Turkish Journal of Agriculture and Forestry Turk J Agric For

(2015) 39: 515-530

http://journals.tubitak.gov.tr/agriculture/
© TÜBİTAK
Review Article doi:10.3906/tar-1406-176

Modernization in plant breeding approaches for improving

biotic stress resistance in crop plants
Babar HUSSAIN*
Department of Plant Breeding and Genetics, University of Agriculture, Faisalabad, Pakistan

Received: 30.06.2014 Accepted: 06.01.2015 Published Online: 08.07.2015 Printed: 30.07.2015

Abstract: Biotic stresses are a potential threat to global food security. The origin of new pathogens and insect races due to climatic and
genetic factors is a major challenge for plant breeders in breeding biotic stress resistant crops. Yield losses due to biotic stresses have
resulted in 800 million people underfed in the world. Reduced yield due to biotic stresses and increasing food demand put international
food security at risk as 70% more food will be required in 2050. This review describes and compares the conventional and molecular
genetics methods being used for breeding biotic stress resistant crops. In the past, classical breeding approaches like introduction,
hybridization, composite crossing, multiline, and backcross breeding were utilized for this purpose. However, these methods were
slow, expensive, and hectic for developing resistance in crops. Furthermore, breakdown of resistance due to fast evolving pathogens
could not be coped with using these time consuming methods. Therefore, molecular genetics approaches like mutation, marker assisted
selection (MAS), genomics, recombinant DNA technology, targeted induced local lesions in genome (TILLING), and virus induced
gene silencing (VIGS) were adapted by breeders to develop effective resistance in crop plants in a shorter time. TILLING, being a
nontransgenic method, is expected to become the most powerful tool for this purpose.

Key words: Gene pyramiding, MAS, QTLs, TILLING, transgenic, VIGS

List of Abbreviations: MAS: marker assisted selection; TILLING: Targeted Induced Local Lesions IN Genome; VIGS: virus induced gene
silencing; NILs: near isogenic lines; ROS: reactive oxygen species; QTLs: quantitative trait loci; RNAi: RNA interference; BC: backcross;
SAR: systemic acquired resistance; EMS: ethyl methyl sulfonate; MMS: methyl methyl sulfonate; miRNA: micro RNA, siRNA: short
interfering RNA, hpRNA: hairpin RNA; LRRs: lucine rich repeats; UV: ultraviolet radiation; SNPs: single nucleotide polymorphism

1. Introduction resistance to biotic and abiotic stresses were lost in this

About 5000–10,000 years ago, domestication of crop plants process. Similarly, selection for some desired traits in all
resulted in conversion of plants from wild to cultivated crop plants resulted in loss of useful genetic diversity/
forms. As humans selected fruits and crop plants on the variation. Genetic erosion caused the loss of primitive land
basis of sweeter fruits or prolific seed production, so races, wild relatives of crops, and cultivated varieties as
these random selections led to genetic erosion of many these were replaced by newer more reproductive cultivars
crop species. Most crops grown worldwide are the direct (Sharma et al., 2013). Likewise, loss of alleles during
result of selection in that era. It is thought that more breeding procedures is also considered genetic erosion
than 900 cultivated plant species were lost during the (Portis et al., 2004). Therefore, the loss of genetic diversity
domestication process as these crops were not preferred is not merely measured in terms of species extinctions, but
by the people of those times (Hammer and Khoshbakht, it also occurred at varietal and allele levels during selection
2005). For example, quinoa (Chenopodium quinoa) and cycles by plant breeders.
maca (Lepidium meyenii Walp.) were not domesticated It is quite evident that modern cultivars are less
in those times, but their recent domestication has found tolerant to biotic and abiotic stresses as compared to their
them very useful crops for their nutrition and medicinal wild relatives and available land races because of genetic
benefits, respectively (wan de Wouwet al., 2009). The erosion of useful genes during the course of evolution and
green revolution led to the development of new cereal selection for high yield (Portis et al., 2004; Reif et al., 2005).
types that had dwarfism and fertilizer responsiveness as As the focus of most of plant breeders was to improve
their most prominent traits. However, genes conferring the crop plants for higher yield, breeding for biotic and
* Correspondence: babar2331@gmail.com
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abiotic stresses was not focused on much in the remote 3.1. Introduction of exotic lines
past. However, three historical crop elimination events due Maize leaf blight disease caused by Cochliobolus
to biotic stresses i.e. potato blight in Ireland during the heterostrophus pathogen eliminated the corn crop from
1840s, and coffee rust in Brazil and maize leaf blight in the southern American regions during 1970. The complete crop
USA during the 1970s (Rogers, 2004) attracted the plant failure was due to the narrow genetic base of corn grown in
breeders towards stress breeding. For many years, plant that region (Ullstrup, 1972). This epidemic was due to the
breeders focused on breeding for biotic stresses (mostly development of a new race of pathogen called Race T, which
diseases) but climate change and consequently appearance had T-cms virulence gene. Therefore, a new type of plant
of abiotic stresses (heat, chilling, drought, and salinity) has material called Texas cytoplasm was introduced, which
shifted the plant breeders’ priority towards breeding for contained a mitochondrial sterility gene that helped to
abiotic stresses recently. develop hybrid seed. These hybrids were high yielding due
to hybrid vigor and were disease resistant. This resulted in
2. Importance of biotic stresses replacement of the old susceptible cultivars and 80% corn
Biotic stresses are the damage caused by other living cultivars in the USA were derived from T-cytoplasm as
organisms like viruses, fungi, bacteria, nematodes, insects, parent by the end of the 1970s (Strange, 2005). In the same
and weeds to plants. These stresses are of historical way, Shah et al. (1982) reported that potato germplasm
significance unlike the abiotic stresses that appeared to imported from the USA, India, and the Netherlands
be important recently due to climate change. There are showed promising resistance against potato leaf roll and
some historical events when biotic stresses (diseases) led blight diseases. Therefore, introduction is an important
to complete failure of the crops, resulting in famine in way to enhance the genetic diversity of crop plants when
those regions; examples include potato blight in Ireland, local germplasm lacks resistance. The introduction can be
coffee rust in Brazil (Rogers, 2004), and maize leaf bight of exotic cultivars through a multinational company or
in the USA (Ullstrup, 1972). The Great Bengal Famine in from a foreign gene bank. Wolfe (1993) reported genetic
1943 is another example of crop failure due to diseases diversity as the most effective method of durable resistance
(Padmanabhan, 1973). All of these events led to millions in crop plants. Efficacy of introduction for development of
of deaths and migration of people to other regions. Biotic disease resistant cultivars was also reported by Simmonds
stresses like insects and diseases cause considerable (1993).
reduction in grain yield, i.e. only diseases reduce 10% In this regard, a recent and important example is the
global food production, leaving 800 million people introduction of Bt-cotton in Pakistan and India. Farmers
underfed (Christou and Twyman, 2004). have adopted Bt-cotton promptly as it gives higher yield due
One of the most important factors that will play a to its resistance to chewing insects, especially bollworms.
role in disease spread is climate change. As temperature Other advantages include no or fewer sprays required to
is expected to increase in the near future, diseases caused control insects. Hence, a large area of the Pakistani cotton
by thermophilic bacteria are expected to appear. There belt has been under introduced transgenic Bt-cotton and
are chances that diseases may appear earlier during the indigenous non-Bt cotton is being replaced by the farmers.
crop season. Another important factor is that both insects As a result the area under Bt-cotton is increasing every
and pathogens change their races very rapidly, making year in the cotton belt of Punjab province in Pakistan
resistance a nondurable process. Similarly, nonavailability (Multan, Vehari, Bahawalpur, Toba Tek Singh, Jhang, and
of durable resistance sources makes the development Faisalabad districts) and 70% of wheat growing and 28% of
of crop plants resistant to biotic stresses a difficult job sugarcane growing farmers are interested in shifting from
(Strange, 2005). However, one or more genes provide non-BT to BT-cotton (Sabir et al., 2011).
resistance to plants against biotic stresses. Therefore, this 3.2. Hybridization and cultivar development
genetic basis can be exploited by plant breeders to develop Hybridization is used by plant breeders to make disease
resistance in crop plants against diseases and insect pests. and insect resistant hybrids and cultivars. The purpose
of hybridization is to combine the genes of higher yield
3. Conventional breeding methods for biotic stresses and disease and insect resistance from different sources.
The choice of methods and strategies for inducing and Brahim and Barrett (1991) developed a hybrid of barley
improving crop plant resistance against biotic stresses by composite crosses between various inbred lines of
mainly depends upon the availability of resistance sources. sorghum that was resistant to powdery mildew. Various
The breeding strategies can be divided into conventional disease resistant hybrids and cultivars of crop plants have
and modern methods. The conventional plant breeding been developed by plant breeders through conventional
methods played an important role in the development of hybridization and subsequent selection. Lasani-2008 is one
biotic stress resistant cultivars and the various methods of the best examples of cultivar development for disease
used for this purpose are discussed below. resistance; it is resistant to Ug-99 stem rust pathotype.

