Combined thermotherapy and cryotherapy: an effective method for the eradication of three common viruses in cultivated apples

doi:10.21203/rs.3.rs-35214/v1

Download PDF

Research

Combined thermotherapy and cryotherapy: an effective method for the eradication of three common viruses in cultivated apples

https://doi.org/10.21203/rs.3.rs-35214/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Background

Apple virus disease is a serious problem affecting apple quality and crop production efficiency, and solutions are being actively and urgently explored. In particular, the cultivation of virus-free seedlings is being increasingly explored as an important technical method for facilitating the virus-free breeding of excellent apple cultivars. In this study, the apple cultivars ‘Ruiyang’, ‘Ruixue’, ‘Rui Xianghong’, and ‘Qinyue’ were used as source materials to compare the effectiveness of thermotherapy and combined thermo- and cryotherapy in eradicating apple chlorotic leaf spot virus (ACLSV), apple stem grooving virus (ASGV), and apple stem pitting virus (ASPV).

Results

Viral presence in the in vitro shoots of the four apple cultivars was detected using a reverse transcription-polymerase chain reaction. All four cultivars were found to carry ASGV and ACLSV, and ‘Rui Xianghong’ and ‘Qinyue’ additionally carried ASPV. In vitro shoots of the four cultivars were subjected to varying periods of thermotherapy (0, 2, 4, 6, and 8 weeks). Shoot tips were then removed and half were cultured directly, and the remainder were cultured after cryotherapy treatment; their survival and regeneration rates were then evaluated to determine the efficiency of virus eradication after 8 months of subculture. We showed that the eradication of viruses in each of the cultivars and their survival rate are both the highest after 4 weeks of thermotherapy. When combined with cryotherapy, application of this period of thermotherapy achieved a 100% eradication of ASPV and ACLSV, and over 50% of ASGV.

Conclusions

This study demonstrated an effective methodology for developing both new virus-free plant materials and new apple cultivars that will support sustainability of the apple production industry in China and globally.

Plant Molecular Biology and Genetics

Plant Physiology and Morphology

Apple

Thermotherapy

Cryotherapy

Virus eradication

Apple chlorotic leaf spot virus

Apple stem grooving virus

Apple stem pitting virus

Apple (Malus × domestica Borkh.) is a globally important cash crop. It is particularly valuable in China, which is both the largest producer and consumer of apples in the world. A total of 2.07 million hectares in China is dedicated to apple cultivation; the output of 39.23 million tons accounts for nearly 50% of global output [1]. In this context, apple viral diseases, which are among the main factors restricting healthy and sustainable development in apple cultivation [2], have received much attention. Viral infection of an apple tree damages normal cell proliferation, therefore seriously restricting the growth potential and other physiological mechanisms; it also reduces apple quality and yield, with consequent adverse implications for the apple industry [3–5]. Viruses can spread from infected plants to their offspring through asexual reproduction, or to healthy plants via insect vectors [6]. Pesticide spraying is ineffective once a plant has become infected [7, 8]. The focus has therefore been on cultivating virus-free apple seedlings as an effective and core technology in the global control of apple viral diseases [9–12].

Viruses in fruit trees, such as apple stem pitting virus (ASPV), apple stem grooving virus (ASGV), and apple chlorotic leaf spot virus (ACLSV) are often present as complexes [13]; this presents a challenge to researchers working on the development of effective ways to produce virus-free apple plants. For example, ASGV is more difficult to eradicate than ASPV or ACLSV [14–19]. Methods of producing virus-free plants that have been considered include chemotherapy [20], cryotherapy [21, 22], micrografting [8, 20, 23], shoot tip culture (STC) [24, 25], and thermotherapy [10, 25, 26].

