Role of Antisense and RNA interference

1. Presented By:
Mariya Zaman
Ph.D (Biotech.)
B.A.C.A., AAU
Submitted To:
Dr. Akarsh Parihar Sir
Associate Professor
B.A.C.A., AAU
Role of Antisense and RNAi-based
Gene Silencing in Crop ImprovementWel Come

2. Outlines
Introduction of Antisense & RNAi Technology
Discovery of RNAi
Types of RNAs
Mechanism of Antisense & RNAi Technology
Importance of RNAi Technology
Pros & Cons of RNAi Technology
Application of RNAi Technology
CRISPR Technology
Case studies
Conclusions
Future Thrust
2

3. Antisense RNA is a single-stranded RNA that is complementary to a
messenger RNA (mRNA) strand transcribed within a cell.
They are introduced in a cell to inhibit the translation machinery by base
pairing with the sense RNA and activating the RNase H, to develop a
particular novel transgenic.
The first natural antisense RNAs were discovered in 1981 independently in
Tomizawas and in Nordstro¨ms laboratories
Small plasmid-encoded
RNA regulators control the copy numbers of the
Escherichia
coli plasmids ColE1 and R1, respectively
Antisense RNAs are small, diffusible, highly structured
RNAs that act via sequence complementarity on target
RNAs called sense RNAs.
Antisense RNAs are encoded in cis, i.e. They are transcribed
from a promoter located on the opposite strand of the same
DNA molecule, and are, therefore, fully complementary to
their target RNAs.
Entails post transcriptional inhibition of target RNA function.
Antisense RNA

4. Mechanism of Antisense Technology
4

5. Characteristics of Antisense RNA
Naturally occurring antisense RNAs are between 35 and 150 nt long and comprise
between 1-4 stem-loops.
•Efficient antisense RNAs have 5–8 nt GC-rich loops.
•Stems that are important for metabolic stability are often (if > 10 bp)
interrupted by bulges to prevent dsRNase degradation and to facilitate melting
upon antisense/sense RNA interaction.
•Some antisense RNAs (those involved in plasmid copy number control and post
segregation killing) are unstable, others (most chromosomally encoded and a
few phage and transposon antisense RNAs) are stable.
•Almost all naturally occurring antisense-RNA regulated systems have been
found mostly in prokaryotes, and only a few systems are known from
eukaryotes and one from archaea.

6. Antisense-RNA regulated systems in prokaryotes
Inhibition of
primer formation
Inhibition of synthesis of a leader peptide
MicF RNA, ompF-RNA
DicF RNA
RNAIII

7. Principal target sites
• Transcription
1. DNA triple helix formation
2. Hybridization to DNA loops
3. Hybridization to nascent RNA
• RNA processing
4. RNA splicing
5. RNA transport
• Translation
6. Inhibition of initiation factor
binding
7. Inhibition of ribosome
assembly
8. Inhibition of movement

8. RNA
Coding RNA
mRNA
Non Coding RNAs
Constituent/
Transcription
RNAs
rRNA tRNA
Regulatory/
Small RNAs
siRNA
miRNA
(stRNA)
snoRNA snRNA
RNA Family
RNA interference
8

9. RNA interference
• RNA interference (RNAi) is an evolutionary
highly conserved defense mechanism of post-
transcriptional gene silencing (PTGS) occurring
naturally against double stranded RNA (dsRNA)
that causes sequence-specific gene regulation
resulting in inhibition of translation or
transcriptional repression of mRNA sequences.
9

10. 10

11. 11

12. Mechanism of RNAi Technology
12

13. 13
Structure of siRNA

14. miRNA
Mechanism
TRBP
Ran
GTP
14

15. 15

16. Summary of Players
• Drosha and Pasha are part of the “Microprocessor” protein
complex (~600-650kDa)
• Drosha and Dicer are RNase III enzymes
• Pasha is a dsRNA binding protein
• Exportein 5 is a member of the karyopherin
nucleocytoplasmic transport factors that requires Ran & GTP
• Argonautes are RNase H enzymes
16