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Pakistani plant breeders working at Ayyub Agricultural develop resistance genotypes; e.g., Shabbir et al. (2014)
Research Institute (AARI), Faisalabad, took the Pakistan screened chick pea germplasm for resistance to the gram
based wheat germplasm to Uganda, where it was screened pod borer and found significant genetic variation in gram
for resistance to stem rust. The selected parents from as 3 genotypes showed stable resistance against the pod
the screened genotypes were crossed and high yielding borer. The Table lists the cultivars of various crop plants
and stem rust resistant plants were selected in successive released in various parts of the world for disease and insect
generations. The resultant cultivar Lasani-2008 is highly resistance.
resistant to the Ug-99 race of stem rust (Singh et al., 3.3. Backcross breeding
2011). The first step in this procedure is to identify the Backcross breeding is one of the most commonly used
resistance source to select the parents to be crossed to methods to insert single disease or insect resistant genes

Table. Different disease resistant cultivars released across the globe.

Cultivar Year of Release Origin Disease/Insect resistance

Novaspy 1986 Canada   Apple scab
McShay 1988 USA   Apple scab
Primevère 1997 Canada   Apple scab
Lasani-2008 2008 Pakistan Wheat stem rust (Ug99)
Golden Gopher 2009 USA Watermelon mosaic virus
Silver Slicer 2009 USA Cucumber mosaic virus
CaledoniaResel-L 2010 USA Wheat fusarium head blight
NARC-11 2011 Pakistan Wheat Stem Rust (Ug99)
Atlantic ----- USA Common bean mosaic virus
Honey Gold ----- USA Common bean mosaic virus
Senator ----- USA Summer squash powdery mildew
Black Pride  ----- USA Eggplant verticillium wilt
Pik-Red ----- USA tomato fusarium wilt
Pilgrim ----- USA Tomato fusarium wilt
Kaseberg 2013 USA Wheat stripe rust
VSM (HD 2733) 2001 India Wheat rusts
Urja (HD 2864) 2004 India Wheat brown and black rust
HD 2967 2010 India Wheat leaf blight
HD 3043 2012 India Wheat stripe and leaf rust
Pusa Sugandh-5  2005 India Rice brown spot, leaf folder and blast
Pusa Composite 4 2005 India Maize stalk borer
Pusa 1088 2005 India Chickpea fusarium wilt
Pusa 5023 2012 India Chickpea fusarium wilt
PARC-298 2005 Pakistan Rice bacterial leaf blight
PARC-299 2005 Pakistan Rice bacterial leaf blight
PARC-301 2005 Pakistan Rice bacterial leaf blight
Basmati-370 1933 Pakistan Rice bacterial leaf blight
Super Basati 1996 Pakistan Rice bacterial leaf blight
Pusa Vishal 2001 India Mungbean yellow mosaic virus
Pusa 9814 2006 India Mosaic virus, soybean mosaic virus
Eagle-10 2011 Kenya Wheat stem rust
Robin 2012 Kenya Wheat stem rust

