Effects of D and V cryo-plate methods for plum pox virus eradication from two plum cultivars

Abstract

Conservation of plant species, particularly of those important for agriculture, has now reached a very high importance in order to ensure sustainable utilization of biological resources by preventing further losses of plant diversity. The most advanced method for plant conservation is cryopreservation. Cryotherapy that relies on cryopreservation procedure, alone or in combination with other techniques, can be used for pathogen elimination from infected plants. Viral diseases pose a significant threat to the agriculture, decreasing yields and crop quality. In this work, two cryotherapy methods (D and V cryo-plate) were evaluated for plum pox virus (PPV) eradication from autochthonous plums ‘Crvena Ranka’ and ‘Belošljiva’, widely present under different local names on the Balkan peninsula. Nine different cryo-plate treatments were performed per each genotype. Cryotherapy was efficient in PPV eradication from plum ‘Crvena Ranka’ in three V cryo-plate treatments, but failed to eliminate PPV from genotype ‘Belošljiva’.

Key Message

Eradication of plum pox virus from infected plums is possible by cryotherapy methods, but the efficiency is highly dependent on the treatment applied and the given genotype.

Introduction

The conservation of genetic diversity and its sustainable use are essential to satisfy a number of challenges facing humanity, from dealing with the predicted climate changes to achieving food security despite a still growing world population. The new biotechnology-based conservation techniques, including different in vitro techniques, are complementary approaches to conventional ex situ conservation methods and represent safety precaution against accidental loss of plant germplasm collections (Engelmann 2004). In recent years, cryopreservation, i.e., the storage of plant material at ultra-low temperature in liquid nitrogen (LN, -196°C) has become a very important tool for long-term conservation of plant germplasm. It is the only safe and cost-efficient option for the long-term conservation of various categories of plants, including non-orthodox seed species, vegetatively propagated plants, rare and endangered species and biotechnology products (Cruz-Cruz et al. 2013). Wide application of plant cryopreservation depends on the availability of efficient, reproducible and robust cryopreservation protocols applicable to different plant species. Numerous protocols have been or are being developed for different crops: slow cooling, pregrowth-desiccation, vitrification, encapsulation-dehydration, droplet vitrification, V and D cryo-plate (Jiroutova and Sedlak 2020). Adjustments of these protocols to the gene bank level are necessary to exploit all the advantages of cryopreservation. One of the main goals of cryopreservation is to simplify the procedure and to minimize the use of expensive equipment and tools to make this technique available to the laboratories in developing countries (Baucaud et al. 2002). It has been proven as efficient conservation method for genetic resources of a range of vegetatively propagated crops (yam, potato, banana, cassava, taro, sweet potato) (Benito et al. 2004).

