Somatic Embryogenesis: A Tool for Fast and Reliable Virus and Viroid Elimination for Grapevine and other Plant Species

Somatic embryogenesis (SE) is a widely used technique in plant biotechnology, and it can be a possible tool for virus and viroid elimination. This review highlights the advantages and limitations of production of pathogen-free plants using somatic embryogenesis. Beside the well-known meristem cultures, chemotherapy, thermotherapy and cryotherapy, SE is a very effective virus and viroid elimination method. Production of virusand viroid-free plants is categorized based on the latest virus taxonomy. The available information on virus and viroid spread in calli and the effect of SE on morphological and genetic stability of the regenerated plants are presented in details. A number of factors which could affect the efficiency of this technique are also pointed out. Based on the collected and analysed data, SE can be a useful option for virus and viroid elimination.


Introduction
The backbone of economical crop production is high-quality propagating material. However, climate change and ongoing globalization are driving crucial changes on crop diseases including viruses through the spread of the host plants and vectors, the intraspecific and interspecific competition, and the latitudinal movements of the pathogens. These changes may affect the occurrence of different viral entities in plantations and in wild plant populations [1]. Climate change has also a strong influence on food security, but the resilience of agro-ecosystems can be increased by appropriate variety utilization [2].
In case of vegetatively propagated plants, suitable plant protection techniques are not available against viruses and viroids, thus different tissue culture-based methods including somatic embryogenesis (SE) are used for the sanitation of candidate materials. The different stages of plant breeding (crossbreeding, clonal selection etc.) require virus-and viroidfree propagation material, but using only one type of the tissue culture-based elimination treatments results in a good chance of failing, due to the significant differences in the genetic background of diverse genotypes or the metabolism modifying effects of mixed infections in the mother plants. Thus, these conditions are able to alter the effectiveness of methods based on tissue culture, such as meristem and shoot tip cultures, thermotherapy, chemotherapy, or cryotherapy [3][4][5].
The phenomenon of somatic embryogenesis (SE) was described at the end of 1950s [6], and became a widely used technique in plant biotechnology for different purposes [7,8], such as the controlled alteration of genetic background, fast propagation or isolation of different mutations, but as well as an effective method for virus and viroid elimination used mainly in the case of vegetatively propagated plants. This tissue culture method has been mostly successfully applied on avocado (Persea americana, Lauraceae), black pepper (Piper nigrum, Piperaceae), cassava (Manihot esculenta, Euphorbiaceae), different citrus species (Citrus spp., Rutaceae), cocoa (Theobroma cacao, Malvaceae), garlic (Allium sativum, Amaryllidaceae), grapevine (Vitis spp., Vitaceae), and sugarcane (Saccharum spp., Poaceae) to eliminate 31 different viral entities causing serious diseases (Table 1).  [26] Up until now numerous experiments have been presented by different laboratories. In this work we review the main results, advantages and disadvantages of this virus elimination method to support the varietal exchange adopting to climate change. In the case of grapevine, the published information is based partially on our unpublished data.
The greatest advantage of SE is its outstanding efficiency and reliability in production of virus-free plants. However, the genotype dependence of this method and the likely higher rate of possible mutations may be a limiting factor in some cases.

Detection of Viruses and Viroids in Embryogenic Callus and Somatic Embryos
Limited data are available about the transmission of viral entities in callus and embryo cultures; however, the different factors (age of calli, culture maintenance time, secondary embryogenesis etc.) that affect the spread of the viruses and viroids during SE are crucial to the effectiveness of eradication.

