Virus-free Planting Material Research Articles

Detection of Viruses and Elimination of Sweet Potato Feathery Mottle Virus in High-Yielding Varieties of Sweet Potato (Ipomoea batatas) from Ethiopia

Sweet potato (Ipomoea batatas L. Lam.) is a major source of food in many parts of Ethiopia. In recent years, viral diseases have become the main threat to sweet potato production in Ethiopia. Previous virus survey studies carried out from 1986 to 2020 reported eight viruses infecting sweet potato in Ethiopia. Consequently, obtaining and multiplying virus-free planting materials have been difficult for farmers and commercial multipliers. This study was conducted to detect viruses infecting the five sweet potato varieties used as source plants and compare the virus elimination efficiency between meristem cultures from untreated and heat-treated mother plants and production of virus-free sweet-potato-planting materials. Seven common viruses were tested for, using grafting to Ipomoea setosa, enzyme-linked immunosorbent assay (ELISA) and reverse-transcription polymerase chain reaction (RT–PCR) before and after elimination procedures as screening and confirmatory methods. The sweet potato feathery mottle virus (SPFMV) elimination efficiencies of meristem cultures from untreated (grown at 25 ± 1 °C) and heat-treated (grown at 39 ± 1 °C) potted plants of sweet potato varieties were evaluated and compared. Sweet potato feathery mottle virus (SPFMV) was detected in 12 of the 15 source plants tested. Triple infections of SPFMV, sweet potato chlorotic stunt virus (SPCSV), and sweet potato virus C (SPVC) were detected in one of the fifteen plants. This study reports the detection of SPVC for the first time in sweet potato plants from Ethiopia. The cutting of meristems from heat-treated plants further increased the percentage of virus-free plantlets by ca 10% to ca 16%, depending on the plant variety. Elimination efficiency also seemed to vary among varieties: the greatest difference was observed for ‘Tola’, and the least difference was observed for ‘Guntute’. The present study provided protocols for detecting viruses and generating virus-free sweet-potato-planting materials in Ethiopia.

Modeling the transmission dynamics of banana bunch top disease in banana plants

Banana Bunch Top Disease (BBTD) is a severe viral infection that poses a significant threat to banana production in tropical regions. Caused by the Banana Bunchy Top Virus (BBTV) and transmitted primarily by the banana aphid (Pentalonia nigronervosa), BBTD results in stunted growth and poor fruit production, leading to substantial economic losses. This paper presents a mathematical model to analyze the transmission dynamics of BBTD in banana plants and evaluate various control strategies using the Next Generation Matrix approach to compute the basic reproduction number R0. The study reveals that effective control of aphid populations, timely removal of infected plants, and the use of virus-free planting material significantly reduce the spread of BBTV and improve plant health. Increasing the removal rate γ and managing the transmission rate β are crucial strategies for controlling the disease, while elevated aphid infection rates λ and progression rates σ exacerbate its spread. Numerical simulations highlight the effectiveness of targeted interventions, including the application of pesticides and the introduction of resistant banana varieties. The study recommends that, in addition to current control measures, more focus should be placed on comprehensive monitoring programs, early detection of symptoms, and community education on BBTD management strategies. Enhanced efforts in these areas are crucial to reducing the incidence of BBTD and improving the resilience of banana crops.

Direct regeneration-mediated in vitro propagation and microtuberization of potato: An appraisal of the past ten-year period

Potato (Solanum tuberosum L.) is one of the most important food crops widely cultivated worldwide due to its edible and nutrition-rich tubers. It is a temperate crop; however, currently, it is grown in a wide range of areas with distinctive climatic conditions. Traditionally, potato is propagated by whole or cut tubers and true potato seeds. Owing to the low rate of multiplication and high cost of cultivation to produce virus-free planting material during traditional propagation, the in vitro technique becomes an alternative approach to conventional propagation. Over the past ten years, significant advancements have been made in the optimization of protocols for several varieties/cultivars of potato via a direct regeneration-mediated approach. The present short-review illustrates the analysis of an array of significant published findings on various physical and chemical variables that are responsible for fruitful in vitro direct regeneration and microtuberization of potato. Furthermore, this short-review describes a number of undiscovered areas, research gaps, and major problems that might be accounted for advancing noteworthy ingenious approaches associated with potato biotechnology in the near future. The current short-review further addresses salient recommendations and conclusions (based on achievements during the last ten-year period), paving the way for potato researchers to work on several unexplored areas in the coming years.

