JIA-2018-09

2018 HU Guo-jun et al. Journal of Integrative Agriculture 2018, 17(9): 2015–2023 healthy during pre-culture (Table 2). The heights of plants for the two cultivars showed hardly any difference among the five pre-culture periods, but the leaves of plants pre-cultured for 13 d (P-13d) was slightly bigger than that of P-1d (Fig. 1). Moreover, as the period of pre-culture time extended, the increase in size of basal callus was more obvious in the cultivar Pink Lady. After 13 d of pre-culture, there were no new shoots observed in both cultivars (Fig. 1). 3.2. Effect of pre-culture on apple virus titer The presence of ACLSV and ASGV in Pink Lady and Huafu plants was detected using a standard RT-PCR after 1, 4, 7, 10 and 13 d of pre-culture. Non-specific bands were rarely observed, and no amplicons were generated for the negative control samples. We observed that targets of ACLSV and ASGV showed little difference among the five periods of pre-culture (Fig. 2). The relative quantities of ACLSV and ASGV in the two cultivars were verified by qRT-PCR, and the results of the titers of the two viruses lacked any increasing or decreasing trends with duration of pre-culture, which indicated that the regularity of virus-titer change in different periods was unclear (Fig. 3). 3.3. Effect of thermotherapy on growth of pre-culture apple plantlets The survival rate of in vitro Pink Lady and Huafu plants decreased as the duration of thermotherapy increased, but the pre-culture treatment generally had positive effects on P-1d P-4d P-7d P-10d P-13d P-1d P-4d P-7d P-10d P-13d A B Fig. 1 Growth of in vitro apple plants after pre-culture for 1, 4, 7, 10 and 13 d (P-1d, P-4d, P-7d, P-10d, and P-13d), respectively. A, in vitro Pink Lady plants. B, in vitro Huafu plants. Fig. 2 Agarose gel electrophoresis of reverse transcription PCR (RT-PCR) products of Apple chlorotic leaf spot virus (ACLSV) and Apple stem grooving virus (ASGV) in the five different periods of in vitro pre-culture and an internal control. M, DNA marker II (Tiangen, Beijing, China); lane 1, no template control; lane 2, negative control; lane 3, no positive control; lanes 4–8, pre-culture for 1, 3, 7, 10 and 13 d, respectively. The primer sets for ACLSV and ASGV detection were A53/A52 and H5873/C6396, respectively. M 1 2 3 4 5 Pink Lady Huafu 6 7 8 M 1 2 3 4 5 6 7 8 ACLSV ASGV Internal control bp bp bp 500 300 100 500 300 100 500 300 100