Virus-Free Strawberry Seedling Production: Advances in Elimination Technologies, Diagnostics and Standardized Propagation Systems — Oak Academic Publishing
Research ArticleOpen AccessGoogle Scholar indexed
Virus-Free Strawberry Seedling Production: Advances in Elimination Technologies, Diagnostics and Standardized Propagation Systems
College of Biological Sciences and Technology, Yili Normal University, Yining, China
,
College of Biological Sciences and Technology, Yili Normal University, Yining, China
,
College of Biological Sciences and Technology, Yili Normal University, Yining, China
,
College of Biological Sciences and Technology, Yili Normal University, Yining, China
,
College of Biological Sciences and Technology, Yili Normal University, Yining, China
,
Key Laboratory of Plant Resources Protection and Utilization in Xinjiang Yili Valley, Yining, China
1 College of Biological Sciences and Technology, Yili Normal University, Yining, China
2 College of Biological Sciences and Technology, Yili Normal University, Yining, China
3 College of Biological Sciences and Technology, Yili Normal University, Yining, China
4 College of Biological Sciences and Technology, Yili Normal University, Yining, China
5 College of Biological Sciences and Technology, Yili Normal University, Yining, China
6 Key Laboratory of Plant Resources Protection and Utilization in Xinjiang Yili Valley, Yining, China
Strawberry is a globally important berry crop, but clonal propagation through runners facilitates the vertical transmission and accumulation of viruses and viroids, leading to seedling degeneration, reduced vigour, poor fruit quality, fruit deformity and yield losses. Strawberry viral diseases are difficult to control because of their high diversity, frequent latent and mixed infections, non-specific symptoms and long-distance dissemination through infected planting materials. In this review, “virus-free seedling” refers to planting material certified negative, within the detection limits of specified assays, for a defined panel of strawberry viruses and viroids at designated propagation stages; it does not imply freedom from all pathogens. Virus-free seedling production, primarily based on shoot-tip culture and supported by thermotherapy, chemotherapy, cryotherapy, anther culture, rapid propagation and molecular diagnostics, is an effective strategy for restoring seedling health and improving propagation quality. Recent advances in high-throughput sequencing, viromics, RT-PCR, RT-qPCR, multiplex PCR, isothermal amplification, lateral-flow assays and protected nursery systems have accelerated the shift from experience-based propagation to standardized, diagnostic-guided and certification-oriented production. This review summarizes the occurrence characteristics of strawberry viral diseases, the theoretical basis of virus elimination, key elimination technologies, molecular diagnostics, quality assessment, rapid propagation and industrial applications. It also proposes an integrated framework linking virus elimination, hierarchical diagnostics, genetic fidelity assessment, protected nursery management, vector control and traceable certification, providing a reference for standardized production, quality control and industrial application of virus-free strawberry seedlings.
Lee, Y.H., Yeoung, H.S., Mezzetti, B. and Yeoung, Y. (2026) Strawberry Propagation: Progress on Propagation Methods, Environmental Regulation, and Disease Management Strategies over the Past 20 Years. Horticulturae , 12, Article 351. https://doi.org/10.3390/horticulturae12030351
Li, Y. (2025) Analysis of the Cultivation and Application of Drugfree Strawberry Seedlings. Seed Science & Technology , 43, 119-121.
