumaria, S.; Tandon, P. High Frequency Regeneration Protocol for Dendrobium nobile: A Model Tissue Culture Approach for Propagation of Medicinally Important Orchid Species. S. Afr. J. Bot. 2016, 104, 232–243. https://doi.org/10.1016/j.sajb.2015.11.013
Faria, R. T.; Rodrigues, F. N.; Oliveira, L. V. R.; Müller, C. In Vitro Dendrobium nobile Plant Growth and Rooting in Different Sucrose Concentrations. Hortic. Bras. 2004, 22(4), 780–783. https://doi.org/10.1590/S0102-05362004000400023
Nongdam, P.; Tikendra, L. Establishment of an Efficient In Vitro Regeneration Protocol for Rapid and Mass Propagation of Dendrobium chrysotoxum Lindl. Using Seed Culture. Sci. World J. 2014, 2014, 740150. https://doi.org/10.1155/2014/740150
Gonçalves, S.; Romano, A. Dendrobium Micropropagation: A Review. Plant Cell Rep. 2015, 34(6), 949–966. https://doi.org/10.1007/s00299-015-1754-4
Thammasiri, K. Effect of Sucrose Concentration and Seed Maturity on In Vitro Germination of Dendrobium nobile Hybrids. Plant Growth Regul. 2014, 72, 149–155. https://doi.org/10.1007/s10725-013-9856-x
Vijayakumar, S.; Rajalakshmi, G.; Kalimuthu, K. Propagation of Dendrobium aggregatum through the Culture of Immature Seeds from Green Capsules. Lankesteriana 2012, 12(2), 131–135.
Zainuddin, I. M.; Sinniah, U. R.; Mukherjee, A.; Julkifle, A. L.; Khoddamzadeh, A. A.; Subramaniam, S. Seed Viability Testing for Research and Conservation of Epiphytic and Terrestrial Orchids. Bot. Stud. 2022, 63, 5. https://doi.org/10.1186/s40529-022-00333-0
Murashige, T.; Skoog, F. A Revised Medium for Rapid Growth and Bio Assay with Tobacco Tissue Cultures. Physiol. Plant. 1962, 15, 473–497. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x
Pais, A. K.; da Silva, A. P.; de Souza, J. C.; Teixeira, S. L.; Ribeiro, J. M.; Peixoto, A. R.; da Paz, C. D. Sodium Hypochlorite Sterilization of Culture Medium in Micropropagation of Gerbera hybrida cv. Essandre. Afr. J. Biotechnol. 2016, 15(36), 1995–1998. https://doi.org/10.5897/AJB2016.15405
Peiris, S. E.; De Silva, E. D. U. D.; Edussuriya, M.; Attanayake, A. M. U. R. K.; Peiris, B. C. N. CSUP Technique: A Low Cost Sterilization Method Using Sodium Hypochlorite to Replace the Use of Expensive Equipment in Micropropagation. J. Natl. Sci. Found. Sri Lanka 2012, 40(1), 49–54. https://doi.org/10.4038/jnsfsr.v40i1.4168
Yildiz, M.; Ozcan, S. F.; Kahramanogullari, C. T.; Tuna, E. The Effect of Sodium Hypochlorite Solutions on the Viability and In Vitro Regeneration Capacity of the Tissue. Open Biotechnol. J. 2012, 6, 328–331. https://doi.org/10.2174/2210315511202040328
Salazar-Mercado, S. A.; Torres-León, C. A.; Rojas-Suárez, J. P. Cytotoxic Evaluation of Sodium Hypochlorite, Using Pisum sativum L. as Effective Bioindicator. Ecotoxicol. Environ. Saf. 2019, 173, 71–76. https://doi.org/10.1016/j.ecoenv.2019.02.027
Salazar Mercado, S. A.; Maldonado Bayona, H. A. Evaluation of the Cytotoxic Potential of Sodium Hypochlorite Using Meristematic Root Cells of Lens culinaris. Sci. Total Environ. 2020, 701, 134992. https://doi.org/10.1016/j.scitotenv.2019.134992
Gammoudi, N.; Nagaz, K.; Ferchichi, A. Establishment of Optimized In Vitro Disinfection Protocol of Pistacia vera L. Explants Mediated a Computational Approach: Multilayer Perceptron–Multi-Objective Genetic Algorithm. BMC Plant Biol. 2022, 22, 324. https://doi.org/10.1186/s12870-022-03674-x
