RNAi for crop inprovement

1. 623Bir Bahadur et al. (eds.), Plant Biology and Biotechnology: Volume II: Plant Genomics
and Biotechnology, DOI 10.1007/978-81-322-2283-5_31, © Springer India 2015
31.1 Introduction
The crop yield is facing drastic reduction because
of scarcity of land, water resources, climatic
changes, and damages caused by abiotic and
biotic stresses. Increasing population is also
posing the demand for global sustainable agricul-
tural practices. To address this demand,
conventional breeding is being employed from
ages as one of the effective ways to raise quality
plants, but this process is time consuming and
laborious and also has other ecological, physio-
logical, and biological constraints. However,
genetic engineering is playing pivotal role in crop
improvement. Novel traits can be effectively
incorporated into crops through genetic manipu-
lations, leading to increased yield, nutritional
value, and also tolerance to biotic and abiotic
S. Yogindran • M.V. Rajam (*)
Department of Genetics, University of Delhi South
Campus, Benito Juarez Road, New Delhi 110021,
India
e-mail: rajam.mv@gmail.com
31RNAi for Crop Improvement
Sneha Yogindran and Manchikatla Venkat Rajam
Abstract
Scientific breakthroughs bring about significant advances in basic research,
which ultimately lead to their applications for human welfare. One such
discovery is RNA interference (RNAi), a highly conserved gene regula-
tory mechanism that controls the expression of genes at posttranscriptional
level. Since the discovery of RNAi, it has become evident that RNAi has
immense potential for crop improvement. The technology has been
employed successfully to study the gene function and to alter the gene
expression in plants for desired traits. RNAi has proved to be a novel and
potential tool in combating abiotic and biotic stresses. Enriched nutritional
quality, male sterility, delayed ripening, and secondary metabolite manip-
ulation add to the list of successful applications of RNAi technology. This
chapter focuses on the potential of RNAi technology to produce improved
crop varieties.
Keywords
RNA interference • Gene silencing • Transgenic plants • Crop
improvement

2. 624
stresses. But, transgenic crops always had ethical
issues in terms of biosafety especially in case of
edible crops. Hence, safer strategies have to be
developed for crop improvement.
RNA interference (RNAi) technology is
proven to be a potential alternative for crop
improvement, probably with less biosafety issues
as no transgene protein is expressed in transgenic
lines (Rajam 2012). RNAi pathway mainly com-
prises small-interfering RNAs (siRNAs) and
microRNAs (miRNAs). Both siRNAs and miR-
NAs are produced by the cleavage of double-
stranded (dsRNA) precursors by a member of the
RNase III family Dicer and Dicer-like enzyme,
respectively (Bernstein et al. 2001; Hutvagner
et al. 2001). Finally, the small noncoding RNAs
(siRNAs and miRNAs) in association with RNA-
induced silencing complex (RISC), Argonaute
(AGO), and other effector proteins lead to gene
silencing. By this means these small RNAs regu-
late various aspects of plant growth and develop-
ment. This gave biotechnologists an opportunity
to use this easy but highly effective way to modu-
late gene expression to get desired traits (Mittal
et al. 2011). The present chapter therefore focuses
on the discovery of RNAi and its mechanism of
action and applications for crop improvement.
31.2 RNAi: Discovery
and Mechanism of Action
It all began in 1990 when chalcone synthase gene
(CHS A) encoding a key enzyme in anthocyanin
biosynthesis pathway, introduced in Petunia hyb-
rida L. for enhancing anthocyanin pigments, pro-
duced white or chimeric flowers instead of dark
purple flowers in transgenic Petunia. This sug-
gested that the introduced transgene was func-
tionally inactive and also suppressed the
endogenous gene expression and the phenome-
non came to be known as “cosuppression”
(Napoli et al. 1990; Hannon 2002; Campbell and
Choy 2005). A similar phenomenon named
“quelling” was then discovered in the fungus
Neurospora crassa (Romano and Macino 1992;
Cogoni et al. 1996). Later on, the term RNAi
came into picture for the first time when it was
elucidated in the nematode, Caenorhabditis
elegans (Fire et al. 1998).
The process of RNAi in plants is initiated by
21- to 24-nucleotide-long (nt), small interfering
RNAs (siRNAs), which are generated from long
endogenous or exogenous dsRNA molecules
through the cleavage by a ribonuclease III-type
enzyme called Dicer (Hamilton and Baulcombe
1999; Zamore et al. 2000). These siRNAs (21–
24 nt) are then incorporated into a multiprotein
complex called RISC which contains AGO pro-
teins (Baumberger and Baulcombe 2005;
Vaucheret 2008). The ATP-activated RISC
unwinds the double-stranded siRNA. The sense
strand of the siRNA duplex is degraded by RNA
helicase activity and the antisense strand of
siRNA molecule is retained in the RISC complex
(Kusaba 2004). RISC with antisense siRNA then
targets the homologous mRNA by complemen-
tary base-pairing and cleaves the mRNA leading
to inhibition of protein synthesis (Bartel 2004)
(Fig. 31.1).
siRNAs also regulate the gene expression at
transcriptional level by regulating the chromatin
modification. siRNA recruits several DNA- and
histone-modifying proteins including the cytosine
methyltransferase CHROMOMETHYLASE3
(CMT3) and maintains the chromatin in a minimal
transcriptional activity state, leading to transcrip-
tional gene silencing (TGS) (Ossowski et al. 2008;
Fagegaltier et al. 2009; Burkhart et al. 2011).
