Document Type : Research Paper


1 Former Ph. D. Student, Temperate Fruits Research Center, Horticultural Sciences Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran

2 Associate Professor, Department of Horticultural Sciences, University of Tabriz, Tabriz, IRAN.

3 Associate Professor, Department of Tissue and Cell Culture, Agricultural Biotechnology Research Institute of Iran (ABRII), Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran.

4 Ph. D. Candidate, Iranian Research Institute in Grape and Raisin, Malayer University, Malayer, Iran.


This study was aimed to production of healthy primary pear plants for propagation and establishment of a high health maternal orchard in 2014 at Agriculture Biotechnology Research Institute of Iran (ABRII). The experiments were conducted by evaluate the effectiveness of the chemotherapy treatments (Ribavirin (0, 20, 40 and 80 mg/l) and the sizes of apical meristems culturing (less than 0.2 mm, between 0.2 to 0.7 mm and larger than 0.7 mm) on virus eradicate (ACLSV, ASPV, ASGV) from seven pear cultivars ("Abate Fetal", "Beiruti", "Coscia", "Dargazi", "Louise Bonne", "Mellina" and "Spadona"). At first maternal samples were tested for ACLSV, ASGV and ASPV by RT-PCR and all explants had been infected with all three viruses except Fettel and Beirut that were free from ASPV. We performed the chemotherapy treatments then meristems were cultured and grown in vitro. Regenerated shoots from meristem were tested by RT-PCR for all three viruses. The results showed that increasing the concentration of ribavirin and reducing the size of cultivated meristem was effective on all the three viruses’ elimination from explants. Also the responses of studied cultivars and virus varieties were different in treatments of this research. Samples that were diagnosed virus free by using RT-PCR, were proliferated, rooted and transferred into the pots to be used for later propagation and establishment of maternal orchard.


