JIA-2018-09

2020 HU Guo-jun et al. Journal of Integrative Agriculture 2018, 17(9): 2015–2023 regenerated for the two cultivars in the 37°C treatments. 3.4. Effect of pre-culture on survival rate of shoot regeneration In vitro Pink Lady and Huafu plants were regenerated from treated shoot tips (1.0 mm) and the survival rate was calculated (Table 2). The effect of pre-culture duration on regeneration of Pink Lady was not obvious, and no clear trend was observed for this cultivar. Whereas, pre-culture clearly affected regeneration of Huafu plants. The average survival rate of plants regenerated from P-1d and P-4d was 20% lower than that regenerated from P-7d, P-10d and P-13d. 3.5. Effect of thermotherapy on ACLSV and ASGV titers The presence of ACLSV andASGV in the high temperature- treated Huafu plants was detected using a standard RT- PCR when the temperature raised to 27, 30, 33 and 37°C, respectively, and during 37°C for 0, 1, 5, 10, 15, 20, 25, and 30 d, respectively. There was no band for the two viruses after 5 d at 37°C. We speculated that the viral titer would decrease as early as the start of the treatment (Fig. 5). 3.6. Efficiency of virus eradication by thermotherapy combined with shoot tip culture Regenerated plants sub-cultured for three cycles were analyzed by RT-PCR to confirm the elimination of viruses, using two primer pairs for each virus. Plants were judged as virus-free when there were no target products amplified by RT-PCR with four primer pairs. Virus elimination efficiencies differed in the two apple cultivars (Table 4). The average elimination rates of ACLSV and ASGV in regenerated Pink Lady plants was 21.7 and 8.7%, respectively. Only two regenerated plants wereASGV-free. Almost noACLSV was P-1d P-4d P-7d P-10d P-13d CK P-1d A B P-4d P-7d P-10d P-13d CK Fig. 4 Growth of in vitro apple plants after thermotherapy for 30 d. A, in vitro Pink Lady plants. B, in vitro Huafu plants. CK, in vitro apple plants were cultured in a standard growth room; P-1d, P-4d, P-7d, P-10d, and P-13d, pre-cultured for 1, 4, 7, 10, and 13 d, respectively. bp 500 M 1 2 3 4 5 6 7 8 9 10 11 12 13 ACLSV ASGV Internal control 300 100 bp 500 300 100 bp 500 300 100 Fig. 5 Agarose gel electrophoresis of reverse transcription PCR (RT-PCR) products of ACLSV ( Apple chlorotic leaf spot virus ) and ASGV ( Apple stem grooving virus ) in five different periods of thermotherapy and a internal control in Huafu plants. M, DNA Marker II (Tiangen, Beijing, China); lane 1, no template control; lane 2, negative control; lane 3, no positive control; lanes 4–6, temperature at 27, 30, and 33°C, respectively; lanes 7–13, pre-cultured for 1, 5, 10, 15, 20, 25 and 30 d (P-1d, P-3d, P-7d, P-10d, and P-13d) at 37°C, respectively. The primer sets for ACLSV and ASGV detection were A53/A52 and H5873/C6396, respectively.