31.3 Applications of RNAi
Technology in Agriculture
Increasing world population is facing threat due
to huge crop losses because of several factors that
affect the proper production and distribution of
crops. There is an urgent need to address this
problem which can be achieved by amalgamation
of conventional and cutting edge technologies.
RNAi technology has emerged as one of the most
potential and promising strategies for producing
improved quality plants. This biological phenom-
enon has been assessed in a number of plant
systems and has been successfully used to silence
the genes to get better traits. The examples listed
S. Yogindran and M.V. Rajam

3. 625
below illustrate the possibilities of this intrinsic
biological mechanism for commercial
exploitation.
31.3.1 Biotic Stress Tolerance
Total agricultural production is greatly affected
by biotic stresses, which include viral, bacterial,
and fungal pathogens, insect pests, and nematode
parasites. There is a need to address this problem
more efficiently. We have discussed the impor-
tance of RNAi as a promising solution to biotic
stress management.
31.3.1.1 Viral Diseases
Viruses cause major loss of plant productivity
and transmit the disease either directly from par-
ent to progeny or indirectly through insect vec-
tors and hence their control becomes very
difficult. “Pathogen-derived resistance” (PDR),
where resistance to a determined pathogen could
be obtained from its own genetic material, has
been used to develop disease-resistant plants.
This approach comprises (1) the expression of
viral coat protein (CP) and replication-associated
proteins (Reps) and (2) gene silencing by anti-
sense and hpRNA (Shepherd et al. 2009).
RNAi has revealed a way for obtaining virus-
resistant trait in many crop plants. It was first
reported in potato, where simultaneous expres-
sion of both sense and antisense transcripts of the
helper-component proteinase (HC-Pro) gene
showed complete resistance to potato virus Y
(PVY) (Waterhouse et al. 1998). Silencing of
viral coat protein through RNAi is an effective
method for generation of resistant plant that has
been successfully reported in potato and medici-
nally important papaya plants (Missiou et al.
2004; Kertbundit et al. 2007).
An important class of viruses includes gemini-
viruses, the DNA viruses, which are responsible
for a significant amount of crop damage.
Expression of hairpin construct of noncoding
intergenic region of mungbean yellow mosaic
India virus (MYMIV) under the control of 35S
promoter in MYMIV-infected black gram plants
resulted in recovery from the infection which
Fig.31.1 Biogenesis and mechanism of action of siRNAs
31 RNAi for Crop Improvement

4. 626
lasted till senescence (Pooggin et al. 2003). Apart
from that, when siRNAs designed to the replicase
(Rep)-coding sequence of African cassava
mosaic virus (ACMV) were cotransfected with
protoplasts, it showed 99 % reduction of Rep
transcripts and 66 % reduction of viral DNA
(Vanitharani et al. 2003). In another report, trans-
genic cassava lines with high levels of AC1-
homologous small RNAs showed resistance to
ACMV (Vanderschuren et al. 2009).
RNAi-mediated resistance to cassava brown
streak disease (CBSD) in Cassava was first dem-
onstrated by Patil et al. (2011). They observed
resistance against very distant isolates of caus-
ative organism cassava brown streak virus
(CBSV) and cassava brown streak Uganda virus
(CBSUV).
31.3.1.2 Bacterial Diseases
Although considered structurally simple, bacteria
are extremely diverse from a metabolic stand-
point and are found almost everywhere on earth
in vast numbers—from those living in jet fuel and
on the rims of volcanoes to those thriving in
hydrothermal vents deep on the ocean floor. The
first bacterial disease ever discovered was anthrax
(caused by Bacillus anthracis) of cattle and sheep
in 1876. The discovery of anthrax in cattle was
immediately followed by the discovery of fire
blight of pear and apple (caused by Erwinia amy-
lovora) by T. J. Burrill from the University of
Illinois (1877–1885). Escobar et al. (2001)
showed the striking example of bacterial disease
management where RNAi showed a remarkable
type of gene regulation. They developed a crown
gall disease management strategy that targets the
process of tumorigenesis (gall formation) by ini-
tiating RNAi of the iaaM and ipt oncogenes.
