Meristem culture

The excision of shoot tips or meristems (0.1–0.5 mm) is one of the early and frequent strategies used for virus elimination in several crop types (Kartha Reference Kartha1986; Retheesh and Bhat Reference Retheesh and Bhat2010; Hu et al. Reference Hu, Zhang, Dong, Fan, Ren and Zhu2015b). It consists of the apical dome and a few leaf primodia (Grout Reference Grout and Hall1999). Walkey and Webb (Reference Walkey and Webb1968) reported that Cucumber Mosaic Virus (CMV) can invade apical meristems with a progressive increase in gradient of virus concentrations between the dome and the primordia. Similar reports showed that the chances of virus elimination through meristem excision is inversely correlated to the size of the meristems cultured (Faccioli and Marani Reference Faccioli, Marani, Hadidi, Khetarpal and Koganezawa1998) as well as the position of the meristem on the plant (Maruthi et al. Reference Maruthi, Mohammed and Hillocks2013). Helliot et al. (Reference Helliot, Panis, Poumay, Swennen, Lepoivre and Frison2002) reported that conventional meristem excision (1 mm diameter) and culture, did not eliminate CMV in banana plants (0%) however, it produced 76% of Banana Streak Virus (BSV)-free plants. Meristem culture technique was also successfully applied to many other plants to free them of virus contaminations (Mori and Hosokawa Reference Mori and Hosokawa1977; Logan and Zettler Reference Logan and Zettler1985; Sunghun Reference Sunghun and Park2021). Meristem culture alone was sub-optimal in a few cases because of the complexity of the interactions between the infectious viruses and the host plant (Maruthi et al. Reference Maruthi, Mohammed and Hillocks2013). However, meristem excision is recommended as an initial step for virus cleaning of in vitro-grown clonal plants (Mantell et al. Reference Mantell, Haque and Whitehall1980; Quak Reference Quak, De Bokx and van der Want1987). A few reports suggest that meristem culture can result in somaclonal variation or juvenility during its regeneration process into new plant (Gribaudo et al. Reference Gribaudo, Gambino, Cuozzo and Mannini2006). Therefore, genetic assessment of in vitro plants is important for monitoring the state of the germplasm, and Diversity Array Technology Sequencing Single Nucleotide Polymorphism (DArTseq SNP) markers are a useful tool towards confirming the extent of diversity among in vitro clonal collections (Byiringiro et al. Reference Byiringiro, Uchendu, Paliwal and Abberton2025).

Thermotherapy

Thermotherapy (heat treatment) followed by excision of shoot tips or meristems has long been useful for eradication or control of virus infestation in clonal crops. It involves the use of hot water or dry hot air on virus-infested explants (Ten Houten et al. Reference Ten Houten, Quak and van der Meer1968), although not all viruses can be inactivated by heat. The viruses that are readily inactivated by heat treatment are mostly the isometric (spherically shaped) viruses as opposed to the rod-shaped viruses (Ten Houten et al. Reference Ten Houten, Quak and van der Meer1968). Heat therapy is effective because it inhibits viral RNA synthesis and thereby slows or inhibits the movement of viral particles into the meristematic region (Valero et al. Reference Valero, Ibanez and Morte2003). The possibility of using thermotherapy on any crop plant depends on the heat and dehydration tolerance of the crop(s), and the type of virus involved (Okori and Nakabonge Reference Okori and Nakabonge2016). The higher the temperature and longer the exposure period, the better the success rate for virus elimination (Dziedzic Reference Dziedzic2008; Tan et al. Reference Tan, Wang, Hong and Wang2010). For example, a 34–36 °C treatment for 20 days, followed by 0.5 mm meristem excision and culture, resulted in about 45% virus elimination in apple (Hu et al. Reference Hu, Dong, Zhang, Fan, Ren and Zhou2015a). Many cassava accessions respond well to heat treatment. Acheremu et al. (Reference Acheremu, Akromah, Yusuf and Gyasi2015) reported that cassava mosaic virus (CMV) was eliminated from 4 cassava cultivars following treatment of shoots in 35–37 °C for 3–4 weeks. Similarly, at IITA, a thermotherapy of 35–38 °C for 1–2 months (Fig. 3) is effective for cassava (Ferguson et al. Reference Ferguson, Choti and Munguti2020) and yam (Balogun et al. Reference Balogun, Maroya, Augusto, Ajayi, Kumar, Aighewi and Asiedu2017) depending on the accession. In addition, a thermotherapy regime (40 °C for 3 weeks) achieved 50% of virus-free cassava plants in a single variety while 45 °C was lethal for all tested cassava varieties (Mohammed et al. Reference Mohammed, Ghosh and Maruthi2017). Hu et al. (Reference Hu, Dong, Zhang, Fan, Ren and Zhou2015a) reported that temperatures above 38 °C did not allow growth or proliferation of apple shoot tips.