Bui, S.; Kamle, M.; Kumar, P. Evaluation of Sterilants on the Establishment of In Vitro Regeneration Protocol in Litsea cubeba (Lour.) Pers. J. Appl. Nat. Sci. 2025, 17(3), 1240–1244. https://doi.org/10.31018/jans.v17i3.6790
Rodrigues, D. T.; Novais, R. F.; Alvarez Venegas, V. H.; Dias, J. M. M.; Otoni, W. C.; Villani, E. M. A. Chemical Sterilization in In Vitro Propagation of Arundina bambusifolia Lindl. and Epidendrum ibaguense Kunth. Rev. Ceres 2013, 60(4), 447–451. https://doi.org/10.1590/S0034-737X2013000400001
Singh, K. B.; Ali, M. N.; Samanta, S.; Mandal, N. A Comparative Analysis among Different Surface Sterilisation Methods for Rice In Vitro Culture. Int. J. Plant Soil Sci. 2021, 33(17), 148–154. https://doi.org/10.9734/ijpss/2021/v33i1730559
Park, H. B.; An, J.; Bae, K.-H.; Hong, S. H.; Park, H. J.; Kim, S.; Lee, C. W.; Lee, B.-D.; Baek, J. H.; Kim, N. Y.; Hwang, J. E. Asymbiotic Seed Germination and In Vitro Seedling Development of the Endangered Orchid Species Cypripedium guttatum. Plants 2023, 12(22), 3788. https://doi.org/10.3390/plants12223788
Poorter, H.; Niinemets, U.; Ntagkas, N.; Siebenkäs, A.; Mäenpää, M.; Matsubara, S.; Thijs, T. A Meta-Analysis of Plant Responses to Light Intensity for 70 Traits Ranging from Molecules to Whole Plant Performance. New Phytol. 2019, 223(3), 1043–1681. https://doi.org/10.1111/nph.15754
Schumann, T.; Paul, S.; Melzer, M.; Dörmann, P.; Jahns, P. Plant Growth under Natural Light Conditions Provides Highly Flexible Short-Term Acclimation Properties toward High Light Stress. Front. Plant Sci. 2017, 8, 681. https://doi.org/10.3389/fpls.2017.00681
Chiang, C.; Bånkestad, D.; Hoch, G. Reaching Natural Growth: The Significance of Light and Temperature Fluctuations in Plant Performance in Indoor Growth Facilities. Plants 2020, 9(10), 1312. https://doi.org/10.3390/plants9101312
Gago, D.; Landin, M.; Gallego, P. P. Micropropagation of Plum (Prunus domestica L.) in Bioreactors Using Photomixotrophic and Photoautotrophic Conditions. Horticulturae 2022, 8(2), 142. https://doi.org/10.3390/horticulturae8020142
Santos, G. C.; Figueiredo, J. L. A.; Blank, A. F.; Costa, A. S. D.; Soares, F. P.; Arrigoni-Blank, M. D. F. Effect of Light and Sucrose on Photoautotrophic and Photomixotrophic Micropropagation of Physalis angulata. Biosci. J. 2020, 36(3), 777–789. https://doi.org/10.14393/BJ-v36n3a2020-42377
Mishra, B. S.; Singh, M.; Aggrawal, P.; Laxmi, A. Role of Sugar and Auxin Crosstalk in Plant Growth and Development. Physiol. Plant. 2021, 172(2), 1211–1226. https://doi.org/10.1111/ppl.13374
Tong, C.; Zhang, L.; Zhao, Z.; Li, Z.; Zhang, Q.; Ma, Y.; Zhao, J.; Yin, X.; Wang, J.; Zhao, Y.; Liu, C.; Song, B. Long-Distance Transport of Sucrose in Source Leaves Promotes Sink Root Growth by the EIN3-SUC2 Module. PLoS Genet. 2022, 18(3), e1010120. https://doi.org/10.1371/journal.pgen.1010120
Hahn, E. J.; Kozai, T.; Paek, K. Y. High Photosynthetic Photon Flux and High CO₂ Concentration under Increased Number of Air Exchanges Promote Growth and Photosynthesis of Four Kinds of Orchid Plantlets In Vitro. In Vitro Cell. Dev. Biol. Plant 2001, 37, 678–682. https://doi.org/10.1007/s11627-001-0118-7
Shang, W. N.; et al. Effects of Light Quality on Growth, Photosynthetic Characteristics, and Endogenous Hormones in In Vitro-Cultured Lilium Plantlets. Hortic. Environ. Biotechnol. 2022, 63, 469–480.
