Applications

1) Identification of recombinant DNA clone carrying the desired DNA insert

2) Identification and isolation of genes (or) specific sequences for cloning
3) Conformation of integration of the DNA insert in the host genome (by SH) and its
expression in transformed cells (N.H)
4) Development of RFLP maps
5) DNA finger printing for identification of plant varieties criminals, parental
relationship etc.
6) Accurate diagnosis of diseases caused by parasites, pathogens (or) detective viruses
7) Preparation of genome maps of Eukaryotes including man

Lecture No. 25*

PCR – Procedure and applications – Comparison of PCR and gene cloning

The PCR technique for quickly cloning a particular piece of DNA in the test tube
rather than in living cells like E. coli / in -vitro method for the amplification of DNA
fragments. PCR technique is developed by Kary mullis 1985. It is one of the most powerful
molecular biology technique used to multiply minute (or) trace amounts (µg microgram
quantities) of DNA copies of the desired DNA by multiple cycles of cooling and heating in a
reaction catalysed by a heat stable DNA polymerase enzyme. (The PCR is carried out in
vitro). PCR is based on the features of semiconservative DNA replication carried out by DNA
polymerase in prokaryotic and eukaryotic cells.
The PCR utilizes the following :
1) DNA preparation containing the desired segments to be amplified must have known
nucleotide sequence so that oligonucleotide primers can bind and synthesize the
DNA..
2) Two nucleotide primers (about 20 bases long) specific i.e. complementary to the two
5’ - 3’ borders of desired segment
Definition of primer – It is a short sequence (often of RNA) /
an oligonucleotide that pairs with one strand of template DNA and provides a 3’ OH
essential for DNA polymerase to extend the primer through DNA synthesis.
Oligomer primers – Primer length
- Duplex stability
 5’ - … GGCG – 3’ - Non complementary base pairs
5’ - … CCGC – 3’
- No hairpin loops
- Optimal distance between primers
- Sequences with long runs can be avoided
3) Amplificatioin buffer:- which consists of four deoxy nucleoside triphosphate viz. TTP
(Thymidine triphosphate) dCTP (deoxyctosine triphosphate), dATP (deoxy adenosine
triphosphate) and dGTP (deoxy guanosine triphosphate) and a heat stable DNA
polymerase such as “Taq polymerase” (isolated from the bacte rium Thermus
aquaticus).
4) Genomic DNA is normally doulde stranded. DNA amplification by the PCR is carried
out in three general, steps that are repeated for a number of cycles to exponentially
increase the number of copies of a specific target region.
Procedure of PCR
Amplification of DNA is achieved by a repetitive series of cycles involving 3 steps.
1. Denaturation
The DNA double helix separated into two complimentary single strands by heating
reaction mixture to temparature between 90-980C that ensures DNA denaturation.
The duration of this step in the first cycle of PCR is usually, 2 min at 94 0C, but in
subsequent cycles it is of only 1min duration.
2. Annealing
1. The mixture is now cooled to a temparature of generally 40-60oC that permits
annealing of primer to the complimentary sequences in the DNA. The duration of
annealing step is usually 1 min during the first as well as the subsequent cycles of
PCR.
2. The annealed primers are then extended (i.e. synthesis of DNA) with Taq DNA
polymerase.
3. Primer Extension (or) synthesis
It involves heating the mixture to 72oC at which a special polymerase synthesizes the
new DNA strand by using the original strand as the template starting by the primer utilizing
their 31OH free ends and continuing in the 31 direction.
The completion of extension step completes the first cycle 01- amplification and these
three steps are repeated 25-40 times to produce millions of exact copies of the target region of
DNA. Thus at the end of each cycle the number of copies of desired segment becomes twice
the number present at the end of previous cycle. Thus at the end of ‘n’ cylces 2n copies of the
segments are expected.
Incase of automated PCR machines, called thermal cycles, the researcher specify only
the number and duration of cycle etc. after placing the complete reaction mixture for
incubation, and the machine performs the entire operations preciously. After PCR cycles the
amplified DNA segment is purified by gel electrophoresis and can be used for cloning, DNA
sequencing, etc.
Application
 DNA cloning for sequencing
 DNA based phylogenetic studies.
 Functional analysis of gene
 Diagnosis of hereditary diseases
 Identification of genetic finger prints used in forensics and paternity studies
 The detection and Diagnosis of infectious diseases
 It can be used to determine the sex of embryos
Comparison of PCR and gene cloning
Parameter PCR Gene cloning
1. Manipulation In vitro step in vitro and in vivo last
st
2. selectivity of specific 1 step Last step
segments from complex
DNA
3. Biological reagents require DNA segment 2 primers Restriction enzymes ligases
DNTP technique polymers vector DNA, host cells
4. Automation yes No
5. labour intensive no Yes
6. Error probability less More
7. Applications more Less
8. cost less More
9. users skill Not required Required
10. Time required for 4 hrs 2-4 days
conducting a experiment
Lecture No. 26*
Molecular markers – Definition – Brief description of different
types of molecular markers, RFLP, AFLP, RAPD and SSR markers–
Impo rtance, procedure and applications

