This document summarizes the potential of RNA interference (RNAi) technology for crop improvement. It discusses how RNAi was discovered through early experiments in plants in the 1990s. The mechanism of RNAi involves long double-stranded RNA being cleaved by an enzyme called Dicer into small interfering RNAs (siRNAs) that are incorporated into a protein complex called RISC that targets and degrades complementary mRNAs, preventing gene expression. The document outlines several successful applications of RNAi technology for increasing biotic stress tolerance in crops against viruses, bacteria, fungi and insects by silencing key pathogen genes. It also discusses using RNAi to modify other crop traits like nutritional quality and abiotic stress resistance.
Targeting Induced Local Lesions IN Genomes (TILLING) is a combined tool of plant mutagenesis and DNA Biology to investigate useful mutations at Genomic level. First time used for cotton improvement.
Genetic engineering is the best technology that is promoting the world and this technology is applied to many plants, animals and microorganisms. It has wider applications in the field of Biology, Medicine, Industry, Research, Agriculture and many other fields of science. In this research paper I update the Roles of Genetic Engineering in Agriculture, Animals, Human enhancement and Evolution, Bacteriophage Against Infectious Diseases, Medicines, Phage in Infectious Diseases, Biofuels Production and Improve Plant Performance Under Drought.
To handle complex Traits like Yield, different stress we must do modification in DNA molecular breeding techniques help us to do such changes in DNA to archive the Goals.
Targeting Induced Local Lesions IN Genomes (TILLING) is a combined tool of plant mutagenesis and DNA Biology to investigate useful mutations at Genomic level. First time used for cotton improvement.
Genetic engineering is the best technology that is promoting the world and this technology is applied to many plants, animals and microorganisms. It has wider applications in the field of Biology, Medicine, Industry, Research, Agriculture and many other fields of science. In this research paper I update the Roles of Genetic Engineering in Agriculture, Animals, Human enhancement and Evolution, Bacteriophage Against Infectious Diseases, Medicines, Phage in Infectious Diseases, Biofuels Production and Improve Plant Performance Under Drought.
To handle complex Traits like Yield, different stress we must do modification in DNA molecular breeding techniques help us to do such changes in DNA to archive the Goals.
Deployment of broad spectrum resistance against rice blast which includes gene pyramiding, deployment, transgenic approaches, marker assisted back cross breeding, pedigree by using major R genes and QTLs and phytoalexin genes.
Plant Genetic engineering ,Basic steps ,Advantages and disadvantagesTessaRaju
plant genetic engineering,first genetically engineered crop plant,first genetically engineered foods,genome editing,uses of GE,transgenic plants,basic process of plant genetic enginering,advantages and disadvantages of genetic engineering.
Deployment of broad spectrum resistance against rice blast which includes gene pyramiding, deployment, transgenic approaches, marker assisted back cross breeding, pedigree by using major R genes and QTLs and phytoalexin genes.
Plant Genetic engineering ,Basic steps ,Advantages and disadvantagesTessaRaju
plant genetic engineering,first genetically engineered crop plant,first genetically engineered foods,genome editing,uses of GE,transgenic plants,basic process of plant genetic enginering,advantages and disadvantages of genetic engineering.
Applying agricultural biotechnology tools and capabilities to enhance food se...ExternalEvents
Applying agricultural biotechnology tools and capabilities to enhance food security and nutrition from local food crops to stimulate sustainable income opportunities for small holder farmers to reduce poverty presentation by "Howard-Yana Shapiro, Mars Incorporated, Dranesville and
University of California Davis, Davis, United States of America"
Bioinformatics and its Applications in Agriculture/Sericulture and in other F...mohd younus wani
The National Center for Biotechnology Information (NCBI, 2001) defines bioinformatics as the field of science in which biology, computer science, and information technology merge into a single discipline. Fredj Tekaia defines Bioinformatics the mathematical, statistical and computing methods that aim to solve biological problems using DNA and amino acid sequences and related information. Bioinformatics has emerged as an essential field of science that is facilitating biological discoveries since more than a decade. Without the usage of bioinformatics tools it is merely impossible to capture, manage process, analyse and interpret the huge amounts data that is available especially after whole genome sequencing projects. The sequencing of the genomes of plants and animals will have enormous benefits for the agricultural community. Bioinformatics tools can be used to search for the genes within these genomes and to elucidate their functions. This specific genetic knowledge could then be used to produce stronger, drought, disease and insect resistant crops and improve the quality. In agriculture it helps in the insect resistance, improve nutritional quality, rational plant improvement, waste cleanup, climate change studies, and development of drought resistance varieties (Dahiya and Lata, 2017) and in addition to this it also plays an important roles in biotechnology, antibiotic resistance, and forensic analysis of microbes, comparative studies, evolutionary studies and veterinary Sciences.
