Virus Elimination from Ornamental Plants using in vitro Culture Techniques

Snežana Milošević1, Aleksandar Cingel1, Slađana Jevremović1, Ivana Stanković2, Aleksandra Bulajić2, Branka Krstić2 and Angelina Subotić1 1University of Belgrade, Institute for Biological Research „Siniša Stanković“, Department of Plant Physiology, Bulevar Despota Stefana 142, 11060 Belgrade, Serbia (snezana@ibiss.bg.ac.rs) 2University of Belgrade, Faculty of Agriculture, Institute of Phytomedicine, Department of Phytopathology, Nemanjina 6, 11080 Belgrade, Serbia Received: December 20, 2011 Accepted: May 6, 2012


INTRODuCTION
During the past several decades, the frequency of plant diseases caused by viruses has increased worldwide.The growing demands for high quality and disease-free planting material, increased volume of international seed and plant trade, high phytosanitary standards imposed by many countries for plant material imports, as well as the fact that a great number of viruses have a wide range of hosts, emphasize the significance of virus diseases of ornamentals.
The genus Impatiens includes over 900 species growing in tropical and subtropical regions of Africa, Madagascar, North India and Sri Lanka, East Himalayas and Southeast Asia.In South America and Australia, Impatiens spp.are introduced species.Some species have been grown as horticultural plants both in Serbia and worldwide and they have a significant role in improving public greeneries, gardens and balconies (Ferrante et al., 2006;Vujošević et al., 2008).
Viruses spread from mother plants to their progenies by infected cuttings, tubers and other plant vegetative propagation materials.Since the Impatiens species, like most ornamentals in commercial production, are reproduced by vegetative propagation, there is a great probability of virus transmission (Gera and Zeidan, 2006;Wang and Valkonen, 2008).Taking into account the fact that the Impatiens genus is more and more popular in our country, while virus infection is a limiting factor in commercial production, the basic goal of this survey is to gain an insight into possibilities of practicing in vitro culture techniques to eliminate viruses from plant tissue.
Techniques of in vitro culture include a group of significant biotechnological procedures which are successfully applied in many branches of agriculture, including horticulture, for the purpose of producing plants with improved features (Kumar Pati et al., 2006).Methods of plant regeneration in culture in vitro are being applied widely in commercial production of ornamentals worldwide, ensuring elimination of pathogens from infected material and production of disease-free plants (Akin-Idowu et al., 2009).Numerous commercial laboratories worldwide use the potential of in vitro propagation for massive plant production.On the other hand, the application of in vitro culture has been limited by the costs of required equipment, special treatments required during acclimatization and risk related to somaclonal variation and other physiological aberrations (Winkelmann et al., 2006).
Elimination of viruses from plant material includes the following stepdetection and identification of viruses in the initial plant material (by serological and molecular methods, electron microscopy, immunolocalization or biotest), • therapy by applying biotechnological techniques (meristem culture, thermo-therapy, chemotherapy, cryotherapy, micrografting), • testing of the treated plants for the presence of viruses using the same methods as described, • micropropagation of virus-free plants with periodical testing for a possible presence of viruses.

