Somaclonal variations for crop improvement : Selection for disease resistant variants in vitro

veenaanil@ymail.com Abstract Somaclonal variations (SV) are genetic or epigenetic changes induced in plant cell and tissue culture. Induction of somaclonal variation, is an alternate approach to conventional breeding and transgenic approaches to introduce desirable genetic variability in the gene pool. SVs that occur spontaneously in culture induce changes in a range of plant characters. However, the probability of improving a key agronomic trait such as disease resistance can be cumbersome when left to chance alone. The efficiency of developing disease resistant SVs is better with the imposition of an appropriate in vitro selection pressure. Selection agents that have been applied include pathogen elicitors, pathogen culture filtrate and purified pathotoxins. This method of SV selection has been successful in enhancing disease resistance in several crops and it is an accepted biotechnological approach with tremendous potential for crop improvement.


Introduction
Biotic stresses are major constraints that contribute to yield losses, and to the failure of crops realizing their full yield potential.
Breeding is the conventional approach to introduce genetic changes for crop trait improvement including disease resistance.Resistance may arise from the introduction of resistance (R) genes, which remains effective over a period of time until overcome by an ever evolving pathogen, thus limiting the shelf life of the effectiveness of the cultivars.
In addition, breeding approaches can fail due to lack of genetic variability in a crop species or its wild relatives.In this scenario, biotechnological approaches such as the development of transgenic plants and the in vitro selection of somaclonal variations, become viable alternatives to support the breeding programmes for crop improvement.Among these alternatives, transgenic approach is yet to gain public acceptance, and has to adhere to stringent biosafety regulations of the country in which the transgenic crop is developed, tested and cultivated.The somaclonal variant approach is based on changes resulting from internal mutations and thus does not face acceptability issues nor pose any known biosafety concerns.SVs can be considered an alternate source of exploitable variation induced in cell and tissue culture.This mini review briefly highlights the resistance mechanisms existing in plants and details the approaches used in enhancing disease resistant traits in crop plants.The review focuses on somaclonal variations as a potential technology in crop trait improvement in general and more specifically for developing disease resistant variants.

Disease Resistance
Plants, are constantly exposed to various pathogens and pests but very few are successful in establishing an infection and causing disease.Occurrence of disease in nature is rare as plants have evolved multiple, sophisticated and overlapping mechanisms of defense including defensive structures, toxins, antimicrobials, barriers such as callose, suberins, waxes, and more specific adaptive defense such as non-host defense, vertical-race-specific and horizontal multigenic resistance.

Types of disease resistance i) Vertical resistance:
The term was first coined by Vanderplank during 1963 (1) to describe singlegene resistance.In vertical resistance, plant possesses single genes for resistance [example, Resistance (R) genes], while the pathogen possesses single genes for pathogenicity [example Avirulence (Avr) genes], which interact and recognize each other at the protein level.This is known as the gene-for-gene relationship between a pathogen and host, and is the basis of vertical resistance.It is qualitative resistance or race specific resistance regulated by major genes which is effective but can be easily overcome by new races of the pathogen.In other words, the pathogen can mutate its Avr genes to escape recognition by the host R gene, thus by-passing an effective resistance response in the host.
ii) Horizontal resistance: This term was also coined by Vanderplank (1) and represents a quantitative or durable resistance, controlled by several genes.It is also sometimes referred to as generalized resistance.Horizontal resistance and horizontal pathogenicity are entirely independent of each other in genetic terms, that is, there is no gene-for-gene relationship in this phenomenon.The polygenic resistance genes of horizontal resistance provide the plants with defensive structures or toxins that slow down or stop the pathogenic infection.The resistance may not be as precise as the race specific vertical resistance and develops at a slower rate.However it is a durable resistance and does not break down to new races of the pathogen, as does vertical resistance.