Figure 3.

Stem cuttings of Manihot esculenta Crantz undergoing thermotherapy at 38 °C for four weeks at the facility of the Genetic Resources Center, International Institute of Tropical Agriculture.

Heat treatment (30OC) of diseased cassava (Manihot esculenta Crantz) stem cuttings for virus eradication

Cryotherapy

Cryotherapy (treatment in liquid nitrogen at −196 °C, Fig. 4) is an effective technique for virus elimination in tissues of many clonal crops (Brison et al. Reference Brison, De Boucaud, Pierronnet and Dosba1997; Kaya et al. Reference Kaya, Galatali, Guldag, Celik, Naseem and Dandekar2020). The use of cryotherapy for the elimination of viruses was demonstrated with many plant types including potato (Solanum tuberosum L.) (Wang et al. Reference Wang, Liu, Xie and You2006; Bai et al. Reference Bai, Chen, Lu, Guo, Xin and Zhang2012), sweet potato (Ipomea batatas L.) (Wang and Valkonen Reference Wang and Valkonen2008), banana (Musa spp.) (Helliot et al. Reference Helliot, Panis, Poumay, Swennen, Lepoivre and Frison2002) and many horticultural crops (Feng et al. Reference Feng, Wang, Li, Wang, Yin, Cui, Li, Bi, Zhang, Li, Lambardi, Ozudogru and Jain2012). Helliot et al. (Reference Helliot, Panis, Poumay, Swennen, Lepoivre and Frison2002) reported that cryotherapy by cryopreservation of meristems using plant vitrification solution #2 (Matsumoto and Niino Reference Matsumoto and Niino2014) eliminated 30% of CMV and 90% BSV infections from banana plants cv. Williams (AAA, Cavendish subgroup) although several abnormalities were observed in the regenerated cells following cryotherapy. Bayati et al. (Reference Bayati, Shams-Bakhs and Moieni2011) obtained 97% elimination of grapevine virus A (GVA) after cryopreserving shoot tips (0.5–2.0 mm) of grapevine in liquid nitrogen regardless of the cryo-protocol used. In addition, the axillary buds of virus-infected yam (Dioscorea rotundata) were 100% free of Yam Mosaic Virus (YMV) after cryopreservation (Ita et al. Reference Ita, Uyoh, Nakamura and Ntui2020). However, the response of yam to cryopreservation is genotype dependent, with many having low regrowth responses (≥40%) following cryopreservation (Uchendu and Keller Reference Uchendu and Keller2016). Zare Khafri et al. (Reference Zare Khafri, Zarghami, Naderpour, Ahmadi and Mirzaei2024) had similar low regrowth experiences with apricot cultivars following cryopreservation with only 5–10% regrowth for the cultivars ‘Shams’, ‘Qaysi’ and ‘Ordubad’. Details of standard cryogenic protocols applicable to clonal crops including the underlying issues involved with each cryogenic technique has been reported (Reed Reference Reed2001, Reference Reed2011).

Figure 4.

Shoot tips (≤1 mm) of D. rotundata Poir (A) undergoing cryotherapy at − 196 ^OC in liquid nitrogen (B).

Major steps of virus eradication from diseased tissue of yam (D. rotundata Poir) through cryotherapy

Nanotherapy

Nanotechnology involves the use of micro or ultra-fine particles to create or modify materials. An organism produces nanoparticles naturally. Anthropogenic nanoparticles are synthetically produced using various geogenic, biogenic and pyrogenic procedures (Nowack and Bucheli Reference Nowack and Bucheli2007). The anthropogenic nanoparticles can be organic anthropogenic nanoparticles (contains carbon) or inorganic. These materials exhibit special properties such as optical and absorbing capabilities (Aslani et al. Reference Aslani, Bagheri, Julkapli, Juraimi, Hashemi and Baghdadi2014).