Identification of Garlic Viruses Associated with Seed Bulbs and Consumption Bulbs from Several Locations in Indonesia

Virus infection is one of the major constraints in garlic production since the viruses are readily accumulated on vegetative propagation material (bulbs). This research aimed to detect garlic common latent virus (GCLV), shallot latent virus (SLV), onion yellow dwarf virus (OYDV), and leek yellow stripe virus (LYSV) infecting local garlic as seed bulb and imported garlic as consumption bulb. Seed bulb samples were obtained from seed breeders in several garlic growing centers in Indonesia. In contrast, consumption bulb samples were obtained from plant quarantine warehouses and three local markets in Bogor. Some bulb samples were used for morphological observations, and some were germinated in the laboratory until the leaves emerged. Leaves were collected for virus detection by RT-PCR using specific primers for GCLV, SLV, OYDV, and LYSV. Seed and consumption bulbs have differences in their morphological characteristics, especially in the type of neck hardness and the size of the bulb diameter. OYDV and LYSV infections were successfully detected in seed and consumption bulbs, while SLV was only found in consumption bulbs. Nucleotide sequence analysis showed that SLV from consumption bulbs formed one group, GCLV from seed bulbs formed one group, while OYDV and LYSV from seed and consumption bulbs were in different groups, indicating that the viruses came from different strains. Further research through high-throughput detection methods and providing virus-free planting material are needed to anticipate the spread of new strains of garlic viruses in Indonesia.

Application of Biotechniques for In Vitro Virus and Viroid Elimination in Pome Fruit Crops.

Sustainable production of pome fruit crops is dependent upon having virus-free planting materials. The production and distribution of plants derived from virus- and viroid-negative sources is necessary not only to control pome fruit viral diseases but also for sustainable breeding activities, as well as the safe movement of plant materials across borders. With variable success rates, different in vitro-based techniques, including shoot tip culture, micrografting, thermotherapy, chemotherapy, and shoot tip cryotherapy, have been employed to eliminate viruses from pome fruits. Higher pathogen eradication efficiencies have been achieved by combining two or more of these techniques. An accurate diagnosis that confirms complete viral elimination is crucial for developing effective management strategies. In recent years, considerable efforts have resulted in new reliable and efficient virus detection methods. This comprehensive review documents the development and recent advances in biotechnological methods that produce healthy pome fruit plants. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

In vitro propagation of stone fruit rootstock cultivar Evrica 99 and its influence on some phytochemical traits of fresh apricot fruit