17. • Dicer is a ribonuclease (RNase III family) with 4 distinct
domains:
1. Amino-terminal helicase domain
2. Dual RNase III motifs in the carboxy terminal segment
3. dsRNA binding domain
4. PAZ domain (110-130 amino-acid domain present in protein
like PIWI, Argonaute, and Zwille proteins) for protein-
protein interaction.
17

18. siRNA miRNA
Exogenous Endogenous
21-25 nt RNAs ~23 nt RNAs
dsRNA
Initially ssRNA but converted into
hairpin secondary structure
Most commonly responses to foreign
RNA(Viral, transposable elements,
repetitive elements or artificial
transgene insert)
Regulates post-transcriptional gene
expression
100% complementary to target RNA
Not often 100% complementary to
target RNA
Produced in large population Only single small RNA
Effective but not tissue specific
Silencing
Precise and tissue specific Silencing
18

19. Advantages of RNAi Technology
Dominant Phenotypes can be observed in the T1 generation
Partial knockdown
Facilitates the study of essential genes whose inactivation
would lead to lethality or extremely severe pleiotropic
phenotypes
Tissue-specific (miRNA) Off-target effects can be reduced
High-throughput/ Ease
of transfection
High-throughput vectors are designed to make an hpRNAi
construct
Flexible
Individual or multiple genes can be silenced with single
hpRNAi construct.
Better than antisense The highest silencing was obtained with hpRNAi
Cost effective
High specificity Middle region 9-14 are most sensitive
Can be labeled
Stable hpRNAi has been shown to be stably inherited over several
generations
19

20. Pitfalls of RNAi
• Off-target effects
• Inefficacy and instability
• Validation of RNAi knockdown
20

21. Importance of RNAi
• Powerful for analyzing unknown genes in sequenced genomes
• Efforts are being undertaken to target every human gene via
siRNAs
• Faster identification of gene function
• Gene therapy: down-regulation of certain genes/ mutated alleles
• Cancer treatments
 Knock-out of genes required for cell proliferation
 Knock-out of genes encoding key structural proteins
• Agriculture
21

22. 22

23. CRISPR interference:
RNA-directed adaptive immunity in bacteria and archaea
• In many bacteria and most archaea, clustered, regularly
interspaced short palindromic repeats (CRISPRs) are involved
in a more recently discovered interference pathway that
protects cells from bacteriophages and conjugative plasmids.
• CRISPR sequences provide an adaptive, heritable record of
past infections and express CRISPR RNAs — small RNAs that
target invasive nucleic acids.
23Marraffini and Sontheimer, 2011USA

24. Mechanism for CRISPR interference
24
At the molecular level, CRISPR function can be divided into three
phases: 1) Incorporation of new spacers into CRISPR arrays
2) Expression and processing of CRISPR RNAs (crRNAs)
3) CRISPR interference
Features of CRISPR loci
• CRISPRs - white boxes
• Leader sequence - black box that is AT-rich but not conserved
• Non-repetitive spacers -coloured boxes that share sequence identity with fragments
of plasmids & bacteriophage genomes and specify the targets of CRISPR interference.
• CRISPR-associated (cas) genes are conserved, different families and subtypes, &
encode the protein machinery for CRISPR activity

25. 25
1) Incorporation of new spacers into CRISPR arrays
• During the adaptation phase of (CRISPR)
immunity, new spacers derived from the
invading DNA are incorporated into
CRISPR loci.
• Cas1 to generate short fragments of
invading DNA
2) CRISPR interference

