A project on high throughput sequencing (HTS) screening of certified rootstocks for virus-like agents (virus, viroid, phytoplasma) started in 2019. The aim is to acquire apple propagation material free of the virus-like agents with qualitatively new knowledge about its health status using HTS. Twenty-eight M9 rootstock plants labelled as standard CZ, elite, and basis material were purchased from a Czech and two international producers and were HTS tested. The HTS libraries were prepared from enriched double-stranded RNA extracts and processed with NovaSeq 6000. All HTS tested plants were free of Apple mosaic virus (ApMV), Apple stem grooving virus (ASGV), Apple stem pitting virus ( ASPV), Apple chlorotic leaf spot virus (ACLSV), Candidatus Phytoplasma mali (Apple proliferation phytoplasma), and Apple scar skin viroid (ASSVd) and complied with the EPPO certification standard ( Anonymous, 1999). The results were verified using conventional RT-PCR. All elite rootstocks were free of the virus-like agents. In the standard CZ and basis rootstocks, we detected Apple hammerhead viroid-like RNA (AHVd), Citrus concave gum-associated virus (CCGaV) and several sequences of three tentative novel viruses, which are subjects of further studies. CCGaV and AHVd mixed infections were reported in apple in Hungary (Jahan et al., 2019) and CCGaV in USA (Wright et al., 2018). Our results suggest the necessity for more rigorous HTS testing of rootstock source plants to determine their health status.

Genetic resources of clonally propagated fruit species cannot be stored as seed and alternative storage methods that are long-term and effective for a wide range of genotypes are important for breeding teams and genebank curators. Cryopreservation in liquid nitrogen is one of these techniques. In our study, in vitro shoot tips of 2 temperate fruit crops (plum and apple) encapsulated in sodium alginate were successfully cryopreserved using 3 different methods based on vitrification and two-step freezing. Initial in vitro shoots were previously established and multiplied on MS medium in Erlenmeyer flasks with 25 mL of MS medium. Basic shoot tip cultures were cold-hardened for 4 weeks in 4 degrees C. Explants (3 mm) aseptically dissected from cold hardened in vitro cultures were cold treated in MS medium supplemented with 2.0 M sucrose and for 48 h at 4 degrees C. Shoot tips encapsulated in sodium alginate were dehydrated by exposure to a sterile air flow for a period of 8 h. Two freezing methods were tested and compared: fast cooling by direct immersion in liquid nitrogen (vitrification) and two variants of two-step freezing. The best results were obtained with a two-step freezing protocol in variant b) with progressive cooling at a rate of -4 degrees C min(-1) to -40 degrees C followed by immersion in liquid nitrogen. In this variant, total average survival rate used for apple and plum cultivars was 71.0%. High values of survival were also observed in the case of the two-step freezing protocol in variant a) with an average survival of 65.1%. Encapsulated shoot tips immersed directly to liquid nitrogen were cryopreserved with 59.4% survival. Described cryopreservation procedures were successfully applied for 6 cultivars of plum and apple.

Pear is considered a traditional fruit crop in the territory of the Czech Republic. In recent years the acreage of commercial pear plantations in the Czech Republic increased to 750 ha. Of this amount, 580 ha are included in integrated fruit production systems (SISPO). Integrated fruit production means the economic production of commercial quality crops, giving priority to methods that are environment friendly with minimisation of applied chemicals. A long-term breeding program of pear was finalized at the end of the 20th century. Twenty-five newly bred Czech pear cultivars were the result and were included into the national Listing of Plant Varieties. 'Bohemica' (63 ha), 'Erika' (35 ha) and 'Dicolor' (25 ha) pears were the most successfully adopted in commercial integrated fruit production systems. The other new cultivars account less than 5 ha each. Although pear has not been commercially grown on a big scale in the Czech Republic, there is potential for larger commercial fruit production due to favorable climatic and soil conditions. Important growth and fruit characteristics of pear cultivars recommended for commercial plantations are described in the present paper.