Expression of these genes is a prerequisite for
wild-type tumor formation. RNAi constructs, tar-
geting iaaM and ipt gene(s) in Arabidopsis thali-
ana and Lycopersicon esculentum, showed
resistance to crown gall disease (Dunoyer et al.
2007).
Infection by Pseudomonas syringae pv.
tomato in Arabidopsis induced the production of
natsiRNA (nat-siRNAATGB2) which downregu-
lates a PPRL gene that encodes a negative regula-
tor of the RPS2 disease-resistance pathway. As a
result, the induction of nat-siRNAATGB2
increases the RPS2-mediated race-specific resis-
tance against P. syringae pv. tomato in
Arabidopsis (Katiyar-Agarwal et al. 2007).
31.3.1.3 Fungal Diseases
Homology-based gene silencing induced by
transgenes (cosuppression), antisense, or dsRNA
has been successfully demonstrated in many
plant pathogenic fungi, including Cladosporium
fulvum (Hamada and Spanu 1998), Magnaporthe
oryzae (Kadotani et al. 2003), Venturia inaequa-
lis (Fitzgerald et al. 2004), Neurospora crassa
(Goldoni et al. 2004), Aspergillus nidulans
(Hammond and Keller 2005; Khatri and Rajam
2007), Fusarium oxysporum (Singh 2011), and
Fusarium graminearum (Nakayashiki 2005).
The efficient method for rapid characterization of
fungal genes using diced siRNAs has been
reported in model filamentous fungus A. nidulans
(Natarajaswamy et al. 2013).
RNAi-mediated downregulation of GST (glu-
tathione S-transferases) enzyme, which catalyzes
a variety of reactions, resulted in significant
increase in resistance in Nicotiana tabacum
against Phytophthora parasitica var. nicotianae
(Hernández et al. 2009). The transgenic tobacco
plants expressing heterologous (mouse) ornithine
decarboxylase (ODC) antisense RNA have been
shown to have higher resistance to Verticillium
wilt as compared to wild-type plants (Kumria
2000). The expression of dsRNA and antisense
transcripts specific to powdery mildew fungi
Blumeria graminis in wheat and barley affected
the growth of the fungus (Nowara et al. 2010).
Virus-induced gene silencing (VIGS) has been
used to introduce the gene fragments from the
rust fungi Puccinia striiformis f. sp. tritici or P.
graminis f. sp. tritici to plant cells leading to
reduced expression of the corresponding genes in
the rust fungus (Yin et al. 2011).
In cereals, barley stripe mosaic virus (BSMV)-
induced RNAi has emerged as a useful tool for
loss of function studies. Three genes, a MAP
kinase (PtMAPK1), a cyclophilin (PtCYC1), and
calcineurin B (PtCNB), predicted to be involved
in pathogenicity, have been targeted by
S. Yogindran and M.V. Rajam

5. 627
BSMV-mediated host-induced gene silencing
(HIGS) in the wheat leaf rust fungus Puccinia tri-
ticina (Pt). BSMV RNAi constructs were then
inoculated in the wheat plant leaves. Subsequent
Pt inoculation resulted in a suppressed disease
phenotype and reduced endogenous transcript
levels of the targeted fungal genes indicating
translocation of siRNA molecules from host to
fungal cells (Panwar et al. 2013b). The same
group has shown the use of Agrobacterium tume-
faciens-mediated in planta induced transient
gene silencing (PITGS) assay for use in Triticum
spp. (wheat). Agroinfiltration effectively deliv-
ered hairpin silencing constructs of the three
genes mentioned above in wheat, leading to the
generation of fungal gene-specific siRNA mole-
cules in infiltrated leaves and resulting in up to
70 % reduction in transcription of the endoge-
nous target genes (Panwar et al. 2013a).
Fusarium wilt, caused by Fusarium oxyspo-
rum f. sp. cubense (Foc), is among the most
destructive diseases of banana (Musa spp.).
RNAi-mediated knockdown of vital genes of
fungus (velvet and Fusarium transcription factor
1) has shown effective resistance against Foc.
Transformed banana lines were found to be free
of external and internal symptoms of Foc after
6-week-long greenhouse bioassays. The five
selected transgenic lines for each construct
showed resistance to Foc for 8 months postinocu-
lation (Ghag et al. 2014).
31.3.1.4 Insect Attack
Insects can cause considerable damage to crop
plants. By defoliating plants or sucking out their
sap, insects slow down their growth by weaken-
ing and sometimes killing them. The use of RNAi
for the insect control seems to be the consequence
of success of Cry toxins from Bt as an insecti-
cide. The sensation of RNAi in insect control
began when two exciting reports came into scien-
tific community. Mao et al. (2007) targeted cot-
ton bollworm gut-specific cytochrome P450 gene
CYP6AE14, which confers resistance to gossy-
pol, a polyphenol compound of cotton plants.
