Propagation through seed production and vegetative methods are the two commonly used methods for lily plant reproduction [3]. However, reliable in vitro techniques have been widely used in combination with other complementary techniques, including advanced virus detection methods and genetic engineering, in order to expedite lily multiplication, ensure disease-free crops, and shorten breeding programs [1,25,26,27,28,29]. In particular, the detection of viruses during the in vitro culture process holds significant importance, as it plays a vital role in generating virus-free lilies [3].

To control plant viral diseases and produce healthy plants free of viruses, the indexing process in lilies focuses on both axillary and apical meristems of the parent plant, the resulting plant, as well as the plant coming from the seed [30,31]. Viruses can be transmitted through the parent plant or the seed itself to the new plant, making indexing essential. By indexing meristems, newly grown plants, or seeds, experts can evaluate their virus-free status and ensure their health [32,33]. This is particularly crucial for viruses that can be vertically transmitted from the parent plant to the seed. The specific indexing practices may vary based on the type of virus, the plant species, and the desired level of assurance for producing healthy plants free of viruses [30,31,32]. Additionally, producing healthy seeds that have been indexed and shown to be free of known viruses is a common method for controlling plant viral diseases. Indexing methods involve selecting a representative subset of seeds from the batch for testing, typically based on statistical principles to ensure accuracy. Additionally, indexing includes the use of resistant indicator plants or acquiring seed, cuttings, or plants from certified virus-free sources [30,31,32,33]. Furthermore, ongoing investigations are exploring the elimination of lily virus through tissue culture-based methods.

Thermotherapy, meristem tissue culture, and chemotherapy have been developed for lily virus elimination [5,34,35]. Studies show that the efficiency of the therapy depends upon the type of virus, the explants used for culture, and also on the hybrid infected [34,36,37]. In the case of eradicating lily symptomless virus (LSV) from Asiatic hybrids, in vitro culture of regenerated shoots from infected bulbs’ internal scales was employed successfully. However, this method was found to be ineffective in eliminating LSV from L. longiflorum Asiatic hybrids, such as ‘Ace’ and ‘Nellie White’ [36]. The above techniques can be used alone or as modified complex therapies [38,39,40]. Moreover, thermotherapy, meristem tissue culture, and chemotherapy techniques can be applied in in vitro culture systems or ex vitro environments [36].

The use of meristem tissue culture is an important in vitro technique that consists of culturing on a nutrient medium a small (0.1–0.5 mm) piece of tissue removed from apical buds or nodal segments with an axillary bud [35,39]. Research has shown that the probability of obtaining plants without virus infection is inversely proportional to the size of the excised meristem [38]. Virus elimination by meristem tissue culture is challenged by the difficulty of tissue excision, the small size of the excised meristem, and the risk of contamination. These challenges can be overcome through careful handling of the plant material using a sharp, sterile scalpel or razor blade to excise the meristem. This requires skill and experience, as the meristem is a delicate tissue that can be easily damaged. Another solution is to use ex vitro scaling from tissue culture of bulb scales to obtain meristem tips. This method involves growing bulbs in tissue culture, then transferring the bulb scales to a new medium to induce the formation of meristem tips. This can be a more reliable and efficient method than direct meristem excision, as it allows for the production of multiple meristem tips from a single bulb.

In addition, it is highly recommended to use appropriate culture conditions to reduce the risk of contamination throughout the tissue culture process. This includes sterilizing all equipment and culture media and working in a laminar flow hood or other sterile environments [41]. Moreover, meristem tissue culture can be used alone or in combination with other techniques such as chemotherapy, thermotherapy, and cryotherapy to further reduce the risk of virus infection [36,42]. Studies have examined the elimination of lily mottle virus (LMoV) and lily symptomless virus (LSV) from lily plants through meristem tip culture. The results indicate that this technique is effective, cost-effective, and carries a minimal risk of contamination when utilizing meristem tips obtained from ex vitro scaling of bulb scales in tissue culture [36,39].