Nanotechnology accelerates plant growth and development during tissue culture by improving plant access to nutrition (Álvarez et al. Reference Álvarez, Tapia, Vega, Ardisana, Medina, Zamora, Bustamante and Prasad2019). Specifically, Graphene oxide nanoparticles promoted the in vitro plant growth of Arabidopsis spp. (Park et al. Reference Park, Choi, Kim, Gwon and Kim2020). Nair et al. (Reference Nair, Kim and Chung2014) reported that culture medium containing copper oxide nanoparticles (20, 50, 100, 200 and 500 mg/L), regulated the phenotype of mung bean (Vigna radiata L.) seedlings, with a gradual build-up of reactive oxygen species in the roots as the concentration of the nanoparticles increased.

In addition to the nutritional benefits of nanoparticles to the plants, they are also used for secondary metabolite production (Oloumi et al. Reference Oloumi, Soltaninejad and Baghizadeh2015), genetic transformation and as disinfectants (Tariq et al. Reference Tariq, Ilyas, Naz and Javad2020). Nanoparticles such as CuO and ZnO are capable of inhibiting growth of microbes such as B. cinerea, P. expansum, F. graminearum and P. infestans (He et al. Reference He, Liu, Mustapha and Lin2011; Jones et al. Reference Jones, Ray, Ranjit and Manna2008; Dimkpa et al. Reference Dimkpa, McLean, Britt and Anderson2013; Giannousi et al. Reference Giannousi, Avramidis and Dendrinou-Samara2013). For example, when wheat plants were treated with 100 mM ZnO nanoparticles. Results showed that the number of colonies (CFU g⁻¹) of F. graminearum in treatments with ZnO nanoparticles were lower, compared to the control (Savi et al. Reference Savi, Piacentini, de Souza, Costa, Santos and Scussel2015).

Recent whole plant studies show the antiviral properties of nanoparticles (Farooq et al. Reference Farooq, Adeel, He, Umar, Shakoor, da Silva, Elmer, White and Rui2021; Singh et al. Reference Singh, Kuddus, Singh and Choden2022b). Elbeshehy et al. (Reference Elbeshehy, Hassan and Baeshen2022) examined the effects of silver nanoparticles (200, 300 and 400 µg/l) on mosaic infection rate, infection severity, and virus concentrations of infected pepper seedlings. Higher concentrations of silver nanoparticles (300 and 400 µg/l) eliminated or limited the Pepper Mild Mottle Virus (PMMoV) ability to spread systematically within the plant cells. None of the concentrations of the silver nanoparticles tested affected the growth of the healthy plants. Reports show that the nanoparticles are absorbed into plant tissues through the root tips, cortex, lateral root junctures and wounding. They can also enter the shoot region through the cuticle, epidermis, stomata, lenticels and wounds (Ruttkay-Nedecky et al. Reference Ruttkay-Nedecky, Krystofova, Nejdl and Adam2017).

Besides in vitro studies, Siddiqui and Al-Whaibi (Reference Siddiqui and Al-Whaibi2014) tested the effects of nanosilicon dioxide (nSiO2: size- 12 nm) on the germination of tomato (Lycopersicum esculentum Mill. CV Super Strain B) seeds. The authors determined that 8 gL⁻¹ of nSiO₂ significantly improved the percentage of seed germination, germination time, germination index, seed vigour index, seedling fresh and dry weights. There is scarcity of information about the role of nanoparticles on virus elimination from vegetative propagated plants.

Chemotherapy

Unlike many microbes, plant viruses may not be controlled by just spraying chemicals on the plant surface because virus replication is closely linked to the normal metabolic processes of plants. Mendoza-Figueroa et al. (Reference Mendoza-Figueroa, Soriano-García, Valle-Castillo and Méndez-Lozano2014) evaluated the biological activity of peptides and peptidomics against viral proteins involved in infection cycle, such as the capsids or movement proteins that affect viral replication. The authors suggest that peptides provide direct protection through affinity selection against viral proteins and that the peptides were effective due to high specificity to viral targets, and their ability to block the process of replication or viral assembly (Mendoza-Figueroa et al. Reference Mendoza-Figueroa, Soriano-García, Valle-Castillo and Méndez-Lozano2014). Hu et al. (Reference Hu, Dong, Zhang, Fan, Ren and Zhou2015a) reported that all plants treated with Ribavirin for 60 days, survived and that the virus elimination rates were 74.4% for 15 μg/ml and 75.0 % for 25 μg/ml, although beyond 25 μg/ml, Ribavirin was toxic to the plants. Virazole (1-β-D-ribofuranosyl-1, 2, 4-triazole-3-carboxamide) is a highly soluble synthetic nucleoside with biological activity against a broad spectrum of DNA and RNA viruses in vitro (Huffman et al. Reference Huffman, Sidwell, Khare, Witkowski, Allen and Robins1973). Nerway et al. (Reference Nerway, Duhoky and Kassim2020) tested Acyclovir on Dahlia (Dahlia pinnata Cav.) for 30 days and determined that it had virus elimination potential because 80% virus-free plants were obtained with 40 mg/l and 90.67% with 50 mg/l. In addition, the plant survival rates were 89.67% at 40 mg/l and 49% at 50 mg/l. The authors also tested salicylic acid at 30–40 mg/l for 30 days on Dahlia. This produced 75–100% Dahlia mosaic virus (DMV)-free plantlets and 65.33% plant survival.

