Meristem Tip Culture Research Articles

The Elimination of Viroids through In Vitro Thermotherapy and a Meristem Tip Culture from a New Limonime Hybrid (Citrus x limon var. limon (L.) Burm. f. x Citrus latifolia var. latifolia)

Viruses and viroids pose a significant challenge in citriculture, and their control is crucial for plant health. This study evaluated the effectiveness of in vitro thermotherapy combined with a meristem tip culture for eliminating citrus exocortis viroid (CEVd) and hop stunt viroid (HSVd) from a new limonime hybrid (Citrus x limon var. limon x Citrus latifolia var. latifolia). The elimination success was confirmed by RT-PCR assays. The in vitro elimination rate for CEVd during the shoot proliferation stage (43%) was higher than for HSVd (21%). Accordingly, in the subsequent rooting stage, the in vitro elimination rate for CEVd (50%) was higher than for HSVd (33%). Successful CEVd and HSVd eradication at a 100% rate was confirmed in the ex vitro acclimatized plants in the greenhouse. The study also established an efficient micropropagation protocol. The optimal treatment for in vitro shoot induction was 0.5–2 mg L−1 benzyladenine (BA) + 0.5 mg L−1 gibberellic acid (GA3) + 0.25 mg L−1 naphthalene acetic acid (NAA), while for shoot elongation, it was 0.5 mg L−1 BA + 0.5 mg L−1 kinetin (KIN) + 0.5 mg L−1 GA3 + 0.25 mg L−1 NAA. Rooting was best promoted by 1 mg L−1 NAA. This study provides valuable insights for the mass production of viroid-free propagation material in this new lemon x lime hybrid, contributing to the conservation of genetic resources in citrus breeding programs through the combined application of in vitro thermotherapy and an in vitro meristem tip culture, a novel and highlighted achievement reported for the first time in this study.

Tracking Sweet Potato Leaf Curl Virus through Field Production: Implications for Sustainable Sweetpotato Production and Breeding Practices.

Sweet potato leaf curl virus (SPLCV) is a whitefly-transmitted begomovirus infecting sweetpotato and other morning glory (Convolvulaceae) species worldwide. The virus is widespread at the USDA, ARS, U.S. Vegetable Laboratory (USVL), and testing of germplasm maintained in the breeding program indicates nearly 100% infection in storage roots of materials propagated for at least four years. Prior to the public release of new germplasm, viruses must be eliminated via laborious and time-consuming meristem-tip culture. The identification of virus-free seedlings early in the selection process can offer an alternative to meristem-tip culture. In this study, we investigated the transmission of SPLCV over two years of consecutive field plantings (early and late) of sweetpotato. While SPLCV is endemic at the USVL, virus transmission pressure over the typical cultivation season is unknown, and avoidance of virus transmission paired with the selection and maintenance of clean material may be a viable alternative to virus elimination. In 2022, the storage roots of 39 first-year seedling (FYS) selections were tested for SPLCV after early-season cultivation, revealing a single selection (2.6%) with a positive test. Similar testing was conducted in 2023 with no SPLCV-positive FYS selections detected. To further assess SPLCV acquisition in the field, replicated late-season plantings of each selected FYS (n = 37) were monitored from planting to harvest. Testing was conducted at 60 and 120 days after planting (DAP). Approximately 35% of the bulk samples were infected at 60 DAP, and infection increased to 52.3% by 120 DAP. Testing of individuals within selected positive bulked samples did not support 100% infection at harvest. Altogether, these results demonstrate that SPLCV transmission during early planting is sufficiently low to facilitate the maintenance of virus-free selections, offering an alternative to virus cleaning and a cultivation strategy that may be leveraged for production.

Yield performance of virus free sweetpotato (Ipomoea batatas) cultivars in the highlands of Papua New Guinea

ABSTRACT Sweetpotato (Ipomoea batatas (L.) Lam.) is the dominant staple food crop in the Highlands Provinces in Papua New Guinea (PNG), but productivity is constrained by pests and diseases including viral pathogens. Meristem-tip culture and thermotherapy can be used to eliminate viral pathogens in planting material, overcoming productivity constraints caused by viral infections. In this study, the performance of four PNG sweetpotato cultivars grown from virus-cleaned or farmer-sourced planting material were assessed at four production locations in PNG Highlands Provinces. Total root yields were significantly increased in three of the four cultivars by using virus-cleaned material, with root number and root weight increasing as well as root quality. A weak interaction between the origin region of a cultivar and trial site location was observed, suggesting that response to virus-cleaning may be lower when cultivars selected for tolerance to pathogens in a production region are grown in the same region after virus-cleaning. The results demonstrated that under traditional production practices in gardens frequently used for sweetpotato production, the use of virus-cleaned planting material is a viable practice for farmers aiming to transition from subsistence production to commercial production.