Studies have shown the relationship between high temperature and viral RNA silencing, and high temperatures are frequently associated with low virus content [6]. Therefore, thermotherapy is one of the conventional in vitro methods used for virus elimination in different plants [30]. For example, a recent study has explored virus-induced gene silencing (VIGS) and its potential for manipulating gene expression without transgenesis. While VIGS has been widely used in various plant species, its efficiency can be influenced by developmental, physiological, and environmental factors. The researchers investigated the impact of temperature on two forms of gene silencing, ViPTGS and ViTGS, using GFP as a reporter gene in N. benthamiana 16c plants infected with recombinant tobacco rattle viruses (TRVs). They found that high temperatures impair ViTGS due to a lack of secondary siRNA production, hindering the transmission of gene silencing to subsequent generations. Understanding the environmental influence on gene silencing mechanisms could aid in optimizing ViTGS for transgene-free crop improvement. Additionally, this highlights the potential application of thermotherapy in eliminating viruses, as higher temperatures negatively impact ViTGS efficiency [43]. Thermotherapy can be used alone or in combination with other techniques such as meristem tissue culture or chemotherapy to eliminate lily viruses [38]. However, one major challenge that must be addressed is that the extended duration of thermotherapy at a high temperature leads to significant impacts on plant regeneration, growth, and overall survival, thus, an appropriate period must be further researched to establish the plant regeneration time during temperature-based therapy [37,38]. Elimination by meristem tip culture of LSV from Asiatic hybrids has been enhanced by the application of thermotherapy using bulbs obtained ex vitro [36,44]. However, a high-temperature treatment during ex vitro scaling has a negative effect on plant regeneration. Studies have shown that the effectiveness of thermotherapy varies with plant genotype [8,39], which means that different lily varieties may require different temperatures or durations of treatment for effective virus elimination. Therefore, it is important to determine the appropriate conditions for each plant genotype to ensure successful virus eradication.

Chemotherapy is an important in vitro culture method with a defined concentration of antiviral drugs used for the production of virus-free plants. It is used alone or in combination with other therapies [36]. Other chemical control methods involve the use of pesticides to manage the vectors or the virus itself. This can be carried out by using insecticides to regulate the vector insects or by using antiviral compounds to reduce the viral load in infected plants [45]. Although successful in some instances, chemotherapy possesses some challenges and limitations. For example, one of the major challenges in chemotherapy is finding the appropriate concentration of antiviral drugs that can effectively eliminate viruses without adversely affecting plant growth and development. Furthermore, some viruses may develop resistance to antiviral drugs, making chemotherapy ineffective in certain cases [46]. Another challenge is the phytotoxicity of the antiviral drugs used, which can have negative effects on plant regeneration and survival. These effects may include stunted growth, reduced photosynthesis, cellular damage, and even plant death [47]. For example, the application of 2,4-dioxohexahydro-1,3,5-triazine (DHT) or virazole was used to eliminate CMV and LSV infection in L. longiflorum, and the eradication effect of DHT on CMV and LSV is higher than that of virazole [35,39].

To overcome these challenges, researchers have experimented with different antiviral drugs and concentrations to determine the most effective and least toxic ones. They have also investigated the utilization of combinations of antiviral drugs to enhance the effectiveness of treatment and minimize the risk of viral resistance. Furthermore, integrating chemotherapy with other approaches like meristem tissue culture or thermotherapy can enhance virus elimination and shorten the duration of treatment [48].

Previous studies have shown that a combination of callus culture with chemotherapy was used to eliminate CMV from L. longiflorum [34]. When chemotherapy was combined with meristem tip culture, a noticeable decrease in the presence of lily symptomless virus (LSV) and/or tulip breaking virus (TBV) was observed in L. longiflorum ‘Arai’. However, the same technique was ineffective to eliminate the above viruses from the Asiatic hybrid ‘Enchantment’. According to reports, chemotherapy demonstrated higher efficacy when applied to bulbs obtained through in vitro scaling compared to ex vitro scaling [36].

The development of combined in vitro methods for lily virus elimination has shown promising results in effectively eradicating these viruses. However, this development is accompanied by certain challenges that need to be overcome. One of the main impediments is the optimization of each individual method to ensure their synergistic action rather than conflicting with one another in the elimination of lily viruses [36,49].

Researchers such as Masuda et al. (2011) have reported on the application of a combination of chemotherapy methods with callus culture for eliminating cucumber mosaic virus (CMV) in L. longiflorum [39]. Various techniques, including callus culture, meristem culture, and their combinations with thermotherapy or chemotherapy have been developed [34,35]. Notably, the combination of thermotherapy with meristem culture has been studied and found to be more efficient than using these methods individually [35,44]. This involves the excision and propagation of meristems, followed by a second round of meristematic tissue culture and subsequent application of thermotherapy to produce virus-free mother lily plants. In the case of lily symptomless virus (LSV) and lily mottle virus (LMoV) elimination from infected lily bulblets, the establishment of a successful complex therapy involved the combination of meristem tip culture with chemotherapy [39]. This approach effectively eliminated these viruses from the infected lily bulblets.

The development of combined in vitro methods for lily virus elimination requires the careful optimization of each individual method to ensure their synergistic action in targeting and eliminating specific viruses or virus combinations. This optimization process fine-tunes the parameters and conditions of each technique, maximizing their efficacy and minimizing potential drawbacks. Strict contamination control measures and a comprehensive understanding of the biology and behavior of each virus are also crucial aspects to consider. By harmoniously integrating different methods, researchers can achieve maximum success rates in producing virus-free lily plants, contributing to the overall health and resilience of lilies.