Cryotherapy, which uses liquid nitrogen as a coolant, has proved promising as a technique, playing an important role in developing virus-free plant production [27, 28]. In some research, a small number of virus-free cells regenerated as healthy, detoxified plants after liquid nitrogen was used to kill stem tip cells [29, 30]. In garlic (Allium sativum L.), cryotherapy has been shown to eradicate viral complexes successfully [31] and, in combination with STC, to eradicate ASPV and ASGV from M9 and M26 apple rootstocks [32]. The principle behind thermotherapy, which was reported in 1998 as an application suitable for virus eradication, is exposing plant tissue to a high temperature to inhibit viral spread; this minimises the extent to which meristem tissue in the stem tip carries viral particles, thus yielding virus-free seedlings [10]. However, there is growing interest in the effects of combined thermo- and cryotherapy. For example, it is more difficult to eradicate raspberry bushy dwarf virus from the in vitro shoots of raspberry when using only cryotherapy, compared with its use in combination with thermotherapy [29].

In this study, our objective was to compare and evaluate the effectiveness of thermotherapy alone and combined thermo- and cryotherapy in successfully eradicating ASPV, ASGV, and ACLSV from apple plants, using the in vitro shoots of four apple cultivars.

Virus detection in plant materials

The results for the detection of ASPV, ASGV, and ACLSV in the in vitro shoots of four apple cultivars (‘Ruiyang’, ‘Ruixue’, ‘Rui Xianghong’, and ‘Qinyue’) are shown in Fig. 1. Two latent viruses, ASGV and ACLSV, but not ASPV, were detected in ‘Ruiyang’ and ‘Ruixue’. However, in vitro shoots of ‘Rui Xianghong’ and ‘Qinyue’ carried all three latent viruses. This therefore clarified the viruses potentially present in each cultivar, thus making it possible to focus eradication efforts appropriately.

Effects of thermotherapy timings on shoot tip survival and regrowth

Shoot tips were obtained from in vitro shoots that been subjected to thermotherapy but not cryotherapy treatment (treatment -LN), for different periods; the resulting growth is shown in Fig. 2a. Regrowth mainly presented as normal (Fig. 2a-ⅱ) with a few vitrified shoots (Fig. 2a-ⅲ, ⅳ). Normally formed shoots regrew faster; the shoot tips were therefore cultured for 1 (Fig. 2a-ⅰ) and 4 (Fig. 2a-ⅴ, ⅵ) weeks, respectively. Shoot tip growth after the treatment combining thermo- and cryotherapy (treatment + LN) is shown in Fig. 2b; growth was observed to be slower with than without cryotherapy treatment (Fig. 3k), and the size of shoot tip cultured to 1 weeks (Fig. 2b-ⅰ, ⅱ) and 8 weeks (Fig. 2b-ⅲ, ⅳ). Regrowth presented normal shoots (Fig. 2b-ⅲ, ⅳ), leaves (Fig. 2b-ⅴ), vitrified shoots (Fig. 2b-ⅵ, ⅶ), and a callus mass (Fig. 2b-ⅷ).

Survival and regrowth rates after the STC treatment are shown in Fig. 2c. In the -LN treatment, the survival rate of all four cultivars decreased with increasing thermotherapy period. When in vitro shoots were subjected to thermotherapy for up to 8 weeks, the shoot tip regrowth rate for ‘Qinyue’, ‘Rui Xianghong’, Ruixue’, and ‘Ruiyang’ gradually decreased from 72.5%, 59.6%, 69.4%, and 75.6–19.8%, 26.3%, 21.3%, and 35.9%, respectively. In the + LN treatment, the shoot tip survival and regrowth rate decreased gradually with increasing thermotherapy period; the regrowth rate for ‘Qinyue’, ‘Ruixue’, and ‘Ruiyang’ decreased to zero by week 8. However, the important point is that, for any length of thermotherapy treatment, the shoot tip survival and regeneration rates are higher without than with cryotherapy treatment.

Effectiveness of different treatments in virus eradication

The results of reverse transcription-polymerase chain reaction (RT-PCR) detection of in vitro shoots subcultured 10 times are shown in Fig. 4b. Cultivars ‘Qinyue’ and ‘Rui Xianghong’ were subjected to the -LN treatment for 8 weeks, and eradication of ASPV increased from nil to 25.6% and 30%, respectively. There was no eradication of ASGV from ‘Qinyue’, ‘Rui Xianghong’, ‘Ruixue’, and ‘Ruiyang’ at 2 weeks, but the value then increased to 10%, 12.5%, 10.9% and 10%. Eradication of ACLSV gradually increased with prolonged thermotherapy, up to 38.6% (‘Ruixue’).

