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Pustaka 2 - Bioteknologi
Pustaka 2 - Bioteknologi
https://dx.doi.org/10.14719/pst.2018.5.2.382
ISSN: 2348-1900
Mini Review
Department of Plant Biotechnology, Gandhi Krishi Vignana Kendra (GKVK), University of Agricultural Sciences (UAS), Bangalore
560065, India
Keywords
Publisher
Somaclonal variations (SV); biotechnology; crop improvement; in vitro selection, disease
Horizon e-Publishing Group
resistance; culture filtrate; pathotoxins
Citation
Correspondence
Veena S Anil
Anil V S, Bennur S, Lobo S. Somaclonal variations for crop improvement: Selection
for disease resistant variants in vitro. Plant Science Today 2018;5(2):44-54.
[email protected] https://dx.doi.org/10.14719/pst.2018.5.2.382
highlights the resistance mechanisms existing in the coexistence of different plant species in a
plants and details the approaches used in natural ecosystem. During domestication, crops
enhancing disease resistant traits in crop plants. have gained in yield potential, but have lost out on
The review focuses on somaclonal variations as a resistance traits of their wild ancestors. Crop
potential technology in crop trait improvement in disease contributes to an average loss of 26% of
general and more specifically for developing the global crop production annually (2). Crop
disease resistant variants. cultivation relies on a few in-bred disease
resistance genes and on excessive application of
pesticides to manage pathogens. Despite their
Disease Resistance
effectiveness pesticides have deleterious
Plants, are constantly exposed to various environmental consequences and the development
pathogens and pests but very few are successful in of genetically resistant cultivars becomes a
establishing an infection and causing disease. paramount goal. It is indeed a major challenge to
Occurrence of disease in nature is rare as plants attain food security, in the backdrop of population
have evolved multiple, sophisticated and explosion, climate change, soil salinity, drought
overlapping mechanisms of defense including and soil erosion. Developing long-lasting and
defensive structures, toxins, antimicrobials, broad-spectrum disease resistance in crops will
barriers such as callose, suberins, waxes, and contribute in the quest to attain yield stability and
more specific adaptive defense such as non-host food security.
defense, vertical-race-specific and horizontal
multigenic resistance.
Approaches to enhance disease resistance
i) Breeding approach: Plant breeders focus a
Types of disease resistance
significant part of their effort on selection and
i) Vertical resistance: The term was first coined development of disease resistant plant lines. Crop
by Vanderplank during 1963 (1) to describe single- varieties and wild ancestral species with inherent
gene resistance. In vertical resistance, plant disease resistance genes are generally the source
possesses single genes for resistance [example, of resistance in breeding programmes. A disease-
Resistance (R) genes], while the pathogen susceptible desirable crop cultivar is crossed with
possesses single genes for pathogenicity [example a variety with suitable resistance trait to obtain
Avirulence (Avr) genes], which interact and populations that segregate for the traits of the
recognize each other at the protein level. This is parents. Crossing includes cumbersome
known as the gene-for-gene relationship between phenotypic selection, and methods such as marker
a pathogen and host, and is the basis of vertical assisted selection, backcross breeding, pedigree
resistance. It is qualitative resistance or race and bulk methods.
specific resistance regulated by major genes which Breeding for disease resistance has been
is effective but can be easily overcome by new an ongoing process since the domestication of
races of the pathogen. In other words, the plants but it requires persistence, takes many
pathogen can mutate its Avr genes to escape years to develop and the resulting cultivar may
recognition by the host R gene, thus by-passing an revert to susceptibility in a few years. This is
effective resistance response in the host. because pathogens are under natural selection
for enhancing their pathogenicity. Thus with
ii) Horizontal resistance: This term was also time and the right mutations pathogens can
coined by Vanderplank (1) and represents a overcome the plant’s resistance. Moreover new
quantitative or durable resistance, controlled by pathogens maybe introduced to the area,
several genes. It is also sometimes referred to as changes in cultivation practice can trigger
generalized resistance. Horizontal resistance and incidence of new diseases, and sometime
horizontal pathogenicity are entirely independent breeding for other characters can disrupt the
of each other in genetic terms, that is, there is no disease resistance present in the parent
gene-for-gene relationship in this phenomenon. varieties. Many a times the crop species may
The polygenic resistance genes of horizontal have limited genetic variability making breeding
resistance provide the plants with defensive programs unviable. In some instances related
structures or toxins that slow down or stop the wild species may possess the required resistance
pathogenic infection. The resistance may not be as genes, however crossability barriers prevent the
precise as the race specific vertical resistance and use of such putative wild parents. To overcome
develops at a slower rate. However it is a durable some of these short comings and to hasten the
resistance and does not break down to new races process of developing new cultivars,
of the pathogen, as does vertical resistance. conventional breeding is now integrated to other
modern methods such as genetic engineering,
somatic hybridization, double haploid and multi-
Need for Disease Resistant Crops parent advanced generation inter-cross (MAGIC)
Agriculture involves large areas of monoculture of populations (3).