Need for Disease Resistant Crops
Agriculture involves large areas of monoculture of genetically identical crops, which is very unlike the coexistence of different plant species in a natural ecosystem.During domestication, crops have gained in yield potential, but have lost out on resistance traits of their wild ancestors.Crop disease contributes to an average loss of 26% of the global crop production annually (2).Crop cultivation relies on a few in-bred disease resistance genes and on excessive application of pesticides to manage pathogens.Despite their effectiveness pesticides have deleterious environmental consequences and the development of genetically resistant cultivars becomes a paramount goal.It is indeed a major challenge to attain food security, in the backdrop of population explosion, climate change, soil salinity, drought and soil erosion.Developing long-lasting and broad-spectrum disease resistance in crops will contribute in the quest to attain yield stability and food security.

Approaches to enhance disease resistance i)
Breeding approach: Plant breeders focus a significant part of their effort on selection and development of disease resistant plant lines.Crop varieties and wild ancestral species with inherent disease resistance genes are generally the source of resistance in breeding programmes.A diseasesusceptible desirable crop cultivar is crossed with a variety with suitable resistance trait to obtain populations that segregate for the traits of the parents.
Crossing includes cumbersome phenotypic selection, and methods such as marker assisted selection, backcross breeding, pedigree and bulk methods.
Breeding for disease resistance has been an ongoing process since the domestication of plants but it requires persistence, takes many years to develop and the resulting cultivar may revert to susceptibility in a few years.This is because pathogens are under natural selection for enhancing their pathogenicity.Thus with time and the right mutations pathogens can overcome the plant's resistance.Moreover new pathogens maybe introduced to the area, changes in cultivation practice can trigger incidence of new diseases, and sometime breeding for other characters can disrupt the disease resistance present in the parent varieties.Many a times the crop species may have limited genetic variability making breeding programs unviable.In some instances related wild species may possess the required resistance genes, however crossability barriers prevent the use of such putative wild parents.To overcome some of these short comings and to hasten the process of developing new cultivars, conventional breeding is now integrated to other modern methods such as genetic engineering, somatic hybridization, double haploid and multiparent advanced generation inter-cross (MAGIC) populations (3).

ii)
Transgenic approach: Transgenic plants possess genetically engineered recombinant DNA and are considered as genetically modified organisms (GMO).This approach allows the introduction of a new trait that does not occur naturally in the species due to the artificial insertion of a gene or genes to the genome.The transgene may originate from a related plant or from a completely different species (from within and across Kingdoms) or may even be a completely synthetic gene.Cisgenic plants, on the other hand, have inserted gene(s) from the same species, however as the inserted gene is a recombinant DNA they are also considered as GMOs.Inserting a combination of genes (gene stacking) in a plant is more beneficial and productive as the introduced trait may last longer.Most genetically modified plants are generated by using the Agrobacterium tumefaciens mediated transformation method or by the biolistic method (Particle gun method) (4-5) which have both proved effective.
Over the last two decades genetic engineering and transgenic technology have been used in tandem to develop disease resistant crops.Genetic engineering has the potential to solve some of the problems of conventional breeding by inserting multiple genes that can confer long lasting broad-spectrum resistance.Recent advances in molecular techniques have unravelled some of the intricacies of the multifaceted nature of plant resistance mechanisms, which has in turn led to more sophisticated transgenic approaches to enhance resistance.A broader and in depth understanding of plant resistance along with transcriptomics, proteomics, metabolomic and protein interaction studies have thrown up several candidate genes from plants, bacteria, viruses and fungi that potentially can enhance resistance.These candidate genes can be constitutively over expressed, induced to express under biotic threat, tissue-specifically expressed, knocked out or silenced by RNAi to obtain the desired resistance trait.The technology has been successful in several crops and has potential to reduce losses incurred by biotic stress.
There have been instances of transgenics failing to perform, often due to the way in which the gene is expressed.Constitutively over expressed transgenes adversely affect plant size or seed production.Failure could also result due to disruption of an important endogenous gene by insertion of the transgene.In addition, there are concerns that 1) transgenic crops may cause allergies in people, 2) the marker antibiotic resistance genes, integrated in these crops can induce resistance to antibiotics, leading to super bugs, 3) transgenic crops can cause damage to the environment, by affecting beneficial microbiota and insects, and 4) there is the fear that genetic modifications maybe unintentionally transferred to other related species via the pollen.These concerns have led to the establishment of strict guidelines and regulation for the development and release of transgenic crops.These regulations are essential; nevertheless they slow down the development of transgenic crops.Transgenic plants have not been accepted in several societies many a times due to unsubstantiated notions or sentiments, such as reluctance to ingest DNA from virus, bacteria or animal sources.Thus the technology has not made inroads to the larger crop cultivation of several countries.However, transgenic food crops have been used for decades in the Americas without any proven ill-effects, and the advantages of the technology are many.It has great potential for developing superior traits for yield, abiotic and biotic stress tolerance, and can contribute to the much needed world's food stability.
iii) Somaclonal Variant Approach: Tissue culture derived plants are referred to as somaclones and tissue culture derived plants exhibiting divergences are referred to as somaclonal variants (6).Callus cultures can be used to recover somatic mutants because the in vitro culture milieu encourages the division of individual cell and regeneration of whole plant.Somaclonal variants can be somatically or genetically stable.The genetically stable variations can be termed mutations.However, because of the possibility of reversible epigenetic variations this area broadly uses the term 'variations' instead of 'mutations' (7).
Any change in the DNA sequence are heritable and important for crop improvement.On the other hand, epigenetic changes are temporary and reversible and not heritable (8).Genetically stable SVs can result due to point mutations, alterations in chromosome number and structure, recombinations, methylation of DNA sequences, deletions and transpositions in nuclear, mitochondrial or chloroplast genomes (9,10).These genomic changes may result in stable