In the + LN treatment, eradication of ASPV and ACLSV in the cultivars ‘Qinyue’, ‘Rui Xianghong’, ‘Ruixue’, and ‘Ruiyang’ increased rapidly with increasing thermotherapy period. After 4 weeks of thermotherapy, ASPV (Fig. 4a-ⅰ) and ACLSV (Fig. 4a-ⅴ, ⅵ) were eradicated successfully. ASGV eradication was slightly more difficult, however, increasing gradually from 0 to 8 weeks. Nonetheless, at 6 weeks, it was completely eradicated in ‘Ruixue’ and ‘Ruiyang’ and by over 70% in ‘Qinyue’ and ‘Rui Xianghong’. After thermotherapy for 4 weeks combined with cryotherapy, ASGV eradication in vitro shoots had achieved a value of over 50%; the results are shown in Fig. 4a-ⅱ, ⅲ, ⅳ.

In this study, we assessed two effective detoxification methods: thermotherapy and combined thermo- and cryotherapy. These two techniques were applied to in vitro shoots of four apple cultivars subcultured at the age of 2 weeks. Our results determined that combined thermo- and hydrotherapy was more effective in virus eradication than thermotherapy alone, which is in agreement with previous studies [31, 33].

The thermotherapy temperature was alternated between 38 °C in the day and 30 °C at night, rather than imposing a constant high-temperature environment. This benefited the in vitro shoots by relieving the discomfort of the higher temperature at night while not lowering the temperature for too long [34–36].

The viral inhibition under thermotherapy treatment potentially reduces the transport of viral particles to the apical meristem [36, 37], and expands the non-infected area at the stem tip, which is therefore virus-free [10]. The length of the thermotherapy treatment is key to the successful establishment of a detoxification system. When thermotherapy treatment is too short, a large number of virus particles will remain in the shoot tips, therefore preventing full detoxification. However, if thermotherapy is administered for too long, the heat-resistant function of the shoots will begin to decline. Tan et al. [35] found that a combined treatment of thermotherapy for 35 days (at 37 °C) and STC could produce ASGV- and ACLSV-free pear plants from shoot tips of 1.0 mm in length; however, shoot tips longer than 2.0 mm could not produce virus-free plants. In our study, we were able to produce ASPV- and ACLSV-free plants using thermotherapy, a result in agreement with previous reports [35, 38].

The effects of droplet-vitrification and encapsulation-dehydration procedures on post-cryotherapy recovery patterns in apple shoot tips have been compared [39, 40]. These studies have shown that shoot tip regrowth rates obtained by droplet-vitrification is more stable than rates yielded by encapsulation-dehydration throughout the year, i.e. there was no seasonal effect. In our study, we used droplet-vitrification as our cryotherapy programme.

ASGV, ACLSV, and ASPV are found in most apple cultivars in commercial orchards [41]. A previous study suggested that using cryotherapy alone could not eradicate ASGV [18]. Some studies have clearly indicated serious ASGV infection and localisation in the shoot tips and meristematic cells, which may explain why it was difficult to eradicate [18, 33].

Previous reports have shown that shoot tips (about 1 mm in length) can produce virus-free plants after thermotherapy [36], but the survival rate of shoot tips of this length under cryotherapy treatment is very low [19, 29, 33]. In our study, we cut shoot tips to a length of 1.5 mm after thermotherapy; when then combining this treatment with cryotherapy, we showed a strong improvement in virus eradication from the cultivars ‘Ruiyang’ and ‘Ruixue’, which increased with increasing thermotherapy period, consistent with the previous studies [29, 33]. We found that a combined thermo- and cryotherapy treatment of shoot tips (1 mm) in ‘Ruiyang’, ‘Ruixue’, ‘Qinyue’, and ‘Rui Xianghong’ cultivars showed an improved effectiveness in virus eradication, the rate of which increased with increasing thermotherapy period. This provides a basis for promoting healthy and sustainable development of the apple industry, so long as the development and performance of virus-free plants are strictly tested and supervised in the production process.