Cotton bollworm larvae when fed on transgenic
tobacco and Arabidopsis plants that expressed
the CYP6AE14-specific dsRNA showed
sensitivity to gossypol in artificial diets. Whereas,
Baum et al. (2007) fed Western corn rootworm
larvae (WCR, Diabrotica virgifera) 290 different
dsRNAs and observed that 14 of them caused sig-
nificant mortalities at doses ≤5.2 ng/cm2
.
Transgenic corn expressing dsRNA specific to
the gene encoding the A subunit of the V-type
ATPase proton pump showed significant reduc-
tion in WCR-feeding damage. Both reports
showed that RNAi pathway can be exploited to
control insect pests via in planta expression of
dsRNA against well-chosen target genes of
insects (Baum et al. 2007; Mao et al. 2007).
v-ATPaseA gene has also been shown to be a
potent target to control the whitefly population.
Plant-mediated pest resistance was achieved
against whiteflies by genetic transformation of
tobacco expressing siRNAs against the whitefly
v-ATPaseA gene. The transcript level of v-ATPa-
seA in whiteflies was reduced up to 62 % after
feeding on the transgenic plants, leading to their
mortality (Thakur et al. 2014).
Thereafter, a number of reports suggested the
success of this technology for the control of
insect pests. Transgenic tobacco plants express-
ing dsRNA against EcR-USP (ecdysone receptor-
ultraspiracle particle) (Zhu et al. 2012), AChE
(acetylcholinesterase) (Kumar 2011), and HR3
(Xiong et al. 2013) involved in regulation of
molting and development in H. armigera resulted
into resistant plants, with larvae fed upon them
showing developmental deformities and lethality.
3-Hydroxy-3-methylglutaryl coenzyme A reduc-
tase (HMG-CoA reductase, HMGR) gene, a key
enzyme in the mevalonate pathway in insects, has
also been shown to be a potential target for insect
control using RNAi (Wang et al. 2013).
Apart from its application in generating resis-
tant plants, RNAi has been extensively used for
functional studies in insects. The feeding of
dsRNA and siRNA solutions for knockdown of
target pest genes has been successfully shown in
Apolygus lucorum (Zhou et al. 2014), Nilaparvata
lugens (Chen et al. 2010), Bemisia tabaci
(Upadhyay et al. 2011), and Helicoverpa armig-
era (Kumar et al. 2009) which led to a strong
decline in the expression of the target gene and
can be used to explore gene functions. Recently,
31 RNAi for Crop Improvement

6. 628
oral delivery of dsRNA molecules to Spodoptera
littoralis against a gene highly similar to P102 of
Heliothis virescens strongly suppressed the
encapsulation and melanization response, sug-
gesting that the protein is functionally conserved
and plays role in insect immunity (Lelio et al.
2014).
31.3.1.5 Nematode Attack
A number of genera and species of nematodes are
highly damaging to a great range of hosts, includ-
ing foliage plants, agronomic and vegetable
crops, fruit and nut trees, turf grass, and forest
trees. Some of the most damaging nematodes are
root knot (Meloidogyne spp.), cyst (Heterodera
and Globodera spp.), root lesion (Pratylenchus
spp.), spiral (Helicotylenchus spp.), burrowing
(Radopholus similis), bulb and stem (Ditylenchus
dipsaci), reniform (Rotylenchulus reniformis),
dagger (Xiphinema spp.), and bud and leaf
(Aphelenchoides spp.) (Tamilarasan and Rajam
2013). Oral delivery of dsRNA was first demon-
strated in C. elegans (Fire et al. 1998).
Plant-mediated RNAi in plant parasitic nema-
todes through dsRNA targeting has been shown
in RKN which resulted in effective resistance
(Yadav et al. 2006; Huang et al. 2006). The host-
induced RNAi in Arabidopsis thaliana, which is
ahostforthesugarbeetcystnematodeHeterodera
schachtii, led to reduction in the number of
mature nematode females. Although no complete
resistance was observed, the reduction of devel-
oping females ranged from 23 to 64 % in differ-
ent RNAi lines (Sindhu et al. 2009). Fewer
females (reduced by 81–93 %) were observed on
the transgenic roots obtained by hairy root cul-
tures engineered to silence either two of ribo-
somal proteins, a spliceosomal protein or
synaptobrevin, of H. glycines by RNAi (Klink
et al. 2009). Similarly, high reduction in egg pro-
duction was achieved by targeting mRNA splic-
ing factor prp-17 or an uncharacterized gene
cpn-1 (Li et al. 2010).
The success reports of RNAi for controlling
the nematode infection also include the following
research outcomes. Targeted silencing of con-
served region of M. incognita gene acetylcholin-
esterase (AChE) involved in neurotransmission
and also in many cellular processes, through
host-derived RNAi, resulted in reduced fecun-
dity. The enhanced resistance to nematode infec-
tion displayed by different lines strongly suggests
their utilization in nematode control (Tamilarasan
2013). A recent study also demonstrated that
MiASB (M. incognita mitochondrial ATP syn-
thase b subunit) silencing had a positive effect on
the control of root-knot nematodes, and the gene
may be associated with the formation of galls
caused by the nematode (Huang et al. 2013).