Yoon, Y. J.; Murthy, H. N.; Hahn, E. J.; Paek, K. Y. Impact of In Vitro CO₂ Enrichment and Sugar Deprivation on Acclimatory Responses of Phalaenopsis Plantlets to Ex Vitro Conditions. Environ. Exp. Bot. 2009, 65, 271–279. https://doi.org/10.1016/j.envexpbot.2008.08.001
Aluko, O. O.; Li, C.; Wang, Q.; Liu, H. Sucrose Utilization for Improved Crop Yields: A Review. Int. J. Mol. Sci. 2021, 22(9), 4395. https://doi.org/10.3390/ijms22094395
Pasternak, T.; Dudits, D. Plant Growth Regulation in Cell and Tissue Culture In Vitro. Plants 2024, 13(3), 327. https://doi.org/10.3390/plants13030327
Dantas, L. L. G. R.; de Almeida, M.; Teixeira, J. B.; Panis, B.; Swennen, R.; Batista, D. S.; Otoni, W. C. The Impact of Carbon Source on Cell Growth and the Production of Bioactive Compounds in Cell Suspensions of Hancornia speciosa Gomes. Sci. Rep. 2021, 11, 6846. https://doi.org/10.1038/s41598-021-86359-z
Wang, M.; Le Gourrierec, J.; Jiao, F.; Demotes-Mainard, S.; Perez-Garcia, M.-D.; Ogé, L.; Hamama, L.; Crespel, L.; Bertheloot, J.; Chen, J.; Grappin, P.; Sakr, S. Convergence and Divergence of Sugar and Cytokinin Signaling in Plant Development. Int. J. Mol. Sci. 2021, 22(4), 1648. https://doi.org/10.3390/ijms22041648
Abdel-Ghany, A. M.; Al-Faraj, A. A.; Ahmed, H. A. Modified Plastic Net-Houses as Alternative Agricultural Structures for Saving Energy and Water in Hot and Sunny Regions. Renew. Energy 2016, 85, 1135–1143. https://doi.org/10.1016/j.renene.2016.02.084
Abdel-Ghany, A. M.; Al-Helal, M. I.; Alkoaik, F.; Alsadon, A.; Shady, M.; Ibrahim, A. Predicting the Cooling Potential of Different Shading Methods for Greenhouses in Arid Regions. Energies 2019, 12, 4716. https://doi.org/10.3390/en12244716
Tian, J.; Jiang, W.; Si, J.; Han, Z.; Li, C.; Chen, D. Developmental Characteristics and Auxin Response of Epiphytic Root in Dendrobium catenatum. Front. Plant Sci. 2022, 13, 935540. https://doi.org/10.3389/fpls.2022.935540
Qi, Y.; Huang, J.-L.; Zhang, S.-B. Correlated Evolution of Leaf and Root Anatomic Traits in Dendrobium (Orchidaceae). AoB Plants 2020, 12(4), plaa034. https://doi.org/10.1093/aobpla/plaa034
Irmawati, D.; Rahayu, E. S. Development of Photomixotrophic Culture Protocol of Lemongrass through Sucrose Concentration Increase and Light Intensity Decrease. Biosaintifika 2023, 15(2), 212–219. https://doi.org/10.15294/biosaintifika.v15i2.38303
Pepe, M.; Leonardos, E. D.; Marie, T. R. J. G.; Kyne, S. T.; Hesami, M.; Jones, A. M. P.; Grodzinski, B. A Noninvasive Gas Exchange Method to Test and Model Photosynthetic Proficiency and Growth Rates of In Vitro Plant Cultures: Preliminary Implication for Cannabis sativa L. Biology 2022, 11(5), 729. https://doi.org/10.3390/biology11050729