From the point of view of human diet, sweet cherry provides a delicious fruit, which is rich in sugars and minerals. In the Czech Republic, sweet cherries are eaten seasonally out of hand or traditionally used in sponge cakes, desserts, fruit salads, juices, sorbets, ice cream, yoghourts or in distilled beverages. In Central European conditions, the earliest sweet cherries are the first fresh seasonal topfruits. On the other hand, late sweet cherry cultivars are gaining popularity in commercial growing systems due to higher return. There were 850 ha of commercial sweet cherry plantations (mostly late varieties) registered in the Czech Republic in 2018. Numerous landraces of sweet cherry for example 'Chlumecka', 'Horicka Pumra', ' Libejovicka rana', 'Techlovicka' arose in different regions of the Czech Republic from medieval times. They were named after locality of distribution and typical for certain areas. These landraces represent the most valuable part of genetic diversity. They are well adapted to the soil-climatic conditions of a particular region. Displacing traditional landraces in modern agricultural systems and continuing destruction of old plantations and alleys contributed to a considerable loss of genetic diversity in Central Europe. Because of the possible extinction of these valuable genotypes, the program for collecting and long-term conservation of sweet cherry genetic resources has been carried out in Research and Breeding Institute of Pomology in Holovousy Ltd. (RBIPH). Rescue of sweet cherry landraces is described in this presentation. Collecting missions were directed to the regions not influenced by modern agricultural production. Important accessions were localized by global positioning system (GPS) and in situ registered. Sweet cherry landraces showed a high variation in tree size, productivity, ripening time, fruit size, quality, and disease resistance. The most important cultivars found were: 'Karesova', 'Kastanka', 'Napoleonova', 'Thurn Taxis', 'Velka cerna chrupka', 'Ladeho pozdni'. Selected accessions were characterized and transferred to the field genebank of RBIPH. Germplasm preservation of sweet cherry in field conditions is supplemented using biotechnical methods (in vitro cultures, cryopreservation).

The evolution of fruit cultivars is a spontaneous process influenced by many factors and growing traditions. It consists of an initial historic phase of unconscious selection by growers for several hundred or thousand years, followed by intentional selection by breeders in successive breeding steps. Traditional fruit landraces represent a valuable part of genetic diversity, because they differ from their wild progenitors morphologically and genetically and they are not influenced by narrowing of genetic base in modern breeding programmes. Old landraces are usually specific for a county and represent also a cultural heritage of a particular nation. The long-term security of fruit genetic resources collections requires their conservation by several techniques. On farm conservation is an alternative method for preservation of fruit genetic resources in indigenous areas. This method is currently investigated and applied at the Research and Breeding Institute of Pomology Holovousy, Ltd. (RBIPH). Three on farm plantations KRNAP Vrchlabi, "Orchard of Reconciliation" Neratov, and National park Sumava were successfully established in cooperation with RBIPH. At present, there are mostly apple, pear, plum and sweet cherry landraces in these plantations. For the longevity of the plantation, seedling rootstocks were used for production of planting material. On farm collections comprise plants representing a diversity of genes and gene combinations for particular traits for each preserved fruit species. For efficient use of this biodiversity source, comprehensive evaluation of phenotypic and genotypic characters is carried out by the RBIPH. This approach appears to be a promising method as a duplicate long-term conservation method.

Sweet cherry is a vegetatively propagated, perennial plant with high level of heterozygosity and ancient breeding history. Sweet cherries are self-incompatible determined by a gametophytic self-incompatibility system (GSI), controlled by a multi-allelic S-locus. Maintenance and conservation of genetic resources are necessary for future breeding programs, growers and fruit production stability. In present, the utilization of DNA molecular genetic methods is the best suitable method for evaluation of genetic variability and S-genotyping within individual accessions. In our work, we used PCR primer combinations for 20 SSR, 2 EST-SSR and 10 universal or specific Slocus markers for analyses of 123 current, old and local sweet cherry cultivars from Czech genetic resources of Research and Breeding Institute of Pomology in Holovousy. In total, 115 polymorphic markers were amplified by SSR markers which we used for hierarchical cluster analysis of genetic variability. Clustering corresponded as with genealogical and geobotanical characteristics of individual accessions as breeding history of several known accessions. We confirmed previous S-genotyping for 72 accessions excepted Drogans Gelbe, Hedelfinger, Erika, Meckenheimer Fruhe, Alfa, Huldra, Rivan, Vanda and Winkler's Fruhe. It can be due to mislabelling or mistakes in previous analysis. Totally, we detected S-locus combinations in 19 incompatibility group. The most frequent incompatibility groups were III (S3S4), II (S1S3), IV (S2S3), and VI (S3S6).

