Sanitation of grapevines in tissue and organ culture in vitro
Abstract
The article presents the results of a study aimed at sanitizing grapevine from Grapevine leafroll-associated virus 3 (GLRaV-3) using a combination of in vitro thermotherapy and apical meristem culture. Considering the relevance of viral infections in grapevine, which reduce yield, deteriorate berry quality, and impair the technological properties of wine materials, ensuring the sanitary health of planting material is one of the key strategies to enhance productivity and stability in viticulture. Purpose. The study aimed to evaluate the effectiveness of combining in vitro thermotherapy and apical meristem culture for the sanitation of grapevine from leafroll virus and to assess the viability and regenerative potential of the resulting microclones. Microbiological, molecular-biological, and biotechnological methods were employed. The material comprised microclonal plants and apical meristems of the technical grapevine cultivar Odeskyi chornyi, obtained from vines exhibiting typical leafroll symptoms. Viral infection was confirmed by enzyme-linked immunosorbent assay (ELISA). The initial explants were cultured on modified Murashige and Skoog media enriched with phytohormones and vitamins. Results. Two combinations of thermotherapy and apical meristem culture were applied for grapevine sanitation. The first involved thermotherapy of established microclonal plants followed by isolation and culture of apical meristems; the second involved culturing apical meristems followed by thermotherapy of microshoots. Thermotherapy was conducted in a growth chamber for 8 weeks at 37 °C, gradually increased from 25 °C, with a 16-hour photoperiod. The results demonstrated high efficiency of combining both methods for grapevine sanitation from GLRaV-3. The survival rate of initial explants after thermotherapy and apical meristem culture (regardless of process sequence) ranged from 51.5 to 74.0 %, with each explant producing 1.0 to 5.8 shoots, 1.8 to 5.5 cm in length. The proportion of virus-free plants reached 85.0–88.0 %, confirming the high efficacy of the combined approach compared to apical meristem culture alone. High viability and stable morphological characteristics of the regenerated microclones were also observed. Conclusions. The combination of in vitro thermotherapy and apical meristem culture is an effective method for sanitizing grapevine from Grapevine leafroll-associated virus 3 (GLRaV-3), ensuring a high level of virus elimination. The resulting microclonal plants exhibit high survivability, strong regenerative potential, and stable morphological traits.
References
2. Ruiz V. S. Advances in grape culture worldwide. Revista Brasileira de Fruticultura. 2011. Vol. 33. No.1. Р.131–143. https://doi.org/10.1590/S0100-29452011000500016
3. Maliogka V. I., Olmos A., Pappi P. G., Lotos L., Efthimiou K., Grammatikaki G., Candresse T., Katis N. I., Avgelis A. D. A novel grapevine badnavirus is associated with the Roditis leaf discoloration disease. Virus Research, Amsterdam. 2015b. Vol. 203. Р. 47–55. https://DOI: 10.1016/j.virusres.2015.03.003
4. Marcos Fernando Basso, Thor Vinicius Martins Fajardo, Pasquale Saldarelli Grapevine Virus Diseases : Economic Impact and Current Advances in Viral Prospection and Management. Revista Brasileira de Fruticultura. 2017. Vol. 39. No. 1. Р. 2–22. https://doi.org/10.1590/0100-29452017411
5. Al Rwahnih M., Daubert S., Golino D., Rowhani A. Deep sequencing analysis of RNAs from a grapevine showing Syrah decline symptoms reveals a multiple virus infection that includes a novel virus. Virology. 2009. Vol. 387. No. 2. Р. 395–401. DOI: 10.1016/j.virol.2009.02.028
6. Al Rwahnih M., Golino D., Rowhani A. First report of Grapevine Pinot gris virus infecting grapevine in the United States. Plant Disease, Saint Paul. 2016. Vol. 100. No. 5. Р. 1030. https://doi.org/10.1094/PDIS-10-15-1235-PDN
7. Martelli G. P. Directory of virus and virus-like diseases of the grapevine and their agents. Journal of Plant Pathology, Bari. 2014. Vol. 96. No.1. Р.1–136.
