Abstract: Fungal pathogens impose a foremost threat to crop productivity. So, efforts to develop resistance against these pathogens in plants are of great importance. In recent years in vitro selection has emerged as a viable tool for developing resistant germ plasm. Resistant variants can be acquired by applying selective agent such as fungal toxins in the tissue culture media. In toxin based in vitro selection, explants that can withstand selection pressure can survive and only those cells are selected for regeneration. The resistance trait appearing in the regenerated plants may be genetically stable and useful in the crop improvement. Henceforth, the present review focuses on implementation of fungal toxin as a potential tool for in vitro selection and recent progress made towards the development of resistant germ plasm through this technique.
INTRODUCTION
The production and yield of majority of crops are severely influenced by various diseases caused by necrotrophic phytopathogen1. Damages caused by these pathogens results into huge economic losses at global level. Conventional breeding strategies had always played an important role in crop improvement. Though, this traditional method has gained less success and has failed to some extent to generate the desired outcome. Henceforth, it had turned to be essential to set up biotechnological methods for solving such vital problem regarding crop improvement.
Among the biotechnological techniques, (transgenic technique2, elicitors mediated plant defense etc3-5), in vitro culture has turned to be an imperative means to study host pathogen interaction not only at the cellular but also at molecular level6 in a small space under controlled environment condition, which are nearly impossible to regulate with conventional system. Infact plant cells under in vitro culture, respond to various stresses in a pattern analogous to that of an intact plants. This facilitates in vitro cell culture as an ideal tool for understanding the resistance related responses and dynamic changes taking place both at the cellular and subcellular level upon infection with the pathogenic microorganism7.
Pathogenic microbes including fungi produces a wide range of toxins that may either have adverse or stimulatory impact in plant system8. An interesting characteristic of many of those toxins are their capacity to stimulate several resistance related responses in the host9 including (1) Alteration in primary10 and secondary metabolites11, (2) Accumulation of defense related components9,12,13, (3) Hypersensitive responses14 or (4) Resulting to systemic acquired resistance (SAR) and induced systemic resistance (ISR)15. This attribute had encouraged several researchers to utilize fungal toxin as a selective agent for obtaining resistant germ plasm through in vitro approach.
The in vitro culture in conjunction with selective agent offers prospect to isolate cells with desirable trait. Among the selective agents, toxins derived from necrotrophic fungal pathogens have received more consideration because of the simplicity in exposing cells to a controlled and sublethal dosage of isolated toxin16. The sublethal dose of toxin when applied in cell culture media, develops a selection pressure that allows only the resistant cells to survive or divide. These resistant cells can then be subjected to regeneration to obtain the whole plant. On regeneration, in majority of cases the in vitro selected tolerant cells show significant degree of resistance or tolerance not only in response to toxin but also against the pathogen17. Besides all these an importance of this selection method lies on the fact that it significantly shortens the time duration for the selection of desirable character under selection pressure with negligible environmental interference, thereby complementing field selection18. These attributes constitute toxin based in vitro selection strategy a feasible, non-controversial biotechnological means for developing resistant varieties. The present paper summarized the recent knowledge about fungal toxin as a selective agent and its potential application in in vitro selection for inducing resistance against diseases.
Fungal toxin as selective agent: Selection of precise selective agent is the most vital step for establishing a screening system19. In recent years application of pathogen mainly fungal toxin for in vitro selection are more encouraged by researchers. Exploiting toxins for in vitro culture is found to be more in advantageous in comparison to pathogen because cells can be exposed easily and evenly to toxin via dispersing the in vitro cell in toxin solution or by plating them on toxin supplemented media20.
The basic parameter that must be satisfied prior a toxin is implemented as a selective agent are, primarily the toxin produced by the pathogen must be involved with the development of disease. Secondly the toxin should directly act at the cellular level19,21. Lastly one or more compound present in the selective agent must also occur in the infected tissue of the host plant22.
