JIA-2018-09

2021 HU Guo-jun et al. Journal of Integrative Agriculture 2018, 17(9): 2015–2023 eliminated from regenerated Huafu plants, and the average eradication rate of ASGV was 24.2%. The pre-culture scarcely showed any effect on the apple virus elimination rate. The detection results showed that no virus-free plants were found in the two regenerated apple cultivars. 4. Discussion Thermotherapy is a routine method for virus eradication, and this method has been widely used to eliminate viruses from fruit trees and other vegetative plants (Valero et al. 2003; Paprstein et al. 2008; Tan et al. 2010). Woody plants, like apple, are sensitive to prolonged exposure to extreme high temperatures (Paprstein et al. 2008; Tan et al. 2010; Hu et al. 2012). High temperatures significantly affected shoot survival, and the survival rate directly associated with the success of thermotherapy. In this research, we tried to investigate the potential influence of various durations of pre-culture to increase the survival rate of apple plantlets. The success of virus elimination is also related to the concentration of each particular virus in the plant material. It was unclear whether the titers of virus would change during pre-culture, so the relative quantitative concentration of ACLSV and ASGV in the Pink Lady and Huafu plants were analyzed. The results showed no obvious trend of the two virus’s titers in the two cultivars during pre-culture. Thus, we speculated that pre-culture did not affect the virus titers, and the differences among the five periods were due to individual differences between plantlets. It showed that the longer time pre-culture could increase the growth of plants, which were helpful for the plants during high temperature treatment. The temperature range is an important factor affecting shoot survival. Most often, the temperature of thermotherapy ismeasured in a growth chamber. Some authors have shown that the air temperature measured in the growth chamber was 1 or 2°C (Deogratias et al. 1989; Stein et al. 1991) lower than that inside the test tubes within the chamber. When choosing a temperature, there must be a balance between relatively low shoot mortality at higher temperatures and the high percentage of virus-free shoots (Dziedzic 2008). Previous research demonstrated that the survival rates of shoots from apple plants treated at 34 and 36°C was 84.6 and 93.3%, respectively, but almost no shoots survived in the 38°C treatment (Hu et al. 2015a). We chose 37°C to eliminate the apple viruses, and the results indicated that this temperature caused high death rates, thus, the method of pre-culture was applied to determine whether it can mitigate the often fatal effect of high temperatures. The survival rate of P-13d plants was 14 and 51% higher than that of P-1d plants for Pink Lady and Huafu, respectively. However, although the in vitro apple plants were cultured in the same conditions, inherent variability among individuals within each cultivar may be responsible for the differences we observed, for example, in the P-4d plants of Pink Lady and P-10d plants of Huafu. The duration of thermotherapy treatment can affect virus elimination efficiency (Tan et al. 2010; Hu et al. 2012). Dong et al. (2002) found that the elimination rate of an apple virus treated with a high temperature of 37°C for 36 d was clearly higher than that of 27 d. Cheng et al. (2003) had reported that when a 38°C temperature lasted for 25 d, the elimination rates of ACLSV andASGV were 17% higher than that lasted for 20 d. In the present study, the duration of the high temperature treatment was 30 d long, and surviving plants (11.1–82.2%) were found in all pre-culture treatments at the end of treatment. However, the effects of temperature and duration of exposure may be obscured by the intrinsic heat tolerance of the host plant because the level of tolerance depends on species and variety (Hu et al. 2015b). Pink Lady plants were more sensitive to high temperature than that of Huafu. The results showed that pre-culture had little effect on apple virus elimination. Unfortunately, none of the regenerated apple plants were virus-free. In fact, the titers of ACLSV and ASGV during thermotherapy were detected, and no virus was found when thermotherapy lasted for 5 d. The underlying mechanism of thermotherapy’s effect on reducing viral infection has not been completely explained yet. Since Kunkel (1935) established that exposure of virus-infected trees in pots to temperatures ranging between 34.4 and 36.3°C was effective in treating peach tree of either peach yellows or peach rosette, heat treatment for the inactivation of different viruses in plants has become an established practice. One explanatory hypothesis is that heat stress, especially at sublethal temperatures, inhibits virus replication in newly growing plant tissues and thus allows a plant to recover from viral infections (Nyland and Goheen 1969; Mink et al. 1998). Szittya et al. (2003) and Qu et al. (2005) reported that plant-virus interactions are strongly affected by temperature, and high temperatures Table 4 Efficiencies of thermotherapy to generate Apple chlorotic leaf spot virus (ACLSV)- or Apple stem grooving virus (ASGV)-free apple plants Duration of pre-culture 1) Pink Lady Huafu ACLSV ASGV ACLSV ASGV P-1d 0/2 2) 0/2 2) 0/2 2) 0/2 2) P-4d 22.2 (2/9) 22.2 (2/9) 0 (0/5) 0 (0/5) P-7d 1/2 2) 0/2 2) 0 (0/23) 21.7 (5/23) P-10d 33.3 (1/3) 0 (0/3) 0 (0/12) 25.0 (3/12) P-13d 14.3 (1/7) 0 (0/7) 4.2 (1/24) 33.3 (8/24) 1) P-1d, P-4d, P-7d, P-10d, and P-13d, pre-cultured for 1, 4, 7, 10, and 13 d, respectively. 2) Less than three shoots were obtained. Numbers in parentheses indicate virus-free shoots/shoots analyzed.