In conclusion, our study explored two effective methods of eradicating viral infections from apple plants, that is, thermotherapy and combined thermo- and cryotherapy. We demonstrated that combining the application of thermotherapy for 4 weeks with cryotherapy can effectively eradicate ASPV and ACLSV, and is effective in minimising ASGV, which is harder to eradicate completely. This study therefore provides a guaranteed technical method that supports the development of new virus-free apple plant materials and the application and promotion of new cultivars.

Plant material

We selected four apple cultivars based on their strong developmental potential as crops. They were: ‘Ruiyang’ (Fuji × Qinguan, S-SV-MD-009-2018) and ‘Ruixue’ (Fuji × Cripp's Pink, S-SV-MD-010-2018), both of which are registered with the National Forestry and Grassland Administration in China; and ‘Qinyue’ (Fuji × Gala, GPD-2018-610058) and ‘Rui Xianghong’ (Fuji × Cripp's Pink, S-SC-MR-002-2019), which are medium and late ripening cultivars, respectively, with crisp flesh, a rich fragrance, and strong disease resistance. Trees of the four cultivars from which samples originated were all grafted onto 11-year-old M26 rootstock. In March 2017, branches (15 branches per cultivar; approximate length, 30 cm) were removed from the trees in the field and transported to the laboratory. There, they were soaked in a solution of 1% NaClO for 10 min, followed by hydroponic culture at 25 °C (with no further additional nutrient); water was changed daily. Shoot tip samples (length, approximately 1.5 cm) (Fig. 3a) per apple cultivar were extracted from the branches, rinsed under tap water for 30 min, treated with 75% alcohol for 30 s and then with 0.1% HgCl2 (adding 2–3 drops of surfactant [Tween 20]) for 10 min (Fig. 3b). The samples were finally rinsed in sterile water 5–7 times. Shoot tip surfaces in contact with the disinfectant solution were removed and inoculated on to the culture medium (Fig. 3c).

In vitro shoots of the four cultivars were cultured in a shoot maintenance medium [42] comprising Murashige and Skoog basal medium (MS) [43], supplemented by 6-benzyladenine (0.5 mg L^− 1), indole-3-butyric acid (IBA; 0.03 mg L^− 1), sucrose (30 mg L^− 1), and agar (8 mg L^− 1). The medium was sterilised by autoclaving for 20 min at 121 °C after adjusting the pH to 5.8. The culture experiment was performed at 25 ± 2 °C and with a 16/8 h photoperiod (light intensity, 50 µEs^− 1 m^− 2) (Fig. 3d); the subculture period was 4 weeks.

Virus detection

All plant materials were tested twice using RT-PCR. The in vitro shoots for each apple cultivar (Fig. 3e) were analysed to determine viral presence. The regenerated plants were then investigated to assess the virus eradication frequency. After the subculture process had been repeated 10 times (from October 2017 to June 2018), the samples were analysed again to detect the presence of viruses (Fig. 3l).

RNA was extracted from 0.1 g of fresh in vitro shoots and purified, using a TaKaRa MiniBEST Plant RNA Extraction Kit (TaKaRa, Dalian, China). The cDNA was synthesised from 2 mg of total RNA using a PrimeScript RT Master Mix Kit (TaKaRa, Dalian, China). The products of each virus were amplified using the following primer pairs: ASPV (370 bp), (5’-ATGTCTGGAACCTCATGCTGCAA-3’) and (5’-TTGGGATCAACTTTACTAAAAAGCATAA-3’); ASGV (524 bp), (5’-CTGCAAGACCGCGACCAAGTTT-3’) and (5’-CCCGCTGTTGGATTTGATACACCTC-3’); ACLSV (794 bp), (5’-GAGARTTTCAGTTTGCTMGA-3’) and (5’-AGTCTACAGGCTATTTATTATAAGT-3’) [44–46]. The PCR reaction volume was 25 µL, incorporating 2 × Taq Master Mix (12.5 µL) (Kangwei, Beijing, China), 0.8 µL of each primer, 1 µL of cDNA, and 9.9 µL of RNase-free water. The PCR reaction procedure was as follows: 2 min at 94 °C; 30 s at 94 °C; 45 s at 53 °C (ASPV), 57 °C (ASGV), and 54.5 °C (ACLSV); 50 s at 72 °C; 35 cycles of amplification; and a final finally extension of 10 min. The PCR products were validated using agarose (1%) gel electrophoresis, and then visualised under ultraviolet light.