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into a susceptible high yielding cultivar. The high yielding breeding (Browning, 1969; Ashizawa et al., 2001; Mundt,
cultivar is the recipient and the resistant cultivar is the 2002; Sharna et al., 2004; Keneni et al., 2012; Mundt 2014;
donor in this case. The high yielding cultivar (suppose A) Sattari et al., 2014) for the purpose of gene pyramiding
is crossed with the resistant cultivar (suppose B) during against biotic stresses have been used by plant breeders.
the first year. The resultant progeny is called backcross 1 In composite crossing, various crosses are made and
(BC1) and has 50% genetic content of both cultivars. To the resultant F1 hybrids are crossed with each other. The
recover high yielding genes of recipient cultivars, the offspring of this cross are again crossed with each other
recipient cultivar (A) is backcrossed with BC generations and the process of repeated crosses is continued unless
in subsequent years until almost 99% genes of A cultivar and until the genetic constituents of crossed plants are
are recovered in the BC6 generation. This strategy has united in a single genotype. In this way, resistance genes of
been used by plant breeders to develop disease resistant various cultivars are combined in single cultivars. Jackson
cultivars in most cases. Zhang et al. (2000) reported the et al. (1977) made three composite cross populations and
transfer of the cryIA gene of transgenic cotton through tested their resistance against the scald disease causing
backcross breeding. They concluded that a balanced pathogen R. secalis for generations. They concluded that
durable transfer of the Bt gene cryIA gene was observed three composite cross populations maintained their
through backcross breeding, which conferred resistance resistance up to F16, F32, and F47 generations, supporting
against chewing insects. the hypothesis that gene pyramiding confers horizontal
Zhu et al. (2004) studied the transfer of the Bt toxin resistance. Improved resistance in barley against different
gene in wild B. rapa from Bt B. napus through backcrossing. diseases through composite cross breeding has been
Cross between nine B. napus lines with three wild B. rapa reported, e.g., scald resistance (Jackson et al., 1982; Maroof
lines showed that there was successful transfer of the Bt et al., 1983) and resistance against powdery mildew
gene to F1 inter-specific hybrids and BC1 to BC4 generations. and blotch (Maroof et al., 1983). Finckh et al. (2000)
The Bt toxin level in F1 hybrid and backcross generations studied various aspects by gene pyramiding methods
was higher or comparable to that of transgenic B. napus. and especially of composite cross and concluded that
This proved that backcrossing can successfully transfer the composite crosses provide better and durable resistance
Bt gene to wild B. rapa, where it showed stable expression. against pathogens and insect pests. This is contributed by
Mutlu et al. (2005) reported the use of backcross breeding avoiding resistance breakdown as accumulation of various
in which the “XAN-159” donor cultivar was crossed with genes provides a buffering effect against disease and insect
the high yielding “Chase” cultivar of pinto bean and epidemics. The increased resistance is actually contributed
recurrent backcrossing by “Chase” gave higher resistance by increased resistance gene frequency in composite
to pinto bean against Xanthomonas campestris pv. Phaseoli. populations. Moreover, composite cross mixtures
This conferred resistance to common bacterial blight also provide differential adaptation against different
(CBB) and genetic analysis also confirmed the presence of pathotypes. Mixtures are a powerful way of getting a stable
QTL of CBB in backcross generations. yield as these resist biotic and abiotic stresses in a better
3.4. Composite crosses (gene pyramiding) way than their respective pure line. The only trait that may
Resistance in crop plants can be divided into two be compromised in the case of composite cross mixtures is
categories, i.e. vertical and horizontal resistance. Vertical quality, but it can be avoided by careful selection of pure
resistance is controlled by a single gene and is not durable lines to be used in composite crosses or mixtures. Butron
or long lasting as evolving pathogen races make such and Widstrom (2001) developed three corn populations
varieties susceptible (Van derPlank, 1975). On the other by composite crosses and all three showed improved
hand, horizontal resistance is controlled by many genes; resistance against corn ear-feeding insects.
thus it can provide resistance against a range of pathogen Although this method has been used by plant breeders
races, making the resistance durable (Browning, 1969). since the 1960s for biotic resistance development in crop
In this way, gene pyramiding from different sources in plants, it is still being used for the purpose. Sometimes,
single cultivars provides long-lasting resistance against it is combined with molecular breeding techniques like
biotic stresses (particularly diseases) and has become plant marker assisted selection (MAS) in order to make the
breeders’ tool to develop durable disease resistant cultivars selection process easy and effective. Danquah and Barrett
against various races of pathogens. Different strategies like (2002) used restriction fragment length polymorphism
composite crosses (Person et al., 1976; Jackson et., 1977; (RFLP) markers to study the powdery mildew resistance
Jackson et., 1982; Maroof et al., 1983; Finckh et al., 2000; pattern in a barley Cambridge Composite Cross Five
Butron and Widstrom, 2001; Danquah and Barrett, 2002; (CCV) population. Some hordein patterns were conserved
Murphy et al., 2004; Phillips and Wolfe, 2005; Steffan et in the three populations studied and hordein patterns
al., 2011; McDonald, 2014), synthetics, and multiline showed strong correlation with powdery mildew

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resistance. RFLP analysis indicated that 80% samples of a response, all isolines were susceptible to one or two races
F24 population showed the same restriction pattern as of of brown and yellow rusts and when bulked isolines were
“Algerian” cultivars, which is one of the 30 parents used grown; it improved the resistance of multilines against
in this composite cross. This cultivar contributes Mla1 many races of both rusts. The multiline had higher
allele, which confers powdery mildew resistance to barley number of tillers and grain size due to less disease attack
and so selection of this allele has been a predominant as compared to the parent Kalyansona cultivar. Wilson
evolutionary force. Composite cross populations are a et al. (2001) used backcross breeding to develop rust
source of dynamic gene pools as these are highly variable lines and the multiline of pearl millets developed by this
and diverse in terms of the resistant genes present in them classical breeding showed improved rust resistance and
and so they constitute a strong germplasm base for future dry matter production. Mundt (2002) has described the
breeding programs. Another advantage of composites multiline cultivars as an efficient tool for disease and insect
is the possibility of selecting heterogeneous cultivars management. The utility of this method against powdery
(Murphy et al., 2004; Phillips and Wolfe, 2005). In this mildew and rusts has been found in small grain crops.
way, composite crossing is an efficient way of introducing As multilines are mixtures of iso-lines, their usefulness
horizontal disease resistance in crop plants. Steffan et al. increases many fold under epidemiological conditions as
(2011) made 218 crosses of 30 wheat varieties in order to many resistance genes are involved in this case. Blast is
combine the bunt resistance genes in a single population a major rice disease that causes significant yield loss and
and ultimately in a cultivar. Molecular markers were used breeders have developed multilines that showed promising
for studying the polymorphism in F2 and F3 generations. resistance against blast disease (Ashizawa et al., 2001;
After each crossing cycle, the populations were bulked and Ishizaki et al., 2005; Sattari et al., 2014).
subsequent studies showed transfer of resistance genes Introduction of Bt genes into multilines has also been
during this process. McDonald (2014) concluded that with found to be efficient in making crops resistant to insect
the availability of molecular techniques composite crosses pests (Sharma, 2004). Multilines are mixtures of similar
may be used to accumulate the R genes in populations. pure lines having different genes of resistance against
insects and diseases, and so they can resist biotic stresses
3.5. Multiline breeding (gene pyramiding) better than their pure components. Even the multilines
Multiline breeding is also a promising way of horizontal can reduce the severity of biotic stress attacks. If any
resistance development in crop plants (Fleming and iso-line becomes susceptible, it can be removed from or
Person, 1978; Ashizawa et al., 2001; Mundt, 2002; Sharna replaced in the mixture (Keneni et al., 2012). Brunner et al.
et al., 2004; Keneni et al., 2012; Mundt 2014; Sattari et (2012) followed a new procedure of introducing durable
al., 2014). Multiline breeding is also called the “dirty resistance that combines both the conventional breeding
crop” approach as in this approach a high yield cultivar and modern transgenic approach to introduce powdery
is improved by making many iso-lines. The iso-lines are mildew resistant wheat. In their work, multilines were
developed by inserting a single resistance gene in different made by combining near-isogenic lines (NILs) containing
plants by backcrossing and so each component has a different resistance genes. Such NILs were developed that
different resistance gene. Then the iso-lines are bulked had the same genetic background except single R gene.
and the resultant line is called multiline as it contains They used different alleles of locus Pm3 to make NILs
many lines having different resistance genes. This gene of the same origin named Pm3a, Pm3c, Pm3d, Pm3f, or
pyramiding tool helps to combine many resistance Pm3g according to the allele expressed in a particular
genes together, thus providing durable resistance. The line. Although all these transgenic lines showed improved
components of multiline mixtures are morphologically resistance against powdery mildew, when a multiline was
alike plants that may be genetically quite different or a developed by mixing Pm3a, Pm3b, and Pm3d transgenic
single variety can be used for this purpose (Keneni et al., lines, the resistance became more durable than that
2012). Plant breeders have developed many disease and of individual transgenic lines. Multiline cultivars have
insect resistant cultivars through this method and some of been described to be have quantitative resistance against
such examples are discussed below. biotic resistance (Mundt, 2014), which makes it a durable
Marshall and Pryor (1978) studied the efficacy of process of resistance. Although the mechanism of this
multilines for long-term durable resistance by standard gene pyramiding is not yet clear, it has been successfully
procedures and concluded that multilines give stable used in various cases for this purpose. It will be a future
yield due to the presence of many resistant genes. Gill et research goal to develop durable resistance in crop plants.
al. (1980) studied bread wheat multiline KSML3, derived
from Kalyansona cultivar, and concluded that multiline 4. Modern breeding methods for biotic stresses
component isolines showed little variance for agronomic The problems associated with classical breeding
traits, i.e. the multiline was uniform. Regarding disease methods are longer time required to develop resistance

