Chapter-8: Recent technologies in

crop
• Genomic editingimprovisent
:is a Precise manipulation of
genetic sequence of plants and organisms,
including humans, in a lucid, handy, and
error- free way.”
• The classical processes of introducing
changes in the genetic makeup, include:
• Hybridization and
•mutagenesis and,
•In the modern age, rDNA technology
• The technique got a shot in the arm by the discovery of
engineered nucleases (enzymes which cut DNA/RNA) that
have a recognition site of over 14 nucleotides in the genome.
These molecular scissors are primarily of four types:
i) meganucleases;
(ii) zinc finger nucleases (ZFNs);
(iii) Transcription activator like effector nucleases (TALENs),
Iv RNA silencing etc
(v) Targeted mutagenesis: “site-directed mutagenesis”,
(vi) clustered regularly interspaced short palindromic
repeats (CRISPR).
• These nuclease systems may act off-target and,
therefore, have low specificity, stringent selection steps,
and cumbersome handling.
• The latest engineered CRISPR-Cas system has overcome
most of these limitations and has captivated global
attention
8.1 gene silencing by RNA
Interference (RNAi)
• RNA interference (RNAi) or Post-Transcriptional
Gene Silencing (PTGS) is a conserved biological
response to double-stranded RNA that
mediates resistance to both endogenous
parasitic and exogenous pathogenic nucleic
acids,
•This natural mechanism for sequence-specific
gene silencing promises to revolutionize
experimental biology and have important
practical applications in functional genomics,
therapeutic intervention, agriculture and other
areas.
 Endogenous triggers of RNAi pathway
•Endogenous triggers of RNAi pathway include:
 foreign DNA or double-stranded RNA (dsRNA)
of viral origin,
 aberrant transcripts from repetitive sequences
in the genome such as transposons, and
 pre-microRNA (miRNA).
•In plants, RNAi forms the basis of virus-
induced gene silencing (VIGS), suggesting an
important role in pathogen resistance
A simplified model for the RNAi
pathway
• A simplified model for the RNAi pathway is
based on two steps, each involving ribonuclease
enzymes:

• First step, the trigger RNA (either dsRNA or

miRNA primary transcript) is processed into an
short, interfering RNA (siRNA) by the RNase III
enzymes Dicer and Drosha.
• second step, siRNAs are loaded into the effector
complex RNA-induced silencing complex (RISC).
• The siRNA is unwound during RISC assembly
and the single-stranded RNA hybridizes with
mRNA target.
• Gene silencing is a result of nucleolytic
degradation of the targeted mRNA by the RNase
H enzyme Argonaute (Slicer).
• If the siRNA/mRNA duplex contains mismatches
the mRNA is not cleaved. Rather, gene silencing
is a result of translational inhibition
RNAi in experiments and therapeutics: how it works
 8.2 Viral suppressors of RNA silencing
•The infection and replication of viruses in the
host induce diverse mechanisms for combating
viral infection..
•Several viral suppressors of RNA silencing
(VSRs) have been identified from almost all
plant virus genera, which are surprisingly
diverse within and across kingdoms, exhibiting
no obvious sequence similarities
• RNA silencing is a conserved sequence-
specific gene regulation system, which has an
essential role in the development and
maintenance of genome integrity.
• In higher plants and insects, RNA silencing
also operates as an adaptive inducible
antiviral defense mechanism
• The silencing of RNA relies on host- or virus-
derived 21–24 nucleotide long sRNA molecules,
which are the key mediators of RNA silencing-
related pathways in plants and other
eukaryotic organisms .
•In plants, there are two main types of sRNAs:
 miRNAs and
siRNAs,
 but the siRNA class contains several
different types .
• These sRNAs are produced from:
double-stranded RNA (dsRNA) or
from folded structures by Dicer-
like proteins (DCLs), and
 they guide Argonaute (AGO) proteins to
target cognate RNA or DNA sequences
• These endogenous sRNAs play important
roles in many aspects of gene regulation in
plants, controlling developmental
programming or biotic and abiotic stress
responses
• Both cellular and antiviral siRNA
biogenesis often requires RNA-dependent
RNA polymerases (RDRs
 Mechanism of silencing-based antiviral plant
response
• The pathway of antiviral silencing can be divided
into three major steps:
(i) sensing and processing viral RNAs to viral siRNAs;
ii) amplifying vsiRNAs; and
(iii) assembling antiviral RISC and targeting viral RNAs.
• The silencing-based antiviral plant response starts
with the recognition of ds or structured single-
stranded (ss) viral RNA by one or more members of
plant Dicers . The recognized viral RNAs are then
processed by Dicers into vsiRNA
• In plants, two distinct classes of vsiRNAs have
been identified:
• primary siRNAs, which result from the DCL
mediated cleavage of an initial trigger RNA,
and
•secondary siRNAs, which require an RDR
enzyme for their biogenesis .
• In the Arabidopsis model plant, DCL4 and DCL2 are the
most important DCLs involved in virusinduced RNA
silencing and they can process ds or hairpin viral
RNAs into vsiRNAs of 21 and 22 nt, respectively.
• The amplification and high level of vsiRNA
accumulation in many but not all virus
infections depend on the combined activity of
the host-encoded RDRs such as:
 RDR1
 RDR2 and
 RDR6
8.3 TILLING an d ECOTILLING
• It is desirable for the breeders to know relative
value of all alleles for gene of interest since it
will help them to decide which allele for a gene
to be introgressed into the desirable variety.
• Local lesion in genome is called TILLING and
the strategy of targeting induced local lesion in
genomes is called Eco TILLING
• It helps in identifying naturally present SNP and
haplotyping leading to characterization of
alleles.
• Eco TILLING can provide a series of alleles for
the genes which are involved in important
processes of the plant even though the
known variants for these genes have not been
observed through genetic studies
• Two main approaches utilized to link
genotype to phenotype are known as :
Forward genetics and
Reverse genetics.
• Both of these processes aim to determine the
function of a gene / genes through screening
the phenotype or genotype of individual
mutants to ultimately determine how it is
control.
 Forward genetics (phenotype to
genotype
• Forward genetics (phenotype to genotype),
in which one starts with a particular
identified phenotype or biological process
and the gene sequence is ultimately deduced
through screening large numbers of
mutagenized individuals for phenotypic
variations .
• Is a useful approach for genome wide analysis
primarily due to the effort and time involved
to identify each gene coding for a particular
phenotype
Reverse genetics (from genotype to
phenotype