Pathogen Eradication in Garlic in the Phytobiome Context: Should We Aim for Complete Cleaning?

Global food production is challenged by plant pathogens that cause significant crop losses. Fungi, bacteria, and viruses have long threatened sustainable and profitable agriculture. The danger is even higher in vegetatively propagated horticultural crops, such as garlic. Currently, quarantine, rouging infected plants, and control of natural vectors are used as the main means of disease and pest control in garlic crops. Agricultural biotechnology, meristem-tip culture, and cryotherapy offer solutions for virus eradication and for the multiplication of 'clean stocks', but at the same time, impact the symbiotic and beneficial components of the garlic microbiome. Our research involves the first metatranscriptomic analysis of the microbiome of garlic bulb tissue, PCR analyses, and a biological assay of endophytes and pathogens. We have demonstrated that in vitro sanitation methods, such as shoot tip culture or cryotherapy can alter the garlic microbiome. Shoot tip culture proved ineffective in virus elimination, but reduced bacterial load and eliminated fungal infections. Conversely, cryotherapy was efficient in virus eradication but demolished other components of the garlic microbiome. Garlic plants sanitized by cryotherapy exhibited a lower survival rate, and a longer in vitro regeneration period. The question arises whether total eradication of viruses, at the expense of other microflora, is necessary, or if a partial reduction in the pathogenic load would suffice for sanitized garlic production. We explore this question from both scientific and commercial perspectives.

Detection and elimination of grapevine leafroll-associated viruses (GLRaVs) from Vitis vinifera cultivars of Pakistan by meristem tip culture

Detection and elimination of grapevine leafroll-associated viruses (GLRaVs) from Vitis vinifera cultivars of Pakistan by meristem tip culture

Characterization and Elimination of Grapevine fanleaf virus Using Thermotherapy in Combination with Meristem Tip Culture

Characterization and Elimination of Grapevine fanleaf virus Using Thermotherapy in Combination with Meristem Tip Culture

Elimination of Solanum nigrum ilarvirus 1 and Apple Hammerhead Viroid from Apple Cultivars Using Antivirals Ribavirin, Rimantadine, and Zidovudine.

Apple hammerhead viroid (AHVd) was detected in the apple cultivar 'Šampion' and in mixed infection with Solanum nigrum ilarvirus 1 (SnIV-1) in the cultivars 'Selena' and 'Jonagored Supra', using a high-throughput sequencing method. Experiments were conducted to eliminate both pathogens in apples using meristem tip cultures in combination with the antivirotics ribavirin, rimantadine, and zidovudine. Elimination of both pathogens was verified by repeated RT-PCR and qRT-PCR assays after 7-11 months. Elimination of SnIV-1 from all cultivars was successful with each of the three antivirotics at concentrations of 20, 40, and 80 mg L-1. Elimination of AHVd was also achieved, although less effectively and only with ribavirin in the concentration range of 20-160 mg L-1.

Development of Colorimetric Loop-Mediated Isothermal Amplification (LAMP) Technique for Rapid, Sensitive and Convenient Detection of Chrysanthemum Chlorotic Mottle Viroid (CChMVd) in Chrysanthemum

Chrysanthemum chlorotic mottle viroid (CChMVd) is a serious pathogen infecting chrysanthemum worldwide. To improve and enhance the detection procedure, the colorimetric loop-mediated isothermal amplifi cation (LAMP) technique was developed. Six LAMP primers were newly designed and tested the optimal conditions using a recombinant plasmid of CChMVd as a DNA template. The optimal conditions for colorimetric LAMP were incubation at 65°C for 60 min. At these conditions, the ladder-liked pattern LAMP products were detected along with the change of color from pink to yellow in the positive reactions. The sensitivity of colorimetric LAMP was up to 2.18 × 103 copies of recombinant plasmid concentration which was 104-fold greater than that of polymerase chain reaction (PCR). The developed colorimetric LAMP was not cross reacted to other viruse s and viroid. Effi ciency of colorimetric reverse transcription (RT)-LAMP for detecting CChMVd in chrysanthemum plantlets obtained from meristem tip culture was evaluated to verify and ensure CChMVd-free plantlets production. The results showed that 18 chrysanthemum plantlets were free of CChMVd contamination. Therefore, the colorimetric RT-LAMP can be used to detect CChMVd routinely due to its accuracy, rapidness and sensitivity.

