Abstract
Virus and viroid-free apple rootstocks are necessary for large-scale nursery propagation of apple (Malus domestica) trees. Apple stem grooving virus (ASGV) and Apple chlorotic leaf spot virus (ACLSV) are among the most serious apple viruses that are prevalent in most apple growing regions. In addition to these viruses, a new infectious agent named Apple hammerhead viroid (AHVd) has been identified. We investigated whether thermotherapy or cryotherapy alone or a combination of both could effectively eradicate ACLSV, ASGV, and AHVd from in vitro cultures of four apple rootstocks developed in the Cornell-Geneva apple rootstock breeding program (CG 2034, CG 4213, CG 5257, and CG 6006). For thermotherapy treatments, in vitro plants were treated for four weeks at 36 °C (day) and 32 °C (night). Plant vitrification solution 2 (PVS2) and cryotherapy treatments included a shoot tip preculture in 2 M glycerol + 0.8 M sucrose for one day followed by exposure to PVS2 for 60 or 75 min at 22 °C, either without or with liquid nitrogen (LN, cryotherapy) exposure. Combinations of thermotherapy and PVS2/cryotherapy treatments were also performed. Following treatments, shoot tips were warmed, recovered on growth medium, transferred to the greenhouse, grown, placed in dormancy inducing conditions, and then grown again prior to sampling leaves for the presence of viruses and viroids. Overall, thermotherapy combined with cryotherapy treatment resulted in the highest percentage of virus- and viroid-free plants, suggesting great potential for producing virus- and viroid-free planting materials for the apple industry. Furthermore, it could also be a valuable tool to support the global exchange of apple germplasm.
Highlights
Apples (Malus spp.) are one of the most valuable fruit crops and are widely grown around the world [1]
We investigated whether cryotherapy and thermotherapy alone or in combination could effectively eradicate the latent viruses Apple chlorotic leaf spot virus (ACLSV) and Apple stem grooving virus (ASGV) as well as Apple hammerhead viroid (AHVd) from in vitro-cultured apple rootstocks
This research used in vitro plants of CG-series apple rootstocks that were infected with ACLSV (CG 2034), AHVd (CG 4213 and CG 5257), and ASGV (CG 6006), as initially determined by high throughput sequencing (HTS)
Summary
Apples (Malus spp.) are one of the most valuable fruit crops and are widely grown around the world [1]. In 2019, the total apple area planted was 4.7 M hectares, producing about 87 Mt, which represents an increase in production of 35% despite a reduction of 16% of harvested area compared with 1999 [2]. This increase in productivity is primarily attributed to the use of higher density planting systems, made possible through the use of dwarfing rootstocks [3,4]. There are variations in sensitivity to viruses and viroids in the Geneva series rootstocks, but the genetic basis of this resistance is unknown [5]
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