BIOTECHNOLOGICAL METHODS FOR THE SANITATION OF APPLE ROOTSTOCKS AND THE PRODUCTION OF VIRUS-FREE PLANTING

In commercial orchards and nurseries, the spread of viral infections in apple (Malus domestica Borkh.) poses a serious threat to plant phytosanitary status and productivity. The aim of this study was to obtain virus-free apple rootstock planting material and to evaluate the effectiveness of integrated in vitro biotechnological methods of plant sanitation. The objects of the study were apple rootstocks from pomological and in vitro collections in Kazakhstan. The highest viral load was detected in rootstock B-16-20. For sanitation, a combined Thermotherapy + Chemotherapy + SAM method with antiviral agents was applied. The results showed that the effectiveness and phytotoxicity of the methods depend on the combination of techniques and the concentration of the applied agents. The obtained data confirm the high efficiency of integrated biotechnological approaches for obtaining virus-free apple planting material and have practical significance for certified propagation of fruit crops in Kazakhstan.

1. RomadanovaN.V., Kushnarenko S.V. Biotekhnologiya polucheniya bezvirusnyh sazhencev yabloni // Vestnik Karagandinskogo universiteta. Seriya «Biologiya. Medicina. Geografiya». - 2021. - № 3(103). - S. 102-118. http://dx.doi.org/10.31489/2021bmg3/102-118.

2. Umer, M., Liu, J., You, H., Xu, C., Dong, K., Luo, N., Kong, L., Li, X., Hong, N., Wang, G., Fan, X., Kotta-Loizou, I., & Xu, W. (2019). Genomic, morphological and biological traits of the viruses infecting major fruit trees. Viruses, 11(6), 515. https://doi.org/10.3390/v11060515

3. Kang, C. M., Kim, M. J., Hong, J. S., & Jeong, R. D. (2025). Managing plant viruses in tissue cultured apple and grapevine: Strategies for detection and eradication. Plant Pathology Journal, 41(5), 545-565. https://doi.org/10.5423/PPJ.RW.07.2025.0092

4. Jones, R. A. C., & Naidu, R. A. (2019). Global dimensions of plant virus diseases: Current status and future perspectives. Annual Review of Virology, 6(1), 387-409. https://doi.org/10.1146/annurev-virology092818-015606

5. Tatineni, S., & Hein, G. L. (2023). Plant viruses of agricultural importance: Current and future perspectives of virus disease management strategies. Phytopathology, 113(2), 117-141. https://doi.org/10.1094/PHYTO-05-22-0167-RVW

6. Xiao, H., Hao, W., Storoschuk, G., MacDonald, J. L., & Sanfagon, H. (2022). Characterizing the virome of apple orchards affected by rapid decline in the Okanagan and Similkameen valleys of British Columbia (Canada). Pathogens, 11, 1231. https://doi.org/10.3390/pathogens11111231

7. Khan, Z. A., Diksha, D., Thapa, P., Mailem, Y. S., Sharma, S. K., Gupta, N., Kishan, G., Watpade, S., & Baranwal, V. K. (2024). Genome analysis of viruses of Phenuiviridae, Betaflexiviridae and Bromoviridae, and apple scar skin viroid in pear by high-throughput sequencing revealing host expansion of a rubodvirus and an ilarvirus. Physiology and Molecular Plant Pathology, 129, 102196. https://doi.org/10.1016/i.pmpp.2023.102196

8. Kwon, Y. H., Lee, J. K., Kim, H. K., Kim, K. O., Park, J. S., Huh, Y. S., Park, E. K., & Yoon, Y. J. (2019). Efficient virus elimination for apple dwarfing rootstock M.9 and M.26 via thermotherapy, ribavirin and apical meristem culture. Journal of Plant Biotechnology, 46(3), 228-235. https://doi.org/10.5010/JPB.2019.46.3.228

9. Karpushina M. V., Suprun 1.1. Metody i podhody k eliminacii virusov v usloviyah in vitro i in vivo // Plodovodstvo i vinogradarstvo YUga Rossii. - 2020. - № 63(3). - S. 254-269. http://iournalkubansad.ru/pdf/20/03/19.pdf

10. Kushnarenko S.V., Manapkanova U.A., Rymhanova N.K., Turdiev T.T., ZHumabaeva B.A., Aubakirova K.P., Galiakparov N.N. Razrabotka in vitro tekhnologii dlya eliminacii virusa kustistoj karlikovosti maliny // Vestnik Karagandinskogo universiteta. Seriya «Biologiya. Medicina. Geografiya». - 2023. - № 2 (110). - S. 76-84. https://doi.org/10.31489/2023bmg2/76-84

11. Mitrofanova O.V., Mitrofanova I.V., Lesnikova SedoshenkoN.P., Ivanova N.N. Primenenie biotekhnologicheskih metodov v ozdorovlenii rastenij i razmnozhenii bezvirusnogo posadochnogo materiala perspektivnyh cvetochno dekorativnyh kul'tur // Sbornik nauchnyh trudov Gosudarstvennogo Nikitskogo botanicheskogo sada - Nacional'nogo nauchnogo centra RAN. - YAlta, 2014. - T. 138. - S. 5 56. https ://scbook. elpub.ru/i our.

