JIA-2018-09

Journal of Integrative Agriculture 2018, 17(9): 2015–2023 RESEARCH ARTICLE Available online at www.sciencedirect.com ScienceDirect Effect of pre-culture on virus elimination from in vitro apple by thermotherapy coupled with shoot tip culture HU Guo-jun, DONG Ya-feng, ZHANG Zun-ping, FAN Xu-dong, REN Fang, LI Zheng-nan Research Institute of Pomology, Chinese Academy of Agricultural Sciences, Xingcheng 125100, P.R.China Abstract We evaluated the role of pre-culture on survival rate of in vitro apple plants treated by thermotherapy. Two apple cultivars, Malus × domestica cv. Pink Lady and Huafu, were used in the experiment and both have widely grown in China and infected with Apple chlorotic leaf spot virus (ACLSV) and Apple stem grooving virus (ASGV). Results in growth and virus titer of apple plants did not exhibit clear trends during five different periods of pre-culture. Whilst, pre-culture increased the survival rate of the two cultivars during thermotherapy. The survival rate of plants pre-cultured for 13 d (P-13d) was 14 and 51% higher than that of P-1d plants for Pink Lady and Huafu, respectively. Moreover, pre-culture positively influenced 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. The efficiency of virus eradication was determined by reverse-transcription PCR with two primer pairs for each virus, and the detection results showed that pre-culture scarcely affected apple virus elimination. Despite the fact that the two viruses were hardly detected at 5 d of thermotherapy, no virus-free plants were found in the two cultivars of regenerated apple plantlets after 30-d treatment. Keywords: apple, in vitro culture, pre-culture, thermotherapy, virus elimination fruit in 2014 (Hu et al. 2016). Genetic resources are required to breed new apple cultivars in both traditional and genetic engineering breeding programs. An alternative breeding method is vegetative propagation; however, viruses are particularly problematic in vegetatively propagated crops because they are transmitted from generation to generation. Apple chlorotic leaf spot virus (ACLSV, genus Trichovirus , family Betaflexiviridae ), Apple stem pitting virus (ASPV, genus Foveavirus , family Betaflexiviridae ) and Apple stem grooving virus (ASGV, genus Capillovirus , family Betaflexiviridae ) are common viruses and infected plants are generally asymptomatic (Adams et al. 2004). They significantly reduce fruit yield and quality, and commonly occur as mixed infections, causing top-working disease of apple on susceptible rootstocks (Yanase 1983; Desvignes and Boyé 1988). All these viruses are naturally disseminated via infected propagation materials (Yanase et al. 1989; Pasquini Received 8 September, 2017 Accepted 14 November, 2017 HU Guo-jun, E-mail: hugj3114@163.com ; Correspondence DONG Ya-feng, Tel/Fax: +86-429-3598278 , E-mail: yfdong@ 163.com © 2018 CAAS. Publishing services by Elsevier B.V. All rights reserved. doi: 10.1016/S2095-3119(18)61913-6 1. Introduction The apple ( Malus × domestica ) belongs to the family Rosaceae which is one of the most widely cultivated temperate fruit trees. China is one of the main apple-producing countries, producing almost half of the world’s apples with a planting area of 2.3 million ha and a production of 41 million tons of