Background: The cultivation of stone fruits is of primary importance in Armenia. Their fruits contain antioxidants, fiber, potassium, vitamin A, C, E, minerals, etc., which have a beneficial effect on human health and prevent many diseases. The concentration of those components varies depending on ecological factors, cultivar, rootstock, cultural practices, etc. Clonal rootstocks are important for increasing orchard density, tree uniformity, and high yields, and they can also affect fruit quality. In vitro culture is a valuable method for rapid propagation of high-quality, virus free plant material. Objective: The purpose of this study was to develop in vitro production technology for the stone-fruit rootstock cultivar Evrica 99, and to determine if rootstocks affect some fresh fruit traits of ´Yerevani´ and ´Sateni´ apricot cultivars. Methods: The shoot apical meristem and lateral bud served as explants for shoot regeneration. Different sterilizing agents at various periods of exposure were used for the explant surface sterilization. Various concentrations of phytohormones, both individually and in combinations, were employed for in vitro regeneration and rooting of plants. The titratable acidity (TA), dry matter (DM), vitamin C, mineral content, total carotenoids (TC), and sugar contents were evaluated in fresh fruit. Results: The most optimal option for explant surface sterilization was the gradual application of calcium hypochlorite [Ca (ClO)2] (2.0% solution, exposure time 10 min) and ethanol (70% solution, exposure time 20 s), as a result of which we had 75.5% survival rate of explants. The efficient medium for in vitro shoot regeneration was MS supplemented with 6-Benzylaminopurine (BAP) 0.8 mg/l, Kinetin (Kin) 0.2 mg/l, and Gibberellic acid (GA3) 1.0 mg/l. The half-strength Murashige and Skoog (MS) medium containing 0.8 mg/l indole-3-butyric acid (IBA) was optimal for in vitro rooting. Rooted plants were successfully adapted with a survival rate of 85.0%. The defined method can be successfully used for 'Evrica 99' cultivar micropropagation. The results obtained showed that fruit quality strongly depended on both the varieties and the rootstock tested. Conclusion: In the current study, an alternative in vitro propagation technology for rootstock cultivar ´ Evrica 99 was developed by direct organogenesis, enabling mass-scale production of virus-free plants that is suitable for commercial purposes as well. The apricot cultivars ´Yerevani´ and ´Sateni´ grafted on the virus-free rootstock cultivar Evrica 99 showed higher fruit quality traits, which are essential for human health and diet. Keywords: ´Evrica 99, fucnctional foods, in vitro regeneration, micropropagation, plant growth regulators, stone fruit rootstock, tissue culture.

IN VITRO PRODUCTION OF VIRUS-FREE CARNATION (DIANTHUS CARIOPHYLLUS L.) PLANTING MATERIAL

The methods of culture of apical meristems and direct and indirect morphogenesis in vitro were used for production of virus-free planting material of carnation. A scheme for obtaining aseptic material has been developed, which consists of stepwise treatment of explants: Thimerosal - 2 min, 70% ethyl alcohol - 0.5 min and 0.08% AgNO 3 - 1 min, which reduces the level of contamination by fungal infection. Expounded the results of studies of callusogenesis and direct and indirect morphogenesis in the culture of in vitro explants of Dutch carnation, their dependence on the content of growth regulators in the nutrient medium. It was established that there were almost no significant differences in the course of callusogenesis processes within carnation varieties . At the same time, the frequency of callusogenesis was 100%. Under the conditions of indirect morphogenesis realization, it is necessary to take into account the age of callus tissues. The growth and intensive shoot formation of carnations was noted on the Murashige-Skoog nutrient medium supplemented with BAP at a concentration of 0.5 mg/l. The best medium for rooting was the MS medium with half the concentration of macro- and microsalts with the addition of 0.5 mg/l of NAA, which is recommended by us for rooting regenerating carnation plants of various varieties. Peat : perlite in a 1:1 ratio was used as a substrate for the adaptation of regenerating plants . Survival of carnation plants to conditions in vivo for the variety "Raffino Linde" was 90%, while for the variety "Tiya" - 83%, respectively.

Development of individual elements of a protocol for sustainable growth and propagation of garden strawberries (Fragaria ananassa Duch.) under aseptic conditions