26. 26
A) In the defence phase of (CRISPR) immunity, repeats and spacers are
transcribed into a long precursor processed by a complex called CRISPR-
associated complex for antiviral defence (Cascade) in Escherichia coli or
CRISPR-associated 6 (Cas6) in Pyrococcus furiosus, which generates small
CRISPR RNAs (crRNAs).
• Processing occurs near the 3′ end of the repeat sequence, leaving a short (~8
nts) repeat sequence 5′ of the crRNA spacer & more heterogeneous 3′
terminus.
B) RNAs serve as guides for an effector complex (Cas proteins) that recognizes
invading DNA and blocks infection
3) Self versus non-self discrimination during CRISPR immunity
Target DNA
CRISPR DNA
Target Target of non-self DNA
Protection of self DNA
CrRNA
CrRNA
RepeatRepeat Spacer
Non-Complementary
Complementary

27. 27
RNA interference (RNAi) CRISPR interference
siRNAs, miRNAs & piRNAs as
guides for gene regulation and
genome defence
crRNA used
Double-stranded precursors single-stranded precursors
Post-transcriptionally
amplified
Not post-transcriptionally
amplified
Recognize other RNAs Recognize other DNAs
No such integration required
Invasive nucleic acids integrated
into host genome before
defense mechanism

28. 28
Case Study

29. Enhanced Whitefly Resistance in Transgenic Tobacco Plants
Expressing Double Stranded RNA of v-ATPase A Gene
1
Thakur et al., 2014India 29
• Transgenic tobacco lines were developed for the expression of long
dsRNA precursor to make siRNA and knock down the v-
ATPaseA mRNA in whitefly.
• Molecular analysis and insecticidal properties of the transgenic plants
established the formation of siRNA targeting the whitefly v-ATPaseA,
in the leaves.
• The transcript level of v-ATPaseA in whiteflies was reduced up to 62%
after feeding on the transgenic plants.
• Heavy infestation of whiteflies on the control plants caused significant
loss of sugar content which led to the drooping of leaves.
• The transgenic plants did not show drooping effect.

30. Methodology/Principal Findings
30
(A) Construction of v-ATPaseA dsRNA expression cassette in pBI101
(B) Transgenic and control tobacco plants
(C) Selection of T1 seeds on kanamycin medium, showing non-transgenic seedlings
turning white
(D) PCR analysis of transgenic lines
• upper panel - amplification of nptII gene
• lower panel- amplification of v-ATPaseA+RTM1 intron

31. In-vivo bioassay of transgenic and control plants with whitefly
Early Stage of Whitefly infection Loss of Sugar content due to heavy infestation
C
D
31

32. Expression analysis of v-ATPase A specific RNA in
different transgenic lines of tobacco
Dot-blot assay of total RNA from 12 T1 transgenic lines with probe of
(A) v-ATPaseA gene & (B) U6 gene
(C) Dot-blot assay of RNA from control plants (expressing dsRNA of asal gene); RNA
was spotted and hybridised with same probes to show specificity of hybridisation
(D) Northern blot analysis of four selected transgenic lines with v-ATPaseA specific
probe
32

33. The Arabidopsis MicroRNA396-GRF1/GRF3 Regulatory Module
Acts as a Developmental Regulator in the Reprogramming of
Root Cells during Cyst Nematode Infection
Hewezi et al., 2012Iowa (U.S.)
2
33
• The syncytium is a unique plant root organ whose differentiation is induced by
plant-parasitic cyst nematodes to create a source of nourishment.
• Syncytium formation involves the redifferentiation and fusion of hundreds of root cells.
soybean
Cyst nematodes Root nodes infection Female cyst containing eggs

34. 34
• A strong downregulation of Arabidopsis (Arabidopsis thaliana) microRNA396
(miR396) in cells giving rise to the syncytium coincides with the initiation of
the syncytial induction/formation phase and that specific miR396 up-
regulation in the developed syncytium marks the beginning of the
maintenance phase, when no new cells are incorporated into the syncytium.
• miR396 has a role in the transition from one phase to the other.
• Expression modulations of miR396 and its Growth-Regulating Factor (GRF)
target genes resulted in reduced syncytium size and arrested nematode
development.
• Genome-wide expression profiling revealed that the miR396-GRF regulatory
system can alter the expression of 44% of the more than 7,000 genes reported
to change expression in the Arabidopsis syncytium.
• Thus, miR396 represents a key regulator for the reprogramming of root cells
and a powerful molecular target for the parasitic animal to modulate plant
cells and force them into novel developmental pathways.

