Acta agriculturae Slovenica, 118/4, 1–8, Ljubljana 2022 doi:10.14720/aas.2022.118.4.1823 Original research article / izvirni znanstveni članek Field performance of cryopreserved seed-derived tomato plants and post-thaw survival of viral-infected meristems Nadiia SHEVCHENKO 1, 2, Tetiana MIROSHNICHENKO 3, Anna MOZGOVSKA 3, Nataliia BASHTAN 3, Galyna KOVALENKO 1, Tetiana IVCHENKO 3 Received August 12, 2020; accepted September 23, 2022. Delo je prispelo 12. avgusta 2020, sprejeto 23. septembra 2022 1 Institute for Problems of Cryobiology and Cryomedicine of the National Academy of Sciences of Ukraine, Kharkiv, Ukraine 2 Corresponding author, e-mail: nadiyashevchenko79@gmail.com 3 Institute of Vegetable and Melon Growing of the National Academy of Agrarian Sciences of Ukraine, Selektsiine, Kharkiv region, Ukraine Field performance of cryopreserved seed-derived tomato plants and post-thaw survival of viral-infected meristems Abstract: The effectiveness of different cryopreservation techniques of tomato meristems isolated from viral-infected plants ‘Irishka’ cultivar was determined. The pieces of stem were protected with dimethyl sulfoxide and propylene glycol and cooled in vapour phase of liquid nitrogen (–170  °C). For the vitrification and droplet-vitrification protocols, the meristems were treated with loading solution and dehydrated with differ- ent plant vitrification solutions (PVS1 modified, PVS2, 88  % PVS3, PVSN). The samples were placed to sterilized aluminum foil pieces, in 1.2 ml cryovials or in 50 µl aluminum pans for differential scanning calorimetry and were directly immersed into liquid nitrogen. Acсording to the dehydration technique, the meristems were dehydrated with sterile airflow for 120 min. The post-thaw survival rates of meristems (from 34.2 to 78.5 %) were observerved only for 50 µl aluminum pans and airflow de- hydration. We determined the productivity of plants, obtaned from cryopreserved seeds (‘Seven’, ‘Potiron Ecarlate’ and ‘Dru- zhba’ cultivars). We observed increasing in total and market- able yields for the plants grown from the cryopreserved seeds for all the cultivars. Total number of diseased plants decreased by 33 % for ‘Seven’, for ‘Potiron Ecarlate’ it did by 6.7 %, for that of ‘Druzhba’ the total percentage of sick and healthy plants did not differ after seeds cryopreservation. Key words: seed cryopreservation; dehydration; meri- stem cryopreservation; plant vitrification solution; Solanum lycopersicum L.; yield Uspevanje paradižnika v poljskem poskusu, vzgojenega iz za- mrznjenih semen in preživetje z virusi okuženih meristemov po odtajanju Izvleček: V raziskavi je bila določena učinkovitost raz- ličnih metod shranjevanja meristemov paradižnika z zamrzo- vanjem, pridobljenih iz z virusi okužene sorte ‘Irishka’. Koščki stebel so bili zaščiteni z dimetil sulfoksidom in propilen gliko- lom in ohlajeni v parah tekočega dušika (–170 °C). Za vitrifika- cijo je bil uporabljen protokol kapljične vitrifikacije, meristemi so bili obdelani s standardno nosilno raztopino in dehidrira- ni z različnimi vitrifikacijskimi raztopinami za rastlinska tki- va (modificirana PVS1, PVS2, 88  % PVS3, PVSN). Vzorci so bili potem položeni na koščke sterilizirane aluminijeve folije v 1,2 ml epruvetkah za zmrzovanje ali v 50 µl aluminijastih po- sodicah za diferencialno vrstično kalorimetrijo, nakar so bili neposredno potopljeni v tekoči dušik. Glede na dehidracijske tehnike so bili vzorci dehidrirani s sterilnim zrakom za 120 min. Preživetje meristemov po odtajanju (od 34,2 do 78,5 %) je bilo opazovano samo za tiste v 50 µl aluminijastih posodicah, ki so bili dehidrirani z zračnim tokom. Določena je bila produk- tivnost obravnavanih sort, pridobljenih iz semen, shranjenih z zmrzovanjem (‘Seven’, ‘Potiron Ecarlate’ in ‘Druzhba’). Za vse sorte, ki so bile vzgojene iz semen, shranjenih z zmrzovanjem, je bilo ugotovljeno povečanje celokupnega in tržnega pridelka. Število okuženih rastlin, vzgojenih iz semen po shranjevanju z zmrzovanjem, se je za sorto ‘Seven’ povečalo za 33 % in za sorto ‘Potiron Ecarlate’ za 6,7 %. Pri sorti ‘Druzhba’ se celoten odsto- tek okuženih in zdravih rastlin ni razlikoval po shranjevanju semen z zmrzovanjem. Ključne besede: shranjevanje semen in meristemov z zmrzovanjem; dehidracija; raztopine za vitrifikacijo