31.3.2 Abiotic Stress Tolerance
Plants are constantly affected by abiotic factors
such as high salinity, flood, drought, heavy
metal, and variable temperatures which consid-
erably reduce the productivity. Functional
genomics studies have come up with novel genes
involved in stress adaptation in plants, which can
be manipulated to get tolerance (Pardo 2010).
Poly (ADP-ribose) polymerase (PARP) is
induced by stress in animals and is responsible
for energy depletion. The enzyme PARP1 and to
a lesser extent PARP2 are primarily responsi-
ble for stress-induced poly (ADP-ribosyl)ation
activity. Upon induction, polymers of ADP-
ribose are synthesized by a range of nuclear
enzymes using NAD+
as substrate, while over-
production of PARP leads to a rapid breakdown
of the NAD+
pool. ATP molecules are required
for resynthesis of NAD+
, and as a consequence
the cellular ATP is depleted which leads to
necrotic cell death (De Block et al. 2005). Since
PARP1 and PARP2 homologues are found in
plants, PARP was targeted by RNAi for abiotic
stress tolerance in plants (De Block et al. 2005;
Vanderauwera et al. 2007).
RNAi-mediated downregulation of RACK1,
receptor for activated C-kinase 1, which is a
highly conserved scaffold protein with flexible
functions, plays important roles in plant growth
and development indicating its possible role in
drought stress response in rice compared to non-
transgenic plants (Da-Hong et al. 2009).
Similarly, disruption of a rice farnesyltransfer-
ase/squalene synthase (SQS) by maize squalene
S. Yogindran and M.V. Rajam

7. 629
synthase through RNAi improved drought
tolerance at both the vegetative and reproductive
stages (Manavalan et al. 2012). Expression of
OsTZF1, a member of the CCCH-type zinc fin-
ger gene family in rice, was induced by drought,
high-salt stress, and hydrogen peroxide. OsTZF1-
RNAi plants were susceptible to abiotic stress
demonstrating that OsTZF1 positively regulates
high-salt and drought stress tolerance in rice
plants (Jan et al. 2013).
31.3.3 Development of Male Sterile
Plants
A hybrid production system is based on the
mechanism for inducing male sterility in one of
the parental lines so as to ensure purity of the
resultant hybrid seed. Male sterility trait has
been an important aspect in agriculture to
improve the crop productivity by the hybridiza-
tion process (Duvick 1999). Moreover, its value
in gene containment of the genetically modified
crops has increased its importance (Moon et al.
2010). Natural male sterile mutants, cytoplas-
mic male sterile (CMS) mutants, and nuclear
male sterile (NMS) mutants have been used for
the hybrid seed production (Duvick 2005; Wang
et al. 2005).
TA29, an anther-specific gene, is expressed
exclusively in anthers at the time of microspore
development. Downregulation of TA29 of
tobacco (N. tabacum cv. Samsun) by RNAi pro-
duced male sterile lines (Nawaz-ul-Rehman et al.
2007). Nucleases are enzymes playing vital role
in nucleic acid metabolism. Rice transgenic
plants expressing hairpin RNA for OsGEN-L
(OsGEN-like) gene, a new member of the RAD2/
XPG nuclease family, exhibited low fertility and
were male sterile (Moritoh et al. 2005). Silencing
a male-specific gene, Bcp1, in the model host A.
thaliana resulted in male sterile lines. Bcp1, an
anther-specific gene, is active in both diploid
tapetum and haploid microspores. Transgenic
plants were phenotypically indistinguishable
from nontransgenic plants, and by crossing with
nontransgenic fertile pollens, successful seed set
was observed (Tehseen et al. 2010).
S-Adenosylmethionine decarboxylase
(SAMDC), a key gene involved in polyamine bio-
synthesis, when targeted in tapetal tissue of
tomato under the control of tapetal-specific A9
promoter using RNAi, resulted in male sterile
lines. These transgenics had sterile pollen and
failed to set fruits, but female fertility was unaf-
fected as cross-pollination resulted in fruit setting
(Sinha and Rajam 2013).
31.3.4 Nutritional Improvement
Plants provide most of the nutrients required in
the human diet, although the major staple crops
are often deficient in some of these nutrients.