The evaluated sweet cherry genebank was established at the Research and Breeding Institute of Pomology Holovousy Ltd. in 1989. Holovousy has an annual temperature of 8.4°C and precipitation of 663.5 mm. Since its establishment, new accessions have continuously supplemented this collection. The collection (three plants representing each cultivar) was planted at an altitude of 320 m with spacing of 6.0×6.0 m on Prunus avium rootstock. Weed-free strips were maintained under the bushes with the application of herbicide. Regularly cut grass was established between the rows. Here, we report the evaluation of economically important characters in 26 early-ripening sweet cherry cultivars in 2015 and 2016. Fruits of early-ripening sweet cherry cultivars ripened in the 1st and 2nd cherry weeks. These early cultivars ripened at the same time as the reference cultivar ‘Burlat’, or 10 days earlier. Vegetative and fruit characters were evaluated. Phenotypic characterisation revealed high diversity among the analysed germplasm. ‘Merchant’ (8.27 g), ‘Valerij Tschkalov’ (8.24 g), and ‘Burlat’ (7.36 g) had the biggest fruits. In contrast, ‘Fruheste der Mark’ (2.45 g) and ‘Moravska rychlice’ (2.94 g) had the smallest fruits. While ‘Early Rivers’, ‘Burlat’, and ‘Merchant’ ranked the highest for taste, ‘Kisinevskaja’, ‘Moravska rychlice’, ‘Munchenberska’, ‘Fruheste der Mark’, ‘Rivan’, ‘Skierniewice 1’, and ‘Skorospielka’ ranked the lowest. The most important sweet cherry cultivars are described in this paper.

In vitro shoot tips of three temperate fruit crops (apple, pear and sweet cherry) encapsulated in sodium alginate were successfully cryopreserved using two different methods based on vitrification or two-step freezing. Initially, shoot tip cultures were cold-hardened for 4 weeks in 4 degrees C. Explants (3 mm) aseptically dissected from cold hardened in vitro cultures were cold-treated in MS medium supplemented with 2.0 M sucrose and 100 mg L-1 vitamin C for 48 h at 4 degrees C. After encapsulation in alginate gel, embedded shoot tips were dehydrated by exposure to a sterile air flow for a period of 8 h. Three cooling procedures were used: rapid cooling by direct immersion of cryotube with embedded shoot tips in liquid nitrogen (vitrification); and two variants of two-step freezing by progressive cooling (0.5 to 10.0 degrees C min(-1)) before immersion in liquid nitrogen. Thawing was performed by placing the cryovials in a 40 degrees C water bath for approximately 1 min. Survival was defined as the percentage of the total number of shoot tips that showed a green colour after post culture according to an evaluation scale. Average survival rate amounted to 91.3% in the case of two-step freezing by progressive cooling. Encapsulated shoot tips immersed directly into liquid nitrogen (vitrification) were also cryopreserved successfully (on average 71.9% survival). The encapsulation dehydration method using alginate beads appears promising for the cryopreservation of rosaceous fruit species.

Sweet cherry (Prunus avium L.) is a globally important, vegetatively propagated fruit crop. Owing to high returns, sweet cherry is gaining popularity in the commercial orchards of central Europe, including the Czech Republic. In 2016, commercial sweet cherry plantations covered 889 ha in the Czech Republic. Additionally, sweet cherries are regularly grown and extremely popular in home gardens. The evaluated sweet cherry germplasm collection was planted on Prunus avium rootstock at an altitude of 320 m with a spacing of 6.0x6.0 m in 1989. Three trees represented each cultivar in the collection. The average annual temperature and precipitation in the region were 8.4 degrees C and 663.5 mm, respectively. Fruits of the late-ripening sweet cherry cultivars ripened 4 weeks or more after that of the reference cultivar 'Burlat'. Late-ripening sweet cherry cultivars have higher commercial importance than sweet cherry cultivars ripening early in the season. The evaluation of economically important characters in 24 late-ripening sweet cherry cultivars conducted during 2015 and 2016 is reported herein. The evaluated cultivars varied in ripening time and fruit size and quality. The average fruit weight of only the Czech cultivar 'Techlovan' (9.56 g) was higher than 9.00 g. 'Kordia', 'Kristin', 'Summit', 'Sunburst', 'Thurn Taxis', 'Techlovan', and 'Techlovicka' had extremely pleasant taste, overall. 'Ladeho pozdni' was distinctively the latest-ripening cultivar, ripening 11 weeks after that of 'Burlat'. Further, 'Hedelfingen', 'Hudson', 'Kordia', and 'Regina' ripened 6 weeks after 'Burlat'. The most important sweet cherry cultivars are described herein. The obtained phenotypic data will be associated with the results of molecular genetic studies and used later in breeding programmes.