rastlinskih tkiv; Solanum lycopersicum L.; pridelek Acta agriculturae Slovenica, 118/4 – 20222 N. SHEVCHENKO et al. 1 INTRODUCTION Conservation of plant genetic resources is impor- tant not only for biodiversity preservation, but also for supporting the biotechnology and plant breeding pro- grams. In this regard, the cryopreservation is a power- ful tool for a long-term conservation of genetic diversity of plant species and crop improvement, as well it can be considered as an extra strategy even for species with or- thodox seeds (Coste et al., 2015). Recently, cryopreserva- tion has also been used to eliminate various pathogens in numerous plant species, so this was coined as cryo- therapy (Wang and Valkonen, 2008, 2009; Vieira et al., 2015; Shin et al., 2013). However, there are conflicting findings on the effect of cryopreservation on different crops seeds. The field performance of cryopreserved gar- lic plantlets under in vivo conditions showed the superi- ority of the morphological traits that increased gradually with the growth improved the net photosynthetic rate, bulb diameter, bulb mass and clove number per bulb (Liu et al., 2019). Cejas et al. (2012) did not observe any phe- notypic changes during the early germination stages (0- 14 days) of the cryopreserved Phaseolus vulgaris L. seeds; but several convincing effects of seeds cryopreservation were revealed at the biochemical level. Arguedas et al. (2018) reported that the significant differences between adult plants derived from cryopreserved and control maize seeds by leaf indices, internodes and ears numbers, plant height and mass of seeds were not observed in field performance. The wild Solanum lycopersicum Mill. seeds showed that liquid nitrogen (LN) exposure increased the percentage of seed germination on day 5 but on day 7, the conversion into plantlets and the plant fresh mass dif- fered slightly between non-cryopreserved and cryopre- served samples. Several indicative effects of cryopreser- vation were recorded at the biochemical level on day 7 of tomato seeds germination (Zevallos et al., 2016). Seeds cryostorage enhanced subsequent plant productivity in terms of growth, but it reduced the seeds production in Teramnus labialis (L.f.) Spreng (Acosta et al. 2019). For 9 among 11 species of wild plants, germination of seeds was deteriorated after cryopreservation (Ballesteros and Pence, 2017). Tomato (Solanum lycopersicum L.) is one of vegeta- bles having a world economic importance. Accordingly to the report of «Global Tomato Market 2019 – Robust Consumption Growth in China and India Drives the Global Market» the world tomato sales increased by 6.5 % in 2018 compared to the previous year – up to $190.4 bil- lion, tomato production reached 188 million tons (+ 3.5% of 2017). However, a large number of viruses that damage the tomato plants can lead to enormous crop losses. S. lycopersicum L. plants may be infected by a wide range of viruses from different families including Bromoviridae (Cucumber mosaic virus (CMV), Tomato aspermy virus (TAV)); Virgaviridae (Tomato mosaic virus (ToMV); Po- tyviridae (Potato virus Y, M (PVY)), Secoviridae (Tomato ringspot virus (ToRSV), Tobacco ringspot virus (TRSV), Tomato black ring virus (TBRV)); Bunyaviridae (Tomato spotted wilt virus (TSWV)), Alphaflexiviridae (Potato vi- rus Х (PVX), Pepino mosaic virus (PepMV)) and Gemini- viridae (Tomato yellow leaf curl virus (TYLCV)). Today, the novel viruses appear and spread rapidly in tomato culture. In Ukraine Tomato yellow leaf curl virus (TYL- CV), Tomato Torrado virus (ToTV) and PepMV, Tomato brown rugose fruit virus (TBRFV), CMV, ToMV, Tobacco mosaic virus (TMV), TSWV, PVM and PVY have been detected in tomato plants (AlDalain et al., 2014). Creation of the effective cryopreservation protocol can be vital condition for the virus destruction in the to- mato germplasm. The cryopreservation techniques for tomato germplasm were previously described (Kulus, 2019). The different vitrification-based procedures can be identified: encapsulation-dehydration; vitrification; encapsulation-vitrification; dehydration; pregrowth; pregrowth-dehydration and droplet-vitrification (Engel- man, 2004). However, the viability of virus-infected samples after cryopreservation is much lower (Wang et al., 2018.). Therefore, the