Somaclonal variations in crop improvement
Stable SVs have been generated in several plant species (12) and is a way to create variations and broaden the germplasm pool.In fact, somaclonal variation is a simple alternate technology aiding breeders for enhancing genetic variability relatively rapidly in crops that are difficult to breed or have low genetic variability (11,13).
The SV breeding practice has led to several cultivars that have been successfully released with improved traits including plant architecture, disease resistance, yield, appearance, and abiotic stress tolerance.Some examples include Yidan No. 6 maize (Zea mays L.) with improved grain quality, CIMAP/bio-13 aromatic grass (Cymbopogon winterianus Jowitt) with enhanced oil yield, He Zu No. 8 a wheat (Triticum aestivum L.) variant with high yield, rice variant DAMA with resistance to Picularia spp.'Ono' somaclonal variant of sugarcane is resistant to eye-spot disease, Fiji disease and downy mildew, and generated from the susceptible cultivar 'Pindar' (14).A sweet potato somaclonal variant cultivar 'Scarlet' is comparable to the parent cultivar in yield and disease resistance, but shows a more desirable darker and more stable skin colour (15).Somaclonal variants of St. Augustine grass showed stable desirable variations during vegetative propagation (12).In vitro selection of desirable traits that have been commercially exploited in horticulture varieties through somaclonal variations are enumerated in a review by Krishna et al. in 2016 (11).
In India, somaclonal variation has been the biotechnological approach to produce commercial varieties.CIMAP (Central Institute for Medicinal and Aromatic Plants), Lucknow has released 'Bio 13' a somaclonal variant of Citronella java, a medicinal plant.This SV, Bio-13, has 37% higher oil and 39% higher citronellol as compared to control varieties (16).