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cultivars, more effort and labor requirements, transfer Parkland. This conferred resistance against Pseudomonas
of nondesirable genes along with resistance genes by syringae when high amounts of inocula were applied.
hybridization, resistance breakdown due to development of This mutant also overexpressed BI-1 and MLO genes
new pathogen races, nonavailability of resistance sources, involved in other mechanisms of race nonspecific or Bgh
and less understanding of the mechanism of resistance resistance. In this way, the Nec1 barley mutant showed
in conventional methods. Therefore, there was a need to systemic acquired resistance (SAR) due to SA signaling
develop new and efficient modern methods to overcome activation, which may provide resistance against many
the above-mentioned problems. With the advancement other pathogens (Keisa et al., 2011).
of molecular genetics knowledge, many modern methods These disease lesion mimic (Les) phenotypes have
have been developed for this purpose. The modern become a powerful tool to develop stress resistance in
breeding procedures to overcome the problems associated plants. These mutants produce dead cells patches or lesions
with traditional breeding strategies are given below. on plant leaves in the absence of pathogens. As it was
4.1. Mutation breeding thought that these lesions appear in response to pathogens,
When resistance sources are not available in germplasm, they were named disease lesion mimics (Neuffer and
one strategy is to introduce inheritable changes or mutations Calvert, 1975; Walbot et al., 1983). In Arabidopsis,
in crop plants and to select rare mutants having resistance six mutants at four different loci were developed that
to particular stresses. Various mutagenic agents are used produced lesions on leaves. These lesions are not produced
for the purpose and they are divided into two main groups, by the pathogens. These mutants produced chemicals
i.e. physical mutagens (gamma, UV, X-ray irradiation) and that activated systemic acquired resistance or SAR in
chemical mutagens (EMS, MMS, colchicine etc.). Some Arabidopsis. These Isd mutants provided resistance against
novel chemicals like benzothiadiazole (BTH) have also various fungi (Dietrich et al., 1994). It has been found
been used in wheat and tobacco for this purpose. In such that Les mutants in Arabidopsis  are involved in disease
an experiment, BTH was used as an activator of systemic resistance against biotrophic pathogens as biotrophs
acquired resistance (SAR) in Arabidopsis thaliana. cannot survive on necrotic dead cells (Lorrain et al., 2003).
Mutants showed resistance against several pathogens In rice, Les (Spl) mutants activate SAR against rice blast
including Pseudomonas syringae pv tomato, turnip caused by Magnaporth egrisea fungus (Yin et al., 2000;
crinkle virus, and Peronospora parasitica. Mutants showed Zend et al., 2004). In maize, the Rp1 based Les mutant
accumulation of mRNAs of SAR related PR-1, PR-2, and provided resistance against Puccinia sorghi rust pathogen
PR-5 genes, which made the plant resistant to the above- and Cercosporazea maydis  causing gray leaf spot disease
mentioned pathogens (Lawton et al., 1996). However, (Hu et al., 1996; Johal, 2007).
recently, biological mutagens (transposable elements and Various mutants in Arabidopsis and other crops have
T-DNA insertion mutagenesis) have been widely used also been reported and mechanisms of resistance have been
by researchers (Alonso and Joseph, 2006). Zipfel et al. described by using recent molecular biology tools. Genger
(2004) reported development of resistance in Arabidopsis et al. (2008) described defense, no death 1 and 2 (dnd 1 and
by introduction of Flagellin flg22 peptide that was dnd 2) mutants in Arabidopsis thaliana that were involved
independent of jasmonic acid, salicylic acid, and ethylene in resistance gene mediated with reduced hypersensitive
signaling. The fls2 mutants showed enhanced resistance to response (HR). These mutants also showed increased
crude bacterial extracts. Various mutants having disease broad-spectrum resistance regulated by enhanced salicylic
resistance have been developed worldwide. In resistance acid (SA) levels in plants. However, some other mutations
conferring R genes, leucine rich repeats (LRRs) are present like sid2, npr1, and ndr1 that affected signaling and SA
and a mutation in the RPS5 gene of Arabidopsis showed accumulation reduced the resistance of dnd mutants
interaction of the LRR region with other plant proteins. against Hyaloperonospora parasitica and Pseudomonas
The mutant rps5-1 resulted in replacement of lysine with syringae pv tomato but resistance of dnd mutants against
glutamate amino acid in the LRR region and this altered Botrytis cinerea remained unaffected due to sid2, npr1,
plant response against downy mildew and bacteria (Warren and ndr1 mutants. Wawrzynska et al. (2008) reported a
et al., 1998; Huang et al., 2010; Zhu et al., 2013; Wang et loss of function mutation in Enhanced Disease Resistance
al., 2014). In barley, the CNGC4 gene has 67% amino acid 1 (EDR1) gene of Arabidopsis conferring resistance to
similarity with Arabidopsis based AtCNGC4, which is Golovinomyces cichoracearum, which caused powdery
a disease resistance related gated ion channel. A mutant mildew in Arabidopsis. This recessive mis-sense mutation
namely Nec1 in barley was made that had a frame shift was mapped in the “Keep on Going” (KEG; At5g13530)
mutation in the CNGC4 gene, resulting in overexpression gene. However, this KEG gene mutation was seedling lethal
of PR-1 protein. The Nec1 barley mutant produced higher and showed HR to abscisic acid (ABA) and glucose. Huang
amounts of salicylic acid and H2O2 than its parent variety et al. (2010) reported that Arabidopsis mutant chilling-