35. 35
Methodology/Principal Findings
miR396 regulates the expression of 7 Arabidopsis growth-regulating transcription
factor genes (GRF1–4 & GRF7–9) that share the miR396-binding site
•miR396 have two genes 1) miR396a (AT2G10606) 2) miR396b (AT5G35407)
N =nematode
S = syncytium
Histochemical localization of GUS activity directed by miR396 promoters
Transgenic plants expressing constructs containing the regions upstream of
miR396 precursor sequences fused to the GUS reporter gene (PmiR396a:GUS and
PmiR396b:GUS).
Promoter activity of miR396a and miR396b during H. schachtii infection.

36. 36
Thus, miR396 expression changes delineate the syncytium induction/formation
phase, whereby a down-regulation marks the beginning of syncytium
induction/formation and a subsequent strong upregulation coincides with the
transition to the maintenance phase.
The GRF1 and GRF3 promoters showed very strong GUS activity in syncytia at
all stages, with only the GRF3 promoter becoming inactive at the J4 stage
Posttranscriptional regulation of GRF1 and GRF3 by miR396 in response to H.
schachtii infection.
A) Mapping of the cleavage sites of GRF1 and GRF3 using 59 RLM-RACE.

37. 37
B) miR396 mediates the down-regulation of GRF1 and GRF3 expression in
response to H. schachtii infection.
The expression level of pri-miR396a, pri-miR396b, mature miR396, GRF1,
and GRF3 was measured by qPCR in wild-type (Col-0) root tissues.
All transgenic lines overexpressing miR396a (Fig. 4A) or miR396b (Fig. 4B) were
dramatically less susceptible to nematodes than the wild-type control.

38. 38
Transgenic plants overexpressing rGRF1 (E) or rGRF3 (F) revealed reduced
susceptibility to H. schachtii
Overexpression of miR396 and the target genes GRF1 and GRF3 negatively impacts
root development.
A) & B) Transgenic plants overexpressing miR396a (line 22-5; A) or miR396b
(line 15-1; B) develop shorter roots than the wild type (Col-0).
C) Transgenic plants overexpressing rGRF1 (line 18-2) or rGRF3 (line 11-15) develop
shorter roots than the wild type (Col-0).

39. Knockdown of Midgut Genes by dsRNA Transgenic Plant-
Mediated RNA Interference in the Hemipteran Insect
(Nilaparvata lugens)
Leaves turn yellow initially and later brownish due to
drying up of the plants.
Under severe cases field gives a burnt appearance in
concentric circles, known as "Hopper burn".
RNAi is a powerful technique for functional genomics research
against Hemipteran Insect (N. lugens)
Transgenic plants producing dsRNA directed against insect genes have
been reported for lepidopteran and coleopteran insects, showing potential
for field-level control of insect pests.
They damage rice directly through feeding and also by
transmitting two viruses, rice ragge stunt virus and rice
grassy stunt virus.
Up to 60% yield loss is common in susceptible rice
cultivars attacked by BPH (Brown Plant Hopper).
Zha et al., 2011China
3
39

40. Methodology/Principal Findings
The Hemipteran insect brown planthopper (Nilaparvata lugens) is a typical
phloem sap feeder specific to rice (Oryza sativa L.)
Analysis and identification of genes (Nlsid-1 and Nlaub) encoding
proteins that involved in the RNAi pathway in N. lugens insect.
Both genes are expressed ubiquitously in nymphs and adult insects.
Nymphs40