Seri bioinformatics tools and techniques not only facilitated detection of proteomic and genomic diversity among the species/strains, but also resulted in finding a gap in the silkworm genome sequence of a strain that diverged during the course of domestication. Seri-bioinformatics databases are a valuable seri-bioresource. The available online resources on silkworm and its related organisms, including databases as well as informative websites help to make silkworms healthier, more disease resistant and more productive. These databases provides information on gene, protein sequences and diseases and play crucial roles in conservation of the silkworm species and mulberry plants (Singh et al., 216). Bioinformatics approaches give an insight, uncovering the lineage with gene and protein count of B. mori and Drosophila encompass ~18,000 and ~16,000 (Genes) and ~9,000 and ~22,000 (Proteins) respectively (Somshekar and Borgowda, 2013).
RNA interference (RNAi) is a biological process in which RNA molecules inhibit gene expression, typically by causing the destruction of specific mRNA molecules. Historically, it was known by other names, including co-suppression, post-transcriptional gene silencing (PTGS), and quelling. Only after these apparently unrelated processes were fully understood did it become clear that they all described the RNAi phenomenon. Andrew Fire and Craig C. Mello shared the 2006 Nobel Prize in Physiology or Medicine for their work on RNA interference in the nematode worm Caenorhabditis elegans, which they published in 1998. Since the discovery of RNAi and its regulatory potentials, it has become evident that RNAi has immense potential in suppression of desired genes. RNAi is now known as precise, efficient, stable and better than antisense technology for gene suppression. Two types of small ribonucleic acid (RNA) molecules – microRNA (miRNA) and small interfering RNA (siRNA) – are central to RNA interference. RNAs are the direct products of genes, and these small RNAs can bind to other specific messenger RNA (mRNA) molecules and either increase or decrease their activity, for example by preventing an mRNA from producing a protein. RNA interference has an important role in defending cells against parasitic nucleotide sequences – viruses and transposons. It also influences development.
Biotechnology and disease management with special reference toSarda Konjengbam
Plant biotechnology can be defined as the use of tissue culture and genetic engineering techniques to produce genetically modified plants that exhibit new or improved desirable characteristics.
PLANT BIOTECHNOLOGY HELPS PLANT PATHOLOGY IN MANY WAYS.
Similar to Chp%3 a10.1007%2f978 81-322-2283-5-31 (20)
A brief information about the SCOP protein database used in bioinformatics.
The Structural Classification of Proteins (SCOP) database is a comprehensive and authoritative resource for the structural and evolutionary relationships of proteins. It provides a detailed and curated classification of protein structures, grouping them into families, superfamilies, and folds based on their structural and sequence similarities.
Professional air quality monitoring systems provide immediate, on-site data for analysis, compliance, and decision-making.
Monitor common gases, weather parameters, particulates.
THE IMPORTANCE OF MARTIAN ATMOSPHERE SAMPLE RETURN.Sérgio Sacani
The return of a sample of near-surface atmosphere from Mars would facilitate answers to several first-order science questions surrounding the formation and evolution of the planet. One of the important aspects of terrestrial planet formation in general is the role that primary atmospheres played in influencing the chemistry and structure of the planets and their antecedents. Studies of the martian atmosphere can be used to investigate the role of a primary atmosphere in its history. Atmosphere samples would also inform our understanding of the near-surface chemistry of the planet, and ultimately the prospects for life. High-precision isotopic analyses of constituent gases are needed to address these questions, requiring that the analyses are made on returned samples rather than in situ.
Seminar of U.V. Spectroscopy by SAMIR PANDASAMIR PANDA
Spectroscopy is a branch of science dealing the study of interaction of electromagnetic radiation with matter.
Ultraviolet-visible spectroscopy refers to absorption spectroscopy or reflect spectroscopy in the UV-VIS spectral region.
Ultraviolet-visible spectroscopy is an analytical method that can measure the amount of light received by the analyte.
Cancer cell metabolism: special Reference to Lactate PathwayAADYARAJPANDEY1
Normal Cell Metabolism:
Cellular respiration describes the series of steps that cells use to break down sugar and other chemicals to get the energy we need to function.
Energy is stored in the bonds of glucose and when glucose is broken down, much of that energy is released.
Cell utilize energy in the form of ATP.
The first step of respiration is called glycolysis. In a series of steps, glycolysis breaks glucose into two smaller molecules - a chemical called pyruvate. A small amount of ATP is formed during this process.
Most healthy cells continue the breakdown in a second process, called the Kreb's cycle. The Kreb's cycle allows cells to “burn” the pyruvates made in glycolysis to get more ATP.
The last step in the breakdown of glucose is called oxidative phosphorylation (Ox-Phos).
It takes place in specialized cell structures called mitochondria. This process produces a large amount of ATP. Importantly, cells need oxygen to complete oxidative phosphorylation.
If a cell completes only glycolysis, only 2 molecules of ATP are made per glucose. However, if the cell completes the entire respiration process (glycolysis - Kreb's - oxidative phosphorylation), about 36 molecules of ATP are created, giving it much more energy to use.
IN CANCER CELL:
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
This results in only 2 molecules of ATP per each glucose molecule instead of the 36 or so ATPs healthy cells gain. As a result, cancer cells need to use a lot more sugar molecules to get enough energy to survive.
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
This results in only 2 molecules of ATP per each glucose molecule instead of the 36 or so ATPs healthy cells gain. As a result, cancer cells need to use a lot more sugar molecules to get enough energy to survive.
introduction to WARBERG PHENOMENA:
WARBURG EFFECT Usually, cancer cells are highly glycolytic (glucose addiction) and take up more glucose than do normal cells from outside.