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sensitive2 (chs2) showed increased pathogen resistance However, Zhu et al. (2013) concluded that plant
due to accumulation of hydrogen peroxide and salicylic defense response activation compromised plant growth
acid and up-regulation of pathogenesis related (PR) genes. and development in Arabidopsis mutant radical-induced
However, seedling death occurred under low temperature cell death1 (rcd1). The rcd1 showed disturbed reactive
of 4–12 °C. This was due to a single amino acid substitution oxygen species (ROS) production in plants, which resulted
in TIR-NB-LRR (Toll/interleukin-1 receptor-nucleotide- in growth inhibition. Chujo et al. (2014) reported that
binding leucine-rich repeat) R protein called RPP4 phosphomimic based mutation in one of the rice WRKY
(Recognition of Peronospora Parasitica 4). At molecular transcriptional factors (TFs) conferred resistance against
level, this resistance is obtained by increased production rice blast disease. It was found that OsWRKY53 TF
of mRNA transcript from mutated RPP4, which made the worked as a substrate of fungal MAPKs cascade OsMPK3/
plants disease resistant and chilling susceptible. Carstens OsMPK6 and so modifications in this TF made it difficult
et al. (2014) reported that constitutive induced resistance 1 for the fungus to identify this region, which conferred
(cir1) mutant in Arabidopsis showed increased production resistance to rice against Magnaporthe oryzae strain
of salicylic acid (SA), which led to constitutive expression of Ina86-137. In a transient reporter assay consisting of co-
defense genes, making Arabidopsis resistant to biotrophic expression of OsWRKY53 with OsMKK4, trans-activation
pathogens. The characterization of the CIR1 mutant was of OsWRKY53 was increased due to phosphorylation of
done by studying enhanced disease susceptibility1 (EDS1) serine-proline cluster. Even more enhanced resistance
and Phytoalexin deficient4 (PAD4) regularity nodes. It against blast fungus was found in transgenic plants that
was found that both EDS1 and PAD4 regulatory nodes are showed over-expression of a phospho-mimic mutant of
required for expression of CIR1 as it is situated upstream OsWRKY53 TF (OsWRKY53SD) due to even higher up-
of the EDS1-PAD4 regulatory node. Expression analysis of regulation of pathogenesis related protein genes. It can
EDS1 showed that there was increased protein production be concluded from the above discussion that mutation
but mRNA level was unchanged; this suggested that post- breeding not only helps to create useful genetic variation
transcriptional gene silencing (PTGS) of EDS1 through for biotic stress resistance but also helps to understand
CIR1 might be involved in this process. It was further the resistance mechanism in crop plants. Mutagenesis
found that temperature variations greatly influence the combined with molecular genetics has become a powerful
resistance to Pseudomonas syringae pv. tomato (Pst) and tool for crop improvement and has the ability to cope with
the resistance mechanism is activated at lower temperature. the useful genetic diversity during genetic erosion of crop
In barley, brassinosteroid hormones are involved plants.
in the regulation of many growth and developmental 4.2. TILLING
processes. Brassinosteroid insensitive 1 (BRI 1) is a Targeting Induced Local Lesions IN Genomes (TILLING)
receptor present in the cell membrane and is a key player was introduced in 2000. It is a cost effective reverse
in the brassinosteroid signaling cascade. A mutation in the genetics tool that detects point mutations induced
conserved domain of the kinase tail of BRI 1 receptor in artificially usually using chemical mutagens (EMS). EMS
semi-dwarf UZU barley made it resistant to lodging and a is the most efficient mutant and it produces G/C to A/T
range of pathogens. This resistance was due to inducible and transition. TILLING can be used as a functional genomics
constitutive resistance mechanisms and was characterized tool to discover the genes involved in biotic and abiotic
by transcriptomic and biochemical profiling. However, stress tolerance. Eco-TILLING is a kind of TILLING that
some viruses are able to cause infection by disrupting provides the advantage of single nucleotide polymorphism
virus-induced gene silencing (VIGS; see details in section (SNP) in natural mutants to screen the plant populations
4.5.1), which reduces production of mRNAs involved for different biotic and abiotic stresses. Eco-TILLING
in resistance (Ali et al., 2014). However, Goddard et al. was used in barley to study the genetic variation in Mla
(2014) reported that the BRI 1 mutation was conserved and Mlo resistance genes at allele level, which conferred
in both barley and brachypodium. Their experiments on resistance against powdery mildew (Mejlhede et al., 2006).
barley and brachypodium showed that disruption of BRI The genetic variation lost during domestication and other
1 had pleiotropic effects on disease resistance and plant breeding programs has traditionally been recovered by
development. Mutation in BRI 1 made the plants resistant plant breeders through utilizing land races and wild
to a wide range of hemibiotrophic and necrotrophic relatives as parents in breeding procedures. However, a
pathogens but they showed no resistance to biotrophic low success rate and transfer of nondesirable genes along
pathogens. The same effects of this mutation were recorded with resistance genes are problems that can be covered by
in model species Brachypodium distachyon and Hordeum using TILLING (Rashid et al., 2011). It can be used as an
vulgare, indicating the conservation of disease resistance alternate method to introduce genetic diversity in targeted
mechanisms between both species. genes by mutagenesis to overcome these problems.