To manage lily viruses, various approaches can be employed, including producing virus-free bulbs, eliminating infected plants and weeds, utilizing meristem culture, chemotherapy, and thermotherapy. Additionally, strategies such as genetic transformation, quarantine measures, cultural control methods, and biological control methods can be implemented to mitigate the impact of viruses. These approaches offer additional avenues for effectively managing lily viruses, complementing traditional control methods, and contributing to sustainable and integrated disease management strategies in lily cultivation.

Genetic transformation is a promising tool that can be used to enhance the resistance of lily plants to viral infections. By introducing genes that encode resistance factors, it is possible to confer durable resistance to viruses. However, the genetic transformation of lilies is challenging due to their recalcitrance to regeneration, low transformation efficiency, and the lack of a comprehensive genome sequence. Despite these challenges, progress has been made, with successful transformation reported in several species. For example, Gladiolus plants transformed with a defective replicase and protein subgroup II gene were found to be resistant to cucumber mosaic virus (CMV). In another study Azadi et al. (2011) reported the successful transformation of Lilium cv Acapulco with a defective cucumber mosaic virus (CMV) replicase gene (CMV2-GDD) constructed using Agrobacterium tumefaciens that confers resistance to CMV. The results suggested that the two lines have increased resistance to CMV and that the CMV-GDD replicase gene is an effective construct that provides protection against CMV in Lilium [51]. In other studies, Agrobacterium-mediated transformation was used to generate improved lily virus resistance [52,53]. These studies demonstrate the potential of genetic transformation as a tool for improving the resistance of lily plants to viral infections, and further research is needed to optimize transformation protocols and identify new resistance genes.

Quarantine measures involve isolating infected plants and preventing them from coming into contact with healthy plants. This can be carried out by physically separating infected plants, using physical barriers, or restricting the movement of plants and plant material from infected areas [54]. Other strategies include crop rotation, controlling weeds, destroying old crops, avoiding planting new crops on diseased plantings [55], and controlling vectors [56]. All these strategies help in eliminating and controlling lily viruses by reducing viral reservoirs and preventing transmission by carriers such as aphids.

Cultural control methods involve modifying the growing environment to make it less favorable for the virus or its vectors. This can be carried out by selecting disease-resistant varieties, planting at the appropriate time, crop rotation, improving soil fertility and drainage, and using appropriate irrigation and fertilization practices. More than any other form of control, cultural methods emphasize that the objective of horticulture is to produce fruitful, high-yielding crops rather than simply to control plant pathogens. These controls act largely in a preventive manner and are applied in advance of invasion [57]. Moreover, cultural control practices such as sanitation, proper plant hygiene, and the removal of infected plants (roguing) help prevent the buildup of virus reservoirs and decrease the chances of viral spread through vectors or mechanical means [58]. In the case of lily viruses, the use of cultural control methods has been limited due to the lack of known resistant varieties [36]. However, recent studies have shown that certain cultivars of lilies may exhibit some level of resistance to specific viruses [59,60]. Therefore, the selection of resistant varieties can be a potential solution for the cultural control of lily viruses.

Another challenge in the cultural control of lily viruses is the difficulty in early identification of infected plants. This is due to the lack of visible symptoms in some cultivars, which can result in the spread of the virus to other plants before detection [61]. To overcome this challenge, it is recommended to carry out routine virus testing, using methods such as ELISA and reverse transcription loop-mediated isothermal amplification (RT-LAMP) [29]. Improving soil fertility and drainage can also be a solution for cultural control of lily viruses. This can be achieved by ensuring proper crop rotation, use of organic fertilizers, and avoiding overwatering. By improving soil conditions, the plants can be healthier and less susceptible to viral infections [58]. While improving soil fertility alone may not directly eliminate lily viruses, it can contribute to a more robust plant defense system, reducing the severity and impact of viral infections [62,63].

Biological control methods involve the use of natural enemies to control the virus or its vectors. This can be carried out by introducing predators or parasites that feed on the vectors or by using beneficial microorganisms that compete with the virus for resources or produce antiviral compounds [64]. The use of virus-free suckers, entomopathogens, predators, and parasitoids has been applied to control viruses [65,66]. In addition, predators, parasitoids, entomopathogenic fungi, and hyper-parasitoids are also used as good natural enemies to control aphids [67]. Biological control has been used to suppress virus infection in various plants. For example, the use of entomopathogenic fungi has been shown to be effective in controlling viruses from the genera Orthotospovirus, Ilarvirus, Alphacarmovirus, and Machlomovirus that affect a Western flower thrip, Frankliniella occidentalis (Pergande) [68]. These agents can be categorized into two groups: macrobials, which include predators and parasitoids, and microbials, such as fungal pathogens and entomopathogenic nematodes [69]. Notably, macrobials like anthocorid bugs (Orius spp.), green lacewing species, predatory phytoseiid mites, and predaceous laelapid mites have shown significant efficacy in combating thrips infestation at different life stages [70,71,72,73,74,75]. These biological control agents specifically target first instar thrips on foliage and thrips pupae in soil, offering promising potential for managing plant viruses and their vectors [76].