Plums are one of Serbia’s most traditional fruits with more than 48 million of trees (Glišić 2015). Numerous cultivars are present throughout the country, from autochthonous, domesticated to the newly bred ones (domestic and foreign). Autochthonous cultivars are part of country’s intangible heritage, tradition, customs and legacy, as well as cultural identity. These genotypes of immeasurable genetic and cultural value are endangered and suppressed by newly bred and commercially important cultivars. In situ conservation of genetic resources in fruits has a very important place in Serbia with more than 130 accessions of Prunus genus present. Cryopreservation of plant species is already introduced in the country and significant results have been achieved in several fruit species, including blackberry, apple, cherry and plum (Vujović et al. 2011; Ružić et al. 2014; Vujović et al. 2015; Vujović et al. 2020, Vujović et al. 2021a). European plum (Prunus domestica L.), as the leading fruit species in Serbian agriculture, is facing with climate changes and long-term inoculum pressure of numerous pathogens, including viruses. The most devastating viral disease in stone fruits is plum pox virus (PPV, genus Potyvirus, family Potyviridae) causing Sharka disease. Sharka was first described on plums in Bulgaria and since then it has spread to many countries on all continents, except Australia (Barba et al. 2011). PPV has a wide host range among cultivated Prunus species: apricot, peach, plums, sour cherry and sweet cherry (Garcia et al. 2014) and induce symptoms on leaves, fruits, flowers, branches and seeds. In sensitive cultivars, as ‘Požegača’ (present on entire Balkan peninsula), PPV causes fruit deformations and fruit drop, leading to total crop losses. According to biological, serological and molecular characteristics 10 PPV strains have been described so far: PPV-M, -D, -Rec, -EA, -T, -C, -CR, -W, -An and -CV (Chirkov et al. 2018). Three strains (PPV-M, -D and -Rec) are considered as major strains. The virus is efficiently aphid transmitted in a non-persistent manner by more than 20 species over short distances. Infected plant material is the main mode of transmission over long distances. PPV is present in Serbia since 1935 and is considered as the most devastating viral disease of the stone fruits. Other known viruses infecting stone fruits were very rarely detected in plums in the country. According to the estimates, about 70% of the plum trees are infected (Jevremović and Paunović 2014). PPV represents the main threat to the production, but also to the existence of plum genotypes in an open field. Old autochthonous plum genotypes that should be preserved are under highest inoculum pressure. It is of the high importance to analyze and select virus-free (or at least virus-tested) specimens for conservation. If a single virus-free tree of the given genotype does not exist, different methods should be applied to eradicate PPV for further preservation purpose. Thermotherapy based methods include treatment of plants in controlled conditions with the temperature of about 37°C, 16 h light and 8 h dark regime for 2 to 3 weeks. The excision of large (10 mm length) shoot tips after thermotherapy can be applied for virus eradication. Thermotherapy and shoot tip culture were successfully applied for PPV eradication in plum, apricot and peach (Manganaris et al. 2003; Kouburis et al. 2007; Polak and Hauptmanova 2009; Vescan et al. 2011). Chemotherapy is another possibility for virus elimination from infected plants. It implies the application of antiviral medications (as ribavirin) in optimal concentration and duration of treatment. The application of chemotherapy for PPV eradication from several plum cultivars in Europe was already demonstrated (Paunović et al. 2007; Hauptmanova and Polak 2009). Cryotherapy is a novel application of plant cryopreservation for virus, phytoplasma and bacteria elimination from the plant material. The major challenge for cryotherapy application is significantly different response of the genotypes of the same species to this treatment. In Prunus species, the effect of cryotherapy has been evaluated only in interspecies rootstock (Prunus salicina Lindley ‘Methley’ x Prunus spinosa L.) using slow-cooling method (Brison et al. 1997). In these experiments, the complete eradication of the virus was not achieved. There are no studies on other cryotherapy protocols for PPV elimination from infected plums.

In this work, different cryopreservation treatments with D and V cryo-plate methods were evaluated for the eradication of plum pox virus from two autochthonous Prunus domestica L. genotypes.

Materials And Methods

Plant material

Two autochthonous plums ‘Belošljiva’ and ‘Crvena Ranka’ were selected for the experiment. Trees of both cultivars showing Sharka-like symptoms were selected from two orchards. Leaf samples were tested on the presence of seven viruses (plum pox virus, prune dwarf virus, Prunus necrotic ringspot virus, apple chlorotic ringspot virus, apple mosaic virus, plum bark necrosis stem pitting-associated virus and myrobalan latent ringspot virus) and ‘Candidatus phytoplasma prunorum’ as described by Jevremović et al (2021). Two PPV-infected trees (one per cultivar) with PPV-Rec strain and free from other tested viruses and phytoplasma were selected as sources for the explants.

Aseptic cultures of these genotypes were established on Murashige and Skoog (MS) medium (Murashige and Skoog 1962) containing 2 mg l^− 1 N6-benzyladenine (BA), 0.5 mg l^− 1 indole-3-butyric acid (IBA) and 0.1 mg l^− 1 gibberellic acid (GA₃), 30 g l^− 1 sucrose and 7 g l^− 1 agar using the protocol previously described by Vujović et al (2021b). In order to obtain enough PPV-infected inital material for cryopreservation experiments, aseptic shoot cultures were repeatedly subcultured on MS medium with 2 mg l^− 1 BA that was previously determined as the most suitable for multiplication of both genotypes.

Prior to cryopreservation experiments, 1 mm large apical shoot tips, as well as axillary buds (both from basal and upper portion of shoots), were dissected from the shoots and analyzed by Real-time PCR to confirm the presence of PPV.