8. Andret-Link P., Laporte C., Valat L., Ritzenthaler C., Demangeat G., Vigne E., Laval V., Pfeiffer P., Stussi- Garaud C., Fuchs M. Grapevine fanleaf virus: Still a major threat to the grapevine industry. J. Plant. Pathol. 2004. Vol. 86. Р. 183–195. https://www.researchgate.net/publication/242759853
9. Giampetruzzi A., Roumi V., Roberto R., Malossini U., Yoshikawa N., La Notte P., Terlizzi F., Credi R., Saldarelli P. A new grapevine virus discovered by deep sequencing of virus- and viroid-derived small RNAs in Cv Pinot gris. Virus Res. 2012. Vol. 163. 262–268. DOI: 10.1016/j.virusres.2011.10.010
10. Sudarshana M. R., Perry K. L., Fuchs M. F. Grapevine red blotch-associated virus, an emerging threat to the grapevine industry. Phytopathology. 2015. Vol. 105. Р. 1026–1032. https://doi.org/10.1094/PHYTO-12-14-0369-FI
11. Maliogka V. I., Skiada F. G., Eleftheriou E. P., Katis N. I. Elimination of a new Ampelovirus (GLRaV-Pr) and Grapevine rupestris stem pitting associated virus (GRSPaV) from two Vitis vinifera cultivars combining in vitro thermotherapy with shoot tip culture. Scientia Horticulturae, Amsterdam. Vol. 123. 2009. No. 2. Р. 280–282. DOI:10.1016/j.scienta.2009.08.016
12. Hans J. Maree, Rodrigo P. P. Almeida, Rachelle Bester, Kar Mun Chooi, Daniel Cohen, Valerian V. Dolja, Marc F. Fuchs, Deborah A. Golino, Anna E. C. Jooste, Giovanni P. Martelli, Rayapati A. Naidu, Adib Rowhani, Pasquale Saldarelli, Johan T. Burger Grapevine leafroll-associated virus 3. Frontiers in Microbiology. 2013. Vol. 4. No. 82. DOI: 10.3389/fmicb.2013.00082
13. Křižan B., Ondrušiková E., Holleinová V., Moravcová K., Bláhová L. Elimination of Grapevine fanleaf virus in grapevine by in vivo and in vitro thermotherapy. Hort. Sci. (PRAGue). 2009. Vol. 36. No. 3. Р. 105–108. DOI: 10.17221/37/2008-HORTSCI
14. Panattoni A., Triolo E. Susceptibility of grapevine viruses to thermotherapy on in vitro collection of Kober 5BB. Scientia Horticulturae. 2010. Vol. 125. Issue 1. P. 63–67. https://doi.org/10.1016/j.scienta.2010.03.001
15. Luvisi A., Panattoni A., Triolo E. Thiopurine prodrugs for plant chemotherapy purposes. Journal of Phytopathology, Berlin. 2011. Vol. 159. No. 5. Р. 390–392. https://doi.org/10.1111/j.1439-0434.2010.01779.x
16. Borroto-Fernandez E. G., Sommerbauer T., Popowich E., Scharti A., Laimer M. Somatic embryogenesis from anthers of the autochthonous Vitis vinifera cv. Domina leads to Arabis mosaic virus- free plants. European Journal of Plant Pathology, Dordrecht. 2009. Vol. 124. No. 1. Р. 171–174. DOI: 10.1007/s10658-008-9404-0
17. Bayati S., Shams-Bakhsh M., Moiens A. Elimination of Grapevine virus A (GVA) by cryotherapy and electrotherapy. Journal of Agricultural Science and Technology, Tehran. 2011. Vol. 13. No. 3. Р. 442–450
18. AlMaarri K, Massa R, AlBiski F. Evaluation of some therapies and meristem culture to eliminate Potato Y potyvirus from infected potato plants. Plant Biotechnol. 2012. Vol. 29. Р. 237–43. DOI: 10.5511/plantbiotechnology.12.0215a
19. MinRui Wang, ZhenHua Cui, JingWei Li, XinYi Hao, Lei Zhao and QiaoChun Wang In vitro thermotherapybased methods for plant virus eradication. Plant Methods. 2018. Р.1–18. https://doi.org/10.1186/s13007-018-0355-y
20. Chinestraa S. C., Curvettoa N. R., Marinangeli P. A. (2015) Production of virus-free plants of Lilium spp. from bulbs obtained in vitro and ex vitro. Scientia Horticulturae. Vol. 194. P. 304–312. https://doi.org/10.1016/j.scienta.2015.08.015
21. Lozoya-Saldana H, Dawson W. O. Effect of alternating temperature regimes on reduction or elimination of viruses in plant tissues. Phytopathology. 1982. Vol. 72. No. 8. Р. 1059–1064.
22. Dziedzic E. Elimination of Prunus necrotic ring spot virus (PNRSV) from plum ‘Earliblue’ shoots through thermotherapy in vitro. Journal of Fruit and Ornamental Plant Research. 2008. Vol. 6. No. 16. Р. 101–109.
23. Wang Q., Cuellar W. J., Rajamaki M. L., Hirata Y., Valkonen J. P. T. Combined thermotherapy and cryotherapy for efficient virus eradication: relation of virus distribution, subcellular changes, cell survival and viral RNA degradation in shoot tips. Molecular Plant Pathology, Malden. 2008. Vol. 9. No. 2. Р. 237–250. doi: 10.1111/j.1364-3703.2007.00456.x
24. Cheong E. J., Jeon A. R., Kang J. W., Mock R, Kinard G Li R. In vitro elimination of Black raspberry necrosis virus from black raspberry (Rubus occidentalis). Hort. Sci. (Prague). 2014. Vol. 2. Р. 95–99. DOI: 10.17221/266/2013-HORTSCI
25. Andret-Link P., Laporte C., Valat L., Ritzenthaler C., Demangeat G., Vigne E., Laval V., Pfeiffer P., Stussi-Garaud C., Fuchs M. Grapevine fanleaf virus: Still a major threat to the grapevine industry. 2004. J. Plant. Pathol. Vol. 86. Р. 183–195.
26. Зеленянська Н. М. Наукове обґрунтування та розробка сучасної технології вирощування садивного матеріалу винограду : дис.... д-ра. с.-г.. наук : 06.01.08. Одеса, 2016. 502 с.
27. Зеленянська Н. М., Рябий М. І. Застосування методу апікальних меристем для розмноження винограду in vitro. Вісник Уманського національного університету садівництва. 2024. № 2. С. 55–61. DOI https://doi.org/10.32782/2310-0478-2024-2-55-61
28. Guojun Hu, Yafeng Dong, Zunping Zhang, Xudong Fan, Fang Ren Efficiency of chemotherapy combined with thermotherapy for eliminating grapevine leafroll- associated virus 3 (GLRaV-3). Scientia Horticulturae. 2020. Vol. 271. Р. 109462
29. Vanja Miljani´c, Denis Rusjan, Andreja Škvarˇc, Philip pe Chatelet, and Nataša Štajner Elimination of Eight Viruses and Two Viroids from Preclonal Candidates of Six Grapevine Varieties (Vitis vinifera L.) through In Vivo Thermotherapy and In Vitro Meristem Tip Micrografting. Plants 2022. Vol. 11. Р. 1064. https://doi.org/10.3390/plants11081064
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