Radiotherapy

Radiotherapy involves the use of relatively high doses of radiation to kill disease-causing agents such as viruses. Due to the immobile nature of plants, they are sometimes exposed to undesirable environmental conditions such as irradiation. Plants try to adjust their growth and physiology to thrive in such challenging environmental condition (Duarte et al. Reference Duarte, Volkova and Geras’kin2019). The impacts of radiation and plant’s responses to ionizing radiations are not fully known, although it was mentioned that research on plant irradiation began following the discovery of X-rays in 1895 (Geras’kin Reference Geras’kin and Paoletti2024). Irradiation therapy by gamma or X-ray is particularly useful but can cause mutation induction in sterile or clean plant tissues leading to genetic alterations (Ahloowalia Reference Ahloowalia1998). Irradiation is also beneficial for the inactivation of genes inherited from parents after in vitro hybridization of protoplasts and novel gene combination. Radiation of cells at high doses without the application of radio protectors is destructive (Wickramasinghe et al. Reference Wickramasinghe, Udagama, Dissanayaka, Weerasooriya and Goonasekera2022). Abramov et al. (Reference Abramov, Fedorenko and Shevchenko1992) analysed populations of Arabidopsis thaliana (L.) Heynh exposed to radioactive contamination levels ranging from 0.02 to 240 mR h⁻¹ for frequency of embryonic lethal mutations and determined that areas of high levels of radioactive contamination caused more plant mutations compared to areas with less contamination. Thus, irradiation of cells targeted for somatic embryo and plantlet production lead to the formation of stable and solid mutants. Therefore, even though this may be a new option to explore for managing plant viruses, it can have severe consequences on plant growth and development.

Electrotherapy

Virus elimination using an electric field to treat infected plant tissues is another option (Fig. 5A–B). Virus-free plants were obtained 2–3 days after treatment with 1-4A of direct current (DC), pulsed to 6500 V h⁻¹ (Blanchard Reference Blanchard1974). It took an average of 5-20 min to eradicate viruses from infected tissues of potato (Solanum tuberosum L.) using electrotherapy (Singh and Kaur Reference Singh and Kaur2016). In a recent review of protocols, Adil et al. (Reference Adil, Singh, Anjum and Quraishi2022) reported that more than 50% of virus-infected plant tissues were cleaned through electrotherapy using a range of voltage at different time intervals. This method of virus elimination involves connecting the infected tissues directly to the electrodes or immersing them into the electrophoresis buffer containing either; Tris Acetate EDTA (Ethylenediaminetetraacetic acid), Tris Borate EDTA solution, or sodium chloride (NaCl) solution followed by electric supply (5–100 mA) to the electrophoresis tank (Mahmoud et al. Reference Mahmoud, Hosseny and Abdel-Ghaffar2009; Badarau et al. Reference Badarau, Florentina and Chiru2014; Adil et al. Reference Adil, Singh, Anjum and Quraishi2022). The most popularly used electric supply power range for different plant tissues are 10–30 mA for 5–20 min (Adil et al. Reference Adil, Singh, Anjum and Quraishi2022). Lozoya-Saldana et al. (Reference Lozoya-Saldana, Abelló, de la and García1996) reported that exposure of potato stems infected with potato virus X (PVX) to 15 mA for 5 min resulted in 60–100% of cleaned regenerated plantlets. Mahmoud et al. (Reference Mahmoud, Hosseny and Abdel-Ghaffar2009) reported a 100% recovery of PVY-infected potato stems cv. Diamond treated in electric current of 15 mA for 5 min. Meybodi et al. (Reference Meybodi, Mozafari, Babaeiyan and Rahimian2011) reported that current supply of 35 mA for 20 min was most effective electrotherapy for the elimination of PVA and PVY from some infected potato cultivars but was not so effective on a few other cultivars, suggesting that electrotherapy is genotype dependent. The virus-free cultivars had over 60% regeneration rate. In addition, Nerway et al. (Reference Nerway, Duhoky and Kassim2020) reported 85% elimination of Dahlia mosaic virus (DMV) also with a current supply of 35 mA for 20 min and obtained high survival rate (65%). Singh et al. (Reference Singh, Adil and Quraishi2022a) reported that 100% of banana explants exposed to 100 mA for 60 min were cleaned of banana bunchy top virus (BBTV). After an electric shock, the explants are surface sterilized for 1 min in 70% ethanol (v/v), followed by additional 1 min in 0.1% sodium hypochlorite (v/v) before a thorough rinse in distilled water (Mirzaei et al. Reference Mirzaei, Yadollahi, Naderpour, Kermani, Zeinanloo, Eftekhari and Eichmeier2024). The explants are then cultured in a nutrient medium for in vitro regeneration or growth to occur before the virus indexing report is processed (Magyar-Tabori et al. Reference Magyar-Tabori, Mendler-Drienyovszki, Hanasz, Zsombik and Dobranszki2021). Other authors (as recently reviewed by Adil et al. Reference Adil, Singh, Anjum and Quraishi2022), proposed several mechanisms or mode of electrotherapeutic action. It was suggested that the pH alteration in a cell sap using electric current, restrains the multiplication of virus. A rise in potato stem temperature up to 10 °C deactivates virus activity due to denaturation of the protein moiety of viral particles (Lozoya-Saldana et al. Reference Lozoya-Saldana, Abelló, de la and García1996). Bayati et al. (Reference Bayati, Shams-Bakhs and Moieni2011) reached similar conclusion after demonstrating that heat treatment caused the denaturation of the viral nucleoprotein. The plants recovered after an electrotherapeutic regime were not genetically altered (Singh et al. Reference Singh, Kuddus, Singh and Choden2022b).