41. Three genes isolated were highly expressed in the N. lugens midgut and used to
develop dsRNA constructs for transforming rice.
1) NlHT1- hexose transporter gene 2) Nlcar- carboxypeptidase gene
3) Nltry- trypsin-like serine protease gene
Primary expression in midgut tissues with limited transcription in salivary glands, fat body and head
41
A
A) The genes were cloned at the BamHI site of the hygromycin gene expression
cassette in the pCU vector of Agrobacterium
B) Southern blot analysis showed that the PCR-positive plants had 1–3 copies of
the target coding sequences

42. When nymphs were fed on rice plants expressing dsRNA, levels of
transcripts of the targeted genes in the midgut were reduced.
Growth phenotypes of wild type (WT) plants, empty transformation vector plants and transgenic lines
(A) Two-week-old seedlings (B) Four leaf stage Plants (C) Mature plants
RNA blot analysis showed that the dsRNAs were transcribed and some of
them were processed to siRNAs in the transgenic lines.

43. Genome-wide identification of Brassica napus
microRNAs and their targets in response to cadmium
• MicroRNAs (miRNAs) also regulate response to environmental
stresses.
• The toxic heavy metal cadmium (Cd) induces expression of several
miRNAs in rapeseed (Brassica napus).
• miRNA-regulated gene silencing may be involved in plant tolerance
to heavy metals.
• In this study, four small RNA libraries and four degradome libraries
were constructed from Cd-treated and non-Cd-treated roots and
shoots of B. napus seedlings.
• Using high-throughput sequencing, the study identified 84 conserved
and non-conserved miRNAs (belonging to 37 miRNA families) from
Cd-treated and non-treated B. napus, including 19 miRNA members
that were not identified before.
Zhou et al. 2012China 43
4

44. 44
• Some of the miRNAs were validated by RNA gel blotting.
• Most of the identified miRNAs were found to be differentially expressed in
roots/shoots or regulated by Cd exposure.

45. • The study also identified 802 targets for the 37 (24 conserved and 13
non-conserved) miRNA families, from which there are 200, 537, and 65
targets, belonging to categories I, II, and III, respectively.
• In category I alone, many novel targets for miRNAs were identified and
shown to be involved in plant response to Cd.
45

46. The Chloroplast Triggers Developmental Reprogramming
When MUTS HOMOLOG1 Is Suppressed in Plants
• To explore the control of phenotypy in higher plants, they
examined the effect of a single plant nuclear gene on the
expression and transmission of phenotypic variability in
Arabidopsis thaliana.
• MutS HOMOLOG1 (MSH1) is a plant-specific nuclear gene
product that functions in both mitochondria and plastids to
maintain genome stability.
• Genetic hemicomplementation experiments show that
this phenotypic plasticity derives from changes in
chloroplast state.
• The result of MSH1 suppression through RNAi, triggers a
plastidial response process that involves non-genetic
inheritance. Xu et al., 2012China 46
5

47. Loss of MSH1 by RNAi results in programmed phenotypic changes, including
A) Altered phytohormone effects for dwarfed growth & reduced internode
elongation B) altered leaf morphology C) Effects are partially reverted with the
application of GA D) enhanced branching E) reduced stomatal density F) extended
juvenility, with conversion to perennial growth pattern in short days G) delayed
flowering
47

48. Fig. 2. Phenotypic plasticity in the Arabidopsis msh1 mutant.
48

49. Fig. 5. Evidence of transcriptional and
metabolic changes in Arabidopsis msh1
mutant and hemicomplementation lines.
A) & B) quantitative RT-PCR assay
C) RNA gel blot assay
D) Heat map assay of metabolites
Fig. 6. Hemicomplementation analysis of the
Arabidopsis msh1 altered growth phenotype.
Col-0, dual-targeted, and plastid-targeted
MSH1 transgenic lines flowered uniformly,
whereas the msh1 mutant and the
mitochondria-targeted MSH1 transgenic line
showed marked variation for growth,
flowering time, and maturity.
The Observed Developmental Reprogramming Is
the Consequence of Chloroplast Changes
49