determination of the influ- ence of various cryopreservation techniques on the sur- vival rates of the meristems obtained from viral infect- ed plants is a very topical issue. The previous research demonstrated the transmission of TYLCV and ToMV via seeds (AlDalain et al., 2014, Kil et al., 2016), so their cryopreservation is likely to be able to eliminate these types of viruses. Our objectives were to: (a) determine effectiveness of different cryopreservation techniques on post-thaw survival of meristems isolated from viral tomato plants of ‘Irishka’; (b) study germination, total and marketable yield, number of fruits per plant, mass of one fruit, plant height, number of internodes of cryopreserved seed-de- rived tomato plants (‘Seven’, ‘Potiron Ecarlate’, ‘Druzhba’). 2 MATERIALS AND METHODS 2.1 PLANT MATERIAL The in vitro tomato culture was obtained from the field growing infected by CMV, ToMV, PVM, TMV, TY- LCV plants of ‘Irishka’ cultivar, which formed sterile flowers. The parts of the stems with meristems were sur- face sterilized with 30 % commertial bleach (5 % active chlorine) for 25 minutes, then washed 5 times with sterile distilled water for initiation of in vitro cultures. Then they Acta agriculturae Slovenica, 118/4 – 2022 3 Field performance of cryopreserved seed-derived tomato plants and post-thaw survival of viral-infected meristems were transferred into glas vials with agar nutrient medi- um Murashige and Skoog (MS) (Murashige and Skoog, 1962), supplemented with 3  % sucrose without phyto- hormones. The specimens were cultured at 20  ±  2  °C, with 16 hours of light and 8 hours of darkness under 37 μmol×m-2×s-1 light intensity. The explants were propa- gated by micro-grafting every 30 days. 2.2 CRYOPRESERVATION PROCEDURE IN VA- POUR PHASE OF LIQUID NITROGEN For cryopreservation of tomato meristems the method described by Grout et al. (1978) was applied for seedlings of in vitro grown plants. All the leaves were re- moved from regenerated plants; the stems were cut and transferred stepwise at 20 min-intervals through increas- ing the concentrations (5.0, 10.0, 15,0 %) of dimethyl sul- foxide (Me2SO4) and propylene glycol (PG). Afterwards the stems were dried with sterile gauze. The stem pieces from the control plants and those treated with cryopro- tectants were placed into different types of containers. Containers were made from sealed by heat-pulse welding polyimide-fluoroplastic film PMF-351 («Progress», Rus- sia) of 50 µm, or sterile aluminum foil of 14 µm thick- ness. Five stems were placed in each container. Cooling was carried out in the vapours phase at a distance of 15 cm above the surface of the LN. The containers were im- mersed into LN when the temperature in the samples reached –170  ºC and held to 30 min. A two-channel sensor measured the temperature change. After that, the containers were thawed by plunging into water at 40 ºC for 2 min. Then the stems were three times rinsed in fresh MS without Me2SO4 and PG. After rinsing all axillary and apical meristems were dissected from the stems and cul- tured on filter-paper bridges in glass tubes with liquid MS medium. 2.3 PREPARATION TO VITRIFICATION-BASED CRYOPRESERVATION TECHNIQUES The apical and axillary meristems up to 1-2 mm with primordias were isolated from three-week in vitro cultured plants. The isolated samples were transferred into a liquid MS medium, supplemented with 12 % su- crose and exposed at dark for 24 hours. 2.4 DEHYDRATION The meristems were sterile airflow-dehydrated (AD) for 120-min and immersed into LN at a needle tip. After- wards they were warmed in MS medium, supplemented with 10 % sucrose at 25 °C. 2.5 VITRIFICATION The meristems were treated with loading solution (2 M glycerol and 0.4 M sucrose) for 20 min and then transferred in different plant vitrification solutions (PVS) for 40 min at 22 °C. The dehydrated meristems were put into 1.2 ml cryovials («Corning», USA) or 50 µl hermetic aluminum pans for differential scanning calorimetry (DSC) and were directly immersed into LN for 1 hour. The specimens were warmed in water bath at 40 °C for 2 min. The cryoprotectants were washed out by two conse- quent transfers of the meristems on filter papers saturat- ed with MS medium, supplemented with 10 % sucrose. 