RNAi technology has also been used in several
plants to improve their nutritional quality. A
dominant high-lysine maize variant was pro-
duced by knocking out the expression of the
22-kDa maize zein storage protein, a protein that
is poor in lysine content (Segal et al. 2003). A
recessive lysine-rich mutant called opaque 2 (O2)
has been obtained by traditional breeding. The
O2 gene encodes a maize basic leucine zipper
transcriptional factor that controls the expression
of a subset of storage proteins, including the
22-kDa zein storage protein. Opaque 2 mutant
was lysine-rich but showed poor seed quality and
yield. Downregulation of lysine-poor zein gene
via RNAi generated normal and quality seeds
with high levels of lysine without altering the
general functions of O2 (Angaji et al. 2010).
Fatty acid composition of cotton seed oil was
manipulated by hpRNA-mediated gene silencing
of two fatty acid desaturase genes, stearoyl-acyl-
carrier protein D9-desaturase and oleoylphos-
phatidylcholine u6-desaturase. Downregulation
of one gene substantially elevated stearic acid
level from 2 to 3 % up to as high as 40 %, and
silencing of the other gene enhanced oleic acid
content, up to 77 % compared with about 15 % in
seeds of untransformed plants (Liu et al. 2002).
RNAi technology was used to enhance
β-carotene content in potato by silencing the
β-carotene hydroxylase gene (BCH), which con-
verts β-carotene to zeaxanthin. RNAi constructs
having the tuber-specific granule-bound starch
31 RNAi for Crop Improvement

8. 630
synthase (GBSS) promoter and the other
containing the strong constitutive cauliflower
mosaic virus 35S (CaMV 35S) promoter were
introduced into potato by Agrobacterium-
mediated transformation. The transformants
derived from the GBSS construct contained more
β-carotene than CaMV 35S transformants. These
results showed that BCH silencing can increase
the content of carotenoids, β-carotene, and lutein
in potato which will provide a tool for combating
the incidence of vitamin A deficiency in popula-
tions (Eck et al. 2007). Silencing of
DE-ETIOLATED1 (DET1) in Brassica napus
resulted in seeds with increased levels of lutein,
β-carotene, and zeaxanthin relative to nontrans-
genic seeds (Wei et al. 2009). DET1 suppression
also led to reduced levels of sinapate esters
responsible for bitter taste, poor meal palatabil-
ity, and unpleasant flavor to the meat and milk of
animals fed on a B. napus seed meal diet.
Tomatoes are a principal dietary source of
carotenoids and flavonoids, both of which are
highly beneficial for human health.
Overexpression of genes encoding biosynthetic
enzymes or transcription factors has resulted in
tomatoes with improved carotenoid or flavonoid
content, but never with both. Increased nutri-
tional value was obtained by suppressing an
endogenous photomorphogenesis regulatory
gene, DET1, using fruit-specific promoters com-
bined with RNA interference (RNAi) technology.
Both carotenoid and flavonoid contents were
increased significantly, whereas other parameters
of fruit quality were largely unchanged (Davuluri
et al. 2005).
Starch, a major plant carbohydrate, is com-
posed of amylase and amylopectin. Amylose
molecules tend to efficiently form digestion-
resistant complexes when the cooked food under-
goes the process of cooling (Crowe et al. 2000).
To increase the amylase content in wheat, RNAi
constructs designed to silence the genes encoding
the two starch-branching isozymes of amylopec-
tin synthesis were expressed under a seed-specific
promoter which resulted in increased grain amy-
lase content to over 70 % of total starch content
(Regina et al. 2006; Tang et al. 2007). RNAi con-
structs have been used in Zea mays and A. thaliana
to modify the levels of phosphate metabolism
involved in leaf starch degradation. Phosphate
manipulation led to increase in starch content
(Weise et al. 2012).
31.3.5 Flower Color Modification
Floriculture, or flower farming, is concerned with
the cultivation of flowering and ornamental plants
for gardens and for floristry. Flower color modifi-
cation is one of the most desirable traits in floral
industry. RNAi can be used as a tool to silence
the pigment synthesis genes, which can lead to
different flower color patterns. CHI (chalcone
isomerase) gene silencing in tobacco by RNAi
showed decreased pigmentation and change of
flavonoid components in flower petals. Plants
showed yellow coloration due to accumulation of
high levels of chalcone in pollens (Nishihara
et al. 2005).
Nakatsuka et al. (2008) performed RNAi-
mediated suppression of three anthocyanin bio-
synthetic genes—chalcone synthase (CHS),
anthocyanidin synthase (ANS), and flavonoid
3′5′-hydroxylase (F3′5′H)—in gentian plant. In
CHS suppressed transgenics, petals exhibited
pure white to pale-blue color, whereas in ANS
suppressed transgenics, petals were only pale-
blue. Suppression of the F3′5′H gene decreased
delphinidin derivatives and increased cyanidin
derivatives and led to magenta flower colors. The
same group demonstrated that RNAi-mediated
downregulation of anthocyanin 5, 3′-aromatic
acyltransferase (5/3′AT) and flavonoid
3′,5′-hydroxylase (F3′5′H) activities in gentian
plant produced modified flower color (Nakatsuka
et al. 2010).