50. Development of male sterility by silencing Bcp1 gene
of Arabidopsis through RNA interference
• Use of RNA interference (RNAi) technology to silence a male
specific gene, Bcp1 in the model host Arabidopsis thaliana.
• Bcp1 is active in both diploid tapetum and haploid microspores.
• Three batches of explants (A. thaliana) were selected on herbicide
Glufosinate ammonium and putative transgenes were confirmed
through PCR and Southern hybridization.
• The present study resulted in developing male sterile A. thaliana
(Eco. Columbia) line through genetic engineering.
Tehseen et al., 2010Pakistan 50
6

51. • Bcp1 gene can be divided in two parts
– 163bp non conserved region
– 372bp conserved region
Methodology/Principal Findings
• Silencing of Bcp1 gene ,587bp in size, responsible for fertility
They targeted 0.77kb regulatory region of Bcp1 gene via antisense
• Expression of both sense and antisense fragment separated by an intron,
yields more efficient silencing than only antisense
Targeting coding sequence of Bcp1 using RNAi leads to male sterility
• Bgp1,female fertility gene
-- 87 % homology with conserved region
-- To avoid silencing of female part, only non conserved region is targeted
51

52. • 163bp region of Bcp1 gene cloned in both sense and antisense orientation
in pFGC5941
-Cloning of gene in sense and antisense orientation in same vector produce
dsRNA inverted repeat molecule which induce PTGS in plant cells
•Primers design w.r.t dsRNA binary vector pFGC5941
-It has two mcs, bar gene and 35S promoter within left and right borders
-Two mcs flanked by intron of 1.364kb
• Construct was transformed in A.tumefaciens strain LBA4404 by electroporation
Agrobacterium culture was confirmed with PCR amplification
The construct was transformed in Arabidopsis using leaf disc method
52

53. • Formation of sharp bands confirm the presence of transgenes
– Transcribed mRNA of RNAi construct will result in a dsRNA with a hairpin loop
– Resultant dsRNA triggered on the RNAi machinery
• 3 batches of explants were selected on herbicide glufosinate ammonium
Putative transgenic plants confirmed by PCR using bar gene specific primers
and southern hybridisation
It gives amplification along with positive and negative controls
53

54. The Introgression of RNAi Silencing of γ-Gliadins into
Commercial Lines of Bread Wheat Changes the Mixing
and Technological Properties of the Dough
•The effects on dough quality by the RNAi-mediated silencing of γ -
gliadins in commercial bread wheat lines (namely ‘Gazul’, ‘Podenco’ and
‘Arpain’) along with the transgenic line A1152 (cv. Bobwhite) were compared
with their respective wild types.
•The protein fractions were quantified by RP-HPLC, whereas the technological
and mixing properties were assessed by SDSS test and by the Mixograph
instrument.
•The down-regulation of γ-gliadins resulted in stronger dough and a better
tolerance to over-mixing in some transgenic lines.
Gil-Humanes et al., 2012Spain 54
7

55. Methodology/Principal Findings
Wheat gliadin genes occur as tightly-linked clusters located on 1 and 6 chromosomes
ω and γ-gliadins on Chromosome 1
having clusters of genes
• Gli-A1
• Gli-B1
• Gli-D1
LMW-GS genes on the Glu3 loci
α-gliadins on Chromosome 6
having clusters of genes
• Gli-A2
• Gli-B2
• Gli-D2
Down-regulation of γ –gliadins through RNAi approach in three commercial lines and
F1 hybrid obtained
•After three generations of self-pollination, the gliadin and glutenin profiles analyzed by
A-PAGE and SDS-PAGE.
55