DNA Viruses
Secondary embryogenesis was more effective for virus elimination (91-96%) than primary SE (81-85%), when somatic embryos were derived from cocoa swollen shoot virus (CSSV) infected cocoa trees [27], and CSSV infected embryos occurred more frequently if SE was induced from older callus cultures [28]. In other experiments, PCR-based CSSV detection showed 30-60% and 40-50% infection in the induced calluses and primary embryos, respectively. However, only 0-19% of secondary cocoa embryos were PCR-positive, depending on the plant genotype, while CSSV could not be detected in regenerated plantlets [29]. Similarly, subculturing cyclic somatic embryos improved the effectiveness of elimination in the case of piper yellow mottle virus (PYMoV) [30]. In cassava, 87.5% of somatic embryos proved to be African cassava mosaic virus-(ACMV) and East African cassava mosaic virus-(EACMV) free, and elimination efficiency was probably reduced when longer regeneration periods were observed or older calli were used [31]. The average effectiveness of secondary somatic embryogenesis was higher (93.9%) than primary somatic embryogenesis (87.6%) [32]. Based on these results, it is also feasible that the development of somatic embryos from healthy cells is more frequent; as such secondary or cyclic embryogenesis probably increases the ratio of regenerated virus-free embryos.

Closteroviridae
Gambino and coworkers [33] observed that embryogenic callus lines after six-month propagation were free from grapevine leafroll-associated virus-1 (GLRaV-1). In this observation, four-month-old ovary-derived calli were found to be infected by grapevine leafroll-associated virus-3 (GLRaV-3), while eight-month-old calli proved to be virus-free. Anther-derived calli showed lower levels of GLRaV-3 infection after four months [34], and GLRaV-3 was not present in somatic embryos regenerated from calli which also contained infected and healthy cells [33]. In contrast, the translocation of GLRaV-s was observed after around 15 repeated transfers of grapevine callus tissue, but viruses were not detectable beyond that [35]. The reason for the lower level of GLRaV-3 infection in anther derived callus is unknown, since both anthers and ovaries were virus infected. According to the authors, the larger size of the ovaries may lay in the background [34].

Betaflexiviridae
Embryogenic callus lines after six-month propagation were free from grapevine virus A (GVA), and GVA was not present in somatic embryos regenerated from calli containing infected and healthy cells [33]. Four-month-old ovary derived calli were infected by grapevine rupestris stem pitting-associated virus (GRSPaV), while eight-month-old calli were proved to be virus-free. Similarly to GLRaV, anther derived calli showed lower amount of GRSPaV after four months [34]. Long term callus cultures proved to be effective for virus elimination, especially for phloem limited viruses, whose movement may be inhibited or slow in the callus. The required culturing time may depend on the virus species and the proliferation ratio of the callus.

Tymoviridae
In the case of phloem limited grapevine fleck virus (GFkV) only a part of the embryogenic calli contained virus particles and there was a fluctuation in ELISA measurements during the propagation. Interestingly the abnormal embryos were GFkV infected, and their conversion to plants was inhibited. Direct embryogenesis was more effective than indirect embryogenesis for GFkV elimination [36]. For the strictly phloem-limited GFKV, the indirect SE seems to be a good strategy for virus elimination due to the lack of vascular tissues in the embryogenic cell mass. The higher efficiency of direct embryogenesis may be justified by the fact that only healthy cells formed embryos, and the very fast regeneration process limited the probability of GFKV spreading.

Secoviridae
For grapevine fanleaf virus (GFLV), the tested embryos and plantlets were GFLVpositive when SE was applied without heat therapy [37]. This indicates that GFLV was able to spread from cell to cell in the tissue culture. In this experiment, GFLV transmission to embryos was highly effective at room temperature despite the application of indirect embryogenesis, which may be explained by the negative effect of the age of embryogenic cultures. More time (90 days) was needed from the callus stage to somatic embryo germination at room temperature, while the time requirement was 20 days shorter in the case of the heat treatment. Gambino and colleagues [33] found that embryogenic callus lines after six-month maintenance were free from GFLV. In their experiments, only one GFLV infected plant was regenerated from 70 embryos, and most of the GFLV-positive abnormal embryos could not develop into plantlets [33].

Negative Sense RNA Viruses
In the case of citrus psorosis ophiovirus (CPsV) [38] all produced embryogenic callus lines proved to be infected.