2.6 DROPLET-VITRIFICATION The meristems were treated with loading solution (2 M glycerol and 0.4 M sucrose) for 20 min and then transferred in different PVS at 22  °C for 40 min. The dehydrated specimens were placed individually into 10 μl droplets of PVS on a pieces of previously sterilized aluminum foil (15 × 20 × 0.15 mm), which were then directly immersed in LN. The cryopreserved meristems were thawed and the cryoprotectants were washed out by immersion in liquid MS medium, enriched with 12 % sucrose at 24 ºC. 2.7 СOMPOSITION OF PVS Modified PVS 1 (22 % glycerol + 13 % PG + 13 % ethylene glycol + 6 % Me2SO4 and 0.4 M sucrose); PVS 2 (30  % glycerol  +  15  % ethylene glycol  +  15  % Me2SO4 and 0.4 M sucrose) (Coste et.al., 2015); 88 % PVS 3 (44 % glycerol + 44 % sucrose) (Nishizawa et.al., 1993, Vilardo et.al., 2019); PVS N (34 % sucrose + 15 % glycerol + 14 % ethylene glycol) (Vitsenia et.al, 2015). 2.8 POST-THAW CULTURE The post-thaw meristems were plased in semi-solid MS medium, enriched with 3 % sucrose and stored under dark conditions for a week. They were transferred to the agar MS medium, supplemented with 3 % sucrose, 3 mg l-1 gibberellic acid and 0.01 mg l-1 indoleacetic acid. Than explants were cultured at 20 ± 2 °C, with 16 hours of light and 8 hours of darkness under 37 μmol×m-2×s-1 light in- Acta agriculturae Slovenica, 118/4 – 20224 N. SHEVCHENKO et al. tensity. The number of meristems, having a green color for 30 days, determined the survival rate. To examine the influence of pretreatment steps on the explants the part of meristems was treated with dif- ferent ways but excluding low-temperature exposure. Non-cooled and not treated with PVS or AD meristems were assumed as the control. 2.9 SEEDS CRYOPRESERVATION For cryopreservation the tomatoes seeds (‘Seven’, ‘Potiron Ecarlate’ and ‘Druzhba’ ) were placed into 1.5 ml polypropylene cryovials (FLMedical, Italy) and were di- rectly immersed into liquid nitrogen for 2 days. To thaw, the tubes transferred to air. Seeds were sowed on day 7 after cryopreservation. Substrate to obtain tomato seed- lings was potting soil “Rozsada” (Kisson, Ukraine), sup- plemented with coco coir (Ceres, Sri Lanka). 2.10 CHARACTERISTICS OF TOMATO CULTIVARS Three cultivars of tomato were obtained from the Institute of Vegetable and Melon Growing of the National Academy of Agrarian Sciences of Ukraine. The ‘Seven’ is a breeding cultivar of the Institute of Vegetables and Melons, Ukraine; it is resistant to major diseases. The growing season lasts 107-112 days and this variety is of late maturity with determinate plant type. Fruits are flattened, bright-red colored. The mass of one fruit can reach 350 g. The ‘Druzhba’ cv. is a breeding material sourced of the Tiraspol Institute of Vegetables, Moldova; it is espe- cially resistant to late blight. The growing season lasts up to 115 days; it is medium early maturity variety with de- terminate type of plant. Fruits are round, yellowish-or- ange in color. The mass of one fruit can gain up to  100 g. The ‘Potiron Ecarlate’ is a France native cultivar. It is mid early maturity variety with indeterminate type of plant. Their slightly flat and ribbed shape is reminiscent of pumpkin; the skin is two-tone, of yellow and red color. Each fruit can reach a mass of  600 g. 2.11 FIELD RESEARCH Tomato seedlings were grown in a greenhouse with- out heating. The seeds were planted on April 10, 2019. Mass shoots were received after 10-12 days. Seedlings were planted in open ground in the third decade of May. The area of the accounting site was 20 m2. The planting scheme was 70 × 35 cm. Caring for the plants consisted of systematic hoeing of the soil and irrigation (norm 300- 500 m3ha-1). During the growing season, morphological description was performed according to the classifier of the Solanum lycopersicum species. We recorded the seed germination, total and marketable yield, number of fruits per plant, mass of one fruit, plant height, number of in- ternodes and amount of healthy plants. 2.12 STATISTICAL ANALYSIS In all experiments, 10-25 meristems and 100 seeds were used per experimental condition and the experi- ments were replicated 3–5 times.The results were statisti- cally analyzed using Software Past 3. The results are pre- sented as mean and standard deviation. For establishing statistical significance, we used non-parametric Mann- Whitney criterion. The differences were considered sig- nificant at p < 0.05. 