MERISTEM CuLTuRE
The most important technique in micropropagation is meristem proliferation wherein apical buds or nodal segments harbouring an axillary bud are cultured to regenerate multiple shoots without any intervening callus phase (Kumar Pati et al., 2006).Meristem culture is an efficient tool for regeneration, elimination of viruses from infected plants and production of virus-free seed material of different plant species (Grout and Brian, 1999;Faccioli, 2001;Rout et al., 2006).Viruses do not have their own metabolism, and virus replication, which occurs in a host cell, is dependent on the host cell's protein and nucleic acid synthesizing machineries and metabolic pathways.Virus control is difficult because virus replication inhibitors have shown to be toxic to the host cell as well.So, effective control agents (usually chemical agents) exist for most plant diseases, except those caused by viruses.The main reason for use of the meristem culture is the fact that most viruses do not attack the meristem of shoots, since the multiplication of meristem cells is faster than replication of viruses, although more recent studies have shown that the mechanism of gene silencing is in fact the main reason (Foster et al., 2002).
Meristem culture is a procedure which does not differ in general from procedures for shoot reproduction in vitro.However, an important requirement is that initial explants be as small as possible in order to be able to isolate only the apical meristem, rather than permanent cells containing viruses.Morel and Martin (1952) first demonstrated the elimination of viruses from Dahlia using meristem culture.Since then, the use of meristem culture to obtain virus-free ornamental plants has been widely used by numerous groups of researchers (Table 1).
Methods of meristem culture are more appropriate than callus culture because continuous proliferation of undifferentiated tissue during subculturing can increase the risk of genetic and/or epigenetic variations (Nesi et al., 2009).Dahlia, freesia, geranium, lily, etc., have been grown virus-free using meristem culture (Ram et al., 2005).However, production of virus-free plants by using meristem culture has numerous obstacles, such as: meristem size, meristem position on a plant, protocol applied during detection (different sensitiveness of the applied methods), endogen infections and the period of recovery and growth of produced plants (Cha-um et al., 2006).Furthermore, the distribution of viruses in shoot tips may vary depending on a virus-host combination.Therefore, various sizes of shoot tips must be excised and tested to determine the optimal size range supporting efficient virus elimination and also a high rate plant regeneration.Regeneration ability is positively proportional to the size of the shoot tip, but pathogen eradication is more efficient using small shoot tips (Wang et al., 2006).Meristems of greater dimensions regenerate a greater number of plants, while the obtained number of virus-free plants is inversely proportional to the size of meristem (Verma et al., 2004).The risk of infection by a virus also increases with explant size.Apart from the indicated factors, elimination of viruses also depends on their concentration in plant tissue and the physiological condition of the mother plant from which a meristem has been isolated (Verma et al., 2004).Meristem culture is a widely used system of plant virus elimination and conservation of virus-free germplasm.Hosokawa et al. (2004aHosokawa et al. ( , 2004b) ) presented a new method for production of virus-free plants, for which a tip of chrysanthemum, petunia, cabbage or carnation root is used apart from the apical shoot meristem.Root apex is considered to be a suitable organ for meristem culture, since root meristem has a high potential of cell division.
Standardization of medium composition is most important for regeneration of virus-free plants (Kumar et al., 2009).The history of morphogenesis in vitro mostly refers to an optimization of composition of mediums and defining different factors affecting the efficiency of in vitro plant regeneration.In an experiment for I. hawkerii, shoot formation rate was the highest in MS medium (Murashige and Skoog, 1962) with 0.1 µM N-(2-chloro-4-pyridil)-N'-phenylurea-forchlorfenuron (CPPU) (Milošević et al., 2011).A similar result was found by Subotić et al. (2008).They investigated the regeneration of axillary buds of I. walleriana in culture in vitro.The media with 0.1 µM CPPU induced the highest number of axillary buds and roots per explant.According to that result, the responses of Impatiens plants to high concentration of 1-phenyl-3-(1,2,3-tidiazole-5-) urea (TDZ) included fascinating malformations of the stem, manifested as enlargements and flattenings as if several stems were fused (Subotić et al., 2008;Milošević et al., 2011).On the other hand, the effect of CPPU and TDZ on shoot hyperhydricity was significant for I. walleriana and I. hawkerii.Higher concentrations of both cytokinins resulted in the formation of shoots of changed habitus which does not meet the criteria of commercial production.Differences regarding the effect of different cytokinins on the induction and number of adventitious buds may result from their different activities, i.e. different tissue sensitivities to the adenine and urea type of cytokinins (Mok and Mok, 2001).Also, differences in the activity of cytokinins may be due to their various translocation rates to meristematic regions and metabolic processes, in which the cytokinins may be degraded and conjugated with physiologically inert compounds, such as sugars or amino acids.Vitrification of carnation shoots in the presence of a high concentration of 6-benzyleaminopurin (BAP) was described by Radojević et al. (2010) and other research groups.According to their results, BAP concentration higher than 1 mgL -1 increased the number of vitrified shoots.Growth and morphogenesis in vitro are regulated by an interaction and balance between the hormone provide in the medium and those produced endogenously by an explanted tissue (Subotić et al., 2008).These results clearly indicated that the choice of medium was a key step in regeneration of a normal phenotype (Fraga et al., 2004b).
Evidence is growing that the main endogenous plant growth regulators, the cytokinins, are involved in the formation of visible disease symptoms.Cytokinins affect cell division, ageing, development of chloroplast and transportation of nutritive matters (Horgan, 1984), and they also affect the processes of virus infection and symptom development (Jerman et al., 1995).Also, Walkey (1991) and Clarke et al. (1998) suggested that the addition of plant growth regulators to media could be related to a development of plant resistance to virus infection since high concentrations of cytokinins stimulate the host to interfere with the synthesis of virus proteins.Cybularz-Urban and Hanus-Fajerska (2006) monitored the effect of elimination of Cymbidium mosaic virus (CyMV) and Odontoglossum ringspot virus (ORSV) from an orchid hybrid using meristem culture.Meristems with two leaf primordia grown in MS media supplemented with 0.5 mg/l zeatin, 3.2 mg/l kinetin or 0.4 mg/l kinetin were used to induce regeneration, while 0.2 mg/l zeatin or 5 mg/l BAP or 0.4 mg/l kinetin was added to the medium during multiplication.The elimination of CyMV was achieved in the medium for induction with 0.5 mg/l zeatin and 3.2 mg/l kinetin, while ORSV was not eliminated in any treatment.The authors indicated that other researchers (Dhingra et al., 1991;Hu et al., 1994;Mauro et al., 1994) had reported the same conclusion that a concentration of viruses in plant tissue would be reduced by adding growth regulators to the culture media.