Fungal toxin includes a range of secondary metabolites such as low molecular weight peptides, terpenoides and carbohydrates23,24. Based on the specificity toxins are divided into host specific (HST) and non -host specific (nHST) toxin. HSTs affect only a selective plant species. These toxins are produced by various pathotypes of the fungal pathogens such as AAL- , ACT-, AK-, AM-, AF-25,26, Victorin27, HC toxin28, T-toxin29 etc.
Non-specific toxins (nHSTs) exhibit activity at broader range affecting several plants irrespective of whether they are host or non-host of the pathogen26,30. Activities of this toxin are not restricted to phylogenetic specialization and generally rely on concentration24. Few examples of nHSTs are Zinniols, tenuazonic acid26, Brefeldin A (dehydrogenase), curvularin and tentoxin31,32 fusaric acid33,34, fusicoccin8 etc. These nHST exercise their effect by various modes in plants.
Among the above mentioned toxins few toxins was applied as a selective agent for in vitro selection experiments detailed35-53 in Table 1.
| Table 1: | List of fungal toxins used as selective agent for in vitro selection of plant |
In vitro selection techniques: For in vitro selection using fungal toxin as a selective agent several methods are developed. The details of various techniques are discussed below.
Double layer culture method: In this strategy, fungal culture that forms the first layer is overlaid with culture growth medium supplemented with fungicides mycostatin or benlate at fungistatic concentration. Following diffusion of the toxic metabolite from the fungal layer, calli are placed on the upper layer of the medium54. However, this method was further modified by Ahmed et al.55, for in vitro selection of wheat calli to obtain resistant lines to Fusarium toxin. In the modified method the fungal culture was autoclaved to kill the fungal cells. This formed the first layer. This layer was overlaid with the callus growth medium forming the second layer. The calli are then placed on the second layer following diffusion of toxic material from the first layer to the callus growth medium55. Later, Kleijer et al.56 used this method for in vitro selection of Triticale for inducing resistance to head blight disease caused by Fusarium sp. This method was also used for selection of embryogenic calli of carnation cultivars for resistance to toxic metabolites of Fusarium oxysporum by Esmaiel et al.57.
Partitioned culture technique: In this method the adjacent quadrants of the petri dish was used for culture. After mycelial growth, walls between the sectors were pierced for diffusion of the fungal toxic metabolites. Following this, the medium supplemented with fungicides were then poured in the compartments where callus are to be transferred54. Resistant calli were then selected and transferred to toxin free medium for further growth and regeneration.
Gradient system: In gradient method, cultures are exposed to culture media with a gradual increase in concentration of selection agent. Through this system, a sublethal dosage of toxin can be optimized that can further be used for in vitro selection experiment. Gradient system for in vitro selection had been applied in several plants. For instance, Kintzios et al.44 studied in vitro reaction in sunflower by allowing the growth of callus in selection media amended with gradient of Alternaria alternata toxin. Similarly a gradual increase in fusaric acid concentration to cell suspension culture of Gladiolus decreased the cell growth. However, at this sublethal dose plantlets of the selected cell lines were showed significant resistance over that of control58. Gradient method was also applied by Chopra et al.59 for selection of resistance in Brassica using Alternaria brassicae toxin.
Continuous-discontinuous selection method: This technique was adapted by Tripathi et al.60 for in vitro selection of resistant cell lines of onion using purified toxic culture filtrate of Alternaria porri. In this strategy two selection method were used -a continuous method, where four cycles of selection were performed on toxic media (continuous method) and a discontinuous system in which a pause was given after second and third cycle of selection using non-toxic medium. The continuous and discontinuous method proved to be superior as it allowed the callus to regain their regeneration potency60.
Direct selection: This method involves direct exposure of in vitro cells to the selective media amended with toxin. Direct selection proved to give better results in several plants. As for instance, in barley 8-11% resistant calli were obtained when grown in a media supplemented with 0.8 mM fusaric acid after four cycle of selection39. Arcioni et al.,61also implemented this method for in vitro selection of alfalfa calli. Srinath and Padmaja62, for obtaining resistant lines in sunflower, directly exposed callus of sunflower to 40% Alternaria culture filtrate (ACF) supplemented in the culture media.