2.13 WEATHER DATA FOR EXPEREMENTAL RE- GION Some weather data 2019 year and long-term ones were obtained from the experimental area (Kharkiv re- gion), and were listed in Table 1. 3 RESULTS AND DISCUSSION 3.1 CRYOPESERVATION OF MERISTEMS It was shown that regeneration rate made 78 % (from 70 to 83.3 %) for the control group. This parameter did not statistically change for the meristems isolated from stems after cryoprotectant treatment. The post-thaw sur- vival rate was 0 % for two types of containers (Tab. 2). Thus, dissected of meristems from rewarmed stems was not good for in vitro grown plants cryopreservation. For the vitrification-based procedure, preculture of shoot apices with sucrose-enriched medium prior to dehydration with different PVSs has been reported to be effective to improve post-thaw survival of tomato (Kulus, 2019). Meristems were precultured for 24 hour in liquid MS medium, enriched with 12 % sucrose. The results in- dicated the survival rate of meristems after cultivation in this medium did not differ from the control value (Fig. 1). Since vitrification solutions contain high concentra- tions of cryoprotectants such as glycerol, ethylene glycol, propylene glycol or dimethyl sulfoxide, an essential step for successful vitrification is to identify the survival rate Acta agriculturae Slovenica, 118/4 – 2022 5 Field performance of cryopreserved seed-derived tomato plants and post-thaw survival of viral-infected meristems of meristems after PVSs treatment. This index was also determined for the airflow dehydrated meristems. Af- ter treatment of meristems with 88 % PVS 3 the growth recovery ranged from 35.5 to 48.5  %, such differences were significantly lower in comparison with the control group. In other variants, no significant differences were observed; re-growth percentages were between 66.6 and 83.3 % (Fig. 1). We observed same decrease in regenera- tion rate of meristems of the control group. It was most likely related with their reduced viability due to viral in- fection or their damage during isolation. The decrease in the number of viable meristems after 88 % PVS 3 treat- ment may be associated with a toxic effect of high con- centrations of cryoprotectants or with osmotic responses that lead to damage of samples. A reduced exposure time for meristems in this solution is likely capable to obtain a higher regeneration rate. Complete death rates were recorded in a week for all the PVSs treated meristems, wich were cryopreserved by droplet-vitrification and vitrification in cryovials. The death of the meristems immersed into LN on a piece of aluminium foil can be associated with their damage dur- ing the liquid nitrogen boiling or during warming in a nutrient medium through active rehydration. In case of vitrification in cryovials, death was caused by an unbal- anced cooling and heating rate, which can lead to the for- mation of ice crystals. After cryopreservation in aluminum pans for DSC, the survival rates of meristems from 34.2 to 78.5 % were observed. The post-thaw survival rates of meristems were 30–40 % for 88 % PVS 3, 70–78.5 % for modified PVS 1, 60–78.5 % for PVS 2 and 55.5–70 % for PVS N, so, the differences between non-cooled and cryopreserved ex- plants were not significant (Fig. 1). In case of AD the growth recovery of meristems ranged from 63 to 83 %. After cooling at the needle tip, the survival rates did not change significantly (Fig. 1). It should be noted that all survived but non-cooled meristems formed shoots and regenerated in plants within a month. Despite the high level of post-thaw mer- istems survival, we could not achieve the formation of the plants-regenerants. During two weeks of the experi- mental study in the post-thaw conditions, we observed the onset of meristem growth, which stopped but shoots were green. We transferred them to a fresh MS medium but no growth was observed. A month later, the samples remained green but we stopped monitoring them. Selec- tion of phytohormones in the reculture medium or light condition may be necessary. Thus, we have shown that meristems obtained from the virus-infected ‘Irishka’ plants had a reduced viability. Cryopreservation of