Viroids
The persistent presence of avocado sunblotch viroid (ASBVd) was detected by RT-PCR in embryogenic cultures, somatic embryos, and regenerated plants in avocado, and high a sequence variability of this viroid was identified in the regenerated plants, which may suggest that in vitro conditions can stimulate the formation of sequence variants [39]. Despite indirect embryogenesis, all of the regenerated embryos proved to be infected; based on this, the virus titre in nucellar tissues may have been high, and the application of liquid cultures probably stimulated the spread and genetic variability of this viroid.
Hop stunt viroid (HSVd) and grapevine yellow speckle viroid (GYSVd) were also detected in embryogenic and non-embryogenic calli, but regenerated embryos and plants were free from them [40]. Based on this, it is more difficult for HSVd-and GYSVd-infected cells to develop into embryos, similar to some viruses. No viroids (citrus dwarfing viroid-CDVd, citrus exocortis viroid-CEVd and HSVd) were detected by RT-PCR in the greenhouse which maintained regenerated Citrus plants 6 months after grafting. However, 18 months after grafting, one of the three viroids was detected in 3-24.2% of tested plants [41], showing a presumably rising concentration of viroids in some older plants with more developed vascular systems.

Healthy Plant Production: Viruses and Viroids in SE Based Elimination Experiments
Until now elimination efficiency of SE for 25     These species show high diversity in nucleic acid structures with single-(Geminiviridae) or double-stranded DNA (Caulimoviridae), and with positive-sense (Betaflexiviridae, Bromoviridae, Closteroviridae, Luteoviridae, Potyviridae, Secoviridae, Tymoviridae), negative-sense (Aspiviridae), or non-coding single-stranded RNA genomes (Avsunviroidae, Pospiviroidae). These viruses and viroids are responsible along with others, for the cassava mosaic, citrus psorosis, cocoa swollenshoot, garlic mosaic, grapevine infectious degeneration, piper yellow mottle, and sugarcane yellow leaf diseases ( Table 1). The These species show high diversity in nucleic acid structures with single-(Geminiviridae) or double-stranded DNA (Caulimoviridae), and with positive-sense (Betaflexiviridae, Bromoviridae, Closteroviridae, Luteoviridae, Potyviridae, Secoviridae, Tymoviridae), negative-sense (Aspiviridae), or non-coding single-stranded RNA genomes (Avsunviroidae, Pospiviroidae). These viruses and viroids are responsible along with others, for the cassava mosaic, citrus psorosis, cocoa swollenshoot, garlic mosaic, grapevine infectious degeneration, piper yellow mottle, and sugarcane yellow leaf diseases ( Table 1). The majority of viruses examined in SE experiments cause serious symptoms reducing crop yield, but some of them have no known or major effect on crop quality and/or quantity.

DNA Viruses Tested in SE Experiments
ACMV and EACMV represent Geminiviridae with single-stranded DNA genomes. CSSV and PYMoV belong to Caulimovirdae, containing double-stranded DNA. In Geminiviridae, the effectiveness of SE was 100% if immature leaf lobes were used as starting material [42], but in the case of nodal cuttings, the elimination rate was lower: 66-100%, depending on the genotype [32]. In Caulimoviridae, CSSV elimination was 100% with different cocoa genotypes, but in the case of piper the efficiency (55-100%) proved to be genotype dependent (Table 2).

Positive Sense RNA Viruses
In Betaflexiviridae, the elimination ratio of different viruses was 100% or almost 100%, with the exceptions of the carlavirus garlic common latent virus (GCLV, 0-50%) and the foveavirus GRSPaV. In our experiments ( Table 2) GRSPaV eradication proved to depend on genotype and SE effectivity, but for 20 different genotypes published in different papers or tested in our laboratory, this method showed full efficiency, and only in the case of three cultivars was lower efficacy experienced (54-88%). If elimination methods other than SE were applied, GRSPaV proved to be one of the most recalcitrant viruses regarding sanitation [22]. The virus elimination with SE in Bromoviridae (100%), Closteroviridae (90-100%), Luteoviridae (100%), and Tymoviridae (100%) is coherent and highly reliable. However, there are other groups such as Potyviridae (0-62.5%) or Secoviridae (0-100%), where different results are reported regarding elimination efficiency. Nepoviruses (Secoviridae) are not phloem limited; therefore it is not surprising that, in the case of GFLV very different elimination rates were observed [23,25,47]. Interestingly, only one publication was found on the elimination of arabis mosaic virus (ArMV) from grapes with 100% efficiency using direct and indirect SE [43], although ArMV can cause serious economic damage. The regeneration pathway (the ratio of direct or indirect SE) for the acclimatised 46 lines was not clearly published, but the regenerated plants were negative for ARMV tested 1, 4 and 32 months after acclimatisation by DAS-ELISA and immunocapture RT-PCR.