The flower color of Torenia hybrid, an impor-
tant garden plant, was successfully modulated by
RNAi. Downregulation of chalcone synthase (CHS)
gene by using each of the coding region and the
3′-untranslated region of the CHS mRNA as an
RNAi target led to modulation of flower color
from blue to white and pale (Fukusaki et al. 2004).
Roses are the most important cut flower
commercially and have played a major role in
human culture from ancient time. RNAi-mediated
S. Yogindran and M.V. Rajam

9. 631
silencing of the cyanidin genes in rose and
introduction of delphinidin genes produced flowers
that accumulated delphinidin-based anthocyanins
exclusively with a concomitant color change toward
blue (Tanaka et al. 2009; Katsumoto et al. 2007).
31.3.6 Secondary Metabolite
Manipulation
Plant secondary metabolites are economically
important as drugs, fragrances, pigments, food
additives, and pesticides. Secondary metabolite
production, however, sometimes is blocked by
undesirable compounds, which can be suppressed
by RNAi. The versatility of RNAi for controlling
multigenes responsible for metabolite production
has been well recognized as an effective strategy
(Borgio 2009).
The first commercially useful cultivar pro-
duced by RNAi was the rice mutant line LGC-1
(low glutelin content-1), thus making it useful for
patients who must restrict protein intake such as
kidney disease patients (Mochizuki and Hara
2000). LGC-1 and some cultivars developed
using LGC-1 as a cross parent are beginning to be
used for this type of diet therapy (Kusaba et al.
2003). This dominant mutation produced hpRNA
from an inverted repeat for glutelin, the gene for
the major storage protein glutelin, leading to
lower glutelin content in the rice through RNAi.
Gil-Humanes et al. (2008) used RNAi technol-
ogy to silence the expression of specific γ-gliadins
and demonstrated the feasibility of systemati-
cally silencing specific groups of gluten proteins
without affecting fertility, grain morphology, and
seed weight when compared to the control lines.
RNA-mediated suppression of tryptamine
biosynthesis in Catharanthus roseus during hairy
root culture eliminated the production of mono-
terpene indole alkaloids (a class of natural prod-
ucts derived from two starting substrates),
tryptamine and secologanin. To utilize this chem-
ically silent background, they introduced an
unnatural tryptamine analog into the production
media and demonstrated that the silenced C.
roseus culture could produce a variety of novel
products derived from this unnatural starting
substrate (Runguphan et al. 2009).
Transformation of Papaver somniferum with
RNAi construct designed to reduce the levels of
the gene encoding the morphine biosynthetic
enzyme salutaridinol 7-O-acetyltransferase
(SalAT) led to the accumulation of the intermedi-
ate compounds, salutaridine and salutaridinol, in
a ratio ranging from 2:1 to 56:1 (Kempe et al.
2009). California poppy (Eschscholzia califor-
nica) cells were transformed with RNAi con-
struct harboring berberine bridge enzyme (BBE)
gene to suppress the activity of the enzyme and
resulted into reticuline accumulation at a maxi-
mum level (Fujii et al. 2007). The artemisinin
content of transgenic Artemisia annua L plants
was significantly increased by 3.14-fold as com-
pared to untransformed control plants by sup-
pressing the expression of SQS (squalene
synthase), a key enzyme of sterol pathway, by
means of a hairpin RNA (Zhang et al. 2009).
Over 10 % of the coffee on the world market
is shared by decaffeinate coffee (DECAF).
Caffeine is a stimulant of the central nervous sys-
tem, the heart muscle, and the respiratory system
and has a diuretic effect. Its adverse side effects
include insomnia, restlessness, and palpitations.
Modulation of caffeine biosynthesis in planta
was done by suppression of CaMXMT1
(7-N-methylxanthine methyltransferase or theo-
bromine synthase) by the double-stranded RNA
method. The caffeine content of transgenic plants
was reduced by up to 70 %, indicating that it is
possible to produce decaffeinated coffee beans
using RNAi (Ogita et al. 2003, 2004).
Cotton is a major cash crop which produces
fibers and oil. The cotton seeds that remain after
fiber extraction could be extensively used as
sources of protein and calories, but they are
largely underutilized because they contain a toxic
gossypol terpenoid. Gossypol is also produced in
vegetative cotton tissues where it protects cotton
plants from insects and other pathogens.
Transgenic cotton plants expressing RNAi con-
struct of the d-cadinene synthase gene of gossy-
pol synthesis fused to a seed-specific promoter
caused seed-specific reduction of this metabolite,
31 RNAi for Crop Improvement

10. 632
while its content in nonseed tissues was
comparable to the control plants (Sunilkumar
et al. 2006).
Cassava is a major staple food in tropical
countries but contains unnecessary glucosides.