Garden strawberry (Fragaria ananassa Duch.) is one of the most valuable fruits the demand for which in the food market is consistently high. One of the limiting factors for achieving consistently high-quality strawberry yields is the presence of diseases caused by bacteria, phytoplasmas, viruses, and viroids. In order to intensify the technology of garden strawberries cultivation, the problem of production in significant volumes of genetically constant material free from pathogens is essential. Biotechnological methods are currently relevant technologies that allow mass production of planting material with high phytosanitary and genetic quality. The purpose of the study is to update the protocol for microclonal propagation of garden strawberries to obtain virus-free planting material. The research was conducted in the micropropagation laboratory of LLC «Blahodatne» (ТеvittaTM) Cherkasy region, Ukraine using the «Alba» and «Present» strawberry cultivars. A series of experiments were conducted according to the «step by step» principle on two types of explants: buds and meristems. The determinants for obtaining aseptic cultures from bud and meristem explants were investigated. The trophic influence was studied in media with different mineral content (at the multiplication stage) and sucrose concentrations during rhizogenesis. Among the phytohormonal determinants during the multiplication stage, the best combination among those investigated was the use of substances with cytokinin activity consisting of BAP at 0.2 mg/l and kinetin at 0.8 mg/l. The addition of 0.1 ml/l of «Gibb plus preparation» (GK4 + GK7) was effective for the reproduction rate increasing. Growing of donor explants in media with BAP at 0.2 mg/l, kinetin at 0.3 mg/l, and adenine at 0.5 mg/l, compared to the control (BAP at 1.0 mg/l) improved rhizogenesis in regenerants. The highest root formation rates were observed in the variant with 4 % of sucrose (40 g/l). Key words: propagation; microclonal propagation; aseptic culture; trophic and hormonal determination.

РЕЗУЛЬТАТЫ АПРОБАЦИИ МОДИФИЦИРОВАННОЙ ПИТАТЕЛЬНОЙ СРЕДЫ В ПРОМЫШЛЕННОЙ ТЕХНОЛОГИИ ПРОИЗВОДСТВА БЕЗВИРУСНОГО ПОСАДОЧНОГО МАТЕРИАЛА ЗЕМЛЯНИКИ МЕТОДОМ IN VITRO

The article presents information on the testing of modified nutrient media used in the technology of micropropagation of strawberry varieties at the stages of multiplication and rhizogenesis in comparison with the traditional nutrient medium according to Murashiga and Skoog (MS). According to the results of studies conducted in the laboratory of agricultural biotechnology of the Stavropol State Agrarian University, the use of a modified nutrient medium at the stage of multiplication contributed to an increase in the multiplication coefficient of microcuttings relative to similar results of the control medium in the experiment by 0.4-2.5 units. Studies conducted at the stage of rhisigenesis showed a similar picture. In all considered strawberry varieties on the modified nutrient medium, more intensive root formation was noted, which led to a significant advantage of the results of the modified nutrient medium relative to the control on the 28th day of rhizogenesis by 15.0-18.7 %. Thus, it can be stated that the replacement of inorganic salts of microelements that are part of the nutrient medium with complexonates of microelements with a carboxyl-containing ligand - EDTA, contributed to a better development of experimental plants, an increase in the yield and quality of produced virus-free planting material of the studied industrial strawberry varieties.

An Efficient Protocol for In vitro Regeneration and Acclimatization of Banana (Musa spp.) cv. Grand Naine

Aim: The study was undertaken with a view to standardize a protocol for in vitro regeneration of Banana cv. Grand naine using shoot tip of sucker as an explant
 Methodology: In present study, explants sterilized with different sterilizing agents such as Tween-20 (1%), Bavistin (0.5-1%), Streptomycin sulphate (250 mg/L), Ascorbic acid (150 mg/L) + Citric acid (100 mg/L), HgCl2 (0.1%) and 70% ethanol.Sterilized explants were cultured on Murashige and Skoog (MS) medium supplemented with different concentrations of plant growth hormones for shoot initiation BAP alone (1.5, 2.0, 4.0, 6.0 mg/L) and BAP (3.0 mg/L) in combination with IAA and IBA (2.0 mg/L), elongation BAP (3.0 mg/L) and NAA (1.0, 1.5, 2.0 mg/L) and rooting IAA (1.0, 1.5 mg/L) and IBA (1.0, 1.5 mg/L). Primary and secondaryhardening was done in potting mixture containing autoclaved black soil: vermicompost: cocopeat (1:1:1) and garden black soil, cocopeat and red soil (1:1:1) respectively.
 Results: In present investigation 1% Bavistin (fungicide) showed maximum respond to prevent fungal contamination. Highest shoot initiation (100%) was observed on a MS medium fortified with BAP (1.5 mg/L). Maximum shoot length (10.7 cm) was recorded on a MS medium supplemented with BAP (3.0 mg/L) + NAA (2.0 mg/L) + Activated charcoal. Maximum root initiation was observed on half strength MS medium supplemented with IAA (1.5 mg/L). In vitro regenerated plantlets hardened on the mixture of autoclaved black soil: vermicompost: cocopeat (1:1:1). After 14 weeks In vitro plantlets transferred in green house for acclimatization where, 80% survival rate was recorded.
 Conclusion: Regeneration protocol was successfully standardized. Therefore, itcan be used for large scale propagation of healthy, disease and virus free planting material and In vitro propagation helps to meet higher demand of healthy planting material within shorter period.