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4.3. Molecular breeding and genomics Chu et al. (2009) identified five QTLs, namely QLr.fcu-
In classical breeding, selections were made on 3BL, QLr.fcu-3AL, QLr.fcu-6BLQLr.fcu-4DL, and QLr.fcu-
morphological bases that were highly influenced by 5BL, linked to leaf rust resistance in wheat. In wheat, QTL
the environment. This created confusion in selection mapping helped to identify the QTLs linked to yellow,
of desirable parents for breeding programs. However, leaf, and stem rust resistance. They identified 10 QTLs for
the discovery of DNA based markers like RAPD, AFLP, yellow rust resistance including five major QTLs (QYr.sgi-
PFLP, and SNPs linked to various economically important 2B.1, Lr34/Yr18, QYr.sgi-4A.1, QYr.sgi- 2B.1a, and QYr.sgi-
traits has provided the opportunity to plant breeders to 4A.2). The seven leaf rust QTLs mapped were wPt2633–
select their desired parents in an efficient way. This type psp3152, barc4–wPt0103, wPt5556–wPt6278, wPt1325–
of selection is called marker assisted selection and it has wPt3045, ksm25m50b–wPt8721, barc352–gwm111, and
the advantage that DNA or molecular markers are not wmc323–gwm537. Stem rust QTLs were barc183–wPt3879
influenced by the environment, making the selection and wPt7181–psp3152 (Prins et al., 2011). Wang et al.
process accurate and efficient (Hussain et al., 2012). Simons (2013) identified 54 QTLs in F2 population for thrips (two
et al. (1998) described the additional benefit of MAS like QTLs), tomato spotted wilt virus (15 QTLs), and leaf
germplasm can be screened for various disease resistance spot (37 QTLs). Out of 23 QTLs in F5, one was for thrips,
genes simultaneously and linkage of these markers to nine were for tomato spotted wilt virus, and 13 were for
target alleles and SNPs can even identify single nucleotide leaf spots. Identification of major QTLs may lead to the
polymorphism. Various molecular markers linked to development of disease resistance cultivars.
disease resistance have been utilized by plant breeders to 4.4. Transgenic approach
select disease resistance cultivars. RFLP markers helped to When resistance genes are not found in a particular species
identify five genes, i.e. Dm3, Dml, Dm4, Dm13, and Dm5/8, or even in its wild relatives and land races, resistance cannot
conferring resistance to powdery mildew in lettuce (Landry be introduced through conventional hybridization. In this
et al., 1987). RFLP and RAPD markers helped to locate the situation, genes of resistance are introduced from unrelated
shs gene linked to head smut resistance in sorghum (Oh species through recombinant DNA technology to overcome
et al., 1994). In tomato, AFLP markers helped to identify the genetic barriers. Foreign genes are transferred to crop
Ve1 and Ve2 genes linked to resistance against Verticillium plants using different transformation tools like gene gun or
dahlia, Tm1, Tm2, and Tm22 genes linked to resistance particle bombardment, electroporation, floral dip (direct
against tomato mosaic virus genes, Mi1-2 gene linked to transformation methods), and Agrobacterium mediated
Meloidogyne incognita resistance, and I and I2 genes linked transformation (in direct transformation methods). The
to Fusarium oxysporumf. sp. Lycopersici resistance (Arens discovery of the ability of A. tumefaciens to transfer its
et al., 2010). T-DNA to its host has been utilized for transformation
In some cases, disease resistance is controlled by by biologists and it is the method used extensively for
more than one gene, making its inheritance complex. transformation. This approach has been used by plant
Such traits are called quantitative traits and the regions breeders to introduce entirely new genes in plants and
in the genome where genes of a specific trait are located the organisms with foreign genes are called genetically
are termed quantitative trait loci (QTLs). QTL analysis to modified organisms or GMOs. The development of disease
identify the regions of the genome linked to biotic stress and insect resistance in plants is the most important
resistance is named QTL mapping and various QTLs application of the transgenic approach. Although proper
linked to disease resistance have been discovered by plant bio-safety protocols are followed for GMO testing, they are
breeders, which has helped in the efficient selection of considered dangerous due to their unnatural origin. These
desirable plants (Hussain et al., 2012). QTLs linked to issues have limited development of the transgenic approach
Fusarium head blight (Yang et al., 2005), foliar disease in developed countries, which indirectly affects food
(Chu et al., 2008), and leaf rust resistance (Huang et security in developing countries, where such innovations
al., 2003) have been mapped in wheat. In Arabidopsis, 3 are badly needed to ensure food security of the masses.
QTLs namely RPW10, RPW11, and RPW12 were mapped The global area under transgenic crops increased 40 times
on chromosome number 3, 5, and 4, respectively, which during 1996 to 2003, i.e. an increase from 1.7 million ha to
provided resistance against powdery mildew in A. thaliana 67.7 million ha was observed (James, 2003).
(Wilson et al., 2001). Similarly, six QTLs (RFO1-RFO6) in 4.4.1. Transgenic approach for disease resistance
Arabidopsis were mapped that provided resistance against For disease resistance, candidate genes are those involved
Fusarium oxysporum. RFO1 and RFO2 were present on the in plant microbe interaction and limit the virulence traits of
1st chromosome while RFO3 and RFO4 were mapped on the pathogens, e.g., pathogen cell wall degrading enzymes
3rd and 4th chromosomes, respectively, and chromosome and toxins. Such genes are introduced to plants that
5 had RFO5 and RFO6 QTLs (Diener et al., 2005). enhance the production of plant defense molecules like