Otto Heinrich Warburg (; 8 October 1883 – 1 August 1970) In 1931 was awarded the Nobel Prize in Physiology for his "discovery of the nature and mode of action of the respiratory enzyme.
WARNBURG EFFECT : cancer cells under aerobic (well-oxygenated) conditions to metabolize glucose to lactate (aerobic glycolysis) is known as the Warburg effect. Warburg made the observation that tumor slices consume glucose and secrete lactate at a higher rate than normal tissues.
Richard's entangled aventures in wonderlandRichard Gill
Since the loophole-free Bell experiments of 2020 and the Nobel prizes in physics of 2022, critics of Bell's work have retreated to the fortress of super-determinism. Now, super-determinism is a derogatory word - it just means "determinism". Palmer, Hance and Hossenfelder argue that quantum mechanics and determinism are not incompatible, using a sophisticated mathematical construction based on a subtle thinning of allowed states and measurements in quantum mechanics, such that what is left appears to make Bell's argument fail, without altering the empirical predictions of quantum mechanics. I think however that it is a smoke screen, and the slogan "lost in math" comes to my mind. I will discuss some other recent disproofs of Bell's theorem using the language of causality based on causal graphs. Causal thinking is also central to law and justice. I will mention surprising connections to my work on serial killer nurse cases, in particular the Dutch case of Lucia de Berk and the current UK case of Lucy Letby.
Introduction:
RNA interference (RNAi) or Post-Transcriptional Gene Silencing (PTGS) is an important biological process for modulating eukaryotic gene expression.
It is highly conserved process of posttranscriptional gene silencing by which double stranded RNA (dsRNA) causes sequence-specific degradation of mRNA sequences.
dsRNA-induced gene silencing (RNAi) is reported in a wide range of eukaryotes ranging from worms, insects, mammals and plants.
This process mediates resistance to both endogenous parasitic and exogenous pathogenic nucleic acids, and regulates the expression of protein-coding genes.
What are small ncRNAs?
micro RNA (miRNA)
short interfering RNA (siRNA)
Properties of small non-coding RNA:
Involved in silencing mRNA transcripts.
Called “small” because they are usually only about 21-24 nucleotides long.
Synthesized by first cutting up longer precursor sequences (like the 61nt one that Lee discovered).
Silence an mRNA by base pairing with some sequence on the mRNA.
Discovery of siRNA?
The first small RNA:
In 1993 Rosalind Lee (Victor Ambros lab) was studying a non- coding gene in C. elegans, lin-4, that was involved in silencing of another gene, lin-14, at the appropriate time in the
development of the worm C. elegans.
Two small transcripts of lin-4 (22nt and 61nt) were found to be complementary to a sequence in the 3' UTR of lin-14.
Because lin-4 encoded no protein, she deduced that it must be these transcripts that are causing the silencing by RNA-RNA interactions.
Types of RNAi ( non coding RNA)
MiRNA
Length (23-25 nt)
Trans acting
Binds with target MRNA in mismatch
Translation inhibition
Si RNA
Length 21 nt.
Cis acting
Bind with target Mrna in perfect complementary sequence
Piwi-RNA
Length ; 25 to 36 nt.
Expressed in Germ Cells
Regulates trnasposomes activity
MECHANISM OF RNAI:
First the double-stranded RNA teams up with a protein complex named Dicer, which cuts the long RNA into short pieces.
Then another protein complex called RISC (RNA-induced silencing complex) discards one of the two RNA strands.
The RISC-docked, single-stranded RNA then pairs with the homologous mRNA and destroys it.
THE RISC COMPLEX:
RISC is large(>500kD) RNA multi- protein Binding complex which triggers MRNA degradation in response to MRNA
Unwinding of double stranded Si RNA by ATP independent Helicase
Active component of RISC is Ago proteins( ENDONUCLEASE) which cleave target MRNA.
DICER: endonuclease (RNase Family III)
Argonaute: Central Component of the RNA-Induced Silencing Complex (RISC)
One strand of the dsRNA produced by Dicer is retained in the RISC complex in association with Argonaute
ARGONAUTE PROTEIN :
1.PAZ(PIWI/Argonaute/ Zwille)- Recognition of target MRNA
2.PIWI (p-element induced wimpy Testis)- breaks Phosphodiester bond of mRNA.)RNAse H activity.
MiRNA:
The Double-stranded RNAs are naturally produced in eukaryotic cells during development, and they have a key role in regulating gene expression .
Nutraceutical market, scope and growth: Herbal drug technologyLokesh Patil
As consumer awareness of health and wellness rises, the nutraceutical market—which includes goods like functional meals, drinks, and dietary supplements that provide health advantages beyond basic nutrition—is growing significantly. As healthcare expenses rise, the population ages, and people want natural and preventative health solutions more and more, this industry is increasing quickly. Further driving market expansion are product formulation innovations and the use of cutting-edge technology for customized nutrition. With its worldwide reach, the nutraceutical industry is expected to keep growing and provide significant chances for research and investment in a number of categories, including vitamins, minerals, probiotics, and herbal supplements.