Traditional Approaches and Emerging Biotechnologies in Grapevine Virology

Environmental changes and global warming may promote the emergence of unknown viruses, whose spread is favored by the trade in plant products. Viruses represent a major threat to viticulture and the wine industry. Their management is challenging and mostly relies on prophylactic measures that are intended to prevent the introduction of viruses into vineyards. Besides the use of virus-free planting material, the employment of agrochemicals is a major strategy to prevent the spread of insect vectors in vineyards. According to the goal of the European Green Deal, a 50% decrease in the use of agrochemicals is expected before 2030. Thus, the development of alternative strategies that allow the sustainable control of viral diseases in vineyards is strongly needed. Here, we present a set of innovative biotechnological tools that have been developed to induce virus resistance in plants. From transgenesis to the still-debated genome editing technologies and RNAi-based strategies, this review discusses numerous illustrative studies that highlight the effectiveness of these promising tools for the management of viral infections in grapevine. Finally, the development of viral vectors from grapevine viruses is described, revealing their positive and unconventional roles, from targets to tools, in emerging biotechnologies.

Potato viruses in Kazakhstan and methods for obtaining virus-free seed material

Potato (Solanum tuberosum L.) is a staple food worldwide, including in Kazakhstan. The potato yield in 2021 in the country was only 20 tonnes per ha, which is a rather low indicator. One of the main reasons for the insufficient yield of potatoes in Kazakhstan is the low quality of seed material, and the main requirement for high-quality seed material is the absence of viral diseases. Viral diseases in crops are a major impediment to sustainable potato production, as they cause large losses in crop quantity and quality. To date, 40 viral potato diseases have been discovered worldwide. Depending on the infection of viral diseases, the yield can be reduced by up to 90% in crop production. In this review, we discuss in detail the current state of potato viral diseases in Kazakhstan and characterise the most common potato viruses in the country, including potato virus M (PVM), potato virus (PVS), potato virus X (PVX), potato virus Y (PVY) and potato leafroll virus (PLRV). To ensure food security of the country and prevent the spread of infection with viral diseases, popular methods (ELISA, RT-PCR and detection of specific types of targets using microarrays) for the early detection of potato plant viruses in seed and field material are considered. For the mass production of virus-free potato plant material, the use of temporary immersion bioreactors is discussed, as well as the use of modern genetic engineering methods to obtain plant varieties resistant to the most common viral diseases.

Вплив діяльності профільних наукових установ та державної фінансової підтримки на стан садівничої і виноградно-виноробної галузі України