Biochemical characterization of disease resistance during in vitro selection: When in vitro plant cells are subjected to selective pressure, several defense related molecules induced that lead to resistance in the in vitro tissue63. The tolerance in these calli or cells can be studied by analysis of various biochemical alterations including oxidative stress, activation of defense enzymes and secondary metabolites. The oxidative damage induced by fungal toxic metabolites leads to activation and deactivation of various antioxidant enzymes that play a considerable role in defense mechanism64,65. The antioxidant enzymes include peroxidase (POD), ascorbate peroxidase (APX), superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GR)64. Besides these, enzymes like β-1, 3-Glucanase and PAL are also induced in response to toxin in calli under in vitro culture9. For instance, Singh et al.66 reported an increase in peroxidase and β-1,3-Glucanase activities in calli of in vitro selected resistant cell line of chickpea. Kumar et al.65 examined antioxidant enzymes in fungal culture filtrate of Alternaria carthami in tolerant plant of safflower cv.NARI. Among enzymes, an increase in POD and SOD activity and a slight decrease in CAT level were observed in the FCF (Fungal culture filtrate) tolerant plants. An increase in PAL, peroxidase and phenolic acid in the in vitro selected calli of Medicago sativa resistant to Fusarium phytotoxin was observed by Hrubcova et al.67.
Henceforth disease resistance during in vitro selection can be characterized through increased expression of PR proteins, antifungal compounds and production of phytoalexin in plants65,68. Still only those in vitro cells exhibiting quick activation of defense mechanism in a sustained way stay viable and survive during recurrent procedure of in vitro selection63. The plants that are regenerated from these in vitro cells may comprise increased level of defense genes expression in a constitutive way making plants more resistant in comparison to the parent63.
Application of in vitro selection for crop improvement: Use of fungal toxin as selection agent for screening in vitro cultures is now well established. In the past few decades most of the researches based on in vitro selection strategy for crop improvement are published. Some examples of the application of toxin-based in vitro selection for crop improvement are summarized as follows.
For instance, in maize, resistance was induced using T-toxin produced by Helminthosporium maydis. The resultant regenerates showed resistance to pathogen69. Sacristan70, applied toxins as a selection agent and screened resistance in Brassica napus to fungal pathogen Phoma lingam. Behnke71 selected Solanum tuberosum calli tolerant to the toxic filtrate of Phytophthora infestans. The resultant calli showed resistance to all four pathotypes of the pathogen. Tobacco callus expressed resistance to toxin from Alternaria alternata (leaf spot disease). The plants regenerated from these calli exhibited resistance to the diseases72. Chawla and Wenzel39 using purified culture filtrate (PCF) produced by Helminthosporium sativum, screened callus of barley and wheat for their resistance. The regenerants from those callus lines survived toxin treatment and in vivo testing against pathogen, revealing majority of plants were less sensitive. Gentile et al.73 regenerated lemon resistant to the disease “mal secco” caused by Phoma tracheiphila by screening cell lines of nucellar origin using partially purified toxin produced by the fungal pathogen. Remotti et al.40 effectively selected cell lines of Gladiolus×Grandiflorus cv. Peter and regenerated plants resistant to toxin-fusaric acid produced by the fungal pathogen Fusarium oxysporum. Nyange et al.74 established the selective impact of filtrate of fungus Colletotrichum kahawae on in vitro culture of cell and protoplast of Coffea arabica. Yang et al.75 selected F. graminearum tolerant plantets of Triticum aestivum by successfully in vitro screening using deoxynivalenol as selection agent. Srinath and Padmaja62, obtained plantlets of chickpea (Cicer arietinum) from selected calli resistant to culture filtrate of F. oxysporum showed resistance to wilt. Jayasankar et al.7 demonstrated that the selected lines of grapevines in response to culture filtrate of Elsinoe ampelina inhibited growth of E. ampelina and Fusarium oxysporum, expressing resistance in the whole plant. Thakur et al.76, applied in vitro strategy for inducing resistance