Thermotherapy and treatment

In vitro shoots of the four cultivars were subcultured for 2 weeks, and then moved into a growth chamber (RXZ-500D-LED, Ningbo, China) under light conditions of 50 µE s^− 1 m^− 2 (Fig. 3f). The temperature in the chamber was set to alternate between 38 °C during the day (6:00 am to 22:00 pm) and 30 °C at night (22:00 pm to 6:00 am). At 0, 2, 4, 6, and 8 weeks of thermotherapy, 100 shoot tips of each cultivar were cut under an anatomical microscope to a length of approximately 1.5 mm (four to five leaf primordia) (Fig. 3g). The shoot tips were divided into two groups: 50 shoot tips for direct STC without cryotherapy (-LN) (Fig. 3h), and 50 for cryotherapy treatment (+ LN) (Fig. 3j). All experiments were repeated in triplicate.

Cryotherapy

Feng et al. [39] describe the procedure for droplet-vitrification. First, shoot tips were cultured under stable conditions on a basic medium (BM) for 1 day, then transferred to the MS solution (0.8 M sucrose, 2 M glycerol), also for 1 day. The shoot tips were transferred into droplets containing 6 uL plant vitrification solution 2 (PVS2) [47] on aluminium foil (5 × 1.5 cm) (Fig. 3i), and frozen in liquid nitrogen for 1 h. The PVS2 solution was composed of 30% glycerol (wt/vol), 15% dimethylsulfoxide (wt/vol), 15% ethylene glycol (wt/vol), and sucrose (0.4 M) in MS (pH 5.8). The shoot tips were then removed quickly from the liquid nitrogen and placed into an MS unloading solution (containing 1.2 M sucrose) for 20 min at a temperature of 20 ± 2 °C. Finally, the shoot tips were placed into a BM medium in dark culture conditions for 3 days, after which the shoot tips were transferred to light culture conditions. Within 3 days of the dark culture treatment, the shoot tips were transferred to the new medium twice, after being frozen for 12 h and 36 h, to reduce browning. After regenerating into normal shoots, they were subcultured every 4 weeks.

Virus-free plants higher than 2 cm (Fig. 3l) were selected from all four cultivars and observed to grow healthily. Prior to rooting, they were inoculated onto 1/2 MS medium with IBA concentrations of 0.9 mg L^− 1, 0.75 mg L^− 1, 0.9 mg L^− 1, and 1.1 mg L^− 1 for the ‘Ruiyang’, ‘Ruixue’, ‘Rui Xianghong’, and ‘Qinyue’ cultivars, respectively. When the root length of the in vitro shoots reached approximately 2 cm, plants were transferred to the seedling refinement room. The cap of each tissue culture bottle was opened for 1–2 days, until no fungus was visible in the bottle, prior to transplanting. After the seedlings were removed from the culture medium to be transplanted, culture medium attached to the roots of the seedlings was washed with clean water (Fig. 3m) and transferred to a nutrition bowl containing peat soil, perlite, and vermiculite (volume ratio of 3:0.5:0.5). The transplanted seedlings were cultured for 7 days (Fig. 3n) in low light (< 20 µEs^− 1 m^− 2), and then gradually transitioned to normal light levels (50 µEs^− 1 m^− 2) (Fig. 3o).

Assessment of shoot survival and regrowth

The survival and regrowth rate of the shoot tips were evaluated after the -LN (Fig. 3h) and + LN treatments (Fig. 3j). Survival rate was expressed as the percentage of the total number of tips that were green after 7 days of STC in both treatments (-LN and + LN). Shoot tips can grow new leaves within 8 weeks, and elongation of the shoot segments is considered to indicate regeneration. The normal regenerated in vitro shoots were analysed to assess any content of ASGV, ASPV, or ACLSV after 10 months, and the level of virus eradication in each cultivar was calculated.