Cryopreservation using aluminum cryo-plates

After dissection, both apical shoot tips and axillary buds (1 mm long) were pre-cultured for 1 day at 23°C in the dark on solidified MS multiplication medium with 0.3 M sucrose. Following pre-culture, explants were carefully placed individually in each of the 12 wells of aluminum cryo-plates previously filled with 2% (w/v) sodium alginate in calcium-free MS basal medium with 0.4 M sucrose (about 4 µl). For polymerization, a calcium solution containing 0.1 M calcium chloride in MS basal medium with 0.4 M sucrose was poured on the aluminum plates until shoot tips were fully covered. Calcium solution was removed after 20 min and cryo-plates with adhering explants transferred to loading solution.

In D cryo-plate procedure loading treatment included application of solution comprising 2 M glycerol and 0.4 M sucrose in liquid MS medium (LS1 solution; Nishizawa et al. 1993) for 30 min at room temperature. Following osmoprotection, dehydration step included desiccation of the shoot tips attached to the cryo-plates in closed 100 ml glass containers over 40 g of silica gel at 23°C for 2, 2.5 and 3 h.

In V cryo-plate protocol, osmoprotection was done using solution containing 1.9 M glycerol and 0.5 M sucrose (C4 solution; Kim et al. 2009a) for 30 min at room temperature. Dehydration was performed at room temperature using three types of plant vitrification solutions (PVSs): original PVS2 solution (13.7% (w/v) sucrose, 30.0% (w/v) glycerol, 15% (w/v) ethylene glycol and 15% (w/v) dimethylsulfoxide) (Sakai et al. 1990); slightly modified PVS2 solution (PVS A3–22.5% (w/v) sucrose, 37.5% (w/v) glycerol, 15% (w/v) ethylene glycol and 15% (w/v) dimethylsulfoxide) (Kim et al. 2009b), each applied for 20 and 40 min; and PVS3 solution (50% (w/v) glycerol and 50% (w/v) sucrose) (Nishizawa et al. 1993) applied for 60 and 80 min.

After dehydration in both protocols, cryo-plates with adhering shoot tips were transferred to 2 ml uncapped cryo-tubes held on cryo-canes and directly immersed in liquid nitrogen (LN) where they were kept for at least 1 h. Rewarming of samples was performed by rapid transfer of the aluminum cryo-plates in an unloading solution containing 1 M sucrose for 15 min (D cryo-plate protocol) or 0.8 M sucrose for 30 min (V cryo-plate protocol) at room temperature. Thereafter, explants were transferred onto the regrowth medium, cultivated in the dark for seven days, and then under standard conditions (growth chamber at 23 ± 1°C, with 16 h photoperiod under 54 µmol m^− 2 s^− 1 light intensity). MS medium containing 2 mgl^− 1 BA was used as regrowth medium for both genotypes.

Each step in the cryopreservation procedure included controls: loading control – shoot tips exposed to loading solution, but neither dehydrated nor cryopreserved; dehydration controls – after loading explants were dehydrated with PVS or desiccated and directly unloaded without immersion in LN.

Assessment of shoot regrowth and data analysis

Regrowth was evaluated eight weeks after samples retrieval from LN by counting the number of explants developed into viable shoots. Each experimental treatment was performed in three independent replicates, with 10 − 12 explants per replicate. Statistical analysis was performed by one-directional analysis of variance (ANOVA) and least significant differences were calculated at 95% significance by Duncan’s Multiple Range Test using the statistical software package Statgraphics 18 Centurion (Statgraphics Technologies Inc., The Plains, Virginia, USA). Data presented as percentages (Table 1) were subjected to arcsine transformation prior to analysis of variance.

In vitro rooting and acclimatization

Shoots regenerated from control and cryopreserved explants were cultivated separately on multiplication medium for four successive subcultures (each subculture lasted four weeks). In addition, within single cryo-treatment each regenerated shoot was designated as separate line and separately multiplied. In fifth subculture shoots were transferred to a rooting medium (half-strength MS medium containing 1 mg l^− 1 1-naphthaleneacetic and 0.1 mg l^− 1 GA₃). In vitro rooted plantlets were removed from culture vessels, washed carefully with water, transferred to plastic pots containing sterile soil substrate (Klasmann-Deilmann GmbH, Germany) and acclimatized on a ‘mist’ bench in a greenhouse for two weeks (Mist system type ‘Electronic leaf’, MC Company, Belgrade).