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.
References
Angaji SA, Hedayati SS, Poor RH, Poor SS, Shiravi S,
Madani S (2010) Application of RNA interference in
plants. Plant Omics J 3:77–84
Bartel DP (2004) MicroRNAs: genomics, biogenesis,
mechanism and function. Cell 116:281–297
Baum JA, Bogaert T, Clinton W, Heck GR, Feldmann P
et al (2007) Control of coleopteran insect pests through
RNA interference. Nat Biotechnol 25:1322e–1326e
Baumberger N, Baulcombe DC (2005) Arabidopsis
ARGONAUTE1 is an RNA slicer that selectively
recruits microRNAs and short interfering RNAs. Proc
Natl Acad Sci U S A 102:11928–11933
Bernstein E, Caudy AA, Hammond SM, Hannon GJ
(2001) Role for a bidentate ribonuclease in the initia-
tion step of RNA interference. Nature 409:363–366
Borgio JF (2009) RNA interference (RNAi) technology: a
promising tool for medicinal plant research. J Med
Plant Res 3:1176–1183
Burkhart KB, Guang S, Buckley BA, Wong L, Bochner
AF, Kennedy S (2011) A pre-mRNA-associating fac-
tor links endogenous siRNAs to chromatin regulation.
PLoS Genet 7:e1002249
Campbell TN, Choy FYM (2005) RNA interference: past,
present and future. Curr Issues Mol Biol 7:1–6
Chen J, Zhang D, Yao Q, Zhang J, Dong X, Tian H, Chen
J, Zhang W (2010) Feeding-based RNA interference
of a trehalose phosphate synthase gene in the brown
planthopper, Nilaparvata lugens. Insect Mol Biol
19:777–786
Cogoni C, Irelan JT, Schumacher M, Schmidhauser T,
Selker EU, Macino G (1996) Transgene silencing of
the al-1 gene in vegetative cells of Neurospora is
mediated by a cytoplasmic effector and does not
depend on DNA-DNA interactions or DNA methyla-
tion. EMBO J 15:3153–3163
Crocker W, Hitchcock AE, Zimmerman PW (1935)
Similarities in the effects of ethylene and the plant
auxins. Contrib Boyce Thompson Inst 7:231–248
Crowe TC, Seligman SA, Copeland L (2000) Inhibition of
enzymic digestion of amylose by free fatty acids in
vitro contributes to resistant starch formation. J Nutr
130:2006–2008
Da-Hong L, Hui L, Yan-li Y, Ping-ping Z, Jian-sheng L
(2009) Down regulated expression of RACK1 gene by
RNA interference enhances drought tolerance in rice.
Rice Sci 16:14–20
Davuluri GR, van Tuinen A, Fraser PD (2005) Fruit-
specific RNAi mediated suppression of DET1
enhanced carotenoid and flavonoid content in toma-
toes. Nat Biotechnol 23:890–895
De Block M, Verduyn C, De Brouwer D, Cornelissen M
(2005) Poly (adp-ribose) polymerase in plants affects
energy homeostasis, cell death and stress tolerance.
Plant J Cell Mol Biol 41:95–106
Dunoyer P, Himber C, Ruiz-Ferrer V, Alioua A, Voinnet O
(2007) Intra- and intercellular RNA interference in
Arabidopsis thaliana requires components of the
microRNA and heterochromatic silencing pathways.
Nat Genet 39:848–856
Duvick DN (1999) Heterosis: feeding people and protect-
ing natural resources. In: Coors JG, Pandey S (eds)
Genetics and exploitation of heterosis in crops.