Figure 5.

Exposure of clonal plant tissue, shoot tips (A) to electromagnetic field (B).

Genetic engineering

Genetic introduction of resistant genes into susceptible crops is a known strategy to control plant viruses. Positive results including partial or full resistance and tolerance to diseases following genome editing have been reported (Zhao et al. Reference Zhao, Yang, Zhou and Zhang2020; Robertson et al. Reference Robertson, Burger and Campa2022). Resistant genes sometimes are over-expressed in natural ways in a few organisms following conventional breeding methods (Mendoza-Figueroa et al. Reference Mendoza-Figueroa, Soriano-García, Valle-Castillo and Méndez-Lozano2014). Under field application, transgenic varieties of papaya showed a high resistance to the Hawaiian strains of papaya ring spot virus (PRSV) compared to the susceptible varieties (Ferreira et al. Reference Ferreira, Pitz, Manshardt, Zee, Fitch and Gonsalves2002; Mishra et al. Reference Mishra, Gaur, Patil, Gaur, Petrov, Patil and Stoyanova2016). The transgenic technology works by the insertion of the PRSV’s coat protein (CP) into the plant tissues (Tripathi et al. Reference Tripathi, Suzuki and Gonsalves2007) or by insertion of a gene fragment of the pathogen into the target crop to become a transgene with resistant attributes bestowed through RNA interference (Mishra et al. Reference Mishra, Gaur, Patil, Gaur, Petrov, Patil and Stoyanova2016).