dehydrated with different PVS mer- istems by droplet-vitrification or vitrification in cryovials did not result in any survival rate. Freezing the pieces of stem under the PG and Me2SO4 protectionin in vapour phase of LN followed by immersion into liquid nitrogen Climate conditions April May June July August September Rainfall, mm 25.5 58.5 14.0 51.0 7.5 32.0 Rainfall, long-term average, mm 40.8 55.5 65.0 73.3 41.9 48.8 Average daily temperature, ºС 10.3 17.9 24.0 21.5 21.5 15.7 Average daily temperature long-term average, ºС 9.6 16.5 20.2 21.3 19.8 14.1 Maximum temperature, ºС 26.0 30.0 34.0 32.0 33.0 29.0 Long-term maximum temperature, ºС 30.0 33.0 38.0 36.5 37.5 31.8 Minimum temperature, ºС -4.0 4.0 10.0 12.0 6.0 -5.0 Long-term minimum temperature, ºС -11.0 -6.8 1.0 6.0 1.5 -6.0 Minimum temperature of soil surface, ºС -4.0 4.0 10.0 12.0 6.0 -5.0 Table 1: Climate conditions of the Ukrainian Eastern forest-steppe Variants Regeneration rate, % Control PG Me2SO4 Non-frozem 78.31 ± 5.24 76.66 ± 5.47 71.4 ± 4.066 Frozen in aluminium foil containers 0 0 0 Frozen in polyimidofluoroplastic containers 0 0 0 Table 2: The regeneration rates of tomato meristems after PG, Me2SO4 pretreatment and cryopreservation in vapour phase of liquid nitrogen Acta agriculturae Slovenica, 118/4 – 20226 N. SHEVCHENKO et al. was also ineffective. The use of hermetic aluminum pans for DSC allows us to get a high level of the preserved meristems, independently of PVSs used. Dehydration of the meristems in the airflow and cooling at the needle tip by direct immersion into LN also made it possible to obtain a high survival rate. 3.2 CRYOPRESERVATION OF SEEDS Cryopreservation of plant materials in LN has been described as a suitable technique to conserve genetic re- sources of several species. However, the potential effects of LN in subsequent plant growth in field should be stud- ied before large-scale implementation of cryopreserved germplasm banks. In our work, we investigated the effect of LN on the growth and development of plants of three tomato cultivars in field. For ‘Seven’ all the studied economically valuable features such as marketable yield and general productiv- ity were significantly higher for the plants grown from the cryopreserved seeds. The increase in total and mar- ketable yields compared with the control group was 351 and 268 % respectively. The productivity from each plant was increased almost in five times, because the mass of one fruit and the number of them were higher (Tab. 3). For ‘Potiron Ecarlate’ the marketable yield increased in 220 %, the mass of one fruit was significantly lower for plants grown from cryopreserved seeds; the number of fruits per plant was significantly higher (Tab. 3). It was shown that for the ‘Druzhba’ the data of to- tal and marketable yield increased in 27.8 and 71.9  % respectively, number of fruits per plant was significantly higher (Tab. 3). The height of plants and number of internodes for all the cultivars did not change significantly. However, there was a tendency to an increase in these indices for the plants grown from frozen seeds (Tab. 3). During the growing season in 2019 in Ukraine, we observed many infected plants with both viral and fun- gal diseases. It was established that the treatment of seeds with LN led to a decrease in the number of plants infect- ed by viruses. The total number of infected plants grown from the cryopreserved seeds decreased by 33 % for the ‘Seven’ , for ‘Potiron Ecarlate’ it did by 6.7 %, for the ‘Dru- zhba’ the total percentage of sick and healthy plants did not differ (Tab. 3). Our results indicate that the germination of tomato seeds after cryopreservation did not change compared to the control. The tomato seeds of relatively small size can be cryopreserved without sophisticated pretreatment, re- quired for more differentiated tissues. They are described as both desiccation and liquid nitrogen tolerant. Up-to- date, seeds of several tomato cultivars were successfully stored in LN. Storage periods ranging from 180 to 1,095 days resulted in germination rates of 99 % when water content of 6–7 % fresh mass. If the moisture content was at 8.7 %, the germination rate of rewarmed seeds fell to 84 % (Grout and Crisp 1995). On the other hand, Mon- toya et al. (2000) found that 69–88 % of