Data analysis

Data were sorted using Microsoft Office Excel software (2007) and plotted using the ggplot2 function in R software (version 3.6.3).

ACLSV: Apple chlorotic leaf spot virus; ASGV:Apple stem grooving virus; ASPV:Apple stem pitting virus; IBA:Indole-3-butyric acid; MS:Murashige and Skoog medium; PVS2:Plant vitrification solution 2; RT-PCR:Reverse transcription-polymerase chain reaction.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Availability of data and materials

The datasets and materials used and (or) analysed during the current study are available from the corresponding author on reasonable request

Competing interests

The authors declare they have no competing interests.

Funding

This work was supported by The Modern Agro-industry Technology Research System of China (CARS-27).

Authors’ contributions

LL, YY and ZZ conceived the research; LL designed and performed the experiments, analyzed data and drafted the manuscript; SL and WY participated in coordination of the study; CX, YL and JY managed the plant in the field; YY and ZZ designed and supervised of review; LL, YY and ZZ wrote the paper. All authors read and approved the final version of the manuscript.

Acknowledgements

The authors are grateful to Prof. Qiaochun Wang and Dr. Minrui Wang for providing help with the cryotherapy procedure.

FAO (Food and Agriculture Organization of the United Nations). FAOSTAT Production Database. 2018. Http://faostat.fao.org. Accessed 12 May 2020.
Zhang LZ, Wei YN, Wang HX, Wang L. Current Situation of Apple Virus Disease in China and Research Progress of Detection Technology. Journal of Ludong University. 2019;35(2):116–21.
Converse RH. Latent Viruses: harmful or harmless. Hort Science. 1985;20(5):845–8.
Cieślińska M, Rutkowski KP. Effect of Apple chlorotic leaf spot virus on yield and quality of fruits from ‘Golden Delicious’ and ‘Sampion’ apple trees. Acta Hortic. 2008;781:119–24.
Barba M, Ilardi V, Pasquini G. Control of pome and stone fruit virus diseases. Adv Virus Res. 2015;91:47–83.
Kumar S, Singh L, Ram R, Zaidi AA, Hallan V. Simultaneous detection of major pome fruit viruses and a viroid. Indian J Microbiol. 2014;54(2):203–10.
Németh M. Virus, mycoplasma and rickettsia diseases of fruit trees. Dordrecht: Martinus Nijhoff Publishers; 1984.
Laimer M, Barba M. Elimination of systemic pathogens by thermotherapy, tissue culture, or in vitro micrografting. In: Hadidi A, Barba M, Candresse T, Jelkmann W, editors. Virus and virus-like diseases of pome and stone fruits. American Phytopathological Society. St. Paul: APS Press; 2011. pp. 389–93.
Bhojwani SS, Razdan MK. Plant Tissue Culture: Theory and Practice, a Revised Edition. Amsterdam: Elsevier Science BV; 1996.
Mink G, Wample R, Howell W. Heat treatment of perennial plants to eliminate phytoplasmas, viruses and viroids while maintaining plant survival. In: Hadidi A, Khetarpal RH, Koganezawa H, editors. Plant Virus Disease Control. American Phytopathological Society. St. Paul: APS Press; 1998. pp. 332–45.
Hadidi A, Barba M. Economic impact of pome and stone fruit viruses and viroids. In: Hadidi A, Barba M, Candresse Th, Jelkman W, editors. Virus and Virus-Like Diseases of Pome and Stone Fruits. St. Paul: APS Press; 2011. pp. 1–7.
Massart S, Jijakli MH, Kummert J. Apple stem grooving virus. In: Hadidi A, Barba M, Candresse Th, Jelkman W, editors. Virus and Virus-Like Diseases of Pome and Stone Fruits. St. Paul: APS Press; 2011. pp. 29–33.