Somatic embryogenesis

Somatic embryogenesis is the production of embryos from somatic (non-reproductive) cells such as found in the leaves, stems, etc. It have been reportedly used for decades as a virus cleaning or sanitation tool for clonal crops such as cassava and sugarcane (ElSayed et al. Reference ElSayed, Komor, Boulila, Viswanathan and Odero2015; Legg et al. Reference Legg, Kumar, Makeshkumar, Tripathi, Ferguson, Kanju, Ntawuruhunga and Cuellar2015) including the elimination of sugarcane yellow phytoplasma (Parmessur et al. Reference Parmessur, Aljanabi, Saumtally and Dookun-Saumtally2002). The production of this type of embryo can be through direct or indirect (callus formation) embryogenesis. Both the direct and indirect methods are effective for virus/viroid elimination although in the case of indirect somatic embryogenesis, cleaning may be a little problematic due to a lower plant regeneration potential (Olah et al. Reference Olah, Turcsan, Olah, Farkas, Deak, Jahnke and Sardy2022), in addition to the risk of genetic instability (Etienne et al. Reference Etienne, Guyot, Beulé, Breitler, Jaligot, Loyola-Vargas and Ochoa-Alejo2016). Somatic embryos develop from single cells; these are unlikely to harbour disease pathogens since it bypasses the vascular bundles. The level of efficiency for elimination of a broad spectrum of genetically diverse viruses and viroids using somatic embryogenesis has been reported (Olah et al. Reference Olah, Turcsan, Olah, Farkas, Deak, Jahnke and Sardy2022). The authors showed that the potential of using somatic embryogenesis for virus elimination was nearly 100% with 16 viruses although in many cases, the effectiveness of somatic embryogenesis depended on the plant genotype, infection level and the virus/viroid entities involved (Olah et al. Reference Olah, Turcsan, Olah, Farkas, Deak, Jahnke and Sardy2022). In addition, secondary embryogenesis was more effective for virus eradication (91–96%) than primary embryogenesis (81–88%) (Mutai et al. Reference Mutai, Wagacha and Nyaboga2017; Olah et al. Reference Olah, Turcsan, Olah, Farkas, Deak, Jahnke and Sardy2022). Nkaa et al. (Reference Nkaa, Ene-Obong, Taylor, Fauquet and Mbanaso2013) reported that the use of immature cassava leaf lobes resulted in 100% of clean plants. Also, Damba et al. (Reference Damba, Quainoo and Sowley2013) reported that about 88% of cassava somatic embryos produced from infected leaves were free of African cassava mosaic virus (ACMV) and East African cassava mosaic virus (EACMV).The success rate for use of nodal cuttings as explants ranged from 66-100% (Mutai et al. Reference Mutai, Wagacha and Nyaboga2017).

Common combinatory techniques

Viral problems are often symptomatic (Fig. 6A–D) but they also may be asymptomatic and mechanically transmitted through generations without caution. Viral diseases significantly decrease the yield and productivity of plants and is worse when it is a mixed infection involving multiple viruses (Wang et al. Reference Wang, Ma, Zhang, Wu, Wu, Wang and Li2011). In such cases, combined therapies are more suitable for effective removal. Combined therapies were used on apricot cultivars (Prunus armeniaca L.) infected with apple chlorotic leaf spot virus (ACLSV), apple mosaic virus (ApMV), and tobacco ring spot virus (TRSV). The authors reported that the highest virus elimination rates (90–100%) occurred with the combination of thermotherapy (38 °C for 7 days) and shoot tip culture (1.1–2.0 mm) (Zare Khafri et al. Reference Zare Khafri, Zarghami, Naderpour, Ahmadi and Mirzaei2024). Thermotherapy followed by meristem excision and culture have long been in use for cleaning infected tissues of clonal crops (Ten Houten et al. Reference Ten Houten, Quak and van der Meer1968; Ng and Hahn Reference Ng and Hahn1985). Mirzaei et al. (Reference Mirzaei, Yadollahi, Naderpour, Kermani, Zeinanloo, Eftekhari and Eichmeier2024) reported that meristem alone did not free olive cultures (cv Meshkat) with mixed infections; Arabis mosaic virus (ArMV), Cherry leaf roll virus (CLRV), CMV and Strawberry latent ringspot virus (SLRSV) but a combination of shoot tip (1 mm) and electrotherapy (35 mA, 100 V, 30 min.) removed all viruses. The benefits of combined techniques are that a larger shoot tip (≥1 mm) can be used to increase the chances of growth on nutrient medium instead of the popular 0.1 –0.25 mm which often leads to poor regrowth. Balogun et al. (Reference Balogun, Maroya, Augusto, Ajayi, Kumar, Aighewi and Asiedu2017) applied thermotherapy (36 ± 0.5 °C) at 16 h photoperiod for 21 days, followed by meristem excision on one-week old in vitro plantlets of Dioscorea spp. infected with YMV, Yam Mild Mosaic Virus (YMMV), CMV and Badnavirus (BV). One hundred percent (100%) of the samples with YMMV and 73% of the samples with YMV were recovered while the treatment effect on BV was 24% and 25% for mixed infections of BV and YMV. Hu et al. (Reference Hu, Dong, Zhang, Fan, Ren and Zhou2015a) reported that by combining thermotherapy (36 °C) and chemotherapy (ribavirin 25 μg/ml), the virus elimination efficiency was significantly enhanced (95.0%).

Figure 6.

Symptoms of virus infestation (A) Banana streak, (B) Banana Bract Mosaic, (C) Yam Mosaic and (D) Cassava Mosaic.