Selection pressure to induce disease resistance in vitro
The conventional and more cumbersome method of obtaining SVs involves the field screening of resistance in a large population of plants, raised through in vitro callus cultures.A more targeted approach is the regeneration of disease resistant plants by generating resistant callus cultures selected on fungal toxin or culture filtrates (20)(21)(22).Somaclonal variation has applications in plant breeding and genetic improvement and generation of such novel variants can be enhanced by  providing a suitable in vitro selection pressure (23).Exposure to phytopathotoxin, pathogen-wall material or secreted elicitors, has been a proven selection pressure to obtain disease resistant somatic variants of several crop plants (11,24).In such a selection method, the cultures are selected by gradually increasing concentrations of culture filtrate or phytotoxin or by a constant challenge with either.Following such selection (Fig 1 ), the calli can be screened further on medium containing a higher concentration of the toxin or culture filtrate.The stability of the putative cultures can be tested by passing the cultures through several subcultures in recovery medium, devoid of the selection pressure.This is followed by screening of the recovered calli on phytopathotoxic medium (Fig. 2).The ability to survive and grow on such screening medium will give an indication of the stability of the adaptation observed in the putative SVs.Somaclonal breeding programmes have been effective in developing disease-resistant crops (25,26,22,27).Phytopathotoxins have been used as a proven selection pressure in callus cultures.The scientist Carlson in 1973 (21) was the first to report in vitro selection of callus for breeding purpose.Maize plants resistant to Helminthosporium maydis were generated by Gengenbach et al. (1977) (28), using culture filtrate selection pressure on callus cultures.Later, Behnke (1979) (29) regenerated Late Blight resistant potato plants from culture filtrateexposed callus (22,29).Tomato (Lycopersicon esculentum) plants with improved resistance to Fusarium (Fusarium oxysporum) wilt were obtained by exposing calli to fusaric acid (30).Potato plants resistant to Early Blight (caused by Alternaria solani) and Late Blight (caused by Phytophthora infestans) were regenerated from protoplasts of potato (S. tuberosum L.) varieties 'Rssset Burbank' (31) and 'Bintje' (32).This approach has also been used in rice to select for resistance against brown spot disease (33).Cerato and co-workers have shown that plants regenerated from potato cells selected in vitro with culture filtrate of Phytophthora infestans exhibited improvement in resistance as compared with the source plants (34).
In India, the selection pressure approach has been used in groundnut against Carcosporidium peronatum (35), and in chickpea cell lines against Fusarium oxysporum sp.cicero (36) (Helianthus annuus L.) to Alternaria helinathi was improved by exposure of callus cultures to Alternaria culture filtrates (39).Bhardwaj et al. in 2012 (40) obtained resistant mutants of ginger (Zingiber officinale Rosc.) against wilt pathogen (Fusarium oxysporum f. sp.zingiberi Trujillo) by in vitro selection approach.More, recently, Krishna et al. (2016) (11) and Dehgahi and Joniyas, in 2016 (24) have reviewed the success of somaclonal variants and the use of pathogen toxin, wall elicitors and culture filtrate as a selection pressure to generate disease resistance in different crops.

Molecular detection of somaclonal variants
Efficient detection of alterations is essential to identify somaclonal variants that might possess useful agronomic traits.SVs have been detected and analyzed using different methods, including morphological, physiological, resistance evaluation, cytological, biochemical and molecular methods.At the molecular level, variations arise from changes in chromosome number or structure, or from subtle changes in the DNA itself (41).
Molecular analysis of variations at the DNA level is sensitive and will enable the detection of changes that are not obvious at the morphological level.Moreover, molecular techniques enable detection of variants in the callus or juvenile stages as opposed to morphological and physiological methods wherein regenerated or adult plant response are measured.Most importantly molecular detection methods will help identify genetically stable variations among a group of SVs that might have arisen due to either epigenetic changes or due to subtle but stable divergence at the DNA level.Molecular methods are useful tools to analyze the degree of divergence in SVs from the source calli or plant material.
Random Amplified Polymorphic DNA (RAPD) markers are the most commonly employed markers used to detect genetic variations in somaclonal variants (42,43).In addition ISSR markers (44), AFLP, RFLP and microsatellite DNA markers have been used for molecular analysis of variations induced in tissue culture (Table 2) (45).
For effective detection and evaluation of somaclonal variants, it is preferable to use a multi thronged approach as shown in Table 2. Somaclonal variants selected specifically for disease resistance are detected and evaluated using a combination of biochemical methods, tissue culture screening, disease resistance assays, cell viability tests and molecular marker methods.The selected putative SV calli are first screened on media containing toxic levels of the phytopathotoxin or CF and selected based on the response of the SV compared to the source calli (Fig 2 ).Further the calli cells may be analyzed by vital stains such as Trypan blue to evaluate the percentage viability in the calli in the screening plates or even after direct exposure to the pathogen.The calli can further be evaluated for the levels of defense enzyme activity such as superoxide dismutase, catalase, peroxidase as a response to exposure to the toxin or the pathogen.The shortlisted putative somaclonal variants showing significant resistance response to the pathogen or its elicitor, are further analyzed using molecular marker methods to ascertain that indeed the resistance adaptation observed is due to genetic changes.