The apple fruit is the most economically important fruit species in the temperate climatic zone of central Europe. In 2012, a project was initiated to test the resistance of apple landraces and older cultivars to fire blight. The aim of this study was to develop a protocol for rapid in vitro shoot multiplication of selected apple cultivars. Successful in vitro propagation, which can be carried out all year round, could provide an alternative method to produce stock material of particular cultivars for experiments with artificial inoculation in safe and controllable laboratory conditions. Selected genotypes were successfully established in vitro using 0.15% mercuric chloride as disinfectant solution. To determine favourable conditions for shoot initiation and development, six proliferation MS media containing 1, 2 and 4 mg L-1 BAP (6-benzylaminopurine), 0.5 and 1 mg L-1 TDZ (thidiazuron) or 10 mg L-1 2iP (6-(gamma,gamma-dimethylallylamino) purine) were tested. All apple genotypes in this study responded favourably to micropropagation in MS medium. The multiplication rate varied depending on the cultivar and concentration of cytokinins between 1.1 and 7.1. The highest proliferation rate was obtained for apple cultivar 'Hvezdnata reneta' which produced 7.1 in vitro shoots (longer than 10 mm) on MS medium containing 1 mg L-1 TDZ. On the contrary, the lowest proliferation rate 1.1 in our experiments was obtained for cultivar `Kminova reneta' on MS medium with 10 mg L-1 2iP. Our results confirmed the preliminary findings which indicated that TDZ was an important plant growth regulator for proliferation and growth in apple micropropagation. However, some abnormalities such as swollen shoots shorter than 10 mm with narrow leaves were observed during shoot multiplication on MS media with TDZ. The cytokinin BAP gave satisfactory results for multiplication in a concentration of 4 mg L-1. All cultivars had well-developed leaves on media containing BAP.

The research and Breeding Institute of Pomology (RBIP) Holovousy Ltd. holds a fruit germplasm of about 2,500 accessions in field collections. Unfortunately trees in these collections are at permanent risk from environmental conditions, pests and diseases. Germplasm preservation of vegetatively propagated fruit species in field collections may be supplemented using biotechnological methods. In vitro storage under slow-growth conditions is one of these techniques. The experiment with medium term storage of in vitro cultures of strawberry, apple, pear and sweet cherry at a low above zero temperature of 4.0°C was used in RBIP Holovousy Ltd. as a duplicate method to field collections. In vitro cultures were established on MS medium supplemented with 1 mg L(-1) 6-benzylaminopurine (BAP) in aluminium-capped 100-mL Erlenmeyer flasks with five explants 25 mL(-1) of medium. The experiment was successful and viable shoot tips were obtained at the end of storage period. The best overall results were obtained awith sweet cherry rootstock genotype P-HL-A, where 92.0% explants survived without an intervening subculture after seven months. In contrast, the lowest survival was observed with sweet cherry P-HL-C, where only 2.0% of shoots survived 7 months from the beginning of experiment. Remaining genotypes of fruit species used in the experiment had intermediate results. The storage of in vitro cultures at 4°C appears to be a promising technique as a duplicate short- or medium- term conservation method for fruit germplasm, which can considerably reduce labour and material costs. Moreover, in vitro collections are year-long accessible, and enable germplasm distribution among germplasm curators and the user community.