• the gene sequence is known and mutants

are screened to identify individuals with
structural alterations in the gene of
interest
• This approach is generally less
time demanding than forward
genetics
• Some of the reverse genetic strategies
employed in plants and animals include
:
homologous recombination,
Agrobacterium mediated
insertional mutagenesis,
 transposon tagging,
RNAi (RNA interference) or PTGS
(post transcriptional gene silencing),
and
chemical mutagenesis
• Tilling accomplished by :
• pooling chemically induced
mutagenized plants together,
• amplifying the region of interest, creating
heteroduplexes among the pooled DNA,
and
• performing dHPLC (denaturing high
performance liquid chromatography)
to detect the mutants by
chromatographic alterations
 The creation of a mutagenized
population
• The first step in TILLING is the creation of a
mutagenized population, which is often
accomplished by treatment with a chemical
mutagen such as EMS
• Many plant species are well suited for this
strategy because they can be self-fertilized and
seeds can be stored for long periods of time.
• In plants, seeds are treated with EMS and grown
out to produce M1 plants, which are
subsequently self-fertilized to produce the M2
generation.
• Leaf tissues from M2 plants are collected for
DNA extraction and then used for
mutational screening
• To avoid sampling of the same mutation only
one M2 individual from each M1 is chosen for
DNA extraction .
• The M2 progeny can be self-fertilized and the
resulting M3 seed can be preserved in long
term storage
• EMS has been widely used as a chemical
mutagen in TILLING in both plant and animal
studies to generate mutant populations,
although other chemical mutagens can be
effective
•EMS typically produces transition mutations (G/C
: A/T) because it alkylates G residues and the
alkylated G residue pairs with T instead of the
conventional base pairing with C.
 Amplifying the region of interest.

• Once the pooled DNA is arrayed into 96 well

microtiter plates, pooled samples are amplified
using primers targeting the gene of interest.
• the forward and reverse primers are
differentially 5’ end labeled with IRD700 and
IRD800 dye labels for fluorescent detection
at
~700 nm and ~800 nm, respectively
• Next, heteroduplexes and homoduplexes
are formed from the PCR products of pooled
samples (consisting of mutants and the wild
type) by heating (denaturing) and cooling
(annealing).
• •The endonuclease enzyme CEL I is applied and a
short incubation is required for the enzymatic
reaction to progress.
• •CEL I, isolated from celery, not only
specifically recognizes mismatches in the
heteroduplex, but it also cleaves DNA on the 3’
side of the mismatch .
 separation on a denaturing
polyacrylamide gel
• After the enzyme incubation period, detection
of any digested fragments occurs by
separation on a denaturing polyacrylamide gel
attached to a LI-COR 4300 DNA analysis system
8.4 CRISPR SYSTEM
• THE END OF THE CHAPTER-8