Negative and positive trends in the efficiency of growing fruit and berry crops and grapes in Ukraine in different periods of time are analyzed. Information is provided on the scientific support of activities of industry market participants. The main areas of research of specialized scientific institutions of the Institute of Horticulture NAAS of Ukraine and the National Scientific Centre «V.Ye. Tairov Institute of Viticulture and Winemaking» NAAS of Ukraine, as well as the implementation of their results in production. Prospective technologies for obtaining virus-free basic clones in industrial horticulture and viticulture, their improvement in in vitro culture, and subsequent accelerated reproduction using classical and biotechnological methods are described. An analysis of the production of planting material in domestic nurseries was conducted and problematic issues of market participants in horticulture and viticulture were identified. Calculations of the necessary volumes of production of horticultural and viticultural products to saturate the domestic food market with competitive products and fully meet the needs of the population according to physiological standards, as well as to expand their export in the volumes specified in the Sectoral Development Programs of the sectors are highlighted. The need to complete the construction of the biotechnological greenhouse complex for the cultivation and accelerated replication of basic virus-free seedlings and the reconstruction of the Biotechnological complex for the production of virus-free planting material of grapes is substantiated. The diagrams present the volumes of planting perennial plantations and production of fruit and berry products, which were obtained, including thanks to state financial support and due to the intensification of production, which was carried out on the basis of scientific research. The volumes and trends of state financial support of industries are analyzed. The role of central executive bodies and specialized scientific institutions of Ukraine in the development of the horticultural and grape-growing industries of Ukraine is highlighted. An explanation has been given regarding the expediency of planting rootstocks and grafts in domestic nurseries that provide branch enterprises with planting material. A conclusion was made regarding the need to implement measures to solve problematic issues. Key words: orchards, berry orchards, vineyards, planting material, state support, grant support, scientific and technical research, scientific institutions.

Effect of Chitosan Nanoparticles (CS-NPs) on In Vitro Regeneration Response and Production of Potato virus Y (PVY)-Free Plants of Potato

Potato virus Y (PVY) causes serious loss in the yield and quality of potatoes. The effect of chitosan nanoparticles (CS-NPs) on the regeneration response and production of PVY-free plants under in vitro conditions was studied. Murashige and Skoog (MS) medium supplemented with 0.1 mg L−1 Gibberellic acid (GA3), 0.1 mg L−1 Naphthalene acetic acid (NAA) and 500 mg L−1 malt extract was used for regeneration of plantlets from sprouts. Double Antibody Sandwich Enzyme-Linked Immunosorbent Assay (DAS-ELISA) and Reverse Transcript Polymerase Chain Reaction (RT-PCR) was used for virus indexing of the mother plant and in vitro-regenerated plantlets. Explants of PVY positive potato plants were cultured on same medium amended with 100, 200, 250, and 300 mg L−1 of (CS-NPs). Shoot regeneration decreased from 100 to 200 mg L−1 as the concentrations of antiviral (CS-NPs) up to 250 mg L−1. It was decreased with the increase in the concentration of the antiviral (CS-NPs) up to 300 mg L−1. Antiviral (CS-NPs) at the concentration of 250 mg L−1 showed a positive effect on shoot regeneration. In vitro-regenerated plantlets were virus free and tested negative in both ELISA and RT-PCR. The level of 100 mg L−1 of (CS_NPs) produced 38.8% PVY-free plants and 30.50% of cultures showed shoot regeneration. The level of 200 mg L−1 of (CS-NPs) produced 49.6% PVY-free plants while 46.8% of cultures showed shoot regeneration. The level of 250 mg L−1 of (CS-NPs) was the most effective and produced 100.0% PVY-free plants and 100.0% of cultures showed shoot regeneration. Histopathological changes simultaneously with elucidation of resistance and growth enhancement were evident in the treated plants with (CS-NPs) than those untreated control. In conclusion, (CS-NPs) treatment is an effective tool to produce PVY-free sprouts explants and has potential for producing virus-free planting material for the potato industry.

