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.
Table 1
Regrowth of control (-LN) and cryopreserved (+ LN) shoot tips of plums ‘Crvena Ranka’ and ‘Belošljiva’ using V cryo-plate and D cryo-plate methods.
Treatment | Regrowth (%) |
‘CrvenaRanka’ | ‘Belošljiva’ |
-LN | +LN | -LN | +LN |
Loading control (LS1) | 100.0 a | - | 100.0 a | - |
Loading control (C4) | 100.0 a | - | 100.0 a | - |
LS1–2 h desiccation | 50.0 b | 25.0 cd | 70.0 c | 25.0 gh |
LS1–2.5 h desiccation | 20.0 cd | 16.7 d | 30.0 g | 20.6 h |
LS1–3 h desiccation | 20.0 cd | 25.0 cd | 30.0 g | 33.3 fg |
C4–PVS2 20 min | 100.0 a | 12.4 de | 90.0 b | 0.0 j |
C4–PVS2 40 min | 20.0 cd | 5.5 e | 90.0 b | 41.7 ef |
C4–PVSA3 20 min | 100.0 a | 5.5 e | 90.0 b | 11.3 i |
C4–PVSA3 40 min | 40.0 bc | 16.7 d | 100.0 a | 45.7 e |
C4–PVS3 60 min | 40.0 bc | 40.0 bc | 60.0 d | 25.0 gh |
C4–PVS3 80 min | 20.0 cd | 16.7 d | 90.0 b | 61.3 d |
| P ≤ 0.05 | P ≤ 0.05 |
Mean values for regrowth in each genotype followed by the same letter are not significantly different according to Duncan’s Multiple Range Test at the level of significance P ≤ 0.05. |
LS1 – Loading solution comprising 2 M glycerol and 0.4 M sucrose; C4 – Loading solution comprising 1.9 M glycerol and 0.5 M sucrose; PVS2 – Plant vitrification solution comprising 13.7% sucrose, 30.0% glycerol, 15% ethylene glycol and 15% dimethylsulfoxide; PVS A3 – Plant vitrification solution comprising 22.5% sucrose, 37.5% glycerol, 15% ethylene glycol and 15% dimethylsulfoxide; PVS3 – Plant vitrification solution comprising 50% glycerol and 50% sucrose. |
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.
Table 2
Frequency of plum pox virus eradication by cryotherapy in plum ‘Belošljiva’
Treatments | Subculture after regrowth | After acclimatization |
1 | 2 | 3 | 4 |
LS1 loading control | 5/5* | 5/5 | 5/5 | 5/5 | 5/5 |
2 h desiccation − LN | 0/5 | 5/5 | 5/5 | 5/5 | 5/5 |
2 h desiccation + LN | 0/9 | 9/9 | 9/9 | 9/9 | 9/9 |
2.5 h desiccation − LN | 0/5 | 5/5 | 5/5 | 5/5 | 5/5 |
2.5 h desiccation + LN | 0/7 | 1/7 | 7/7 | 7/7 | 7/7 |
3 h desiccation − LN | 0/5 | 5/5 | 5/5 | 5/5 | 5/5 |
3 h desiccation + LN | 0/12 | 12/12 | 12/12 | 12/12 | 12/12 |
C4 loading control | 5/5 | 5/5 | 5/5 | 5/5 | 5/5 |
PVS2 20 min − LN | 0/5 | 5/5 | 5/5 | 5/5 | 5/5 |
PVS2 20 min + LN | - | - | - | - | - |
PVS2 40 min − LN | 0/5 | 5/5 | 5/5 | 5/5 | 5/5 |
PVS2 40 min + LN | 0/15 | 13/15 | 15/15 | 15/15 | 15/15 |
PVS A3 20 min − LN | 0/5 | 5/5 | 5/5 | 5/5 | 5/5 |
PVS A3 20 min + LN | 0/4 | 3/4 | 4/4 | 4/4 | 4/4 |
PVS A3 40 min − LN | 0/5 | 5/5 | 5/5 | 5/5 | 5/5 |
PVS A3 40 min + LN | 0/16 | 16/16 | 16/16 | 16/16 | 16/16 |
PVS3 60 min − LN | 0/5 | 5/5 | 5/5 | 5/5 | 5/5 |
PVS3 60 min + LN | 0/9 | 8/9 | 9/9 | 9/9 | 9/9 |
PVS3 80 min − LN | 0/5 | 5/5 | 5/5 | 5/5 | 5/5 |
PVS3 80 min + LN | 0/19 | 12/19 | 19/19 | 19/19 | 19/19 |
LS – loading solution; PVS − plant vitrification solution; -LN − dehydration controls (non-frozen explants); +LN − cryopreserved explants. *Numbers indicate number of PPV-infected samples/number of samples tested. |
Table 3
Frequency of plum pox virus eradication by cryotherapy in plum ‘Crvena Ranka’.
Treatments | Subculture after regrowth | After acclimatization |
1 | 2 | 3 | 4 |
LS1 loading control | 5/5* | 5/5 | 5/5 | 5/5 | 5/5 |
2 h desiccation − LN | 5/5 | 5/5 | 5/5 | 5/5 | 5/5 |
2 h desiccation + LN | 6/9 | 9/9 | 9/9 | 9/9 | 9/9 |
2.5 h desiccation − LN | 0/5 | 5/5 | 5/5 | 5/5 | 5/5 |
2.5 h desiccation + LN | 0/6 | 1/6 | 3/6 | 6/6 | 6/6 |
3 h desiccation − LN | 0/5 | 5/5 | 5/5 | 5/5 | 5/5 |
3 h desiccation + LN | 0/9 | 6/9 | 8/9 | 9/9 | 9/9 |
C4 loading control | 5/5 | 5/5 | 5/5 | 5/5 | 5/5 |
PVS2 20 min − LN | 0/5 | 5/5 | 5/5 | 5/5 | 5/5 |
PVS2 20 min + LN | 0/4 | 0/4 | 0/4 | 0/4 | 0/4 |
PVS2 40 min − LN | 0/5 | 5/5 | 5/5 | 5/5 | 5/5 |
PVS2 40 min + LN | 0/2 | 0/2 | 2/2 | 2/2 | 2/2 |
PVSA3 20 min − LN | 0/5 | 5/5 | 5/5 | 5/5 | 5/5 |
PVSA3 20 min + LN | 0/2 | 0/2 | 2/2 | 2/2 | 2/2 |
PVSA3 40 min − LN | 0/5 | 3/5 | 4/5 | 5/5 | 5/5 |
PVSA3 40 min + LN | 0/6 | 0/6 | 3/6 | 6/6 | 6/6 |
PVS3 60 min − LN | 0/5 | 5/5 | 5/5 | 5/5 | 5/5 |
PVS3 60 min + LN | 0/12 | 0/12 | 0/12 | 0/12 | 0/12 |
PVS3 80 min − LN | 0/5 | 4/5 | 5/5 | 5/5 | 5/5 |
PVS3 80 min + LN | 0/6 | 0/6 | 0/6 | 0/6 | 0/6 |
LS – loading solution; PVS − plant vitrification solution; -LN − dehydration controls (non-frozen explants); +LN – cryopreserved explants. *Numbers indicate number of PPV-infected samples/number of samples tested. |
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.