Sweet cherry is a vegetatively propagated, perennial plant with high level of heterozygosity and ancient breeding history. Therefore, it is necessary to keep, conserve and evaluate known genetic resources for future breeding programs and fruit production stability. In present, the utilization of DNA molecular genetic methods is the best suitable method for evaluation of individual accessions, so that we eliminated duplications and characterized genetic relationships. Recently, there are many different molecular methods but the most reliable are SSR markers. In our work, we used PCR primer combinations for 20 SSR and 2 EST-SSR markers for analyses of 82 current, old and local sweet cherry cultivars from Czech genetic resources of the Research and Breeding Institute of Pomology in Holovousy. In total, 113 polymorphic markers were amplified which we used for hierarchical cluster analysis of genetic variability. The resulting dendrogram corresponded with genealogical and geobotanical characteristics of individual accessions which were divided into five clusters. The breeding history of several known accessions was also proved.

Sweet cherries are self-incompatible determined by a gametophytic self-incompatibility system (GSI). The self-incompatibility is controlled by a multi-allelic Slocus and includes two genes coding for the synthesis of proteins responsible for the incompatibility response. The knowledge about the S-allele constitution of cultivars is very important for fruit growers and breeders. Recently, molecular methods have been developed to distinguish the S-alleles in sweet cherries and different molecular primers for the PCR-based identification of all S-alleles were designed during the last years. In our work, we analyzed S-locus genotypes by 10 universal and specific PCR primer combinations within 82 current, old and local sweet cherry cultivars from Czech genetic resources of Research and Breeding Institute of Pomology in Holovousy. We confirmed previous S-genotyping for 46 accessions except 'Droganova', 'Hedelfingen', 'Erika Fruhe Meckenheimer', 'Huldra', 'Rivan', 'Vanda' and 'Winkler'. It can be due to mislabeling or mistakes in previous analyses. Newly, S-genotyping was determined for 36 accessions when we found 4 new S-loci combinations. Totally, we detected S-locus combinations in 21 incompatibility groups. The most frequent incompatibility groups were III (S3S4), IV (S2S3), VI (S3S6) and II (S1S3) with 13, 11, 9 and 8 genotypes, respectively.

The aim of the present study was to determine whether chemotherapy with ribavirin can be used to eliminate Prune dwarf virus (PDV) from in vitro grown plants of infected sweet cherry cultivars ‘Tamara’ and ‘Amid’. Initial in vitro cultures were grown in Erlenmeyer flasks with 25 mL of MS medium (Murashige and Skoog, 1962), to which 1.5 mg L(‑1) BAP (6-benzylaminopurine) had been added. All cultures were cultivated in controlled environment chambers at 22±1°C. As soon as enough shoots were developed, individual shoots (5-10 mm in length) were excised from stock collections and transferred to treatment media with ribavirin. Ribavirin was added in concentration of 20 mg L(‑1) after autoclaving to the same MS medium as for multiplication. After a subculture period of 4 weeks, the apical part of the axis (about 3 mm in length comprising the apical meristem plus two-three primordial leaves) was dissected under a laminar flow hood and transferred to a fresh multiplication MS medium with 1.5 mg L(‑1) BAP for regeneration. The chemotherapy technique was able to eliminate PDV in high percentage according to the cultivar, ranging between 100.0 and 96.4% for cultivars ‘Tamara’ and ‘Amid’, respectively. In the course of chemotherapy cycles, ribavirin concentrations of 20 mg L(‑1), ‘Amid’ plants did not display symptoms of phytotoxicity and appeared vigorous and healthy. In contrary, 35% of ‘Tamara’ in vitro plants did not survive the ribavirin treatment. The obtained results demonstrate the effectiveness of the method to eliminate PDV by a combination of in vitro cultures, chemotherapy with ribavirin and subsequent removing of apical meristematic region.

Landraces of cultivated plants originated from the beginning of agriculture through selection from wild ecotypes and cultivated by farmers in their domestic region were investigated with the goal to conserve fragments of still existing fruit landraces, including restoration of variable countryside with scattered and roadside trees, wind-breaking and snow-catching functions including fruit consumption. Individual trees were identified, attributed to cultivars, localized by GPS and mapped in the Geobase Map of the Czech Republic. The inventory listed 388 fruits out of total number of 2866 accessions. It is a basis for searching lost and extinct materials. The database was analyzed for cultivars, regionality and their longevity by the length of registration. The best indigenous materials were proposed for in situ conservation mainly in the territory of National Parks and Protected Landscape Areas.