Jelkmann W. 1994. Nucleotide sequences of apple stem pitting virus and of the coat protein gene of a similar virus from pear associated with vein yellows disease and their relationship with potex- and carlaviruses. J GenVirol. 1994;75 (7):1535–1542.
Campbell A. Heat Sensitivity of Some Apple Viruses. Tagungsber. 97. Berlin: Dal DDR; 1968. pp. 311–6.
Cropley R. Comparison of some apple latent viruses. Ann Appl Biol. 1968;61:361–72.
Knapp E, Hanzer V, Weiss H, Camara M, Weiss B, Wang Q, Katinger H, Laimer M. New aspects of virus elimination in fruit trees. Acta Hortic. 1995;386:409–18.
Wang L, Wang G, Hong N, Tang R, Deng X, Zhang H. Effect of thermotherapy on elimination of Apple stem grooving virus and Apple chlorotic leaf spot virus for in vitro-cultured pear shoot tips. Hort Science. 2006;41:729–32.
Li BQ, Feng CH, Hu LY, Wang MR, Wang QC. Shoot tip culture and cryopreservation for eradication of Apple stem pitting virus (ASPV) and Apple stem grooving virus (ASGV) from apple rootstocks ‘M9’ and ‘M26’. Ann Appl Biol. 2016;168:142–50.
Wang MR, Li BQ, Feng CH, Wang QC. Culture of shoot tips from adventitious shoots can eradicateApple stem pitting virusbut fails in Apple stem grooving virus. Plant Cell Tiss Org. 2016;125:283–91.
Postman JD, Sugar D. Elimination of viruses from the USDA Pyrus germplasm collection. Acta Hortic. 2002;596:529–30.
Wang QC, Panis B, Engelmann F, Lambardi M, Valkonen JPT. Cryotherapy of shoot tips: a technique for pathogen eradication to produce healthy planting materials and prepare healthy plant genetic resources for cryopreservation. Ann Appl Biol. 2009;154:351–63.
Wang QC, Valkonen JPT. Cryotherapy of shoot tips: novel pathogen eradication method. Trend Plant Sci. 2009;14:119–22.
Faccioli G, Marani F. Virus elimination by meristem tip culture and tip micrografting. In: Hadid A, Khetarpal RK, Koganezawa H, editors. Plant Virus Disease Control. The American Phytopathological Society. St. Paul: APS Press; 1998. pp. 389–93.
Wang B, Ma YL, Zhang ZB, Wu ZM, Wu YF, Wang QC, Li MF. Potato viruses in China. Crop Prot. 2011;30:1117–23.
Panattoni A, Luvisi A, Triolo E. Elimination of viruses in plants: twenty years of progress. Span J Agri Res. 2013;11:173–88.
Zhang LM, Wang QM, Liu QC, Wang QC. Sweetpotato in China. In: Loebenstein G, editor Sweetpotato biology. Berlin: Springer. 2008;321 – 54.
Romadanova NV, Mishustina SA, Gritsenko DA, Omasheva MY, Galiakparov NN, Reed BM, Kushnarenko SV. Cryotherapy as a method for reducing the virus infection of apples (Malus sp.). Cryo Lett. 2016;37:1–9.
Wang MR, Yang W, Zhao L, Li JW, Liu K, Yu JW, Wu YF, Wang QC. Cryopreservation of virus: a novel biotechnology for long-term preservation of virus in shoot tips. Plant Methods. 2018;14(1):47.
Wang QC, Cuellar WJ, Rajamäki ML, Hirata Y, Valkonen JPT. Combined thermotherapy and cryotherapy for efficient virus eradication: Relation of virus distribution, subcellular changes, cell survival and viral RNA degradation in shoot tips. Mol Plant Pathol. 2008;9:237–50.
Feng CH, Wang RR, Li JW, Wang B, Yin ZF, Cui ZH, Li BQ, Bi WL, Zhang ZB, Li MF, Wang QC. Production of pathogen-free horticultural crops by cryotherapy of in vitro-grown shoot tips. Protocols for Micropropagation of Selected Economically-Important Horticultural Plants. Humana Press. 2013;463–482.
Vieira RL, Silva AL, Zaffari GR, Steinmacher DA, Freitas HP, Guerra MP. Efficient elimination of virus complex from garlic (Allium sativum L.) by cryotherapy of shoot tips. Acta Physiol Plant. 2015;37:1733.
Li BQ, Feng CH, Hu LY, Wang MR, Wang QC. Shoot tip culture and cryopreservation for eradication of Apple stem pitting virus (ASPV) and Apple stem grooving virus (ASGV) from apple rootstocks ‘M9’ and ‘M26’. Ann Appl Biol. 2016;168:142–50.
