Presented By:

Mariya ZamanPh.D

(Biotech.)B.A.C.A., AAU

Submitted To:Dr. Akarsh Parihar SirAssociate Professor

B.A.C.A., AAU

Role of Antisense and RNAi-based Gene Silencing in

Crop ImprovementWelCome

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OutlinesIntroduction of Antisense & RNAi TechnologyDiscovery of RNAiTypes of RNAsMechanism of Antisense & RNAi TechnologyImportance of RNAi TechnologyPros & Cons of RNAi TechnologyApplication of RNAi TechnologyCRISPR TechnologyCase studiesConclusionsFuture Thrust

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-encodedRNA regulators control the copy numbers of the

Escherichiacoli 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

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Mechanism of Antisense Technology

Characteristics of Antisense RNANaturally 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.

Antisense-RNA regulated systems in prokaryotes

Inhibition of primer formation

Inhibition of synthesis of a leader peptide

MicF RNA, ompF-RNADicF RNA

RNAIII

Principal target sites

• Transcription1. DNA triple helix formation2. Hybridization to DNA loops3. Hybridization to nascent RNA

• RNA processing4. RNA splicing5. RNA transport

• Translation6. Inhibition of initiation factor binding7. Inhibition of ribosome assembly 8. Inhibition of movement

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RNA

Coding RNA

mRNA

Non Coding RNAs

Constituent/ Transcription

RNAs

rRNA tRNA

Regulatory/ Small RNAs

siRNA miRNA (stRNA) snoRNA snRNA

RNA Family

RNA interference

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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.

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Mechanism of RNAi Technology

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Structure of siRNA

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miRNA Mechanism

TRBP

Ran

GTP

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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

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• Dicer is a ribonuclease (RNase III family) with 4 distinct domains:

1. Amino-terminal helicase domain2. Dual RNase III motifs in the carboxy terminal segment3. dsRNA binding domain4. PAZ domain (110-130 amino-acid domain present in protein

like PIWI, Argonaute, and Zwille proteins) for protein-protein interaction.

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siRNA miRNAExogenous 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

Pre-miRNA

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Advantages of RNAi TechnologyDominant Phenotypes can be observed in the T1 generation

Partial knockdownFacilitates 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 labeledStable hpRNAi has been shown to be stably inherited over several

generations

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Pitfalls of RNAi

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

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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

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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.

Marraffini and Sontheimer, 2011USA

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Mechanism for CRISPR interference

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

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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

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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

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RNA interference (RNAi) CRISPR interference

siRNAs, miRNAs & piRNAs as guides for gene regulation and

genome defencecrRNA used

Double-stranded precursors single-stranded precursors

Post-transcriptionallyamplified

Not post-transcriptionallyamplified

Recognize other RNAs Recognize other DNAs

No such integration requiredInvasive nucleic acids integrated

into host genome before defense mechanism

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Case Study

Enhanced Whitefly Resistance in Transgenic Tobacco Plants Expressing Double Stranded RNA of v-ATPase A Gene

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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.

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Methodology/Principal Findings

(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

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In-vivo bioassay of transgenic and control plants with whiteflyEarly Stage of Whitefly infection Loss of Sugar content due to heavy infestation

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D

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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

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.)

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• 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

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• 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.

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Methodology/Principal FindingsmiR396 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 =nematodeS = 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.

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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.

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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.

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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).

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

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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.

Nymphs

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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

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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

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.

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

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• 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.

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• 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.

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

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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

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

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Fig. 5. Evidence of transcriptional and metabolic changes in Arabidopsis msh1 mutant and hemicomplementation lines.A) & B) quantitative RT-PCR assayC) RNA gel blot assayD) 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

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

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• 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

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•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 electroporationAgrobacterium culture was confirmed with PCR amplification The construct was transformed in Arabidopsis using leaf disc method

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• 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 ammoniumPutative transgenic plants confirmed by PCR using bar gene specific primers

and southern hybridisation It gives amplification along with positive and negative controls

The Introgression of RNAi Silencing of γ-Gliadins intoCommercial Lines of Bread Wheat Changes the

Mixingand 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

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Methodology/Principal Findings

Wheat gliadin genes occur as tightly-linked clusters located on 1 and 6 chromosomes

ω and γ-gliadins on Chromosome 1having 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-D2Tightly linked

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.

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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.

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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.

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• 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.

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Biosynthetic pathway

Of

flavonoid

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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.

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Host flowerviolet-coloured

transgenic flower98% delphinidin

Flower and petal colour comparison

Transgenic roses exhibited paler flower colour

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.

• 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.

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(si RNA)

Production of picotee-type flowers in Japanese gentian by CRES-T

A B

Wild type flower‐Solid colorsuppression of pigment production generates picotee type flower

Nakatsuka et al., 2011Japan

CRES-TChimeric repressor gene-silencing technology

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• (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.

• In contrast, the ‘early’ flavonoid biosynthetic genes, such as CHS and FNSII, were not affected.

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• 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

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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

AnthocyaninsFlavones

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Conclusions

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-1Host 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-2miR396- a key regulator, provides the nematode with a single molecular target to wield power over a substantial proportion of syncytium developmental events.

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Case-3These 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-4Identification of many high-quality target transcripts will help in the regulatory mechanism for plant tolerance to Cd.Case-5Suppression of MSH1, which occurs under several forms of abiotic stress, triggers a plastidial response process that involves non-genetic inheritance.

Conclusions

Case-6Bcp1 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.

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Case 7The down-regulation of γ-gliadins resulted in stronger doughs and a better tolerance to over-mixing in some transgenic lines.Case-8Spectral 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

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Future Prospects and New Avenues

•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.

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• 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

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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