Banerjee, D., Singh, V. and Thakur, R. (2023) Micro Propagation on Strawberry: A Review. E 3 S Web of Conferences , 453, Article 01019. https://doi.org/10.1051/e3sconf/202345301019
Li, D., Sujata, S., Kang, K., Pang, H., Li, Y., Hou, C., et al . (2024) Polysaccharide Peptide Treatment Eliminates Strawberry Viruses and Promotes Strawberry Plant Growth and Rooting in Tissue Culture Media. Plant Disease , 108, 2027-2033. https://doi.org/10.1094/pdis-10-23-2226-re
Diaz-Lara, A., Stevens, K.A., Klaassen, V., Hwang, M.S. and Al Rwahnih, M. (2021) Sequencing a Strawberry Germplasm Collection Reveals New Viral Genetic Diversity and the Basis for New RT-qPCR Assays. Viruses , 13, Article 1442. https://doi.org/10.3390/v13081442
Nunes-Leite, L., Liefting, L.W., Waite, D.W., Khan, S. and Thompson, J.R. (2024) High-Throughput Sequencing Methods for the Detection of Two Strawberry Viruses in Post-Entry Quarantine. Viruses , 16, Article 1550. https://doi.org/10.3390/v16101550
Koloniuk, I., Přibylová, J., Čmejla, R., Valentová, L. and Fránová, J. (2022) Identification and Characterization of a Novel Umbra-Like Virus, Strawberry Virus A, Infecting Strawberry Plants. Plants , 11, Article 643. https://doi.org/10.3390/plants11050643
Fránová, J., Lenz, O., Přibylová, J., Čmejla, R., Valentová, L. and Koloniuk, I. (2021) High Incidence of Strawberry Polerovirus 1 in the Czech Republic and Its Vectors, Genetic Variability and Recombination. Viruses , 13, Article 2487. https://doi.org/10.3390/v13122487
Medberry, A.N., Srivastava, A., Diaz-Lara, A., Rwahnih, M.A., Villamor, D.E.V. and Tzanetakis, I.E. (2023) A Novel, Divergent Member of the Rhabdoviridae Family Infects Strawberry. Plant Disease , 107, 620-623. https://doi.org/10.1094/pdis-05-22-1078-sc
Wang, Y., Zeng, X., Xiao, G., Zhang, D., Wen, X., Zhou, X., et al . (2024) Development of a Multiplex RT-PCR Detection for Six Viruses Infecting Strawberry. Viruses , 16, Article 1858. https://doi.org/10.3390/v16121858
Cultrona, M., Bonini, N., Margaria, P., Menzel, W., Pacifico, D. and Tessitori, M. (2024) First Report of Strawberry Polerovirus 1 in Strawberry in Italy. Plant Disease , 108, Article 1122. https://doi.org/10.1094/pdis-01-24-0239-pdn
Yan, B., Lu, M., Han, J., Cao, Y., Yan, F. and Song, X. (2025) Molecular Insights and Diagnostic Advances in Strawberry-Infecting Viruses. Frontiers in Microbiology , 16, Article 1655696. https://doi.org/10.3389/fmicb.2025.1655696
Mozafari, A.A., vafaee, Y., Hajizadeh, M., Saed-Moucheshi, A., Shahidi, P., Zandan, N.G., et al . (2022) Strawberry Crinkle (SCV) and Strawberry Mottle (SMoV) Viruses Affect Yield and Physio-Biochemical Responses of Three Strawberry Cultivars under Different Seasonal Conditions. Molecular Biology Reports , 49, 8651-8661. https://doi.org/10.1007/s11033-022-07701-w
Koloniuk, I., Matyášová, A., Brázdová, S., Veselá, J., Přibylová, J., Fránová, J., e t al . (2022) Transmission of Diverse Variants of Strawberry Viruses Is Governed by a Vector Species. Viruses , 14, Article 1362. https://doi.org/10.3390/v14071362
Dholi, P., Khatiwada, P., Basnet, B. and Bhandari, S. (2023) An Extensive Review of Strawberry (Fragaria × Ananassa) Diseases and Integrated Management Approaches: Current Understanding and Future Directions. Fundamental and Applied Agricu lture , 8, 655-667. https://doi.org/10.5455/faa.136385
Bradamante, G., Mittelsten Scheid, O. and Incarbone, M. (2021) Under Siege: Virus Control in Plant Meristems and Progeny. The Plant Cell , 33, 2523-2537. https://doi.org/10.1093/plcell/koab140
Mori, K. and Hosokawa, D. (1977) Localization of Viruses in Apical Meristem and Production of Virus-Free Plants by Means of Meristem and Tissue Culture. Acta Horticulturae , 78, 389-396. https://doi.org/10.17660/actahortic.1977.78.49
Han, R., Chang, J., Zhao, J., et al . (2022) Establishment of Tissue Culture and Rapid Propagation System of Strawberry Stem Tips. Journal of Shanxi Agricultural Sciences , 50, 15-21.