American Society of Agronomy/Crop Science Society
of America, Madison, pp 19–29
Duvick DN (2005) The contribution of breeding to yield
advances in maize (Zea mays L.). Elsevier Academic
Press, San Diego
Eck JV, Conlin B, Garvin DF, Mason H, Navarre DA,
Brown CR (2007) Enhanced beta-carotene content in
potato via RNAi silencing of the beta-carotene
hydroxylase gene. Am J Potato Res 84:331–342
31 RNAi for Crop Improvement
12. 634
Escobar MA, Civerolo EL, Summerfelt KR, Dandekar
AM (2001) RNAi-mediated oncogene silencing con-
fers resistance to crown gall tumorigenesis. Proc Natl
Acad Sci U S A 98:13437–13442
Fagegaltier D, Bouge AL, Berry B, Poisot E, Sismeiro O,
Coppee JY, Theodore L, Voinnet O, Antoniewski C
(2009) The endogenous siRNA pathway is involved in
heterochromatin formation in Drosophila. Proc Natl
Acad Sci U S A 106:21258–21263
Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE,
Mello CC (1998) Potent and specific genetic interfer-
ence by double stranded RNA in Caenorhabditis ele-
gans. Nature 391:806–811
Fitzgerald A, Van Kha JA, Plummer KM (2004)
Simultaneous silencing of multiple genes in the apple
scab fungus Venturia inaequalis, by expression of
RNA with chimeric inverted repeats. Fungal Genet
Biol 41:963–971
Fujii N, Inui T, Iwasa K, Morishige T, Sato F (2007)
Knockdown of berberine bridge enzyme by RNAi
accumulates (S)-reticuline and activates a silent path-
way in cultured California poppy cells. Transgenic
Res 16:363–375
Fukusaki E, Kawasaki K, Kajiyama S, An CI, Suzuki K,
Tanaka Y, Kobayashi A (2004) Flower color modula-
tions of Torenia hybrida by downregulation of chal-
cone synthase genes with RNA interference. J
Biotechnol 111:229–240
Ghag SB, Shekhawat UKS, Ganapathi TR (2014) Host-
induced post-transcriptional hairpin RNA-mediated
gene silencing of vital fungal genes confers efficient
resistance against Fusarium wilt in banana. Plant
Biotechnol J 12(5):541–553
Gil-Humanes J, Pisto’n F, Hernando A, Alvarez JB,
Shewry PR, Barro F (2008) Silencing of g-gliadins by
RNA interference (RNAi) in bread wheat. J Cereal Sci
48:565–568
Goldoni M, Azzalin G, Macino G, Cogoni C (2004)
Efficient gene silencing by expression of double
stranded RNA in Neurospora crassa. Fungal Genet
Biol 41:1016–1024
Gupta A, Pal RK, Rajam MV (2013) Delayed ripening
and improved fruit processing quality in tomato by
RNAi-mediated silencing of three homologs of
1-aminopropane-1-carboxylate synthase gene. J Plant
Physiol 170:987–995
Hamada W, Spanu PD (1998) Co-suppression of the
hydrophobin gene Hcf-1 is correlated with antisense
RNA biosynthesis in Cladosporium fulvum. Mol Gen
Genet 259:630–638
Hamilton AJ, Baulcombe DC (1999) A species of small
antisense RNA in posttranscriptional gene silencing in
plants. Science 286:950–952
Hammond TM, Keller NP (2005) RNA silencing in
Aspergillus nidulans is independent of RNA-
dependent RNA polymerase. Genetics 169:607–617
Hannon GJ (2002) RNA interference. Nature
418:244–251
Hernández I, Chacón O, Rodriguez R, Portieles R, Pujol
YLM, Borrás-Hidalgo O (2009) Black shank resistant
tobacco by silencing of glutathione S-transferase.
Biochem Biophys Res Commun 387:300–304
Huang GZ, Allen R, Davis EL, Baum TJ, Hussey RS
(2006) Engineering broad root-knot resistance in
transgenic plants by RNAi silencing of a conserved
and essential root-knot nematode parasitism gene.
Proc Natl Acad Sci U S A 103:14302–14306
Huang Y, Mei M, Zhenchuan Mao Z, Lv S, Zhou J, Chen
S, Xie B (2013) Molecular cloning and virus-induced
gene silencing of MiASB in the southern root-knot
nematode, Meloidogyne incognita. Eur J Plant Pathol
138:181–193
Hutvagner G, McLachlan J, Pasquinelli AE, Balint E,
Tuschl T, Zamore PD (2001) A cellular function for
the RNA-interference enzyme Dicer in the maturation
of the let-7 small temporal RNA. Science
293:834–838
Jan A, Maruyama K, Todaka D, Kidokoro S, Abo M et al
(2013) OsTZF1, a CCCH-tandem zinc finger protein,
confers delayed senescence and stress tolerance in rice
by regulating stress-related genes. Plant Physiol
161:1202–1216
Jørgensen K, Bak S, Busk PK, Sørensen C, Olsen CE,
Puonti-Kaerlas J, Moller BL (2005) Cassava plants
with a depleted cyanogenic glucoside content in leaves
and tubers. Plant Physiol 139:363–374
Kadotani N, Nakayashiki H, Tosa Y, Mayama S (2003)
RNA silencing in the pathogenic fungus Magnaporthe
oryzae. Mol Plant Microb Interact 16:769–776
Katiyar-Agarwal S, Morgan R, Dahlbeck D, Borsani O,
Villegas JA, Zhu J, Staskawicz BJ, Jin H (2007) A
pathogen-inducible endogenous siRNA in plant
immunity. Proc Natl Acad Sci U S A 103:47–52
KatsumotoY, Mizutani M, FukuiY, Brugliera F, Holton T
(2007) Engineering of the rose flavonoid biosynthetic
pathway successfully generated blue-hued flowers
accumulating delphinidin. Plant Cell Physiol
48:1589–1600
Kempe K, Higashi Y, Frick S, Sabarna K, Kutchan TM
(2009) RNAi suppression of the morphine biosyn-
thetic gene salAT and evidence of association of path-
way enzymes. Phytochemistry 70:579–589
Kertbundit S, Pongtanom N, Ruanjan P, Chantasingh D,
Tanwanchai A, Panyim S (2007) Resistance of trans-
genic papaya plants to papaya ringspot virus. Biol
Plant 51:333–339
Khatri M, Rajam MV (2007) Targeting polyamines of
Aspergillus nidulans by siRNA specific to fungal orni-
thine decarboxylase gene. Med Mycol 45:211–220
Klink VP, Kim K-H, Martins V, MacDonald MH, Beard
HS, Alkharouf NW, Lee S-K, Park S-C, Matthews BF
(2009) A correlation between host-mediated expres-
sion of parasite genes as tandem inverted repeats and
abrogation of development of female Heterodera gly-
cines cyst formation during infection of Glycine max.