Diversity of Viruses and Viroids Infecting Taro in Kenya Based on Small RNA Sequencing and PCR Detection

Purpose: Viral diseases cause severe yield losses and quality decline in crops worldwide. Despite their economic significance, the occurrence and distribution of the major viruses and viroids infecting Taro in Kenya remain poor, limiting the development of robust disease management strategies to mitigate their spread. This study thus aimed to identify the viruses and viroids infecting Taro in Kenya as a basis for developing effective management strategies to support the prevention and control of Taro viruses.
 Methodology: Viral surveys and sampling were conducted across nine Taro-growing counties with diverse agroecological conditions in Kenya to determine the incidence and distribution of viruses affecting Taro. Leaf and whole plant samples of symptomatic edible and wild Taro were collected for PCR, RT-PCR, and small RNA sequencing assays to determine the diversity of viruses and viroids infecting Taro.
 Results: Disease-like symptoms, including stunting, leaf rolling, shrinkage, deformed leaves with mosaic and yellow veins, and dwarfism, were observed. An overall mean disease incidence of 32-60% was recorded in all sites surveyed. Small RNA sequencing revealed the presence of both DNA and RNA viruses. Detected DNA viruses included the Taro Bacilliform Virus (TaBV) and Taro Bacilliform CH Virus (TaBCHV), badnaviruses specific to Taro, the sweet potato Badnavirus B, sugarcane bacilliform virus, and sweet potato leaf curl virus. The RNA viruses included the sweet potato feathery mottle and Phaseolus vulgaris alphaendornavirus. A Citrus exocortis viroid was also detected. Interestingly, the wild relatives of Taro displayed very few viral sequence hits. This study reports the Taro viruses and viroids circulating in Kenya and is the first to describe the incidence, distribution, and sequence variability of TaBV in Kenya.
 Recommendations: Future studies should focus on developing effective management strategies to support the prevention and control of Taro viruses, including genetic resources for virus-Taro interactions, removing infected crops, controlling insect vectors, and developing virus-free planting materials.

Eradication of Potato Virus S, Potato Virus A, and Potato Virus M From Infected in vitro-Grown Potato Shoots Using in vitro Therapies.

Certain viruses dramatically affect yield and quality of potatoes and have proved difficult to eradicate with current approaches. Here, we describe a reliable and efficient virus eradication method that is high throughput and more efficacious at producing virus-free potato plants than current reported methods. Thermotherapy, chemotherapy, and cryotherapy treatments were tested alone and in combination for ability to eradicate single and mixed Potato virus S (PVS), Potato virus A (PVA), and Potato virus M (PVM) infections from three potato cultivars. Chemotherapy treatments were undertaken on in vitro shoot segments for four weeks in culture medium supplemented with 100 mg L−1 ribavirin. Thermotherapy on in vitro shoot segments was applied for two weeks at 40°C (day) and 28°C (night) with a 16 h photoperiod. Plant vitrification solution 2 (PVS2) and cryotherapy treatments included a shoot tip preculture followed by exposure to PVS2 either without or with liquid nitrogen (LN, cryotherapy) treatment. The virus status of control and recovered plants following therapies was assessed in post-regeneration culture after 3 months and then retested in plants after they had been growing in a greenhouse for a further 3 months. Microtuber production was investigated using in vitro virus-free and virus-infected segments. We found that thermotherapy and cryotherapy (60 min PVS2 + LN) used alone were not effective in virus eradication, while chemotherapy was better but with variable efficacy (20–100%). The most effective result (70–100% virus eradication) was obtained by combining chemotherapy with cryotherapy, or by consecutive chemotherapy, combined chemotherapy and thermotherapy, then cryotherapy treatments irrespective of cultivar. Regrowth following the two best virus eradication treatments was similar ranging from 8.6 to 29% across the three cultivars. The importance of virus removal on yield was reflected in “Dunluce” free of PVS having higher numbers of microtubers and in “V500’ free of PVS and PVA having a greater proportion of microtubers > 5 mm. Our improved procedure has potential for producing virus-free planting material for the potato industry. It could also underpin the global exchange of virus-free germplasm for conservation and breeding programs.

Is It Possible to Produce Certified Hazelnut Plant Material in Sicily? Identification and Recovery of Nebrodi Genetic Resources, in vitro Establishment, and Innovative Sanitation Technique From Apple Mosaic Virus.