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saponins, ROS, phytoalexin, and antimicrobial peptides. diseases in Arabidopsis thaliana and over-expression
These are antimicrobial proteins that confer resistance to of this gene in transformed wheat conferred resistance
pathogens by attacking their virulence factors. Such gene against Fusarium graminearu (Makandar et al., 2006).
introductions by transgenic approach confer resistance to Similarly, rice transgenic having NH1 gene, an NPR1 gene
plants against different diseases (Strange, 2005). In rice, orthologue, provided resistance against Xanthomonas
the afp gene of Aspergillus giganteus was introduced whose oryzae pv Oryzae causing bacterial leaf blight disease
product AFP protein is an antifungal compound. This gene in rice (Chern et al., 2005). Various transgenic crops
showed stable integration and was inherited by the next transformed with the AtNPR1 gene have been developed
generations. This provided resistance against Magnaporthe that have resistance against various pathogens. Such plants
grisea  fungus, which caused rice blast disease (Coca et include grapefruit resistant to Xanthomonas citri subsp.
al., 2004). Wheat transformed with a viral gene encoding citri (Zhang et al., 2010); cotton resistant to Fusarium
KP4 protein conferred 10%–30% resistance against oxysporum f.sp. vasinfectum and nematodes (Parkhi et
Ustilago maydis causing wheat smut (Schlaich et al., 2007). al., 2010); carrot resistant to Botrytis cinerea, Alternaria
Similarly, the thionin gene transferred in rice from oats radicina, and Xanthomonas hortorum (Wally et al. 2009);
provided resistance against Burkholderiap lantarii  (Iwai tomato resistant to Stemphylium solani, F. oxysporum,
et al., 2002). Sugarcane transformation by detoxifying and X. campestris (Lin et al., 2004); rice resistant to
agents degraded toxins and provided resistance against Xanthomonas oryzae and Fusarium verticillioides (Quilis et
various pathogens (Zhang et al., 1999). A synthetic al., 2008); and tobacco resistant to Meloidogyne incognita
peptide named D4E1 was introduced in cotton and poplar, (Priya et al., 2011).
providing resistance against Thielaviopsis basicola fungus 4.4.2. Transgenic approach for insect resistance
in cotton (Rajasekaran et al., 2007) and some bacteria in One of the most important traits that have been improved
poplar (Mentag et al., 2003; Montesinos, 2007). Apple through the transgenic approach is insect resistance.
was transformed with a grapevine gene stilbene synthase Transfer of insecticidal protein coding genes present in
that codes for a phytoalexin, namely resveratrol, found Bacillus thuringiensis (a gram positive, naturally occurring
in grapevine but absent in apples. Apple pathogens were soil-borne bacterium) to crop plants has conferred
unable to degrade the resveratrol due to the absence of resistance against chewing type insects. Crops transformed
any mechanism for this purpose. Resveratrol presence in with Bacillus thuringiensis based genes are termed Bt crops
transgenic apple was confirmed by chemical analysis. This and Bt cotton, maize, and eggplant are the most noteworthy
conferred resistance against fungal pathogens (Szankowski examples of such transgenic crops. These transgenic plants
et al., 2003). produce toxic proteins that damage the insect gut region,
Sometimes, R genes are transformed to a new species resulting in insect death. This also lowers the cost of
or even genus to activate a general resistance mechanism production of crops as no sprays of pesticides/insecticides
in crop plants. An R gene, Vf, was transferred from wild to kill chewing Lepidoptera insects are required. Kumar
apple (Malus floribunda) to Malus domestica, cultivated et al. (2008) reported additional benefits of Bt crops that
apples, which conferred resistance against Venturia included higher yield due to no or less damage to crop due
inaequalis (Belfanti et al. 2004). A maize based R gene Rxo1 to chewing insects, and their environment friendliness and
was transformed to rice that conferred resistance against hygienic nature as compared to insecticides that pollute the
bacterial blight of rice caused by Xanthomonas oryzae environment due to toxic residues of insecticides on edible
(Zhao et al., 2005). However, R gene transfer between plant parts. Bt-cotton transformed with the cry1Ac gene
closely related species gives better results (Ayliffe et al., was grown in a field for years and still even soil did not
2004). Rpi-blb2, an NB-LRR R-gene, was transformed show traces of Bt toxins, showing their environmentally
to cultivated potato type from wild potato type Solanum friendly nature (Head et al., 2002). These benefits resulted
bulbocastanum, providing resistance against an Oomycete, in wide acceptance of Bt crops by farmers despite the
Phytophthora infestans (van der Vossen et al. 2005). In this concerns shown by some people about their bio-safety
way, various resistance genes can be transferred to crop (Sabir et al., 2011).
plants during independent events, which may provide Bt crops transformed with a Bacillus thuringiensis
horizontal resistance to crop plants. gene have been used in many regions of the world over
Disease resistance by transgenic approach can also the past 30 years after the development of Bt corn in the
be achieved in crop plants by introducing such genes mid 1980’s. Lepidopteran pests of various crops including
that activate the plant signaling pathways on pathogen vegetable and food crops are controlled by this transgenic
infections, making them resistant to various pathogens approach. Bt genes have even been useful against beetles,
simultaneously. The NIM1 or NPR1 gene is a very black flies, and nuisance mosquitoes. The higher efficacy
important part of signaling pathways against several of its toxic proteins has made them an integral part of

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IPM procedures. Five major classes of Bt (cry) genes are many crops to target and silence the pathogen genes. These
cry1, cry2, cry3, cry4, and cyt1. Cry1 and cry2 are useful transgenic plants target the pathogen genome by using
against Lepidoptera, cry3 provides resistance against artificial miRNA, hairpin RNA, sense/antisense RNA, or
Coleoptera (beetles), and cry4 and cyt1 develop resistance siRNA (Simon-Mateo and Garcia, 2011).
against Diptera (mosquitoes and black flies). These toxins RNA silencing based resistance has mostly been
are not contact toxins but have to be eaten by the insect reported against RNA viruses and rarely against DNA
along with plant parts. Most of the toxins have a core viruses. Transgenic mung bean formed by bombardment
portion about half the toxin size that digests the mid gut of its leaves with hpRNA construct containing mung
of the insect, resulting in insect death. In the USA, the bean yellow mosaic virus (DNA geminivirus) promoter
CrylAc gene has been transferred into cotton to make it conferred resistance against MYMV (Pooggin et al.,
tolerant to the tobacco budworm, CrylAb was engineered 2003). Resistance against bean golden mosaic virus (a
in corn to provide resistance against the European corn DNA geminivirus) was developed by expressing an
borer (Ostrinia nubilalis), and potatoes were cloned with hpRNA transgene obtained from AC1 sequence coding for
the Cry3A gene to check the population of the Colorado replicase enzyme (Aragao and Faria, 2009). Therefore, the
potato beetle (Federici, 1998). Bt eggplant made by ‘Event geminiviruses can be silenced at post- or transcriptional
EE-1’ produces crystalline (Cry) protein toxin expressed in level. Similarly, resistance against potato spindle tuber
all plants and confers resistance to eggplant against insects viroid (PSTVd) was achieved when potato was transformed
(Seralini, 2009; Hanur, 2011). Zhang et al. (2000) reported with a viroid hairpin RNA (Schwind et al., 2009). In
the Bt gene cryIA provided resistance against chewing cassava, transgenic plants were developed to generate
insects when cotton was transformed with this gene. Such siRNAs by inserting the virus coat protein sequence of
insect resistance has also been developed in B. rapa from Ugandan cassava brown streak virus. In normal cassava
B. napus by transferring the cry1c gene (Zhu et al., 2004). plants, disease appeared 6 months after planting while
4.5. RNAi mediated gene silencing transgenic plants having the viral coat sequence showed
RNAi silencing has been utilized against viruses, bacteria, a 3-month delay in disease occurrence and 98% of clones
fungi, and nematodes for development of biotic stress of the 718-001 line remained disease-free even after 11
resistant plants. months of planting. RT-PCR results showed that Ugandan
4.5.1. Virus induced gene silencing (VIGS) cassava brown streak virus was found in leaves of 0.5%
RNA interference was once considered a gene expression of transgenic and 57% of nontransgenic plants. Similarly,
regulation mechanism in eukaryotes as it involved 90% of nontransgenic storage roots in cassava had necrosis
degradation of mRNA resulting in inhibition of translation due to attack of Ugandan cassava brown streak virus
after transcription, and so was termed post-transcriptional (UCBSV) and cassava brown streak virus (CBSV). On the
gene silencing or RNA interference (RNAi). The mechanism other hand, the 718-001 line had 95% of its roots free from
of RNAi starts with degradation of dsRNA by Dicer UCBSV and CBSV symptoms, showing the effectiveness of
enzyme into miRNAs or siRNAs of 21–24 nucleotides. RNAi in viral disease control (Ogwok et al., 2012). RNAi
These smaller RNAs are recruited by RISC complex (an has been used to induce immunity against a wide range
effecter complex) to their target sequence of mRNAs, of viruses like cassava mosaic virus (Vanderschuren et al.,
which results in its degradation (Ding, 2010). In addition 2009), rice dwarf virus (Takumi et al., 2008), bean golden
to its gene regulatory role, siRNA based RNAi also plays mosaic virus (Bernstein et al., 2001), tomato leaf curl virus
a role in innate antiviral defense in plants and the process (Shelly et al., 2010), stripe mosaic virus in barley (Cakir
is termed virus induced gene silencing (VIGS) (Beclin and Tor, 2010), and mung bean yellow mosaic virus (Haq
et al., 2002; Ding, 2010). When a virus attacks the plant, et al., 2010).
plant machinery targets the viral dsRNA formed during 4.5.2. RNAi in bacteria
host machinery based viral replication and converts it into Now, it is a documented fact that RNA mediated silencing
virus-derived siRNAs. Host RISC complex now recruits is not limited to developing resistance against viruses only
these vsiRNAs to the viral genome, resulting in inhibition but it also confers resistance against other biotic stresses
of viral protein translation (Ding, 2010). Many viruses like bacteria, fungi, insects, and nematodes. Niblett and
have VSR or viral suppressor of RNA silencing protein to Bailey (2012) reported that siRNA targeting essential
encounter the plant defense mechanism. VSR suppresses genes of pathogens and insect pests provided resistance
the gene silencing mechanism either by modifying to date palm against a wide range of pests including
components of RANi or by binding siRNA (Duan et al., Staphylococcus bacterium, red palm weevil, Helicoverpa
2012). Keeping in view the mechanism of siRNA based and Diabrotic insects, Fusarium oxysporum, Albedinis
RNA silencing, transgenic plants have been developed in fungus, and Heterodera and Meloidogyne nematodes.