Planta 230:53–71
S. Yogindran and M.V. Rajam
13. 635
Kumar M (2011) RNAi-mediated targeting of acetylcho-
linesterase gene of Helicoverpa armigera for insect
resistance in transgenic tomato and tobacco. PhD the-
sis. University of Delhi, New Delhi
Kumar M, Gupta GP, Rajam MV (2009) Silencing of
acetylcholinesterase gene of Helicoverpa armigera by
siRNA affects larval growth and its life cycle. J Insect
Physiol 55:273–278
Kumria R (2000) Modulation of polyamine biosynthesis,
plant regeneration and stress responses in transgenic
rice and tobacco by introduction of ornithine decar-
boxylase gene. PhD Thesis, University of Delhi,
Delhi
Kusaba M (2004) RNA interference in crop plants. Curr
Opin Biotechnol 15:139–143
Kusaba M, Miyahara K, Iida S, Fukuoka H, Takano T,
Sassa H, Nishimura M, Nishio T (2003) Low glutelin
content 1: a dominant mutation that suppresses the
glutelin multigene family via RNA silencing in rice.
Plant Cell 15:1455–1467
Lelioa ID, Varricchioa P, Priscoa GD, Marinellia A et al
(2014) Functional analysis of an immune gene of
Spodoptera littoralis by RNAi. J Insect Physiol
64:90–97
Li J, Todd TC, Oakley TR, Lee J, Trick HN (2010) Host-
derived suppression of nematode reproductive and fit-
ness genes decreases fecundity of Heterodera glycines
Ichinohe. Planta 232:775–785
Liu Q, Singh SP, Green AG (2002) High-stearic and high-
oleic cottonseed oils produced by hairpin RNA-
mediated posttranscriptional gene silencing. Plant
Physiol 129:1732–1743
Luo Z, Zhang J, Li J, Yang C, Wang T et al (2013) A
STAY-GREEN protein SlSGR1 regulates lycopene
and b-carotene accumulation by interacting directly
with SlPSY1 during ripening processes in tomato.
New Phytol 198:442–452
Manavalan LP, Chen X, Clarke J, Salmeron J, Nguyen HT
(2012) RNAi-mediated disruption of squalene syn-
thase improves drought tolerance and yield in rice. J
Exp Bot 63:163–175
Mao YB, Cai WJ, Wang JW, Hong GJ, Tao XY et al
(2007) Silencing a cotton bollworm P450 monooxy-
genase gene by plant mediated RNAi impairs larval
tolerance of gossypol. Nat Biotechnol 25:1307e1313
Missiou A, Kalantidis K, Boutla A, Tzortzakaki S, Tabler
M, Tsagris M (2004) Generation of transgenic potato
plants highly resistant to potato virusY (PVY) through
RNA silencing. Mol Breed 14:185–197
Mittal P,Yadav R, Devi R, TiwariA, Upadhey SP, Ghoshal
SS (2011) Woundorous RNAi- gene silencing.
Biotechnology 10:41–50
Mochizuki T, Hara S (2000) Usefulness of low protein
rice on diet therapy in patients with chronic renal fail-
ure. Jpn J Nephrol 42:24–29
Moon HS, Li Y, Stewart CN Jr (2010) Keeping the genie
in the bottle: transgene biocontainment by excision in
pollen. Trends Biotechnol 28: 3–8
Moritoh S, Miki D, Akiyama M, Kawahara M, Izawa T,
Maki H, Shimamoto K (2005) RNAi-mediated silenc-
ing of OsGEN-L (OsGEN-like), a new member of the
RAD2/XPG nuclease family, causes male sterility by
defect of microspore development in rice. Plant Cell
Physiol 46:699–715
Nakatsuka T, Mishibaa KI, Abe Y, Kubota A, Kakizaki Y,
Yamamura S, Nishihara M (2008) Plant biotechnol-
ogy, flower color modification of gentian plants by
RNAi-mediated gene silencing. Plant Biotechnol
25:61–68
Nakatsuka T, Mishiba KI, Kubota A, Abe Y, Yamamura S,
Nakamura N, Tanaka Y, Nishihara M (2010) Genetic
engineering of novel flower colour by suppression of
anthocyanin modification genes in gentian. J Plant
Physiol 167:231–237
Nakayashiki H (2005) RNA silencing in fungi: mecha-
nisms and applications. FEBS Lett 579:5950–5970
Napoli C, Lemieux C, Jorgensen R (1990) Introduction of
a chimeric chalcone synthase gene into Petunia results
in reversible co-suppression of homologous genes in
trans. Plant Cell 2:279–289
Natarajaswamy K, Naorem A, Rajam MV (2013)
Targeting fungal genes by diced siRNAs: a rapid tool
to decipher gene function in Aspergillus nidulans.