12. EPPO. (2009). Certification scheme for pathogen-tested material of Malus, Pyrus and Cydonia (PM 4/27(1)). European and Mediterranean Plant Protection Organization. Retrieved August 22, 2025, from https://www.eppo.int

13. EPPO. (2021). PM 3/76 (2) Trees of Malus, Pyrus, Cydonia and Prunus spp.: Inspection of places of production. EPPO Bulletin, 51, 354-386. https://doi.org/10.1111/epp.12771

14. Mekuria, G., Ramesh, S. A., Alberts, E., Bertozzi, T., Wirthensohn, M., Collins, G., & Sedgley, M. (2003). Comparison of ELISA and RT-PCR for the detection of Prunus necrotic ring spot virus and prune dwarf virus in almond (Prunus dulcis). Journal of Virological Methods, 114, 65-69. https://doi.org/10.1016/i.iviromet.2003.08.014

15. Cho, I. S., Kim, J. H., Kim, H., Choi, H., & Lim, H. S. (2016). Deep sequencing analysis of apple infecting viruses in Korea. The Plant Pathology Journal, 32(5), 441. https://doi.org/10.5423/PPJ.OA.04.2016.0104

16. Kinoti, W. M., Ratti, C., Constable, F. E., & Jones, R. A. C. (2018). The incidence and genetic diversity of apple mosaic virus (ApMV) and prune dwarf virus (PDV) in Prunus species in Australia. Viruses, 10(3), 136. https://doi.org/10.3390/v10030136

17. Hu, G. J., Li, Y., Yang, X., Zhang, Z., & Zhang, J. (2021). First report of apple rubbery wood virus 2 infection of apples in China. Plant Disease, 105(2), 519. https://doi.org/10.1094/PDIS-07-19-1451-PDN

18. Baric, S. (2012). Quantitative real-time PCR analysis o f ‘Candidatus Phytoplasma mali’ without external standard curves. Erwerbs-Obstbau, 54(3), 147-153. https://link.springer.com/article/10.1007/s10341-012-0166-7

19. Zikeli, K., Kegler, H., & Rott, M. (2025). Prevalence, genetic diversity, and molecular detection of the apple hammerhead viroid in Germany. Frontiers in Microbiology, 16, Article 1592572. https://doi.org/10.3389/fmicb.2025.1592572

20. Murashige, T., & Skoog, F. (1962). A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiologia Plantarum, 15(3), 473-497. https://doi.org/10.1111/i.1399-3054.1962.tb08052.x

21. Sharma, S., Singh, B., Rani, G., Zaidi, A. A., Hallan, V. K., Nagpal, A. K., & Virk, G. S. (2008). In vitro production of Indian citrus ringspot virus (ICRSV) free Kinnow plants employing thermotherapy coupled with shoot tip grafting. Plant Cell, Tissue and Organ Culture, 92, 85-92. DOI: https://doi.org/

22. Hu, G.-J., Zhang, Z.-P., Dong, Y.-F., Fan, X.-D., Ren, F., & Zhu, H.-J. (2015). Efficiency of virus elimination from potted apple plants by thermotherapy coupled with shoot-tip grafting. Australasian Plant Pathology, 44, 167-173. https://link.springer.com/article/10.1007/s13313-014-0334-3

23. Verma, N., Ram, R., & Zaidi, A. A. (2005). In vitro production of Prunus necrotic ringspot virus-free begonias through chemo- and thermotherapy. Scientia Horticulturae, 103(2), 239-247. https://doi.org/10.1016/i.scienta.2004.05.005

24. Paprstein, F., Sedlak, J., Svobodova, L., Polak, J., & Gadiou, S. (2013). Results of in vitro chemotherapy of apple cv. Fragrance - Short communication. Horticultural Science (Prague), 40(4), 186-190. https ://hortsci.agriculturei ournals. cz/artkey/hor-201304-0008

25. Kabylbekova, B., Nurseitova, T., Yussupova, Z., Turdiyev, T., Kovalchuk, I., Dolgikh, S., Soltanbekov, S., Seisenova, A., & Madenova, A. (2025). Application of in vitro techniques for elimination of Plum pox virus (PPV) and Apple chlorotic leaf spot virus (ACLSV) in stone fruits. Horticulturae, 11(6), 633. https://doi.org/10.3390/horticulturae11060633

26. Paunovic, S., Ruzic, D., Vujovic, T., Milenkovic, S., & Jevremovic, D. (2007). In vitro production of Plum pox virus-free plums by chemotherapy with ribavirin. Biotechnology & Biotechnological Equipment, 21, 417-421. https://doi.org/10.1080/13102818.2007.10817486