Meristem tip culture in Phragmites australis and genetic fidelity study using SRAP markers

The improved sugarcane varieties grown in and around the world are majorly evolved through interspecific hybridization of Saccharum species.  Only few promising hybrids have been developed from allied genera such as Erianthus and Sclerostachya. Several other genera, including Phragmites, Vetivera, Narenga and Neyurida, deserve further research. Successful exploitation of these allied genera resources require careful characterization, evaluation and conservation using different methods including tissue culture techniques. Among them, Phragmites australis is used to remediate waste water and remove heavy metals. In this study, meristem tip culture was standardized for five clones of Phragmites to evaluate their response and growth. Among the five clones of Phragmites studied, two clones (IND 89-711 and IND04-1326) showed better regeneration and growth. The in vitro derived plants were compared with the mother plants using four SRAP markers to check their genetic fidelity. Monomorphic bands were obtained in both the in-vitro and mother plants. No polymorphism was detected, thus proving the genetic uniformity of the micropropgated plants. Keywords: Allied genera, Phragmites, Saccharum spontaneum, Apical meristem, Genetic fidelity

Garlic (Allium spp.) viruses: detection, distribution and remediation attempts in a European garlic collection

Garlic is an important vegetable crop in numerous countries used as food and natural based medicine. Similar to the majority of vegetatively propagated plants, garlic may be affected by several viruses that can cause severe crop losses. The present study aimed to screen 105 garlic accessions (mother plants) from 5 European countries (Germany, Czech Republic, Poland, Italy, and France) for possible presence of Onion yellow dwarf virus (OYDV), Leek yellow stripe virus (LYSV), Garlic common latent virus (GCLV) and Shallot latent virus (SLV). The occurrence of three Allexiviruses (GarV-A, GarV-B and GarV-C) in mixed assays was also investigated. Meristem-tip culture assays were performed in order to attempt eradication of the studied viruses. Garlic viruses identification was made by ELISA and RT-PCR. ELISA outcomes showed that all 105 garlic accessions were infected by different virus combinations. The OYDV and LYSV were identified, by ELISA, in all countries at 96% and 88,6% respectively and by RT-PCR at 99% and 96%. Furthermore, GCLV and SLV were detected by ELISA in about 88% and by RT-PCR at 89% and 90%, respectively with the exception of the studied Allexiviruses which were not amplified by RT-PCR with ALLEX1/ALLEX2 primers. Smaller meristem size (0,3-1,5 mm) led to better virus elimination efficiency (29%) compared to 8% obtained for the larger size (2-2,5 mm). The outcomes were opposite (16% vs. 90%) for plants regeneration. Virus elimination efficiency was linked to the virus type, e.g., OYDV and LYSV were eradicated at 90% while GCLV and Allexiviruses were difficult to eliminate (57,4% and 55,6% of eradication). Given the economic relevance of garlic crops worldwide and the frequently reported incidence of viral infections, it is important to make virus-free germplasm available. Therefore, investigating the garlic germplasm sanitary status and constantly improving it is of crucial importance aiming to increase the overall garlic production.

Protocol optimization for elimination of sugarcane bacilliform virus and rapid propagation of virus-free sugarcane using meristem tip culture

Protocol optimization for elimination of sugarcane bacilliform virus and rapid propagation of virus-free sugarcane using meristem tip culture

Detection and elimination of viruses infecting sweet potatoes in Hungary

AbstractSweet potato has been grown in Hungary for the last three decades, and its popularity is increasing among farmers and consumers. Its production is hampered by pests and diseases due to poor agricultural practices, such as the use of virus‐infected propagation materials. We tested the presence of 15 viruses by PCR and quantitative PCR in 110 sweet potato plants collected from seven regions in Hungary. Seven viruses in single or multiple infections associated with a wide range of foliar symptoms were detected: sweet potato chlorotic stunt virus (SPCSV), sweet potato virus G (SPVG), sweet potato virus C (SPVC), sweet potato feathery mottle virus (SPFMV), sweet potato virus 2 (SPV2), sweet potato leaf curl virus (SPLCV), and sweet potato pakakuy virus (SPPV). This is the first report on the occurrence of the begomovirus SPLCV in sweet potatoes in Hungary. The infectivity and identity of these viruses were confirmed through bioassays (grafting to Ipomoea setosa) and sequencing of the PCR‐amplified sections of their genomes, respectively. Due to the necessity for virus‐free sweet potato propagation material in Hungary, virus elimination was carried out successfully in five out of six genotypes important for Hungarian farmers using heat treatment and meristem tip culture. All five viruses detected in the plants before heat treatment were removed except SPPV, which persists after heat treatment. Production and strict regulation of virus‐free sweet potato propagation materials are recommended to avoid exacerbating the virus situation and protect Hungarian farmers from further losses.