Plum pox virus detection

Viral status of in vitro shoots originated from both control (non-frozen explants) and cryopreserved explants of plums ‘Belošljiva’ and ‘Crvena Ranka’ was evaluated by a Real-time reverse transcription-polymerase chain reaction (Real-time RT-PCR). A number of 1285 samples (730 of plum ‘Belošljiva’ and 555 of ‘Crvena Ranka’) were taken and tested from in vitro shoots during four multiplication stages and two months after acclimatization of rooted plants in the greenhouse. Control shoots regenerated from non-frozen explants (loading controls and dehydration controls) were grouped and 5 samples (each taken from several plantlets) were collected and tested per each treatment in each subculture as well as after acclimatization. Shoots regenerated from cryopreserved explants were designated as separate lines and tested as separate samples in the first subculture after regrowth. The numbers of tested plants per each cry-treatment are presented in the Tables 2 and 3. In the second, third and fourth subculture of multiplication in vitro and after acclimatization, leaves taken from several shoots belonging to each regenerating line within single cryo-treatment were grouped and tested as one sample. In that way number of tested samples originated from cryopreserved shoots was constant during subculturing and after acclimatization.

The extraction of total nucleic acids (TNA) was done from 0.2 g of fresh in vitro shoots with 2% CTAB buffer as described by Li et al (2008). Extracted TNA was used for Real-time RT-PCR assay using TaqMan Universal PCR master mix (Applied Biosystems, USA). Samples were tested using primers and TaqMan probes reported by Olmos et al (2005). The 20 µl reaction mixture was composed as follows: 1 µM P241 primer, 0.5 µM each of P316D and P316M primers, 200 nM TaqMan PPV-DM probe, 2×TaqMan Universal PCR Master Mix (Applied Biosystems, USA), 6.25 U 1×MultiScribe RT (Applied Biosys-tems, USA) and 10U RNase Inhibitor Mix (Applied Biosystems, USA), and 5 µl RNA template. The RT-PCR was performed with the following thermocycling parameters: 15 min at 48°C, 10 min at 95°C, and 40 cycles of 15 s at 95°C and 60 s at 60°C. Data acquisition and analysis were conducted in a StepOnePlus™ Real-Time PCR System (Applied Bio-systems, USA) and StepOne™ Software package.

Results

Virus detection in initial material

Shoot cultures of PPV-infected plums prepared for cryopreservation process were tested on the plum pox virus presence prior cryotherapy. Three types of the material were tested per each genotype: axillary buds taken from basal and upper portion of the shoots (separately) and apical shoot tips. A number of 24 samples per cultivar were analyzed by Real-time PCR. All tested samples proved to be PPV-infected confirming that PPV-infected tissue was used for cryotherapy.

Effect of cryopreservation on plantlets regrowth

Cryopreservation/cryotherapy was conducted in nine different treatments (Table 1). Regrowth of control shoot tips in both plums was similar or significantly higher comparing to cryopreserved shoot tips in all treatments (Table 1). Very low percentages of regrowth of plum ‘Crvena Ranka’ explants (below 10%) were recorded after dehydration with PVS2 for 40 min and PVS A3 for 20 min. Regrowth ranging between 10% and 20% was achieved in four out of nine treatments, while 25% of regrowth in cryopreserved shoot tips was achieved both after 2 h and 3 h of desiccation. Explants dehydrated for 60 min with PVS3 solution gave the highest regrowth (40%) after retrieval from LN (Fig. 1a).

Genotype ‘Belošljiva’ displayed slightly higher regeneration capacity after cryopreservation. In D cryo-plate protocol, regrowth of cryopreserved shoot tips ranged between 20.6% and 33.3%. In V cryo-plate protocol, dehydration with both original and modified PVS2 solution for 20 min gave no or very low regrowth (11.3%). Prolonged dehydration with those PVSs resulted in significant increase in regeneration ability to 41.7% (PVS2) and 45.7% (PVS A3). The highest regrowth in this genotype was achieved after dehydration with PVS3 solution for 80 min (61.3%; Fig. 1b) that was significantly higher than for 60-min PVS3 treatment (25.0%) as well as for all other treatments.