PLoS One 8(10):e75443
Nawaz-ul-Rehman MS, Mansoor S, Khan AA, Zafar Y,
Briddon RW (2007) RNAi-mediated male sterility of
tobacco by silencing TA29. Mol Biotechnol
36:159–165
Nishihara M, Nakatsuka T,Yamamura S (2005) Flavonoid
components and flower color change in transgenic
tobacco plants by suppression of chalcone isomerase
gene. FEBS Lett 579:6074–6078
Nowara D, Gay A, Lacomme C, Shaw J, Ridout C et al
(2010) HIGS: host-induced gene silencing in the obli-
gate biotrophic fungal pathogen Blumeria graminis.
Plant Cell 22:3130–3141
Ogita S, Uefuji H, Yamaguchi Y, Koizumi N, Sano H
(2003) RNA interference: producing decaffeinated
coffee plants. Nature 423:823
Ogita S, Uefuji H, Morimoto M, Sano H (2004)
Application of RNAi to confirm theobromine as the
major intermediate for caffeine biosynthesis in coffee
plants with potential for construction of decaffeinated
varieties. Plant Mol Biol 54:931–941
Ossowski S, Schwab R, Weigel D (2008) Gene silencing
in plants using artificial microRNAs and other small
RNAs. Plant J 53:674–690
Panwar V, McCallum B, Bakkeren G (2013a) Endogenous
silencing of Puccinia triticina pathogenicity genes
through in planta-expressed sequences leads to the
suppression of rust diseases on wheat. Plant J
73:521–532
PanwarV, McCallum B, Bakkeren G (2013b) Host-induced
gene silencing of wheat leaf rust fungus Puccinia tri-
ticina pathogenicity genes mediated by the Barley
stripe mosaic virus. Plant Mol Biol 81:595–608
31 RNAi for Crop Improvement
14. 636
Pardo JM (2010) Biotechnology of water and salinity
stress tolerance. Curr Opin Biotechnol 21:1–12
Patil BL, Ogwok E, Wagaba H, Mohammed IU et al
(2011) RNAi-mediated resistance to diverse isolates
belonging to two virus species involved in Cassava
brown streak disease. Mol Plant Pathol 12:31–41
Pooggin M, Shivaprasad PV, Veluthambi K, Hohn T
(2003) RNAi targeting of DNA virus in plants. Nat
Biotechnol 21:131–132
Rajam MV (2011) RNA interference: a new approach for the
control of fungal pathogens and insects. In: Proceedings
of the national symposium on genomics and crop
improvement: relevance and reservations. Acharya NG
Ranga Agricultural University, Hyderabad, pp 220–229
Rajam MV (2012) Host induced silencing of fungal
pathogen genes: an emerging strategy for disease con-
trol in crop plants. Cell Dev Biol 1:1, http://dx.doi.
org/10.4172/2168-9296.1000e118
Regina A, Bird A, Topping D, Bowden S, Freeman J et al
(2006) High-amylose wheat generated by RNA inter-
ference improves indices of large-bowel health in rats.
Proc Natl Acad Sci U S A 103:3546–3551
Romano N, Macino G (1992) Quelling: transient inactiva-
tion of gene expression in Neurospora crassa by trans-
formation with homologous sequences. Mol Microbiol
22:3343–3353
Runguphan W, Maresh JJ, O’Connor SE (2009) Silencing
of tryptamine biosynthesis for production of non natu-
ral alkaloids in plant culture. Proc Natl Acad Sci U S
A 106:13673–13678
Segal G, Song R, Messing J (2003) A new opaque variant
of maize by a single dominant RNA-interference-
inducing transgene. Genetics 165:387–397
Shepherd DN, Martin DP, Thomson JA (2009) Transgenic
strategies for developing crops resistant to geminivi-
rus. Plant Sci 176:1–11
Sindhu AS, Maier TR, Mitchum MG, Hussey RS, Davis
EL, Baum TJ (2009) Effective and specific in planta
RNAi in cyst nematodes: expression interference of
four parasitism genes reduces parasitic success. J Exp
Bot 60:315–324
Singh N (2011) Genetic engineering of tomato for fusar-
ium wilt resistance by in planta RNAi-mediated
silencing of fungal ornithine decarboxylase gene. PhD
thesis. University of Delhi, New Delhi
Sinha R, Rajam MV (2013) RNAi silencing of three
homologues of S-adenosylmethionine decarboxylase
gene in tapetal tissue of tomato results in male sterility.
Plant Mol Biol 82:169–180
Sunilkumar G, Campbell LM, Puckhaber L, Stipanovic
RD, Rathore KS (2006) Engineering cottonseed for use
in human nutrition by tissue-specific reduction of toxic
gossypol. Proc Natl Acad Sci U S A 103:18054–18059
Tamilarasan (2013) RNAi knockdown of acetylcholines-
terase gene of Meloidogyne incognita for nematode
resistance in tobacco and tomato. PhD thesis.
University of Delhi, New Delhi
Tamilarasan S, Rajam MV (2013) Engineering crop plants
for nematode resistance through host-derived RNA
interference. Cell Dev Biol 2:2 http://dx.doi.
org/10.4172/2168-9296.1000114
TanakaY, Brugliera F, Chandler S (2009) Recent progress
of flower colour modification by biotechnology. Int J
Mol Sci 10:5350–5369
Tang G, Galili G, Zhuang X (2007) RNAi and microRNA:
breakthrough technologies for the improvement of
plant nutritional value and metabolic engineering.