Adams, M. J., Antoniw, J. F., Bar-Joseph, M., Brunt, A. A., Candresse, T., Foster, G. D., Martelli, G. P., Milne, R. G., & Fauquet, C. M. (2004). The new plant virus family Flexiviridae and assessment of molecular criteria for species demarcation. Archives of Virology, 149(8), 1045–1060.
Chiari, A., & Bridgen, M. P. )2002(. Meristem culture and virus eradication in Alstroemeria. Plant Cell, Tissue and Organ Culture, 68(1), 49-55.
Cieslinska, M. )2002(. Elimination of apple chlorotic leaf spot virus (ACLSV) from pear by in vitro thermotherapy and chemotherapy. Acta Horticulturae, (ISHS), 596, 481–484. .2002.596.80.
Cieslinska, M. )2007(. Application of thermotherapy and chemotherapy in vitro for eliminating some viruses infecting Prunus sp. fruit trees. Journal of Fruit and Ornamental Plant Research, 15, 117–124.
Deng, X. Y., Hong, N., Hu, H. J., & Wang, G. P. )2004(. Detection of latent viruses in Pyrus pyrifolia by IC-RT-PCR and TC-RT-PCR. Journal of Fruit Science, 21, 569-572. 
Hauptmanova´, A., & Pola´k, J. (2011). The elimination of Plum pox virus in plum cv. Bluefree and apricot cv. Hanita by chemotherapy of in vitro cultures. Horticultural Science (Prague), 38(2), 49–53.
Hosokawa, M. )2008(. Leaf primordia-free shoot apical meristem culture: a new method for production of viroid-free Plants. Journal of the Japanese Society for Horticultural Science, 77(4), 341-349. /jjshs1.77.341.
Hu, G., Dong, Y., Zhang, Z., Fan, X., Ren, F., & Zhou, J. (2015). Virus elimination from in vitro apple by thermotherapy combined with chemotherapy. Plant Cell, Tissue and Organ Culture (PCTOC), 121(2), 435-443.
James, D. (2001). Long term assessment of the eff ects of in vitro chemotherapy as a tool for Apple stem grooving virus elimination. Acta Horticulturae (ISHS), 550, 459–462.
Kazemi, N., Zaree Nahandi, F., Habashi, A., & Dadpour, M. (2017). Assessment of the efficacy of thermotherapy and Meristem culture using molecular and serological techniques to optimize production of healthy pear plantlet. Thesis for the degree of Ph.D. University of Tabriz.
Komorowska, B., Malinowski, T., & Michalczuk, L. (2010). Evaluation of several RT-PCR primer pairs for the detection of Apple stem pitting virus. Journal of Virological Methods, 168(1-2), 242–247. /j.jviromet.2010.04.024.
Manganaris, G. A., Economou, A. S., Boubourakas, I. N., & Katis, N. I. (2003). Elimination of PPV and PNRSV through thermotherapy and meristem-tip culture in nectarine. Plant Cell Reports, 22(3), 195-200.
Mathioudakis, M. M., Maliogka, V. I., Dovas, C. I., Paunovi´c, S., & Katis, N. I. (2008). Reliable RT-PCR detection of Apple stem pitting virus in pome fruits and its association with quince fruit deformation disease. Plant Pathology, 58(2), 228–236.
Masoomi-Aladizgeh, F., Jabbari, L., Khayam Nekouei, R., & Aalami, A. (2016). A simple and Rapid system for DNA and RNA isolation from diverse plants using handmade Kit. Nature, Protocol Exchange.
Paprstein, F., Sedlak, J., Svobodova, L., Polak, J., & Gadiou, S. (2013). Results of in vitro chemotherapy of apple cv. Fragrance.  Horticultural Science(Prague), 40(4), 186–190.
Prokhnevsky, A. I., Peremyslov, V. V., Napuli, A. J., & Dolja, V. V. (2002). Interaction between long-distance transport factor and Hsp70- related movement protein of Beet yellows virus. Journal of Virology, 76(21), 11003–11011. DOI: 10.1128/JVI.76.21.11003-11011.2002.
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(4), 417-421.
Quecini, V., Lopes, M. L., Pacheco, F. T. H., & Ongarelli, M. D. G. (2006). Ribavirin, a guanosin analogue mammalian antiviral agent, impairs Tomato spotted wilt virus multiplication in tobacco cell cultures. Archives of Phytopathology and Plant Protection, 41(1), 1-13.
Ramgareeb, S., Snyman, S. J., Antwerpen, T. V., & Rutherford, R. S. (2009). Elimination of virus and rapid propagation of disease-free sugarcane (Saccharum spp. cultivar NCo376) using apical meristem culture. Plant Cell, Tissue and Organ Culture (PCTOC), 100(2), 175-181.
Rana, T., Chandel, l.V., Kumar, Y., Ram, R., Hallan, V. & Zaidi, A.A., (2010). Molecular variability analyses of Apple chlorotic leaf spot virus capsid protein. Journal of Biosciences, 35(4), 605-615. 10.1007/s12038-010-0069-4.
Retheesh, S.T. & Bhat, A.I. (2010). Simultaneous elimination of Cucumber mosaic virus and Cymbidium mosaic virus infecting Vanilla planifolia through meristem culture. Crop Protection, 29(10), 1214-1217.
Sareila, O., Hohkuri, M., Wahlroos, T., & Susi, P. (2004). Role of viral movement and coat proteins and RNA in phloem-dependent movement and phloem unloading of tobamoviruses. Journal of Phytopathology, 152(11-12), 622–629.
Sharma, S., Singh, B., Rani, G., Zaidi, A. A., Hallan, V., Nagpal, A., & Virk, G. S. (2007). Production of Indian citrus ringspot virus-free plants of Kinnow employing chemotherapy coupled with shoot tip grafting. Journal of Central European Agriculture (JCEA), 8(1), 1–8.
Shim, H. K., Min, Y. J., Hong, S. Y., Kwon, M. S., Kim, H.R., Choi, Y. M., Lee, S. C., & Yang, J. M. (2004). Nucleotide sequences of a Korean isolate of Apple stem grooving virus associated with black necrotic leaf spot disease on pear (Pyrus pyrifolia). Molecules & Cells, 18(2), 192-199.
Simpkins, I., Walkey, D.G.A., & Neely, H.A. (1981). Chemical suppression of virus in cultured plant tissues. Annals of Applied Biology, 99(2), 161–169.
Sun, Q., Sun, H., & Bell, R. L. (2009). Effect of polyvinyl alcohol on in vitro rooting capacity of shoots in pear clones (Pyrus communis L.) of different ploidy. Plant Cell, Tissue and Organ Culture (PCTOC), 99(3), 299- 304.
Tan, R. R., Wang, L. P., Hong, N., & Wang, G. P. (2010). Enhanced efficiency of virus eradication following thermotherapy of shoot-tip cultures of pear. Plant Cell, Tissue and Organ Culture (PCTOC), 101(2), 229-235.
Tatineni, S., Afunian, M. R., Hilf, M. E., Gowda, S., Dawson, W. O., & Garnsey, S. M. (2009). Molecular characterization of Citrus tatter leaf virus historically associated with Meyer lemon trees: complete genome sequence and development of biologically active in vitro transcripts. American Phytopathological Society, 99(4), 423-431.
Valero, M., Ibanez, A., & Morte, A. (2003). Effects of high vine yard temperatures on the Grape vine leaf roll associated virus elimination from Vitis vinifera L. cv. Napoleon tissue cultures. Scientia Horticulturae, 97(3-4), 289–296.
Wang, L. P., Wang, G. P., Hong, N., Tan, R. R., Deng, X. Y., & Zhang, H. (2006). Effect of thermotherapy on elimination of Apple stem grooving virus and Apple chlorotic leaf spot virus for in vitro-cultured pear shoot tips. Hort Science, 41(3), 729–732.