Effect of cryotherapy on PPV eradication

A total number of 1285 samples of plums ‘Belošljiva’ and ‘Crvena Ranka’ were tested. In vitro shoots regenerated from control and cryopreserved explants were continuously tested during multiplication (four subcultures) and two months after acclimatization on the PPV presence with Real-time PCR. Results were analyzed for each performed treatment and each subculture after regrowth. Cryotherapy treatment was considered as successful only when all tested samples were found to be PPV-free.

Performed nine cryotherapy treatments in plum ‘Belošljiva’ were not able to produce PPV-free plants (Table 2). From the second subculture after regrowth plantlets proved to be PPV-infected. After acclimatization, PPV infection was also confirmed in all obtained plants. PPV was detected both in control and in cryopreserved plants.

Three cryotherapy treatments (PVS2 20 min + LN, PVS3 60 min + LN and PVS3 80 min + LN) in plum ‘Crvena Ranka’ produced PPV-free plants (Table 3). Not a single tested sample from the plants in these treatments was found to be infected during all four subcultures and after acclimatization in the greenhouse. In all other treatments, PPV was detected up to the fourth subculture and afterwards in the acclimatized plants.

Discussion

In this study two PPV-infected plum cultivars ‘Belošljiva’ and ‘Crvena Ranka’ were used as source materials for cryopreservation using D and V cryo-plate methods. These two cryogenic procedures using aluminium cryo-plates are based on PVS2-vitrification dehydration (V cryo-plate) or air dehydration of explants (D cryo-plate) (Matsumoto 2017). Both protocols appear promising for cryopreservation of both herbaceous and woody plants including fruit-tree species after appropriate modifications. Niino et al (2019) emphasized main advantages of the V and D cryo-plate methods such as easy and quick handling of shoot tips attached to cryo-plates, reduced possibility of injuring and losing explants, achieving ultra-rapid cooling and warming, and better control of dehydration. Many successful results have been obtained with different plant species (Niino et al. 2019). As regards plum genotypes, these methods were previously employed for cryopreservation of cherry plum (P. cerasifera), plum cultivar ʻPožegačaʼ (Vujović et al. 2011) and virus-free autochthonous cultivar ʻCrvena Rankaʼ (Vujović et al. 2021a). However, regrowth rates obtained with PPV-infected cryopreserved explants of ʻCrvena Rankaʼ were markedly lower in comparison with those obtained using the same treatments (both V cryo-plate and D cryo-plate) in virus free explants (Vujović et al. 2021a). These differences were especially noticeable for PVS A3-based dehydration in V cryo-plate procedure. Although the cryopreservation efficiency depends on many factors, the physiological state of the mother plants is a key factor for the success of cryopreservation. Thus, viral infection may significantly affect physiological condition of in vitro plantlets which could lead to significant decrease in regeneration ability of explants isolated from these shoots. Reduced in vitro growth of virus-infected tissue compared to non-infected tissue has already been reported in different plant species including fruit tree species such as strawberry (Tsao et al. 2000). On the other side, although belong to the same species, studied plum genotypes also displayed differences regarding cryopreservation ability under the same experimental conditions. Autochthonous plum ʻBelošljivaʼ seems to be more tolerant to biochemical and osmotic toxicity of plant vitrification solutions used in V cryo-plate experiments than ʻCrvena Rankaʼ. Regrowth rates higher than 40% (which could be considered successful) were achieved with three (40-min PVS2 and PVS A3 treatments and 80-min PVS3 treatment) out of six dehydration treatments in ʻBelošljivaʼ, while ʻCrvena Rankaʼ gave satisfactory regeneration only after 60-min PVS3 treatment. Regarding D cryo-plate experiments, both genotypes displayed similar regeneration ability being under 30%.

The size of excised shoot tips and the virus location in the infected shoot tips may affect the success of virus eradication by shoot tip culture (Bettoni et al. 2022). Isolation of 1 mm apical shoot tips as well as of axillary buds from PPV infected in vitro plum shoots completely failed to eradicate virus that was confirmed by Real-time PCR prior cryopreservation experiments. Consequently, regrowing plants from loading and dehydration controls were completely infected. Bettoni et al (2022) also concluded that the 1 mm shoot tips were too large for achieving efficient virus eradication per se (without any additional virus eradication treatment) in potato.