56. Glutenin content increased and the ratio Gli/Glu reduced in the transgenic lines,
provoking an increase of the dough strength and a decrease of the extensibility.
Analysis of grain composition and quality parameters
•The analysis of the variance (ANOVA) and LSD analysis of homogeneous groups showed
significant differences in all the fractions of gliadins and glutenins.
•SDSS volume showed an increase in all the transgenic lines w.r.t. the wild types, resulting in
significantly higher volumes for transgenic lines G613, G626, G845 and G664.
•In all parameters of Mixograph, no significantly different observed in the average of
transgenic lines(except G622 & G626) to wild types, indicating that the mixing properties
were not affected by the silencing of the γ-gliadins in the transgenic lines with different
HMW-GS profile. 56

57. 57

58. Engineering of the Rose Flavonoid Biosynthetic
Pathway Successfully Generated Blue-Hued Flowers
Accumulating Delphinidin
Katsumoto et. al (2007)Japan
o Rosa hybrida lacks violet to blue colour due to the absence of
flavonoid 3’,5’-hydoxylase (F3’5’H) enzyme which produces
delphinidin-based anthocyanins.
o Other factors such as co-pigments and vacuolar pH also affect
flower colour.
o Expression of the viola F3’5’H gene  accumulates (~95% high)
delphinidin  a novel bluish flower colour.
58
8

59. • For more exclusive and dominant accumulation of
delphinidin irrespective of the hosts, the endogenous
dihydroflavonol 4-reductase (DFR) gene was down-
regulated and overexpressed the Iris3hollandica DFR
gene in addition to the viola F3’5’H gene in a rose
cultivar.
• The resultant roses exclusively accumulated delphinidin
in the petals, and the flowers had blue hues not achieved
by hybridization breeding.
• Moreover, the ability for exclusive accumulation of
delphinidin was inherited by the next generations.
59

60. Bi
o
sy
n
t
h
et
ic
p
at
h
w
a
y
O
f
fl
a
v
o
n
oi
d
60

61. WKS77 WKS82 WKS100
WKS116 WKS124 WKS140
Rose Varieties transformed with pSPB130 and their flower colour changed are shown
Schematic representation of T-DNA region of binary vectors constructed for colour
modification  for the constitutive over expression of the viola F3’5’H BP40 gene
and the torenia 5AT gene in rose.
61

62. Host flower
violet-coloured
transgenic flower
98% delphinidin
98% delphinidin Madam Violet Seiryu
 The vector pSPB919 is to down-regulate the endogenous rose DFR gene using RNA
interference (RNAi) and to over express the iris DFR and the viola F3’5’H genes. 62

63. • Northern blot analysis of LA/919-4-10.
• The expected sizes of the transcripts of
viola F3’5’H BP40 (1.8 kb) and iris DFR (1.7
kb) genes & smaller size was detected for
rose DFR mRNA (A).
• A rose DFR probe detected about 23 bp
small sized RNA, which was supposed to be
a degraded endogenous rose DFR transcript
with RNAi (B).
• Delphinidin contents was confirmed
in all transgenic (KmR) progeny of
LA/919-4-10.
• The flowers of F1 and F2 progeny
contained exclusively delphinidin.
63
(si RNA)

64. Production of picotee-type flowers in Japanese
gentian by CRES-T
A B
Wild type flower‐Solid color suppression of pigment
production generates picotee
type flower
Nakatsuka et al., 2011Japan
CRES-TChimeric repressor gene-silencing technology
9
64

65. • (CRES-T) is an efficient gene suppression system which
worked successfully in Japanese gentian.
• A chimeric repressor of the anthocyanin biosynthetic
regulator gene GtMYB3, under the control of the
Arabidopsis actin2 promoter, was introduced into blue-
flowered gentian.
• Of 12 transgenic lines, 2 exhibited a picotee flower
phenotype with a lack of pigmentation in the lower part of
the petal.
• HPLC analysis showed that the petals of these lines
contained less anthocyanin and more flavone than the
wild-type.
• The expressions of ‘late’ flavonoid biosynthetic genes,
including F3H, F35H, DFR and ANS, were strongly
suppressed in petals of these transgenic plants.
65