Du, L., Ma, Z., Zhang, Y., et al . (2024) Establishment of a Detoxification and Rapid Propagation System for Red Strawberry Tissue Culture. Hans Journal of Agricult ural Sciences , 14, 1083-1087. https://doi.org/10.12677/hjas.2024.1410136
Naing, A.H., Kim, S.H., Chung, M.Y., Park, S.K. and Kim, C.K. (2019) In Vitro Propagation Method for Production of Morphologically and Genetically Stable Plants of Different Strawberry Cultivars. Plant Methods , 15, Article No. 36. https://doi.org/10.1186/s13007-019-0421-0
Wöhner, T. and Höfer, M. (2023) Evaluation of Cryotherapy and Meristem Isolation from Stolons to Eliminate Viruses in Fragaria Germplasm. Journal of Plant Patholo gy , 106, 729-735. https://doi.org/10.1007/s42161-023-01570-3
Tian, R., Chen, S., Guo, J., Liu, K., Li, Z., Meng, L., et al . (2026) Establishing a Virus-Free Rapid Propagation System for Strawberry ‘Miaoxiang 7’ through Anther Culture. Horticulturae , 12, Article 227. https://doi.org/10.3390/horticulturae12020227
Liang, Z., Huo, H., Jia, M., et al . (2022) Study on Tissue Culture and Rapid Propagation of Stem Tips for Suizhu Strawberry. Journal of Shanxi Agricultural Sciences , 50, 613-619.
Alavijeh, M.K., Bayat, H., Kianpour, D., Kalantari, S. and zarei, A. (2025) Optimization of in Vitro Propagation and Virus Eradication Using Meristem Culture and Thermotherapy in Two Geranium Species Pelargonium × hortorum (‘Zonal’) and Pelargonium × d omesticum (‘Regal’). BMC Plant Biology , 25, Article No. 9. https://doi.org/10.1186/s12870-024-06027-y
Szabó, L.K., Desiderio, F., Kirilla, Z., Hegedűs, A., Várallyay, É. and Preininger, É. (2023) A Mini-Review on in Vitro Methods for Virus Elimination from Prunus sp. Fruit Trees. Plant Cell , Tissue and Organ Culture ( PCTOC ), 156, Article No. 42. https://doi.org/10.1007/s11240-023-02670-9
Zhang, A., Bettoni, J.C., Shi, X., Liu, Y., Yang, B. and Liu, Z. (2024) In Vitro Chemotherapy-Based Methods for Virus Elimination from Actinidia Macrosperma. Scientia Horticulturae , 337, Article 113543. https://doi.org/10.1016/j.scienta.2024.113543
Turcsan, M., Jaksa-Czotter, N., Nagyne Galbacs, Z., Olah, K., Olah, R., Varallyay, E., et al . (2025) Effects of Chemotherapy on the Elimination of Various Viruses and Viroids from Grapevine. Horticulturae , 12, Article 46. https://doi.org/10.3390/horticulturae12010046
Bettoni, J.C. and Volk, G.M. (2024) Cryotherapy Using Shoot Tip Cryopreservation. In: Volk, G.M., Ed., Training in Plant Genetic Resources : Cryopreservation of Clonal Propagules , Colorado State University, 30-37.
Ramlal, A., Quan, P.W., Rajendran, A. and Subramaniam, S. (2025) Impact of Biophysicochemical Factors on Micropropagation, Haploidy and Doubled Haploidy in Strawberry ( Fragaria sp.): A Critical Revisit. Current Plant Biology , 44, Article 100555. https://doi.org/10.1016/j.cpb.2025.100555
Zhao, X., He, C., Gao, D., Xu, T., Li, X., Liu, J., et al . (2022) Construction of Infectious cDNA Clone of Brassica Yellows Virus Isolated from Strawberry and Establishment of Taqman RT-qPCR. Plants , 11, Article 3380. https://doi.org/10.3390/plants11233380
Xu, T., Gao, D., Wu, M., Wang, H. and He, C. (2025) Establishment of Singleplex and Duplex TaqMan RT-qPCR Detection Systems for Strawberry Mottle Virus (SMoV) and Strawberry Vein Banding Virus (SVBV). Plants , 14, Article 2330. https://doi.org/10.3390/plants14152330
Maina, S., Donovan, N.J., Plett, K., Bogema, D. and Rodoni, B.C. (2024) High-Throughput Sequencing for Plant Virology Diagnostics and Its Potential in Plant Health Certification. Frontiers in Horticulture , 3, Article 1388028. https://doi.org/10.3389/fhort.2024.1388028