Jørgensen et al. (2005) used RNAi to prevent pro-
duction of the cytochrome P450 enzyme that
makes the first committed step in the biosynthesis
of linamarin and lotaustralin, and generated
transgenic cassava (Manihot esculenta) plants
with elimination of cyanogenic glucosides in the
leaves (<1 % of nontransgenic amounts) and a
92 % reduction of cyanogenic glucoside amount
in tubers.
31.3.7 Enhanced Fruit Shelf Life
Fruit ripening has received considerable attention
because of the dramatic changes in the metabolic
processes that take place before and after this
event, as well as due to its commercial impor-
tance. Fruits are an important dietary supplement.
The quality of fruit is determined by a wide range
of desirable characteristics such as nutritional
value, flavor, processing qualities, and shelf life.
The massive losses accrue during transportation
and post-harvest handling of the fruit which run
into billions of dollars worldwide. Therefore,
there is a need to increase the shelf life of fruits
so as to minimize the agronomic loss.
Ethylene, unlike the rest of the plant hormone
compounds, is a gaseous hormone inducing sev-
eral responses during ripening through a signal-
ing cascade (Crocker et al. 1935). The shelf life
of tomato has been increased by targeting the
genes coding for ethylene biosynthesis pathway.
The dsRNA of tomato ACC oxidase expression
cassette was successfully introduced into tomato
cultivar Hezuo 906 under the control of cauli-
flower mosaic virus 35S promoter by A.
tumefaciens-mediated transformation.
Transgenic plants produced had fruits having
traces of ethylene and had a prolonged shelf life
of more than 120 days with similar levels of total
soluble sugar, titratable acid, amino acids, and
total soluble solids as the control plants (Xiong
et al. 2005). Similarly, delayed ripening tomatoes
were generated by silencing three homologues of
1-aminocyclopropane-1-carboxylate (ACC) syn-
thase (ACS) gene, catalyzing the rate-limiting
step in ethylene biosynthesis during the course of
ripening, using RNAi technology. The chimeric
RNAi-ACS construct designed to target ACS
homologues effectively repressed the ethylene
production in tomato fruits. Fruits from such
lines exhibited delayed ripening and extended
shelf life for 45 days, with improved juice quality
(Gupta et al. 2013).
Recently, SlSGR1 (encoding a STAYGREEN
protein that plays a critical role in the regulation
of chlorophyll degradation in tomato leaves and
fruits)-repressed lines reduced H2O2 levels and
inhibited ethylene signal transduction during
fruit ripening, promoting the retention of firm-
ness and sustained cell membrane integrity and
resulting in delayed fruit senescence during stor-
age and an enhanced shelf life from 25 to
45–58 days when harvested at the breaker (Br)
stage and stored at room temperature (Luo et al.
2013).
31.3.8 Pros and Cons of RNAi
Technology
RNA silencing has emerged as an area of thor-
ough investigations leading to new discoveries.
RNAi-mediated gene silencing is a valuable tech-
nology for the development of transgenic crop
plants, with a focus on nutritional enrichment and
plant protection from bacteria, nematodes, fungi,
and insects pests, which are two major hurdles in
production and productivity of agriculture crops.
RNAi strategy has certain advantages over other
approaches. For instance, the silencing is
sequence-specific and more than one gene can be
targeted. Additionally, the extent of the gene
silencing can be controlled, so that the essential
genes will only be silenced at desired stage and
tissue. As there is no transgene protein expres-
sion in RNAi approach, there would not be any
extra metabolic load on the transgenic plants.
Further, in the absence of transgene protein,
there is less likelihood of development of resis-
tance by the target pest or pathogen, and RNAi
S. Yogindran and M.V. Rajam

11. 633
plants would pose minimal biosafety issues
(Rajam 2011).
However, there are also some limitations to
RNAi technology. Although it is a method of
sequence-specific targeting, there may be issues
of off-target effects leading to undesirable traits.
There does exist a concern that inadvertent sec-
ondary effects could be generated by using non-
coding small RNA-mediated gene silencing,
especially when this approach is used to engineer
broad spectrum resistance into plants against
pathogens/pests. Delivery methods for the
dsRNA are a limiting step for a number of spe-
cies for which RNAi-based approaches cannot be
used easily. There still remain many significant
challenges in development and commercializa-
tion of GM crops utilizing RNAi-based technol-
ogy. Not only tremendous efforts are required for
achieving scientific breakthroughs but also pro-
motion of public acceptance of GM crops among
other complicated ethical issues has to be taken
care of.
We, hence, conclude that agricultural biotech-
nology, including RNAi technology, would serve
as one of the most important measures for crop
improvement, which will contribute to agricul-
ture productivity to a great extent.
Acknowledgements We thank the University Grants
Commission, New Delhi, for their Special Assistance
Programme and the Department of Science and
Technology (DST), New Delhi, for their FIST and
DU-DST PURSE Programme. INSPIRE fellowship to SY
by DST is acknowledged.
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