In our study nine different cryo treatments were evaluated for the ability to eradicate plum pox virus in each cultivar – three treatments were performed with D cryo-plate and six treatments with V cryo-plate procedure. Treatments with D cryo-plate technique were not successful in PPV eradication in both studied cultivars. The virus was detected in early stages, up to the second subculture after the treatment. All V cryo-plate treatments also failed in PPV eradication in plum ‘Belošljiva’ where PPV was detected yet in the second subculture. None of the analyzed and PPV-infected in vitro plant during subculturing (in the laboratory) and after acclimatization (in the greenhouse) exhibited Sharka-like symptoms. Three V cryo-plate treatments proved to be efficient for PPV eradication in plum ‘Crvena Ranka’. All plants obtained after treatments with solutions PVS2 for 20 min, PVS3 for 60 min and PVS3 for 80 min proved to be PPV-free using Real-time RT-PCR. Also, PPV has not been detected in any tested sample from the plants obtained in these treatments two months of acclimatization in the greenhouse. In all previously published studies on PPV eradication with different methods (Manganaris et al. 2003; Kouburis et al. 2007; Křižan and Ondrušiková 2009; Polak and Hauptmanova 2009), PPV detection from the treated material was performed by ELISA and/or RT-PCR. During our study, the analysis of the cryopreserved material was performed with Real-time PCR assay that is much more sensitive for PPV detection than ELISA and conventional PCR (Olmos et al. 2006). The initial material of plums ‘Belošljiva’ and ‘Crvena Ranka’ was infected with recombinant (PPV-Rec) plum pox virus strain. PPV-Rec strain is a natural recombinant of PPV-M and PPV-D and it is the most prevalent strain in plums and apricots in Serbia (Jevremović 2013; Jevremović and Paunović 2014). In the study of Brison et al (1997), a vitrification protocol was performed for PPV eradication from infected interspecies Prunus rootstock Fereley-Jaspi. The complete eradication of the PPV-M strain was not achieved, but PPV-free plants were obtained with high frequencies (45 − 60%). Different cryotherapy methods, alone or in combination with other techniques, have been successfully applied for virus eradication in some fruit species. Cucumber mosaic virus and banana streak virus with different efficiency were eradicated from banana, 2% and 87%, respectively (Helliot et al. 2002). Combining thermotherapy and cryotherapy, raspberry bushy dwarf virus was eradicated from red raspberry, but survival and regrowth of the plantlets was low (Wang and Valkonen 2009). Latent apple viruses (apple stem pitting virus, apple stem grooving virus and apple chlorotic leafspot virus) were eradicated from several apple cultivars and rootstocks (Bettoni et al. 2015; Zhao et al. 2018; Bettoni et al. 2019; Liu et al. 2021). On the other hand, cryotherapy failed to eradicate fruit tree viruses in some studies. Cryotherapy alone was not efficient to eradicate raspberry bushy dwarf virus from red raspberry (Wang et al. 2008; Pathirana et al. 2019; Mathew et al. 2020), and apple stem grooving virus from apple rootstocks and cultivars (Li et al. 2016; Zhao et al. 2018). The eradication of viruses and other pathogens is depended on many factors, such as cryopreservation method and procedure, susceptibility of the given plant genotype, localization of the virus in plant tissue, growth conditions and other (Brison et al. 1997).

Conclusions

The obtained results showed that performed treatments of cryopreservation were not efficient for PPV eradication from autochthonous plum ‘Belošljiva’. Three V cryo-plate treatments resulted in PPV suppression in plum ‘Crvena Ranka’. These are the first results on the application of D and V cryo-plate methods for PPV eradication from infected plum cultivars. Obtained results are encouraging and showed that the efficiency of methods is highly dependent on the given genotype and applied treatment.

Declarations

Funding

This research was supported by the Science Fund of the Republic of Serbia, PROMIS, #6062279, project Conservation and plum pox eradication from Serbian autochthonous plum genotypes using cryotechniques – CryoPlum and by the Ministry of Education, Science and Technological Development of the Republic of Serbia, contract 451-03-68/2022-14/200215.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Informed consent

Informed consent was obtained from all individual participants included in the study.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

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