Effect of Different Treatments on Producing in vitro Healthy Vitis vinifera L. cv. Halwani Free From Grapevine Leafroll-Associated Virus-3

El-Samin, M., K. El-Maari and F. El-Biski. 2022. Effect of Different Treatments on Producing in vitro Healthy Vitis vinifera L. cv. Halwani Free From Grapevine Leafroll-Associated Virus-3. Arab Journal of Plant Protection, 40(4): 334- 339. https://doi.org/10.22268/AJPP-40.4.334339 This study was carried out during the period 2020-2021, to produce virus-free propagation material from Halwani cultivar of grape infected with Grapevine leafroll-associated virus 3 (GLRaV-3), by using the meristem tip culture technique together with thermal and chemical treatments. The meristem tip of 500 micrometers was excised from infected plants and placed on 1/2 MS medium which contains 0.5 mg/l BAP and 30 g/l sucrose. Virus presence or absence was confirmed by the ELISA test (DAS-ELISA). Results showed that the highest Percentage of virus free plantlets (87.67%) was obtained from the combined treatment, whereas the thermal treatment resulted in the lowest percentage of virus-free plants (56.25%). The results obtained also showed the significant superiority of using meristem-tip culture compared with all other treatments in terms of percent growing plants, which reached 98.13%. This study indicated that there were negative effects of all treatments on plant elongation, especially the thermal treatment, which gave the least plant growth elongation (0.33 cm). Keywords: Grapevine leafroll-associated virus, grape, meristem, thermal treatment, chemical treatment, DAS-ELISA

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.

Accelerated in vitro thermotherapy and indexing against apple chlorotic leaf spot virus in Shiro plum

Virus and virus-like diseases are major production constraints in vegetatively propagated crops; once a plant is infected, it remains so throughout its life. This work was undertaken to accelerate the process of eliminating apple chlorotic leaf spot virus (ACLSV) from explants obtained from an infected plum tree by in vitro heat therapy combined with meristem tip culture. After a minimum of 1 week of in vitro heat treatment followed by microdissection of meristematic tissues, trees were propagated successfully into virus-free plants. The treatment procedure, from infected plant to virus-free plantlets, takes a minimum of 14 weeks and could be applied to other fruit tree crops. The results of a one-step reverse transcription PCR analysis of treated plants after three accelerated growth cycles indicated the absence of ACLSV. Heat treatment of the test plants, followed by three rounds of indexing, was accomplished in about 28 months, a procedure that could take ≥36 months using conventional methods. The techniques described in this work may help to speed up the treatment of infected plants and screening of planting materials. The application of these procedures will benefit regulatory agencies, allow stakeholders to have timely access to virus-tested material, and facilitate domestic and international trade.

Elimination of phytoplasmas in brinjal by meristem-tip culture incombination with thermotherapy

The main aim of the study was to eliminate the phytoplasmas in brinjal using meristem-tip culture in combination with thermotherapy. Brinjal shoot tips were taken from field grown plants and subjected to different temperature/time treatments followed by meristem tip excision and culture in a callus inducing medium. The 45°C for 2 minutes showed the minimum number of days for callus induction and the maximum survival percentage of explants. MS medium with BAP 1 mgl−1 and kinetin 1 mgl−1 recorded the maximum shoot initiation percentage at all temperature treatments. The MS medium with BAP 2 mgl−1, kinetin 1 mgl−1 and NAA 0.5 mgl−1 recorded minimum number of days for multiple shoot formation and produced more shoots per callus clump. For root formation, half concentration in MS medium supplemented with IBA 0.5 mgl−1 and IAA 0.5 mgl−1 recorded lesser number of days for root initiation and produced more roots per shoot. Effective regeneration was recorded at 45°C for 2 minutes. To confirm the elimination of phytoplasmas, DNA was extracted from these plants and from little leaf diseased brinjal plants. The molecular detection was done through PCR and the phytoplasma infected plant sample produced the expected length amplification products while no amplification was observed in the in vitro grown plants.

