Effect of Thermotherapy on Elimination of Apple Stem Grooving Virus and Apple Chlorotic Leaf Spot Virus for In vitro-cultured Pear Shoot Tips

Abstract

<jats:p>Apple stem grooving virus (ASGV) and apple chlorotic leaf spot virus (ACLSV) are two major viruses of pear. In this study, in vitro thermotherapy was carried out at 37°C for 25, 30 and 35 days followed by subculturing of meristem tips of different sizes to eliminate ASGV and ACLSV from pear plants. Virus titers in heat-treated shoot tips were evaluated by ELISA testing of regenerated plants. Results showed that thermotherapy for 35 days significantly decreased the titer of ASGV and ACLSV in cultures regenerated from tips of main and axillary shoots, especially in those from explants 1 mm in length from the tip of meristems. Dot-blot hybridization of biotinylated cDNA probes derived from ACLSV and ASGV was used to detect these viruses in crude tissue extracts of in vitro-grown pear plants. Intense signals were consistently detected in untreated plant samples equivalent to less than 0.5 mg tissue. Comparison of signals from dot-blot hybridization and ELISA absorbance values (A<jats:sub>405</jats:sub>) confirmed that dot-blot hybridization had a higher sensitivity than PAS-ELISA. Dot-blot hybridization could detect viruses with a titer below the threshold level of ELISA. These results indicate that dot-blot hybridization is a useful tool for large-scale surveys of viruses, which facilitates the production of virus-free propagation materials in certification and sanitation programs. Results of PAS-ELISA and dot-blot hybridization showed that high virus elimination efficiency was achieved by a combination of thermotherapy for 35 days and in vitro culture of 1 mm meristem tips.</jats:p>

Results of in vitro thermotherapy of apple cultivars

“…By this method, mixed infection of two viruses ACLSV and ASPV was completely eliminated from apple cultivar 'Idared'. Successful in vitro heat treatments and virus elimination were previously reported for ACLSV in apple (Cieslinska 2002), for ACLSV and ASGV in apple (Knapp et al 1995(Knapp et al , 1997da Câmara Machado et al 1998) and for ACLSV and ASGV in pear (Wang et al 2006). On the other hand, the experiments reported here revealed that virus elimination from some genotypes may be difficult.…”

Virus elimination from in vitro apple by thermotherapy combined with chemotherapy

“…In previous studies, virus-free plants were regenerated from both apical and axillary shoots (Wang et al 2006;Hu et al 2012), but the difference between the two materials was unclear. Our results show that the elimination efficiency was higher for plants regenerated from axillary shoots than from apical shoots.…”

Enhanced efficiency of virus eradication following thermotherapy of shoot-tip cultures of pear

“…Samples that gave positive reactions for three viruses were used as starting materials. The establishment of in vitro cultures was conducted as previously described (Wang et al 2006a). The basic nutrient solution contained the inorganic Murashige and Skoog (MS) and complemented with 0.2 mg l -1 6-benzylaminopurine (BAP), 1.0 mg l -1 3-indole-butyric acid (IBA), 30 g l -1 sucrose and 7.5 g l -1 agar.…”

“…Meristem tips of 1.0, 2.0 and 5.0 mm in length were dissected from treated shoots every 5 days after thermotherapy for 25 days. Previously established treatment at 37°C (Wang et al 2006a) was also employed for comparison of virus eradication efficiency at different thermotherapy temperatures.…”

“…The thermotherapy is most frequently carried out at 36 to 38°C for 21-35 days (Mink et al 1998;Knapp et al 1995;Verma et al 2005). In previous study, we had found a great effect of thermotherapy duration at 37°C on the distribution of both ASGV and ACLSV in tips of in vitro-cultured pear and established a sensitive dot blot hybridization to detect pear viruses (Wang et al 2006a(Wang et al , 2006b. However, the high temperature also had a severe effect on pear plant growth, which decreased the virus-eradication efficacy.…”