Villamor, D.E.V., Keller, K.E., Martin, R.R. and Tzanetakis, I.E. (2022) Comparison of High Throughput Sequencing to Standard Protocols for Virus Detection in Berry Crops. Plant Disease , 106, 518-525. https://doi.org/10.1094/pdis-05-21-0949-re
Ren, J., Zhang, J., Wang, Q., Zhou, Y., Wang, J., Ran, C., et al . (2022) Molecular Characterization of Strawberry Vein Banding Virus from China and the Development of Loop-Mediated Isothermal Amplification Assays for Their Detection. Scientific Rep orts , 12, Article No. 4912. https://doi.org/10.1038/s41598-022-08981-9
Zou, X., Dong, C., Ni, Y., Yuan, S. and Gao, Q. (2022) Rapid Detection of Strawberry Mottle Virus Using Reverse Transcription Recombinase Polymerase Amplification with Lateral Flow Strip. Journal of Virological Methods , 307, Article 114566. https://doi.org/10.1016/j.jviromet.2022.114566
Zou, X., Dong, C., Ni, Y. and Gao, Q. (2022) Rapid Detection of Strawberry Mild Yellow Edge Virus with a Lateral Flow Strip Reverse Transcription Recombinase Polymerase Amplification Assay. Current Microbiology , 79, Article No. 365. https://doi.org/10.1007/s00284-022-03045-7
Yang, X., Feng, J., Xu, K., Han, J., Zhang, M., Cheng, A., et al . (2025) RD21 Enhances Resistance to the Strawberry Vein Banding Virus by Promoting Autophagy‐Mediated Degradation of the Viral Silencing Suppressor p6 Protein. Plant Biotechnology Journ al , 23, 3597-3611. https://doi.org/10.1111/pbi.70168
Fan, L., He, C., Gao, D., Xu, T., Xing, F., Yan, J., et al . (2022) Identification of Silencing Suppressor Protein Encoded by Strawberry Mottle Virus. Frontiers in Plant Scie nce , 13, Article 786489. https://doi.org/10.3389/fpls.2022.786489
Xu, T., Zhan, B., Zhang, Z., Fan, L., Gao, D., Dong, Z., et al . (2025) FaNDUFB 9 Attenuates Strawberry Mottle Virus Infection by Inhibiting the Activity of the Viral Gene Silencing Suppressor, Pro2Glu. Molecular Plant Pathology , 26, e70061. https://doi.org/10.1111/mpp.70061
Neri, J.C., Meléndez-Mori, J.B., Tejada-Alvarado, J.J., Vilca-Valqui, N.C., Huaman-Huaman, E., Oliva, M., et al . (2022) An Optimized Protocol for Micropropagation and Acclimatization of Strawberry ( Fragaria × ananassa Duch.) Variety ‘Aroma’. Agronomy , 12, Article 968. https://doi.org/10.3390/agronomy12040968
Dwiyani, R., Fitriani, Y., Ana, F.G.S. and Bimantara, P.O. (2024) A Study of Acclimatization Media on Strawberry ( Fragaria × a nanassa Duch.) Plantlets Produced from Meristem Culture. AgriHealth : Journal of Agri - Food , Nutrition and Public Health , 5, 85-91. https://doi.org/10.20961/agrihealth.v5i2.85776
Selivanova, M., Aisanov, T., Romanenko, E. and Esaulko, N. (2025) Survival and Development of Strawberry Plants on Various Substrates at the Stage of Adaptation. BIO Web of Conferences , 194, Article 01025. https://doi.org/10.1051/bioconf/202519401025
Grzelak, M., Pacholczak, A. and Nowakowska, K. (2024) Challenges and Insights in the Acclimatization Step of Micropropagated Woody Plants. Plant Cell , Tissue and Organ Culture ( PCTOC ), 159, Article No. 72. https://doi.org/10.1007/s11240-024-02923-1
Nasir, S.M. and Abdulhussein, M.A.A. (2022) Strawberry Shoots Multiplication under Temporary Immersion System (TIS) Using Plant Form Bioreactor. NeuroQuan-tology , 20, 3186-3193.
Lopez, L., Addison, T., Archambault, M., et al . (2023) Pest Management Strategic Plan for Strawberry in North Carolina, Virginia, South Carolina, Georgia, and Florida. National IPM Database. https://ipmdata.ipmcenters.org/source_report.cfm?view=yes&sourceid=2489