Eight Sicilian cultivars of hazelnut (Corylus avellana L.), namely-Curcia, Nociara Collica, Panottara Collica, Panottara Galati Grande, Parrinara, Panottara Baratta Piccola, Enzo, and Rossa Galvagno, registered into the Italian Cultivar Register of fruit tree species in 2017 were selected from Nebrodi area and established in vitro. The aim of the work was to carry out the sanitation of the cultivars and get virus-free plants from the most important viral pathogen threat, the apple mosaic virus. Virus-free plant material is essential for the production of certified plants from Sicilian hazelnut cultivars, complying the CE (cat. CAC) quality and the technical standards established in 2017 for voluntary certification by the Italian Ministry of Agricultural, Food and Forestry Policies (MIPAAF). In this study, we investigated the possibility of establishing in vitro true-to-type and virus-free hazelnut plantlets via the encapsulation technology of apexes. The in vitro shoot proliferation rates were assessed for the different cultivars, sampling periods, temperature treatments, and type of explant used for culture initiation. Viability, regrowth, and conversion rates of both conventional meristem tip culture (MTC) and not conventional (MTC combined with the encapsulation technology) sanitation techniques were evaluated.

Methods applied in the production system of virus free planting material of apple in the Republic of Moldova

Methods applied in the production system of virus free planting material of apple in the Republic of Moldova

Optimization of grapevine clonal micropropagation

Clonal micro-propagation ensures the production of genetically homogeneous, healthy virus-free planting material. This kind of propagation is influenced by genetic, physiological, hormonal, physical factors, and plant genotype. To increase the success of the process, it is necessary to optimize the methods of clonal micropropagation. The technology of clonal micropropagation of grapevine using a culture of apical meristems with a size of 0.1-0.2 mm was developed, the scheme of plant regeneration was improved, a new biotechnological techniques were developed for all stages of propagation. A method of processing of meristems with an ultrahigh frequency (microwave) electromagnetic field (EMF) in combination with a narrow-band laser was developed. Method for improving recovery from viral and bacterial infections using the growth regulator Emistim, Salicylic acid, the antibiotics Gentamicin and Cefotaxime was found. A method of water therapy was developed. The optimal parameters of intensity and duration of illumination were established. Ways of adaptation of healthy plants to non-sterile environmental conditions were optimised. Methods of testing of viral infection was improved. Techniques of planting healthy seedlings in film, greenhouses and open ground were developed. The creation of unique basic vine nursery in Ust-Donetsk region was the result of conducted studies.

Establishment of an Efficient Micropropagation System for Humulus lupulus L. cv. Cascade and Confirmation of Genetic Uniformity of the Regenerated Plants through DNA Markers

The demand for virus-free hop planting material has increased in the last few years due to its multipurpose uses. The present study aimed to establish an effective protocol for clonal propagation of cv. Cascade using only the cytokinins as PGRs in all stages of micropropagation: (i) in vitro culture initiation using single-node micro-cuttings inoculated on modified Murashige and Skoog (MSm) medium solidified with Plant agar and supplemented with 0.5 mg L−1 6-benziyladenine (BA) with 76% recorded viability of nodal explants; (ii) in vitro multiplication of multinodal shoots on MSm medium gelled with Plant agar and supplemented with different types and concentrations of cytokinins: 2 mg L−1 kinetin (KIN), 0.7 mg L−1 1-(2-Chloro-4-pyridyl)-3-phenylurea) (1 CPPU), 2 mg L−1 meta-topoline (mT) and 0.5 mg L−1 BA, which was the best variant for shoot proliferation (9.48 ± 0.78 shoots/explant); (iii) rooting and acclimatization with the best results obtained by ex vitro rooting and acclimatization of plants in the same stage in perlite (96.00 ± 0.60% acclimatized rooted plants with 100% survival under greenhouse conditions). The true-to-type nature of in vitro raised plants with the mother plant was assessed by Random Amplified Polymorphic DNA (RAPD) and Start Codon Target Polymorphism (SCoT) molecular markers, and then their genetic uniformity were confirmed.