genetically identical crops, which is very unlike
ii) Transgenic approach: Transgenic plants species, however as the inserted gene is a
possess genetically engineered recombinant DNA recombinant DNA they are also considered as
and are considered as genetically modified GMOs. Inserting a combination of genes (gene
organisms (GMO). This approach allows the stacking) in a plant is more beneficial and
introduction of a new trait that does not occur productive as the introduced trait may last longer.
naturally in the species due to the artificial Most genetically modified plants are generated by
insertion of a gene or genes to the genome. The using the Agrobacterium tumefaciens mediated
transgene may originate from a related plant or transformation method or by the biolistic method
from a completely different species (from within (Particle gun method) (4-5) which have both
and across Kingdoms) or may even be a proved effective.
completely synthetic gene. Cisgenic plants, on the Over the last two decades genetic
other hand, have inserted gene(s) from the same engineering and transgenic technology have been
Fig 1. Schematic representation for a general methodology to select resistant Somaclonal variants
used in tandem to develop disease resistant crops. release of transgenic crops. These regulations are
Genetic engineering has the potential to solve essential; nevertheless they slow down the
some of the problems of conventional breeding by development of transgenic crops. Transgenic
inserting multiple genes that can confer long plants have not been accepted in several societies
lasting broad-spectrum resistance. Recent many a times due to unsubstantiated notions or
advances in molecular techniques have unravelled sentiments, such as reluctance to ingest DNA from
some of the intricacies of the multifaceted nature virus, bacteria or animal sources. Thus the
of plant resistance mechanisms, which has in turn technology has not made inroads to the larger crop
led to more sophisticated transgenic approaches to cultivation of several countries. However,
enhance resistance. A broader and in depth transgenic food crops have been used for decades
understanding of plant resistance along with in the Americas without any proven ill-effects, and
transcriptomics, proteomics, metabolomic and the advantages of the technology are many. It has
protein interaction studies have thrown up several great potential for developing superior traits for
candidate genes from plants, bacteria, viruses and yield, abiotic and biotic stress tolerance, and can
fungi that potentially can enhance resistance. contribute to the much needed world’s food
These candidate genes can be constitutively over stability.
expressed, induced to express under biotic threat,
tissue-specifically expressed, knocked out or
iii) Somaclonal Variant Approach: Tissue culture
silenced by RNAi to obtain the desired resistance
derived plants are referred to as somaclones and
trait. The technology has been successful in
tissue culture derived plants exhibiting
several crops and has potential to reduce losses
divergences are referred to as somaclonal variants
incurred by biotic stress.
(6). Callus cultures can be used to recover somatic
There have been instances of transgenics mutants because the in vitro culture milieu
failing to perform, often due to the way in which encourages the division of individual cell and
the gene is expressed. Constitutively over regeneration of whole plant. Somaclonal variants
expressed transgenes adversely affect plant size or can be somatically or genetically stable. The
seed production. Failure could also result due to genetically stable variations can be termed
disruption of an important endogenous gene by mutations. However, because of the possibility of
insertion of the transgene. In addition, there are reversible epigenetic variations this area broadly
concerns that 1) transgenic crops may cause uses the term ‘variations’ instead of ‘mutations’ (7).
allergies in people, 2) the marker antibiotic
Any change in the DNA sequence are
resistance genes, integrated in these crops can
heritable and important for crop improvement. On
induce resistance to antibiotics, leading to super
the other hand, epigenetic changes are temporary
bugs, 3) transgenic crops can cause damage to the
and reversible and not heritable (8). Genetically
environment, by affecting beneficial microbiota
stable SVs can result due to point mutations,
and insects, and 4) there is the fear that genetic
alterations in chromosome number and structure,
modifications maybe unintentionally transferred
recombinations, methylation of DNA sequences,
to other related species via the pollen. These
deletions and transpositions in nuclear,
concerns have led to the establishment of strict
mitochondrial or chloroplast genomes (9,10).