Drawbacks of the SV technology
Selection of disease resistance in cell cultures is a rather simple process.However, the regeneration of plantlets from such somaclonal variant calli is usually difficult.The process of selection requires multiple subcultures of callus in medium containing the selection pressure and the phytohormone 2,4-D.Multiple subcultures resulting in prolonged exposure to auxins such as 2,4-D and the stress of the selection itself can result in the cells losing their regeneration potential.Another drawback of this method is the generation of pleotropic effects in regenerated plantlets.This can only be overcome by obtaining mulitiple somaclonal variants, so as to select a few amongst them having only the desirable traits.More focused and specific methods of tissue culture and selection that may overcome such road blocks need to be developed.

Conclusion
Several resistant somaclonal variant lines have been generated using the phytopathotoxin in vitro selection method.This approach is acceptable, simple and has high potential in obtaining desirable variability in crops.Moreover it has the potential of generating novel pathways of resistance that can be further exploited in crop improvement.An understanding of the underlying molecular pathways to resistance in somaclonal variants is lacking.These pathways need to be explored, and candidate genes identified for further exploitation in crop improvement.Only by understanding the many interactions that occur between host plants and pathogens can we utilize cell-culture approach of inducing variations to its fullest potential.

Fig 1 .
Fig 1.Schematic representation for a general methodology to select resistant Somaclonal variants A somaclonal variant of the B. juncea variety 'Varuna' has been released for commercial cultivation as 'Pusa Jai Kisan' (http://nrcpb.org/content/varieties-developed).The new variety has bolder seeds and yield advantage over the parent variety Varuna.Gupta et al. (2002) (17) developed a superior somaclonal variant of Rose-scented Geranium at CIMAP, India.Arun and coworkers (2003) (18) generated SVs (R2, R3 and R4 generations) from immature embryos of two spring wheat varieties, HUW-206 and HUW-234.These SVs displayed improved characters such as resistance to spot blotch disease and enhanced yield over the source varieties.A high sugared and high yielding SV of sugarcane (CoC 671), Co 94012, released as Phule Savitri in Maharastra (19), has better sucrose content and resistance to red rot and smut diseases.

Fig 2 .
Fig 2. Schematic representation for a tissue culture methodology to evaluate stability of somaclonal variants Thakur et al. (2002) (37) carried out in vitro selection and regeneration of carnation (Dianthus cayophyllus L.) plants resistant to culture filtrate of Fusarium oxysporum f. sp.dianthi.Rao et al. (2006) (36) developed Pigeon Pea cell lines and regenerated plantlets from callus tolerant to culture filtrate of Fusarium odum Buttler.Saxena et al. (2008) (38) generated leaf blight-resistant Pelargonium graveolens (rose scented Geranium) plants by selection of callus with culture filtrate of Alternaria alternate.The resistance of sunflower

Table 1 .
Examples of in vitro selection for disease resistance in crop plants

Table 2 .
Examples for molecular detection of Somaclonal variants in different crops