Zhao L, Wang M, Cui Z, Chen L, Volk G, Wang QC. Combining thermotherapy with cryotherapy for efficient eradication of apple stem grooving virus from infected in vitro-cultured apple shoots. Plant Dis. 2018;PDIS-11-17-1753-RE.
Paprstein F, Sedlak J, Polak J, Svobodova L, Hassan M, Bryxiova M. Results of in vitro thermotherapy of apple cultivars. Plant Cell Tiss Org. 2008;94:347–52.
Tan RR, Wang LP, Hong N, Wang GP. Enhanced efficiency of virus eradication following thermotherapy of shoot-tip cultures of pear. Plant Cell Tiss Org. 2010;101:229–35.
Hu G, Dong Y, Zhang Z, Fan X, Ren F, Zhou J. Virus elimination from in vitro apple by thermotherapy combined with chemotherapy. Plant Cell Tiss Org. 2015;121:435–43.
Cooper VC, Walkey D. Thermal inactivation of Cherry leaf roll virus in tissue cultures of Nicotiana rusticaraised from seeds and meristem-tips. Ann Appl Biol. 1978;88:273–8.
Hu G, Hong N, Wang L, Hu H, Wang G. Efficacy of virus elimination from in vitro-cultured sand pear (Pyrus pyrifolia) by chemotherapy combined with thermotherapy. Crop Prot. 2012;37:20–5.
Feng CH, Li BQ, Hu LY, Wang MR, Wang QC. A comparison of two cryogenic protocols in cryopreservation of apple shoot tips. Acta Hortic. 2014;1039:161–6.
Li BQ, Feng CH, Wang MR, Hu LY, Volkb G, Wang QC. Recovery patterns, histological observations and genetic integrity in Malus shoot tips cryopreserved using droplet-vitrification and encapsulation-dehydration procedures. J Biotechnol. 2015;214:182–91.
Desvignes JC. Virus diseases of fruit trees. Certification schemes-pathogen-tested material of Malus, Pyrus and Cydonia. EPPO Bull Ctifl Paris. 1999;29:239–52.
Feng CH, Cui ZH, Li BQ, Chen L, Ma YL, Zhao YH, Wang QC. Duration of sucrose preculture is critical for shoot regrowth of in vitro-grown apple shoot-tips cryopreserved by encapsulation-dehydration. Plant Cell Tiss Org. 2013;112:369–78.
Murashige T, Skoog F. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant. 1962;15:473–97.
MacKenzie DJ, McLean MA, Mukerji S, Green M. Improved RNA extraction from woody plants for the detection of viral pathogens by reverse transcription-polymerase chain reaction. Plant Dis. 1997;81:222–6.
Candresse T, German S, Lanneau M, Dunez J. In vitro translation of apple chlorotic leaf spot virus (ACLSV) RNA[J]. Arch Virol. 1996;141(11):;2031–2043.
Menzel W, Zahn V, Maiss E. Multiplex. RT-PCR-ELISA compared with bioassay for the detection of four apple viruses and time during the certification of plant material. J Virol Methods. 2003;110(2):153–7.
Sakai A, Kobayashi S, Oiyama I. Cryopreservation of nucellar cells of navel orange (Citrus sinensis Osb. var. brasiliensis Tanaka) by vitrification. Plant Cell Rep. 1990;9:30–3.

coverletter.doc

Download PDF

Version 1

posted

You are reading this latest preprint version

Combined thermotherapy and cryotherapy: an effective method for the eradication of three common viruses in cultivated apples

Status:

Version 1

Abstract

Background

Results

Conclusions

Figures

Background

Results

Virus detection in plant materials

Effects of thermotherapy timings on shoot tip survival and regrowth

Effectiveness of different treatments in virus eradication

Discussion

Conclusions

Materials And Methods

Plant material

Virus detection

Thermotherapy and treatment

Cryotherapy

Assessment of shoot survival and regrowth

Data analysis

Abbreviations

Declarations

References

Supplementary Files

Status:

Version 1