guidelines and regulation for the development and
These genomic changes may result in stable
Fig 2. Schematic representation for a tissue culture methodology to evaluate stability of somaclonal variants
alterations, which are transmitted sexually to the In India, somaclonal variation has been the
progeny. Such SV based mutations can be very biotechnological approach to produce commercial
advantageous for improving a cultivar (11). varieties. CIMAP (Central Institute for Medicinal
and Aromatic Plants), Lucknow has released ‘Bio
13’ a somaclonal variant of Citronella java, a
Somaclonal variations in crop improvement
medicinal plant. This SV, Bio-13, has 37% higher oil
Stable SVs have been generated in several plant and 39% higher citronellol as compared to control
species (12) and is a way to create variations and varieties (16). A somaclonal variant of the B.
broaden the germplasm pool. In fact, somaclonal juncea variety ‘Varuna’ has been released for
variation is a simple alternate technology aiding commercial cultivation as ‘Pusa Jai Kisan’
breeders for enhancing genetic variability (http://nrcpb.org/content/varieties-developed). The
relatively rapidly in crops that are difficult to new variety has bolder seeds and yield advantage
breed or have low genetic variability (11,13). over the parent variety Varuna. Gupta et al. (2002)
The SV breeding practice has led to several (17) developed a superior somaclonal variant of
cultivars that have been successfully released with Rose-scented Geranium at CIMAP, India. Arun and
improved traits including plant architecture, coworkers (2003) (18) generated SVs (R 2, R3 and R4
disease resistance, yield, appearance, and abiotic generations) from immature embryos of two
stress tolerance. Some examples include Yidan No. spring wheat varieties, HUW-206 and HUW-234.
6 maize (Zea mays L.) with improved grain quality, These SVs displayed improved characters such as
CIMAP/bio-13 aromatic grass (Cymbopogon resistance to spot blotch disease and enhanced
winterianus Jowitt) with enhanced oil yield, He Zu yield over the source varieties. A high sugared and
No. 8 a wheat (Triticum aestivum L.) variant with high yielding SV of sugarcane (CoC 671), Co 94012,
high yield, rice variant DAMA with resistance to released as Phule Savitri in Maharastra (19), has
Picularia spp. ‘Ono’ somaclonal variant of better sucrose content and resistance to red rot
sugarcane is resistant to eye-spot disease, Fiji and smut diseases.
disease and downy mildew, and generated from
the susceptible cultivar ‘Pindar’ (14). A sweet
Selection pressure to induce disease resistance
potato somaclonal variant cultivar ‘Scarlet’ is
in vitro
comparable to the parent cultivar in yield and
disease resistance, but shows a more desirable The conventional and more cumbersome method
darker and more stable skin colour (15). of obtaining SVs involves the field screening of
Somaclonal variants of St. Augustine grass showed resistance in a large population of plants, raised
stable desirable variations during vegetative through in vitro callus cultures. A more targeted
propagation (12). In vitro selection of desirable approach is the regeneration of disease resistant
traits that have been commercially exploited in plants by generating resistant callus cultures
horticulture varieties through somaclonal selected on fungal toxin or culture filtrates (20-22).
variations are enumerated in a review by Krishna Somaclonal variation has applications in plant
et al. in 2016 (11). breeding and genetic improvement and generation
of such novel variants can be enhanced by
providing a suitable in vitro selection pressure (Helianthus annuus L.) to Alternaria helinathi was
(23). Exposure to phytopathotoxin, pathogen-wall improved by exposure of callus cultures to
material or secreted elicitors, has been a proven Alternaria culture filtrates (39). Bhardwaj et al. in
selection pressure to obtain disease resistant 2012 (40) obtained resistant mutants of ginger
somatic variants of several crop plants (11,24). In (Zingiber officinale Rosc.) against wilt pathogen
such a selection method, the cultures are selected (Fusarium oxysporum f. sp. zingiberi Trujillo) by in
by gradually increasing concentrations of culture vitro selection approach. More, recently, Krishna
filtrate or phytotoxin or by a constant challenge et al. (2016) (11) and Dehgahi and Joniyas, in 2016
with either. Following such selection (Fig 1), the (24) have reviewed the success of somaclonal
calli can be screened further on medium variants and the use of pathogen toxin, wall
containing a higher concentration of the toxin or elicitors and culture filtrate as a selection pressure
culture filtrate. The stability of the putative to generate disease resistance in different crops.