Negative Sense RNA Viruses
Only one negative sense RNA virus, CPsV was attempted to be removed by SE, and CPsV was successfully eliminated from Citrus lemon, C. reticulata and C. sinensis via SE [38,41,44]. We have not found other published results with negative sense RNA viruses. Nevertheless, the effectiveness of CPsV elimination was 100% in these experiments.

Viroids
The elimination of ASBVd representing Avsunviroidae from avocado failed, where the nucellus of immature avocado seeds was used for SE induction. In the case of Apscaviroids, the effectiveness of SE was 64-100% for CDVd and 8.3-100% for GYSVd-1. Citrus barkcracking viroid (CBCVd) belonging to Cocaviroids and CEVd representing Pospiviroids were completely or almost completely removed by SE. In the case of hop stunt viroid (Hostuviroid), the eradication efficacy of SE was between 60% and 100% in Citrus sp., and 0-100% in Vitis sp. One reason explaining the lower elimination rates may be the rapid reproduction and transmission of viroids from cell to cell [25]. Based on these results viroid elimination with SE seems to be an appropriate but less reliable method for Hostuviroids, and based on the very limited data, ineffective for Avsunviroids (Table 3).

Genotype Effect
In the case of the different garlic ecotypes the elimination efficiencies were between 13 and 63%, which shows a strong genotype effect in SE-based elimination [46]. In cassava, the age of the callus tissues and long regeneration period probably reduced the efficiency for the 'Ankrah' cultivar [31]. This phenomenon may be related to the different regeneration capacity and rapidity of the cassava genotypes used under the same culture conditions. The effectiveness of SE proved to be higher in the case of CPsV-free mother plants than in infected ones [38], from which it follows that genotype and the infections act together on the regeneration process. The experienced differences in the effectiveness of elimination of CDVd, CEVd, GLRaV-3, EACMV, GCLV, HSVd and GYSVd-1 on the different cultivars may be caused by similar factors [23,25,32,41,46,48].

Genetic Variability of the Regenerated Plants
Varietal and sometimes even clonal identities of vine-and fruit-propagating material are also of great economic importance. However, the propagation of embryogenic callus cultures in the SE has often been associated with an increased risk of genetic instability in the regenerated plants [57].
During the plant breeding process, the judgment of the genetic fidelity of the SEderived plants to the mother plants could be different. SE could be even a possible way to gain greater somatic variability and, as such, could be a way to establish the starting material for clonal selection. This depends on the initial cells and tissues, as well as the type of SE applied by the breeders.
If SE is used at the end of the breeding process or exclusively for sanitation, then the genetic stability is more important. These genetic alterations can be verified by DNA (mostly SSR) markers, although the resolution of microsatellites is limited compared to the level of genetic polymorphism expected in SE. This type of variety identification is possible for most grape varieties, grape rootstocks and fruit varieties and rootstocks [23,58], but is not available for clones.
In grapevine Bouamama-Gzara and colleagues [52] found that 8% of the regenerated plants showed a phenotypic difference compared to the mother plants using direct embryogenesis from stamens [52]. Plant fidelity (from anthers) morphologically appeared to be true to type and flow cytometry was used to confirm the diploid level of the anther-derived plants [43]. Using immature cut seeds of grapevine for SE, around 10% of the regenerated plants were both virus-free and true to type [23].
In the case of lemon regenerants, ISSR analyses did not find any deviation from the mother plants, but in 50% of the tested Citrus sinensis 'Washington navel 251 plants, genetic polymorphism was identified [41]. A total of 25-35.5% genetic difference was detected by RAPD analysis after indirect SE on stigma-derived plantlets of 'Washington navel 251 orange depending on the primer [44]. Ovule cell cultures of different Citrus cultivars were genetically variable, and, due to this, the obtained virus-free somatic embryos from them were used for breeding purposes [45]. Nucellar embryony was also applied to produce pathogen-free plants of clementine varieties, nevertheless phenotypic differences between the regenerated and the original seed source plants were observed [59]. After indirect embryogenesis juvenile character (thorns) appeared on the stems in the first year on the regenerated Citrus plants [38,55].
Embryogenic cell cultures have been developed for important crops, but their application for propagation is very rare for commercial purposes, with the exception of coffee. In the case of coffee, 1% of the SE-derived plants showed somaclonal variation in field experiments [57]. These results suggest that genetic variability of regenerants depends on the species and the type of the inoculum used for SE. The genetic polymorphism appears to be higher if different parts of immature seeds are isolated for SE induction.