Virus elimination from ornamental plants in Serbia
Among ornamental plants, I. walleriana, I. hawkerii and I. balsamina are particularly popular and widely cultivated in Serbia.Their production is substantial, and a significant part of Impatiens plants produced in Serbia originate from imported seeds and planting material.Despite the fact that active quarantine measures are regularly employed, a small part of the imported planting material and potted plants of Impatiens have been proved to be infected with TSWV (Krstić et al., 2005a(Krstić et al., , 2005b)).During a survey for TSWV on ornamentals in greenhouse production in Serbia (Krstić et al., 2006) growers have been observed to produce their own Impatiens planting material sometimes by using diseased mother plants.For that reason an investigation was conducted in Serbia to establish a successful protocol for virus elimination from TSWV infected Impatiens plants.Milošević et al. (2011) examined a possibility for obtaining virus-free plants from I. hawkerii shoots infected with TSWV using meristem culture.For the investigation, DAS-ELISA and RT-PCR positive plants were selected.Meristem explants (0.3-1.5 mm) from virus-infected shoots were cultured on MS media supplemented with different concentrations of the cytokinins, CPPU or TDZ (0.01-1.0 μM), respectively.Using this protocol, a large number of in vitro shoots were successfully produced from a single explant.Cytokinins showed a stimulatory effect on the length, fresh and dry weights, as well as on the content of plant pigments.By DAS-ELISA and RT-PCR, 80% of the in vitro plantlets were shown to be virus-free, recommending the presented protocol for practical implementation in production of virus-free Impatiens planting material.A similar protocol was tested with ELISA and RT-PCR positive I. walleriana plants bearing characteristic TSWV symptoms (Figure 1) (Milošević et al., unpublished).After employing a slightly modified protocol for virus elimination, 75% of produced regenerated plants proved to be virus-free (Figure 2).It was shown that in vitro culture is an efficient method for elimination of TSWV from Impatiens species.The effectiveness of this virus removal treatment depends on the genotype.The established protocol with both Impatiens sp. is one of the first attempts to obtain virus-free Impatiens plants from infected mother plants in Serbia, and its implementation could be of great importance for the production of these species and ornamentals in general.Domestic production of safe and virus-free planting material of different ornamentals will help reduce the imports of planting material, thus raising biosecurity in Serbia and reducing the number of introduced quarantine and economically harmful organisms.