in two cultivars of Dianthus caryophyllus resulting from callus cultures by applying toxic culture filtrate of F. oxysporum. Nearly 32% of the regenerated plants derived from resistant callus exhibited significant resistance in response to pathogen in field, showing no variation in phenotype in the selected regenerants. Gayatri and Kavyashree77 obtained clones tolerant to root rot disease in turmeric variety Suguna (Curcuma longa) against pure culture filtrate (PCF) of the pathogen Pythium graminicola by method of in vitro selection. The disease tolerant clones showed 90% survival frequency. Regenerants of Pelargonium graveolens (a rose scented geranium) obtained from callus resistant to culture filtrate of Alternaria alternata, showed induced disease resistance in geranium plant at the cellular level16. Valencia et al.78, obtained calli of pineapple treated with various concentrations of Phytophthora cinnamomi crude culture filtrate. Calli resistant to the crude filtrate of pathogen P. cinnamomi were selected followed by regeneration into plantlets subsequent to three selection cycles. Nurcahyani et al.79 applied fusaric acid as selective agent to obtain vanilla plantlets resistant to Fusarium oxysporum. Oxalic acid, a phytotoxin produced by Sclerotium cepivorum was used by Sayed et al.52 for in vitro selection of onion for inducing disease resistance against white rot disease.
Constraints in toxin based in vitro selection: In vitro approach represents a viable integration of traditional breeding method for crop improvement, specifically against necrotrophic phytopathogens. Though several instances are available on in vitro selection for disease resistance but only a few somaclones are declared successful as varieties.
Another major constraints encountered in this method was the reduction or loss of regeneration ability of selected cell lines upon exposure to toxin for a longer period of time. One reason behind this constraint can be cytogenetic alternation that is induced under selection pressure of in vitro cells80. This problem was reported by Nasir and Riazuddin58 in cell lines of Gladiolus upon exposure to fusaric acid for longer duration.
Penetration and transportation of the toxic media into callus is another difficulty. This was resolved by Saxena et al.16 in Pelargonium sp. by allowing the calli to grow in liquid media facilitating maximum transportation of the toxin into the cells.
CONCLUSION AND FUTURE PROSPECTS
For the last few decades the incredible progress made in the field of plant biotechnology for generating resistant genotype is an indication of great enthusiasm in the scientific community on this issue. Fungal toxin based in vitro selection offers a significant means for studying various physiological, biochemical and molecular mechanism in plants. In recent years, extensive advancement has been made concerning the development and isolation of resistant genotypes by using in vitro techniques. In vitro selection is a feasible approach as it save time that is actually required for developing a disease resistant line of commercially important crops. Assessment of these resistant plants obtained by in vitro selection method further need to be evaluated at field level and proper agronomic data should be obtained to certify that this technique can be effectively applied in fields to boost the disease management practices. Apart these a significant feature which make this biotechnological approach more important is that the selection of resistant variants is neither protected under intellectual property regulations, nor it is matter of public safety concerns for development of novel crop cultivars. Thus this method of selection is becoming a promising, non transgenic approach which offers an attractive method for producing improved cultivars. Henceforth, in future toxin based in vitro selection technique in concert with molecular approaches and functional genomics can turn to be a better option that will offer a novel prospect to develop disease resistant plants.
SIGNIFICANCE STATEMENT
The review systematically explains the potential role of fungal toxin in in vitro selection and regeneration of resistant germ plasm. This article will help the researchers to uncover the critical areas of inducing resistance in the in vitro tissue using toxin as a selective agent that many researchers would become enthusiastic to explore this area further. Thus a new avenue might be explored scientifically to develop many new resistant plants.