Metabolism 3:357–369
Tehseen M, Imran M, Hussain M, Irum S, Ali S, Mansoor
S, Zafar Y (2010) Development of male sterility by
silencing Bcp1 gene of Arabidopsis through RNA
interference. Afr J Biotechnol 9:2736–2741
Thakur N, Upadhyay SK, Verma PC, Chandrashekar K,
Tuli R, Singh PK (2014) Enhanced whitefly resistance
in transgenic tobacco plants expressing double
stranded rna of v-ATPase A gene. PLoS One
9(3):e87235
Upadhyay SK, Chandrashekar K, Thakur N, Verma PC,
Borgio JF (2011) RNA interference for the control of
whiteflies (Bemisia tabaci) by oral route. J Biosci
36:153–161
Vanderauwera S, De Block M, Van de Steene N, van de
Cotte B, Metzlaff M, Van Breusegem F (2007)
Silencing of poly(adp-ribose) polymerase in plants
alters abiotic stress signal transduction. Proc Natl
Acad Sci U S A 104:15150–15155
Vanderschuren H, Alder A, Zhang P, Gruissem W (2009)
Dose dependent RNAi-mediated geminivirus resis-
tance in the tropical root crop cassava. Plant Mol Biol
64:549–557
Vanitharani R, Chellappan P, Fauquet CM (2003) Short
interfering RNA-mediated interference of gene
expression and viral DNA accumulation in cultured
plant cells. Proc Natl Acad Sci U S A
100(16):9632–9636
Vaucheret H (2008) Plant ARGONAUTES. Trends Plant
Sci 13:350–358
Wang Y, Xue Y, Li J (2005) Towards molecular breeding
and improvement of rice in China. Trends Plant Sci
10:610–614
Wang Z, Dong Y, Desneux N, Niu C (2013) RNAi silenc-
ing of the HaHMG-CoA reductase gene inhibits ovi-
position in the Helicoverpa armigera cotton bollworm.
PLoS One 8:e67732
Waterhouse PM, Graham MW, Wang MB (1998) Virus
resistance and gene silencing in plants can be
induced by simultaneous expression of sense and
antisense RNA. Proc Natl Acad Sci U S A 95:
13959–13964
Wei S, Li X, Gruber MY, Li R, Zhou R, Zebarjadi A,
Hannoufa A (2009) RNAi-mediated suppression of
DET1 alters the levels of carotenoids and sinapate
esters in seeds of Brassica napus. J Agric Food Chem
57:5326–5333
S. Yogindran and M.V. Rajam
15. 637
Weise SE,Aung K, Jarou ZJ, Mehrshahi P, Li Z, HardyAC,
Carr DJ, Sharkey TD (2012) Engineering starch
accumulation by manipulation of phosphate metabo-
lism of starch. Plant Biotechnol J 10(5):545–554
Xiong A, Yao Q, Peng R, Li X, Han P, Fan H (2005)
Different effects on ACC oxidase gene silencing trig-
gered by RNA interference in transgenic tomato. Plant
Cell Rep 23:639–646
XiongY, Zeng H, ZhangY, Xu D, Qiu D (2013) Silencing
the HaHR3 gene by transgenic plant-mediated RNAi
to disrupt Helicoverpa armigera development. Int J
Biol Sci 9:370–381
Yadav BC, Veluthambi K, Subramaniam K (2006) Host-
generated double stranded RNA induces RNAi in
plant-parasitic nematodes and protects the host from
infection. Mol Biochem Parasitol 148:219–222
Yin C, Jurgenson JE, Hulbert SH (2011) Development of
a host-induced RNAi system in the wheat stripe rust
fungus Puccinia striiformis f. sp. tritici. Mol Plant
Microbe Interact 24:554–561
Zamore PD, Tuschl T, Sharp PA, Bartel DP (2000) RNAi:
double stranded RNA directs the ATP-dependent
cleavage of mRNA at 21 to 23 nucleotide intervals.
Cell 101:25–33
Zhang L, Jing F, Li F, Li M, Wang Y, Wang G, Sun X,
Tang K (2009) Development of transgenic Artemisia
annua (Chinese wormwood) plants with an enhanced
content of artemisinin, an effective anti-malarial drug,
by hairpin-RNA-mediated gene silencing. Biotechnol
Appl Biochem 52:199–207
Zhou YL, Zhu XQ, Gu SH, Cui HH, Guo YY,
Zhou JJ, Zhang YJ (2014) Silencing in Apolygus
lucorum of the olfactory coreceptor Orco gene by
RNA interference induces EAG response declining
to two putative semiochemicals. J Insect Physiol
60:31–39
Zhu JQ, Liu S, Ma Y, Zhang JQ, Qi HS et al (2012)
Improvement of pest resistance in transgenic tobacco
plants expressing dsRNA of an insect-associated gene
EcR. PLoS One 7:e38572
31 RNAi for Crop Improvement