THERMOTHERAPy AND CHEMOTHERAPy
Heat treatment was originally applied by Kassanis in 1949 (Parmessur and Saumtally, 2001) to eliminate viruses from plant tissue.Since then, thermotherapy has been extensively used for elimination of different viruses from various plants.Growing host plants at higher temperatures significantly reduces replication of many plant viruses by disrupting viral ssRNA and dsRNA synthesis.Thermal inactivation point differs for different viruses, and it ranges between 40 and 46 0 C in TSWV (depending on isolates), while it is 90 0 C in TMV.Since high temperature can inhibit virus replication and movement, thermoterapy combined with meristem culture can greatly improve virus elimination efficiency by augmenting the virus-free region of treated shoot tips.Thermotherapy along with meristem culture has been used to raise virus-free carnation, narcissus, chrysanthemum and others (Hakkaart and Quak, 1964).Ram et al. (2005) presented in vitro production of CVB-free chrysanthemum cv.Regol Time by meristem culture, chemotherapy and thermotherapy.No plant was found virus-free using meristem culture alone.Meristem culture and thermotherapy did not give a satisfactory number of virus-free plants.The maximum percentage of CVB-free chrysanthemum was obtained with 2-thiouracil and thermotherapy for 30 days.These results are in contrast with earlier findings of other research groups (Hakkaart and Quak, 1964;Asatani, 1972;Goethals et al., 1973;Paludan, 1973), who had produced 90-100% CVB-free plants using these methods.One of the reasons may be that earlier testing of virus-free plants had mainly been based on biological inoculation.Biological indexing was not found to be a reliable method as most plants that were found negative for the virus were tested positive with RT-PCR (Ram et al., 2005).Another approach to in vitro virus elimination in plants is to supplement the nutrient medium with a chemical of a known ability to prevent virus replication.Incorporation of antiviral compounds into explant and meristem culture media has resulted in a higher percentage of virusfree progeny plants originating from virus-infected explant or meristem donor plants than are produced in tissue cultures that have no chemicals such as ribavirin (virazole), acycloguanosine, azidothymidine, and 2-thiouracil.

CRyOTHERAPy
More recently, prolonged exposure to a low temperature (-196 0 C) followed by shoot tip culture has proved quite successful in virus elimination.Cryotherapy of shoot tips can result in virus-free plants at a high frequency.Thermotherapy had other effects which together with subsequent cryotherapy resulted in virus elimination.When used for cryotherapy, conditions are selected to allow survival of only a limited number of the least differentiated cells and to eliminate a large proportion of virus-infected tissues.Therefore, cryotherapy can result in virus-free regenerants with a much greater frequency than what is typically obtained with a conventional meristem culture (Wang and Valkonen, 2008).Cryotherapy has successfully eliminated pathogens (viruses and bacteria) from numerous plant species: Solanum tuberosum, Ipomea batatas, Vitis vinifera, Citrus spp., Rubus idaeus, Musa spp.(Wang and Valkonen, 2009).The elimination of TSWV by cryotherapy was performed in tobacco (Yordanova et al., 1998), and the procedure is currently developing for the Impatiens species in the Laboratory for Plant Physiology of the Institute for Biological Researches "Siniša Stanković", University of Belgrade.A successful procedure has been developed for immunolocalization of TSWV in I. walleriana plants (Trifunović et al., 2011).Immunolocalization carried out on Pelargonium apices originating from cryopreserved shoot tips, sampled from DAS-ELISA negative plants, showed that they were still virus-infected (Gallard et al., 2011).Using immunolocalization, Pelargonium flower break virus (PF-BV) and Pelargonium line pattern virus (PLPV) could be detected in Pelargonium apices, even in the meristematic dome (Gallard et al., 2011).However, viral particles were more numerous in the basal zone cells than in meristematic cells.These results demonstrate that PFBV and PLPV are present within meristematic cells and that cryopreservation can partly reduce the quantity of these viruses in Pelargonium plants but not eliminate them totally.

CONCLuSION
Diseases caused by viruses are a significant problem constantly present in commercial production of plants, horticultural species in particular.The need for virusfree stocks has therefore become essential.In order to obtain virus-free material, the following methods can be applied: meristem culture, thermotherapy, chemotherapy, cryotherpy or a combination of these several techniques as virus elimination is more effective when different techniques are combined.Investigations in Serbia have begun and the first successful virus elimination protocol using meristem culture has been tested for I. hawkerii and I. walleriana.Efforts should now be made to develop an optimized cryotherapy system that could be useful for elimination of TSWV from Impatiens and other ornamentals species.

Figure 1 .
Figure 1.Symptoms on I. walleriana plants infected with TSWV -(a) necrotic spots, (b) necrotic flecks, line pattern and deformation of leaf, (c) necrosis of petals and sepals, (d) necrosis of steam

Figure 2 .
Figure 2. In vitro propagation of virus-free I. Walleriana -(a) shoot formation on MS media after 7 days, (b) multiplication of shoots on MS supplemented with 0.01 μM CPPU, (c) development of axillary shoot on MS with 1 μM TDZ, (d) axillary shoots on MS with 0.1 μM CPPU, (e) shoot elongation on MS cytokinins-free medium, (f ) plants in acclimatization stage