Embryogenesis Combined with other Treatments
We found a limited number of studies on combined treatments, probably due to the sensitivity of embryogenesis and plant regeneration. PyMoV elimination was 100% effective if cyclic somatic embryos were treated with chemotherapy using 20-30 mg/L ribavirin, but the phytotoxicity of ribavirin reduced the effectiveness of regeneration system, depending on the used concentrations [30]. In the case of the grapevine cultivars 'Gewürztraminer' and 'Rupestris du lot', SE combined with heat therapy showed 100% effectiveness, while SE alone was not able to eliminate GFLV [37,50].

Potential Transferability of Elimination Results by SE to Different Plant Species
In the case of grapevine, SE is a reliable tool for virus or viroid elimination. The most important grapevine viruses and viroids (Tables 1-3), but also minor viruses such as ArMV, GFkV, GFLV, GLRaV-1,2,3, grapevine Pinot gris virus (GPGV), GRSPaV, grapevine rupestris vein feathering virus (GRVFV), grapevine Syrah virus-1 (GSyV-1), GVA, and grapevine virus T (GVT), were successfully eliminated. Furthermore, SE is also a promising elimination method for two viroids (GYSVd-1 and HSVd). Nevertheless, more than 80 viral entities are known in grapevine, and there are no SE experiences with the majority of them yet [25]. The situation for the citrus species is highly similar: important viruses (CPsV, CTV, CVV) and viroids (CBCVd, CDVd, CEVd, HSVd) were successfully eliminated. However, SE proved to be useless to manage ASBVd in avocado. It was effective against CSSV in cocoa, leek yellow stripe virus (LYSV), and onion yellow dwarf virus (OYDV) in garlic, and PYMoV in black pepper, all of which cause crucial serious diseases on these species. Cassava and sugarcane have several important viruses; from these, ACMV and EACMV in cassava, as well as SCYLV in sugarcane, were eliminated by SE.