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Escobar et al. (2001) utilized RNAi technology to shut complementary RNAi constructs. As a result, female
down or silence the expression of two oncogenes (iaaM nematode number reduction up to 64% was observed
and ipt genes) in Lycopersicon esculentum and Arabidopsis (Sindhuet al., 2009). Niblett and Bailey (2012) reported
thaliana. These genes were involved in crown gall that siRNA targeting essential genes conferred resistance
formation under bacterial attack and so silencing of these to date palm against Heterodera and Meloidogyne
genes conferred resistance against crown gall disease. nematodes. Therefore, this method has the potential to
A long siRNA (30–40 nucleotides) was transformed in induce immunity against a range of nematodes.
Arabidopsis thaliana that silenced AtRAP (an important
downregulator of plant defense) and conferred resistance 5. Future challenges and prospects
against P. syringae (Katiyar-Agarwal et al., 2007). This Plant pathogens and insects are living entities that not
shows the effectiveness of RNA mediated silencing for only respond to plant resistance mechanisms but also
developing resistance against bacterial diseases as well. evolve rapidly to avoid the plant resistance response, thus
4.5.3. RNAi in fungi posing a serious threat to global food security. Another
RNAi has also been utilized for developing plants resistant to challenge that biologists face is nonrecognition of many
fungal pathogens. In tomato pathogen C. fulvum, silencing plant pathogens. Similarly, increasing populations of
of a vital gene HCf-1 coding for pathogen hydrophobin insects with increasing temperatures are another issue that
conferred resistance against this pathogen (Spanu, 1997). needs to be given consideration. The scoring methods to
Similarly, cgl1 and cgl2 genes of Cladosporium fulvum document pest damage are qualitative in nature, which
fungus were silenced by transforming tomato with cgl2 results in errors. Another problem in breeding crops for
hairpin, which provided resistance in tomato against this biotic stresses is that insects and pathogen species and
pathogenic fungus (Segers et al., 1999). This method has even races vary from area to area, which makes it difficult
also been utilized to induce resistance against various for a single cultivar to maintain resistance for a long
fungi like Fusarium graminearum (Nakayashiki, 2005), time. Recombination of pathogens is another problem
Magnaporthae oryzae (Chen et al., 2010), Blumeria associated with breeding for biotic stresses, for example,
graminis (Nowara et al., 2010), and Puccinia striiformis appearance of the Burewala strain of CLCUV in Pakistan
(Chuntao et al., 2011). Nowara et al. (2010) developed and Ug99 rust pathogen in Africa, and recombination of
barley resistant to powdery mildew (Blumeria graminis) East African cassava mosaic virus with African cassava
by transforming barley with hairpin RNAi cassettes that mosaic virus gave rise to a new pathogen strain called
targeted vital genes of the fungus and significant disease UgV. This strain along with African cassava mosaic virus
reduction was observed resultantly. has resulted in the occurrence of more infection in crop
4.5.4. RNAi in nematodes plants, showing greater yield losses. Similarly, crop plants
The discovery of RNAi in a nematode C. elegans has have lost considerable genetic diversity during the course
indicated the possibility of exploiting the RNA mediated of evolution, which makes them potentially susceptible
silencing to make crop plants resistant to nematodes. to evolving pathogens and insects. Usually, a few high
Silencing of a gene coding for chitin synthase enzyme was yielding cultivars bred from narrow based germplasm
found in Meloidogyne artiella eggs by dipping eggs in a grown in a particular area having limited resistance genes
gelatinous matrix having dsRNA. The silencing resulted in also results in pest attack.
reduced chitin in the eggshell, leading to delay in hatching Keeping in view the above facts, introduction of
of juveniles (Fanelli et al., 2005). Feeding of dual oxidase such resistance mechanisms to crop plants is important,
based dsRNA to root knot nematode juveniles resulted in targeting some vital pathogen or insect part. Introduction
silencing of vital genes, and female size, number, and egg of chitinase genes coding for chitinase enzymes in plants
production reduction up to 70% were observed (Bakhetia that degrade chitin in the cell wall of fungal pathogens is
et al., 2005). The transgenic soybean having siRNA a general resistance mechanism. In the case of insects, Bt
targeting major sperm protein genes resulted in reduced toxin provides resistance against a wide range of chewing
cyst formation and egg production, making soybean insects. Plant breeders need to broaden the genetic base
resistant to cyst nematodes (Steves et al., 2006). Transgenic and must include wild relatives, landraces, and exotic
tobacco inserted with hpRNA constructs gave resistance germplasm of crops in their hybridization programs, as
against root knot nematodes as these hairpins targeted two these have genes of resistance against various biotic and
vital genes of root knot nematode and reduced their total abiotic stresses. Modern plant breeding methods like
mRNAs production (Fairbairn et al., 2007). The mRNA transgenic approach, TILLING, gene silencing, and VIGS
production of targeted nematode genes was reduced have great potential to be used in future for breeding crops
when the nematodes were fed on transgenic plants having against biotic stresses.

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