Chintakovid, N.; Papathakorn, P.; Chomkhuntod, P.; Roytrakul, S. In Vitro Acclimatization of Curcuma longa under Controlled Iso-Osmotic Conditions. Plant Biotechnol. 2021, 38(4), 433–443. https://doi.org/10.5511/plantbiotechnology.21.0915a
Martins, J. P. R.; Rodrigues, L. C. A.; Rodrigues, M. A.; Taglieber, A. B.; Pasqual, M. Anatomical and Physiological Changes of In Vitro-Propagated Vriesea imperialis (Bromeliaceae) in the Function of Sucrose and Ventilated Containers. Plant Biosyst. 2020, 154(3), 356–366. https://doi.org/10.1080/11263504.2019.1613980
Sasamori, M. H.; Endres Junior, D.; Figueiredo, A. B. S.; Droste, A.; Almeida, R. Optimal Conditions for In Vitro Culture of Cattleya cernua, a Small Orchid Native of Atlantic Forest and Cerrado. Rodriguésia 2021, 72, e01102019. https://doi.org/10.1590/2175-7860202172024
Kocot, D.; Maślanka, P.; Nowak, K. LED Light Quality Drives In Vitro Development of Bletilla striata: Toward Sustainable Orchid Propagation. Sustainability 2026, 18(3), 1025. https://doi.org/10.3390/su18031025
Huo, J.; Lin, Q.; Mo, L.; Zheng, L.; Meng, X.; Song, X.; Liang, J.; Chen, T. The Influence of Varying Wavelengths of LED Light on the Development, Physiological Response, and Metabolic Activities of Micropropagated Dendrobium Hybrid ‘Shuijing’ Plantlets. Horticulturae 2024, 10(8), 774. https://doi.org/10.3390/horticulturae10080774
Shin, K. S.; Park, S. Y.; Paek, K. Y. Physiological and Biochemical Changes during Acclimatization in a Doritaenopsis Hybrid Cultivated in Different Microenvironments In Vitro. Environ. Exp. Bot. 2014, 100, 26–33. https://doi.org/10.1016/j.envexpbot.2013.12.004
Utami, E. S. W.; Lestar, S.; Wahyun, K. D.; Purnobasuk, H. J.; Hariyanto, S.; Istighfari, N.; Murtadlo, A. A. A.; Umamah, S. Improvement of In Vitro Seed Germination and Shoot Development of the Indonesian Endangered Orchid, Dendrobium lineale Rolfe, Using Sucrose and Coconut Water. Scientifica 2026, 2026, 2153196. https://doi.org/10.1155/sci5/2153196
Samala, S.; Sripichitt, P.; Chanprasert, W. Influences of Organic Additives on Asymbiotic Seed Germination of Dendrobium cruentum Rchb. f. for In Vitro Micropropagation. Trends Sci. 2023, 20(4), e6456. https://doi.org/10.48048/tis.2023.6456
Nuammee, A.; Pingyot, T.; Foowan, S.; Pumikong, S.; Rujichaipimon, W.; Sornpood, S.; Panyadee, P. Effect of Substrates of Transplantation of the Rare Epiphytic Orchid Dendrobium farmeri for Conservation. Biodiversitas 2024, 25(2), 708–715. https://doi.org/10.13057/biodiv/d250231