It can be assumed that commercial production of fruit crops will be impacted by climate change. In the Research and Breeding Institute of Pomology Holovousy Ltd. (RBIPH), there are continuous meteorological records since 1955. RBIPH carries out observations in the form of services for the Czech Hydrometeorological Institute and we present an overall analysis of the evolution of climatic conditions in relation with key periods in the sweet cherry growth cycle. The average annual temperature for the whole 55 year period is 8.4 degrees C. The lowest annual temperature (6.2 degrees C) was in year 1956 and on the contrary, the highest in years 2007 and 2008 (10.1 degrees C). There is a visible increase in average annual temperatures in quinquenniums beginning from 1990-1994. Measurement of weather parameters that affect sweet cherry is relevant for serious interpretation of experiments carried out in the field. In particular, the course of temperatures in April and May affect sweet cherry from the point of view of late spring frosts damaging flowers and fruits. Furthermore, precipitation from April to July have a high impact on fruit size and undesirable fruit cracking. There is a visible increase in average temperatures in May in quinquenniums beginning from 1985-1989 and in April in quinquennium 2005-2009. Increasing trend of average annual and month temperatures is in accordance with climate changes and global warming. In contrast to the tendency to warming, a long term trend is hardly discernible in the case of precipitation.

Sweet cherry (Prunus avium L.), member of the subgenus Cerasus, is a globally important vegetatively propagated fruit crop. Unfortunately, trees grown in fields and nurseries are threatened by the Prune dwarf virus (PDV). This virus causes malformations and a decrease in growth vigour and yield. A solution to get rid of the PDV virus in the Czech prospective sweet cherry cultivars 'Tamara' and 'Kasandra' is a part of the global sanitation programme. Tissue culture techniques are an important tool for quick multiplication of requested cultivars and offer the opportunity to improve the health status of micropropagated plants by combining in vitro culture with chemo or thermotherapy. The goal of this study was to identify factors affecting the ability of Czech sweet cherry cultivars to produce shoots under in vitro culture conditions. Six proliferation MS media containing 1, 2 and 4 mg L-1 BAP (6-benzylaminopurine), 0.5 and 1 mg L-1 TDZ (thidiazuron) or 10 mg L-1 2iP (6-(g, gdimethylallylamino) purine) were tested. Values of multiplication rate varied between 1.2 and 4.5. From the tested cytokinins, BAP at a concentration of 4 mg L-1 was found to be more effective than TDZ and 2iP for shoot multiplication. In conclusion, our experiments confirmed that in vitro propagation of selected sweet cherry cultivars can be achieved. The described procedure enabled us to multiply and maintain in vitro plants throughout the year of the two sweet cultivars for further experiments with in vitro chemotherapy.

The goal of this study was to identify factors affecting in vitro shoot and root production of two Czech sweet cherry (Prunus avium) cultivars, 'Amid' and 'Kares Fruhe' under in vitro culture conditions. This information is required prior to experiments with chemotherapy as a method of sanitation from viruses. The two genotypes were successfully established in vitro using mercuric chloride in a concentration of 0.15% as a sterilization solution. The overall rate of contamination was 16.7%. Six proliferation MS media containing 1, 2 and 4 mg L-1 BAP (6-benzylaminopurine), 0.5 and 1 mg L-1 TDZ (thidiazuron) or 10 mg L-1 2iP (6-(g, g-dimethylallylamino) purine) were tested for effects on proliferation, callus formation and shoot morphology. Multiplication rate values were relatively low and varied between 1.1 and 2.1. BAP at 4 mg L-1 was found to be more effective than TDZ and 2iP for shoot multiplication. Rooting was promoted on MS medium supplied with 1 mg L-1 NAA (naphthaleneacetic acid). Root initiation started within 2 weeks. The root induction was relatively low, 45% for 'Kares Fruhe' and 28% for 'Amid'. In conclusion, multiplication and rooting rates were sufficient for in vitro culture establishment, short-term maintenance and in vitro chemotherapy. However, these relatively low rates are not satisfactory for in vitro production of plants on a commercial scale.