Conclusions
SE was successfully used for virus or viroid elimination in seven different economically important families of plants, and the effective removal of sugarcane yellow phytoplasma by SE was also reported [54]. SE was effective on a broad spectrum of different virus and viroid species-all of the examined 25 viruses and 5 from 6 tested viroids were eliminated by this method. In the case of viruses the elimination ratio was almost 100% for 16 viruses, over 50% for 6 viruses and less reliable for 3 viruses (Figure 3). The most recalcitrant viruses belong to the Betaflexiviridae (Carlavirus), Potyviridae (Potyvirus) and Secoviridae (Nepovirus) families. The elimination ratios of the different viroids are not usually lower than the majority of the viruses (Figure 4), and SE is a useful method for their elimination, with the exception of ASBVd (Avsunviroidae). Interestingly, the in vitro conditions increased the number of ASBVd sequence variants, and the presence of this viroid was persistent during the SE. When the presence of the different viruses and viroids was directly checked in embryos, then ASBVd, ACMV, CSSV, EACMV, GFKV, GFLV, and PYMoV were detected in some of the regenerated embryos, but GFKV was detected only in abnormal embryos. The sanitation results of the regenerated plants suggest that even more viruses and viroids are able to infiltrate into somatic embryos, such as GCLV, GLRaV-3, GRSPaV, LYSV, and OYDV viruses, and CDVd, CEVd, HSVd, and GYSVd-1 viroids. Based on these results, it appears that around half of the investigated viruses and all of the examined viroids are able to transfer into the regenerated somatic embryos. Secondary embryogenesis or the cyclic subculturing of somatic embryos was more effective for elimination in the case of ACMV, CSSV, and EACMV. The age of callus tissues and the slow regeneration may have a negative effect on the effectiveness (ACMV, CSSV, EACMV), but subculturing the calli around 6-15 times is able to eliminate the GFLV, GLRaV-s, GRSPaV, and GVA viruses. In the case of GFKV, direct embryogenesis was more suitable for elimination than the indirect one, and direct SE also proved to be highly effective in several other experiments [43,52]. The plant genotype could have a strong effect on elimination ratios, likely through the regeneration efficiency, especially in cassava and garlic, as well as in grapevine and citrus species, depending on the virus.
Chemotherapy or heat treatment combined with SE can improve the elimination effectiveness, but these additional treatments likely reduce the regeneration capacity of calli or somatic embryos.
If the genetic fidelity of the regenerated plants is important for the breeder, then it needs to be considered that ISSR, RAPD and SSR analyses showed 8-90% polymorphism, which is probably inoculum-and species-dependent. Based on the phenotypes of the regenerated plants, 0-1% of the plants typically showed morphological differences. As such these changes are likely causing only minor variation in the economically important properties of the cultivars. However, rigorous phenotypization of the regenerants is strongly recommended, and further experiments are needed to specify the expected value of genetic polymorphism.
The main advantage of SE as a virus and viroid elimination tool lays in its outstandingly high efficiency on a broad spectrum of viral entities of taxonomically diverse origin. SE could be successful even where other methods prove to be ineffective. The main limitation of SE, the genotype dependency, can be reduced by finding the optimal stage of the plant material used for initiation of the callus culture.  Chemotherapy or heat treatment combined with SE can improve the elimination effectiveness, but these additional treatments likely reduce the regeneration capacity of calli or somatic embryos.
If the genetic fidelity of the regenerated plants is important for the breeder, then it needs to be considered that ISSR, RAPD and SSR analyses showed 8-90% polymorphism, which is probably inoculum-and species-dependent. Based on the phenotypes of the regenerated plants, 0-1% of the plants typically showed morphological differences. As such these changes are likely causing only minor variation in the economically important   Chemotherapy or heat treatment combined with SE can improve the elimination effectiveness, but these additional treatments likely reduce the regeneration capacity of calli or somatic embryos.
If the genetic fidelity of the regenerated plants is important for the breeder, then it needs to be considered that ISSR, RAPD and SSR analyses showed 8-90% polymorphism, which is probably inoculum-and species-dependent. Based on the phenotypes of the regenerated plants, 0-1% of the plants typically showed morphological differences. As such these changes are likely causing only minor variation in the economically important

Short Conclusions
Since the aim of the published elimination experiments was usually to remove the identified natural combinations of viruses and viroids, it would be worthwhile to test the viruses and viroids in a systemic way in the future. Based on the experiences up until now, SE, compared to other methods, proved to be very effective for viruses and almost all cases for viroid elimination, but this effectiveness could depend on the plant genotype, the infection level and the virus species. Direct and indirect SE can be equally effective, but in the case of indirect SE, the low propagation ratio and slow regeneration of somatic cultures may reduce the efficiency. Most of the viruses and viroids are probably detectable in the calli, and around half of the virus and viroid species are certainly able to transfer to somatic embryos. In many cases, the infected embryos do not regenerate into plants, or, if they do, occurs with low efficiency. Additional treatments can improve the efficiency of SE, but they are rarely applied. To evaluate the genotype fidelity, further data are needed, but considerable morphological changes are normally rare, while genetic polymorphism, for example, in the SSR, seems to be much more common.
The importance of SE in the production of pathogen-free propagation material could increase further in the future, due to the ongoing research in the field of SE supporting its appropriate and widespread application, and due to the growing challenges of climate change in the agricultural economy.