cryopreserved seeds remained viable, but did not germinate after stor- age in LN. Cryopreservation of tomato meristems in germetic aluminum pans for DSC, allowed us to obtain a high level of survival rate, but no further recovery was observed. Al-Abdallat et al. (2017) reported the same problem of the divergence between survival and recovery rates. De- spite the survival rate of the cryopreserved transgenic Figure 1: Survival rates of tomato meristems after AD and vitrification in 50 µl aluminum pans for DSC: □ – non-cooled, ■ – re- warmed. Note: * – differences are significant if compared with the control group, p < 0.05 Acta agriculturae Slovenica, 118/4 – 2022 7 Field performance of cryopreserved seed-derived tomato plants and post-thaw survival of viral-infected meristems tomato shoot tips reached even 70 %, however, no fur- ther recovery was possible. The authors considered that meticulously plant growth regulators selection, and their concentration optimisation, is required. Additionally, hormonal regulation of tomato explants growth can be altered by the cryopreservation procedure. For example, Grout et al. (1978) reported that viable S. lycopersicum explants (cryopreserved shoot tips) produced shoots di- rectly by typical meristem growth when cultured in the presence of gibberellic acid after rewarming. Without gibberellic acid, the surviving explants produced cal- lus and, subsequently, adventitious shoots. On the other hand, non-cryopreserved plant material produced shoots directly without the requirement for addition any plant growth regulators. In our study, we added 3 mg\l gibber- ellic acid and 0.01 mg/l indoleacetic acid to the reculture medium, but we still did not observe the regrowth of the cryopreserved meristems. Perhaps in order to enhance the uptake of phytohormones, a semi-solid medium, with reduced by half agar concentration, can be applied at the beginning of the recovery culture (Coste et al., 2015). 4 CONCLUSIONS It was shown that the meristems obtained from virus-infected plants ‘Irishka’ had a 78  % viability. The effect of 20 % PG, 20 % Me2SO4, different PVSs and dehy- dration by airflow on the regeneration potential of meris- tems was determined. The significant decrease in the re- generation rate was obtained for meristems treated with 88 % PVS3 (42 % vs 78 %), other variants of pretreatment did not strongly change the meristems regeneration rate. We determined the possibility of tomato meristems cryopreservation by freezing in vapour phase of LN, droplet-vitrification, as well as vitrification in cryovials and in aluminum pans for DSC. Freezing of stems pieces in vitro grown plants after PG and Me2SO4 treatment in vapour phase of LN was ineffective. Cryopreserva- tion of meristems by droplet-vitrification or vitrification into cryovials did not allow receiving any survival rate. Vitrification in aluminum pans for DSC did not change the meristems survival rate if compared with treated but non-cooled explants. Dehydration of the meristems in the airflow and cooling at the tip of the needle by a direct immersion into LN also made it possible to obtain a high survival rate. It was shown that all the studied economically valuable features such as marketable and total yield were significantly higher for the plants grown from the cryo- preserved seeds of ‘Seven’, ‘Potiron Ecarlate’ and ‘Druzh- ba’. The height of plants and number of internodes for all the cultivars did not change significantly; however, there was a tendency to an increase in these indices for the plants grown from the frozen seeds. The total number of infected plants grown from the cryopreserved seeds de- creased by 33 % for the ‘Seven’, for ‘Potiron Ecarlate’ it did by 6.7 %, for the ‘Druzhba’. total percentage of sick and healthy plants did not differ. Thus, the cryotherapy can likely applied for the tomato seeds, but this will demand additional experiments. 5 REFERENCES Acosta, Y., Hernandez, L., Mazorra, C., Quintana. N., Zevallos B. E., Cejas, I., . . . Fontes, D. (2019). Seed cryostorage en- hances subsequent plant productivity in the forage species Teramnus Labialis (L.F.) Spreng. Cryo Letters, 40(1), 36–44. Al-Abdallat, A. M., Shibli, R. A., Akash, M. W., Rabbaa, M., & Al-Qudah, T. (2017). 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