JIA-2018-09

2016 HU Guo-jun et al. Journal of Integrative Agriculture 2018, 17(9): 2015–2023 et al. 1998; Hirata et al. 2003). Apple is vegetatively propagated by grafting, thus resulting in virus transmission from generation to generation. Thermotherapy is an efficient technique to eliminate viruses from infected apple plants (Paprstein et al. 2008; Hu et al. 2015a, b). A higher temperature over an extended time period can reduce the movement of virus particles into the apical meristem by inhibiting viral RNAsynthesis (Valero et al. 2003; Wang et al. 2008). Even if the viruses are not eliminated, higher temperatures can sufficiently slow down their replication and infection rates to allow new shoots to grow (Wang et al. 2008). However, studies have reported that prolonged heat treatments would severely negatively affected the growth and survival of apple plants (Paprstein et al. 2008; Hu et al. 2015b). In previous researches, Hu et al. (2015a) found that the duration of pre-culturemay have effects on the survival of high temperature-treated plants. In this study, we investigated the effects of pre-culture duration on the survival rate and viral elimination in in vitro samples of two apple cultivars that were also exposed to a thermotherapy treatment. We hypothesized that pre-culture and thermotherapy acting together would increase the viral elimination and plant survival. We compared virus concentrations in in vitro apple plants that were pre-cultured at one of five different time periods to determine the percentage of viral elimination. Plant survival was determined by calculating the percentage of initial shoots after thermotherapy. 2. Materials and methods 2.1. Plant materials Two apple cultivars that are widely grown in China, in vitro Malus × domestica cv . Pink Lady and Huafu, were used as the plant materials in the experiment. Shoots of the two cultivars were collected in spring from the Research Institute of Pomology, Chinese Academy of Agricultural Sciences. The presence of ACLSV and ASGV was tested by reverse transcription PCR (RT-PCR). 2.2. In vitro cultures In vitro plantswere cultured onmodifiedMurashige andSkoog (MS) medium containing 1.0 mg L –1 6-benzylaminopurine, 0.2 mg L –1 indole-3-butytric acid, 30 g L –1 sucrose, and 5.6 g L –1 agar. The pH was adjusted to 5.8 with 1 mol L –1 NaOH or HCl before autoclaving at 121°C for 15 min. All cultures were kept in a standard growth room at (24±1)°C under a 16 h light/8 h dark photoperiod with 2 000 lx light intensity. Viable cultures were transferred to fresh MS medium at 30–40 d intervals, and the presence of ACLSV and ASGV in the cultures was reassessed by RT-PCR. 2.3. Isolation of RNA and RT-PCR Total RNA was extracted from apple samples as described by Hu et al. (2017). First-strand cDNA synthesis was conducted in a 20-μL reaction containing approximately 1 μg RNA, 5× M-MLV RT buffer, 0.5 mmol L –1 dNTP mix (Roche Diagnostics, Mannheim, Germany), 5 μmol L –1 random hexamer primers (TaKaRa, Dalian, China), and 5 U μL –1 M-MLV reverse transcriptase (Promega, Madison, WI, USA). The mixtures were incubated at 37°C for 10 min and then at 42°C for 1 h. The PCR was carried out in a 25-μL reaction volume containing 10× PCR buffer with Mg 2+ , 10mmol L –1 dNTPmix, 0.5 μLeach primer (final concentration 1 μmol L –1 ), 0.2 μL rTaq DNApolymerase (U μL –1 ) (TaKaRa), 2 μL template cDNA, and sterile water. Each apple virus was detected using two primer pairs (Table 1). The cycling profile comprised a 3-min incubation at 94°C, followed by 35 cycles of 94°C for 30 s (denaturation), 50–55°C for 30 s (annealing), and 72°C for 30–50 s (extension), with a final extension step at 72°C for 10 min. Amplicons were separated by electrophoresis on 1.2% (w/v) agarose gels, stained with ethidium bromide, and then visualized under ultraviolet light. 2.4. Quantitative real-time PCR (qRT-PCR) Total RNA samples were treated with the RT reagent kit with gDNA Eraser (TaKaRa) to remove genomic DNA and synthesize the first strand cDNA according to the manufacturer’s instructions. In a PCR tube, 2.0 µL 5× gDNA Eraser buffer, 1.0 L gDNA Eraser, and 3.0 µg total RNAwere mixed and then RNase-free dH 2 O was added up to 10 µL, and the solution was then incubated at 42°C for 2 min. Additional reagents of 4.0 µL 5× PrimeScript buffer, 1.0 µL PrimeScript RT Enzyme Mix I, and 1.0 µL RT Primer Mix was then added to the previous tube, then RNase-free dH 2 O was added up to 20 µL, and the solution was then incubated at 37°C for 15 min followed by 85°C for 5 s and stored at −20°C for later use. The titres of ACLSV and ASGV during pre-culture and thermotherapy was quantified using SYBR ® Premix Ex Taq ™ II (TaKaRa) according to the manufacturer’s instructions on the CFX Connect Real-Time System (Bio-Rad, Hercules, CA). The 20 μL reaction mixtures were incubated at 95°C for 2 min for the initial denaturation stage, followed by 40 cycles at 95°C for 10 s, 58°C for 15 s and 72°C for 20 s. The ΔC t method was used to calculate the relative concentration of ACLSV and ASGV during treatment. Each sample was examined in triplicate. The primers used in this study were synthesized by Sangon Biotech (Shanghai, China). Aprimer