English

Sweet potato (Ipomoea batatas L. Lam) has become one of the staple crops in Africa in the last 20 years. In Ethiopia, sweet potato is the second most widely grown root crop and is the first regarding the production per hectare. Thus, there is a great demand of planting material throughout the country. Currently, planting material is usually obtained from own previous season harvest, local markets or from the neighboring fields since no certified clean planting material production scheme has been established in Ethiopia yet. Unfortunately, this practice has contributed to the spread of viral diseases throughout the country. Elimination of viruses from infected plants is a tedious job, which requires efficient methods to eliminate the virus and also to verify that the plants are indeed virus-free. In the case of sweet potato, it was observed that heat treatment, combined with meristem tip culture is an efficient method for virus elimination. Previous findings indicate that reverse transcription (RT) PCR is more efficient than ELISA to verify the efficiency of virus elimination. In this study, the use of next generation sequencing (NGS) was explored as a verification method and compared with RT-PCR. The results show that NGS seems to be more efficient than RT-PCR, although also prone to inconclusive results.   Key words: Viruses, next generation sequencing (NGS), sweet potato, reverse transcription (RT) PCR, badnavirus.

Micropropagation and pathogen elimination in elderberry (Sambucus nigra L.)

To date, more than 75% of Austrian elderberry acreage is planted with cultivar ‘Haschberg’. This leads to high costs during harvest, since most of the berries have to be harvested at the same time and need to be frozen within hours after picking. In order to improve the spectrum of elderberry cultivars micropropagation of five elderberry cultivars was optimized. To meet the requirement for certified planting material, thermotherapy and chemotherapy (ribavirin), followed by meristem tip culture, were succesfully applied to eliminate various plant pathogens from elderberry plants. Virus detection was carried out by RT-PCR and confirmed by electron microscopy. An efficient protocol was developed using the cytokinins, BA for multiplication and meta-Topolin for shoot elongation and rooting of Sambucus nigra L. plants. Two commercial arbuscular mycorrhizal inoculants were evaluated for their potential to improve the process of ex-vitro-acclimatization. Both inoculants significantly enhanced survival rates, shoot length and root colonization levels of elderberry plants. Pathogen free elderberry plants were produced via thermotherapy or chemotherapy followed by meristem culture. Culture media for micropropagation were optimized. Acclimatization of plants was significantly improved by artificial inoculation with AMF products.

Production of virus-free cassava through hot water therapy and two rounds of meristem tip culture

Cassava genetic resources are highly threatened by devastating viral diseases. It is therefore important to conserve the specifically farmer-preferred landraces/cultivars that are highly susceptible. The study assessed the efficiency of hot water therapy and meristem tip culture techniques in the elimination of cassava mosaic disease (CMD) and cassava brown streak disease (CBSD) causal viruses. Stems of symptomatic cassava genotypes confirmed infected with cassava brown streak viruses (CBSV) and cassava mosaic viruses (CMV) were subjected to hot water therapy at varying temperatures (25°C—60°C) followed by two rounds of meristem tip culture (MTC). Data on sprouting in the screen house and response in vitro were recorded and weaned plants were indexed for CMV and CBSV. The results indicated 100% elimination of CMV and a significant reduction in the viral load of CBSV. Thus, these techniques could be integrated into the cassava conservation strategy, for sustainable management of cassava genetic resources.

Efficacy of thermotherapy combined with chemotherapy and meristem tip culture in reducing Cassava brown streak virus in infected cassava

Thermotherapy, chemotherapy and meristem tip culture have been used either alone or in combination to eliminate viruses from plants. . The objective of this study was to determine the efficacy of combining thermotherapy with meristem tip culture and chemotherapy on the reduction of CBSV from infected cassava.Cassava Brown Streak Virus infected cuttings of Guzo variety collected from Coast region of Kenya, were established and maintained in a greenhouse at the Plant Quarantine Station, Kenya Plant Health Inspectorate Service (KEPHIS) in Muguga. Cassava leaves were sampled from eighteen plants and virus indexing done using RT-PCR with virus specific primers. Those confirmed to be positive for CBSV were used as initiation materials for the prospective test plants. From the initiated tissue culture materials, the 2nd subcultures were subjected to heat treatment at 38°C for 21 days. They were later subjected to ribavirin treatment at varying concentrations of (10mg/l, 20mg/l, and 30mgl) then left to establish for 14 days. Meristems of 1mm were excised from heat treated plantlets at 38°C for 21 days and cultured in vitro in modified Murasgige and skoog media. Nodal plantlets of 10mm not subjected to any treatment were used as controls. There was a significant difference (P<0.01) in the number of plants that survived among the treatments. Thermotherapy combined with chemotherapy resulted in complete mortality of plants due to the high stress levels from the high temperatures combined with the phytotoxic effect of the ribavirin. Thermotherapy followed by excision of meristem tips (1mm) resulted in 68.8% shoot survival with 84% being virus-free. Thermotherapy combined with meristem culture was successfully employed to produce CBSV-free cassava plants. Key words: Cassava Brown steak virus, Chemotherapy, meristem culture, Thermotherapy