66. • Expression of flavonoid biosynthetic genes in transgenic
gentian plants.
• The expression levels of GtMYB3-SRDX and endogenous
flavonoid biosynthetic genes were determined by semi-
quantitative RT-PCR analysis in wild-type and GtMYB3-
SRDX expressed transgenic gentian clone nos. 7 & 11
• Schematic representation of pSMABR-AtACT2pro-
GtMYB3-SRDX.
• Bar  herbicide bialaphos resistance gene as a
selectable marker.
• NOSp  promoter of nopaline synthase (NOS) gene
from A. tumefaciens.
• rbcSt  terminator of RuBisCO small subunit 2B gene
from Arabidopsis.
• NOSt  terminator of NOS gene.
• LB  left border; RB  right border.
semi-quantitative RT-PCR analysis
66

67. Flavonoid analysis in the flowers of transgenic gentian plants by HPLC
A & D- wild type
B & E- transgenic
gentian clones no. 7
C & F- transgenic
gentian clones no. 11
Anthocyanins
Flavones
67

68. Conclusions
68
Genetic engineering overcomes almost all the limitations of
traditional breeding approaches.
Recent advances in plant molecular biology provide
opportunities to use techniques of genetic engineering for
improvement of crop plants for disease resistance, toxic
resistance, plant architecture, male sterility, dough quality
parameter, flower colour, etc.
Case-1
Host plant derived pest resistance was achieved at field
level against whiteflies by genetic transformation of tobacco
which generated siRNA against the whitefly v-ATPaseA
gene.
Case-2
miR396- a key regulator, provides the nematode with a
single molecular target to wield power over a substantial
proportion of syncytium developmental events.

69. 69
Case-3
These genes (Nlsid-1 and Nlaub) were identified and
knockdown in N. lugens through RNA interference. The
results demonstrate the potential of this technique at field-
level control of plant-hoppers.
Case-4
Identification of many high-quality target transcripts will
help in the regulatory mechanism for plant tolerance to Cd.
Case-5
Suppression of MSH1, which occurs under several forms of
abiotic stress, triggers a plastidial response process that
involves non-genetic inheritance.
Conclusions
Case-6
Bcp1 gene is responsible for fertile pollen development and
active in both diploid tapetum and haploid microspores.
Silencing Bcp1 gene lead to transgenic male sterile plants.

70. 70
Case 7
The down-regulation of γ-gliadins resulted in stronger
doughs and a better tolerance to over-mixing in some
transgenic lines.
Case-8
Spectral difference in flower colour is mainly determined by
the ratio of different classes of pigments and other factors
and knowledge of flower coloration at the biochemical and
molecular level has made it possible to develop novel
color.
Case-9
(CRES-T) is an efficient gene suppression system which
worked successfully in Japanese gentian.
Conclusions

71. Future Prospects and New
Avenues
71
•Careful consideration of the interplay factors (such as
the selection of the target gene, most effective site within the
gene for knockdown, length of the targeted nucleotide
sequence, and minimization of the off-target effects) is
expected to deliver a competent technology in
insect/pest resistance in the near future.
•The regulatory unit represents a powerful molecular target
for the parasitic animal to modulate plant cells and force
them into novel developmental pathways.
•More research efforts are needed for tolerance of crop
plants toward heavy metal toxicity.

72. 72
• RNAi can be employed to obtain hybrid seeds of
commercially high valued crops.
•New genes should be isolated that will have utility in
floriculture, and new transformation methods for flower
crops should be further optimized.
• More advancement required for dough quality, as the
reduction of γ-gliadins seems not to have a direct effect on
the mixing and bread-making properties, the compensatory
effect on the synthesis of the other prolamins has resulted
in stronger doughs with improved over-mixing resistance in
some transgenic lines.
Future Prospects and New
Avenues

73. Earth is our unique Masterpiece, can neither be replicate,
transcribe nor translate it. We are only regulatory
components of Earth, can save our planet by Downregulating
environmental pollutions and Upregulating natural resources.
- Mariya Zaman
73