cultures can be tested by passing the cultures
through several subcultures in recovery medium,
Molecular detection of somaclonal variants
devoid of the selection pressure. This is followed
by screening of the recovered calli on Efficient detection of alterations is essential to
phytopathotoxic medium (Fig. 2). The ability to identify somaclonal variants that might possess
survive and grow on such screening medium will useful agronomic traits. SVs have been detected
give an indication of the stability of the adaptation and analyzed using different methods, including
observed in the putative SVs. morphological, physiological, resistance
evaluation, cytological, biochemical and molecular
Somaclonal breeding programmes have
methods. At the molecular level, variations arise
been effective in developing disease-resistant
from changes in chromosome number or
crops (25,26,22,27). Phytopathotoxins have been
structure, or from subtle changes in the DNA itself
used as a proven selection pressure in callus
(41).
cultures. The scientist Carlson in 1973 (21) was the
first to report in vitro selection of callus for Molecular analysis of variations at the DNA
breeding purpose. Maize plants resistant to level is sensitive and will enable the detection of
Helminthosporium maydis were generated by changes that are not obvious at the morphological
Gengenbach et al. (1977) (28), using culture filtrate level. Moreover, molecular techniques enable
selection pressure on callus cultures. Later, detection of variants in the callus or juvenile
Behnke (1979) (29) regenerated Late Blight stages as opposed to morphological and
resistant potato plants from culture filtrate- physiological methods wherein regenerated or
exposed callus (22,29). Tomato (Lycopersicon adult plant response are measured. Most
esculentum) plants with improved resistance to importantly molecular detection methods will help
Fusarium (Fusarium oxysporum) wilt were identify genetically stable variations among a
obtained by exposing calli to fusaric acid (30). group of SVs that might have arisen due to either
Potato plants resistant to Early Blight (caused by epigenetic changes or due to subtle but stable
Alternaria solani) and Late Blight (caused by divergence at the DNA level. Molecular methods
Phytophthora infestans) were regenerated from are useful tools to analyze the degree of
protoplasts of potato (S. tuberosum L.) varieties divergence in SVs from the source calli or plant
‘Rssset Burbank’ (31) and ‘Bintje’ (32). This material.
approach has also been used in rice to select for Random Amplified Polymorphic DNA (RAPD)
resistance against brown spot disease (33). Cerato markers are the most commonly employed
and co-workers have shown that plants markers used to detect genetic variations in
regenerated from potato cells selected in vitro with somaclonal variants (42,43). In addition ISSR
culture filtrate of Phytophthora infestans exhibited markers (44), AFLP, RFLP and microsatellite DNA
improvement in resistance as compared with the markers have been used for molecular analysis of
source plants (34). variations induced in tissue culture (Table 2) (45).
In India, the selection pressure approach has For effective detection and evaluation of
been used in groundnut against Carcosporidium somaclonal variants, it is preferable to use a multi
peronatum (35), and in chickpea cell lines against thronged approach as shown in Table 2.
Fusarium oxysporum sp. cicero (36). Thakur et al. Somaclonal variants selected specifically for
(2002) (37) carried out in vitro selection and disease resistance are detected and evaluated
regeneration of carnation (Dianthus cayophyllus using a combination of biochemical methods,
L.) plants resistant to culture filtrate of Fusarium tissue culture screening, disease resistance assays,
oxysporum f. sp. dianthi. Rao et al. (2006) (36) cell viability tests and molecular marker methods.
developed Pigeon Pea cell lines and regenerated The selected putative SV calli are first screened on
plantlets from callus tolerant to culture filtrate of media containing toxic levels of the
Fusarium odum Buttler. Saxena et al. (2008) (38) phytopathotoxin or CF and selected based on the
generated leaf blight-resistant Pelargonium response of the SV compared to the source calli
graveolens (rose scented Geranium) plants by (Fig 2). Further the calli cells may be analyzed by
selection of callus with culture filtrate of vital stains such as Trypan blue to evaluate the
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