Molecular marker is a DNA segment that is readily detected and whose inheritance
can easily be monitored. The use of molecular markers is based on the naturally occurring
DNA polymorphism.
A DNA marker is a small region of DNA showing sequence polymorphism in
different individuals with in a species (or) among different species
The DNA markers are most widely used type of markers predominantly due to their
abundance the first such DNA markers to be used for the fragments produced by the digestion
with restriction endonucleases that means restriction fragment length polymorphism (RFLP)
based genetic markers. A wide range of molecular techniques is now available to detect the
polymorphism at DNA level. These have been grouped into the following.
1) Non PCR based approaches Eg:- RFLP
2) PCR based approaches Eg:- RAPD AFLP SSRS etc (plants)
Restricted fragment length polymorphism (RFLP)
This technique was first developed in 1980 and it was based on Southern hybridization
technique. RFLP is used to describe the variation in the length of fragments obtained from the
digestion of DNA from two or more organisms with the same endonuclease. The variation at
DNA level is assayed by shearing the entire DNA with restriction enzymes. The restriction
sites for a particular enzymes are present at several places through out the entire genome with
the result that a large number of fragments of DNA are produced.
The length of each segment depends on the distance between two adjascent restriction
sites. Plants are able to replicate their DNA with high accuracy and rapidity, but many
mechanisms causing changes in the DNA are operative. Simple base, pairs changes (or) large
scale changes as a result of inversions, translocations, deletions (or) transposition may occur.
This will result in loss (or) gain of recognition site and inturn lead to restriction fragments of
different lengths. These restriction fragments of different lengths beteween the genotypes can
be defected on southern blots and by the use of suitable probe. An RFLP is detected as a
differential movement of a band on the gel lanes from different species and strains. Each such
bond is regarded as single RFLP locus. The pattern of RFLP generated will depend mainly on
 1) The differentiation in DNA of selected strains (or) species
2) The restriction enzymes used
3) The DNA probe employed for southern hybridization
RFLP analysis comprises the following basic steps.
1) Isolation of DNA
2) Cutting of DNA into smaller fragments using restriction endonuclease enzymes
3) Seperation of DNA fragments by gel electrophorosis
4) Transferring of DNA fragments on to a nylon and nitrocellulose membrane filter.
5) Visualization of special DNA fragments by using labeled probes
6) Analysis of results.
Application (or) uses of RFLP
 Identification and isolation of any gene known to be linked with an RFLP locus.
 RFLP are co-dominant markers enabling heterozygotes to be distinguished from
homozygotes
 The method is simple as no sequence specific information is required
 Used to measure genetic diversity between different populations or related species and
also important in studies on evolution
 RFLP’S can be used to supplement regular plant breeding protocols through indirect
selection
 Used as a marker in chromosomal mapping
 Used as a diagnostic tool for diseases.
Limitations
 Requires relatively large amount of highly pure DNA
 Needs a good supply of probes that can reliably detect variation
 Laborious and expensive to identify a suitable marker restriction enzymes combination for
genomic or CDNA libraries
 Time consuming as they are not amenable to automation.
 Required expertise in auto radiography because of using radio actively labeled probes
Random amplified polymorphic DNA (RAPD)
It is a PCR based molecule marker technique. Here, single short oligonucleotide
primers are arbitrarily selected to amplify a set of DNA segments distributed randomly
throughout the genome. RAPD amplification is performed in condition resembling those of
PCR using genomic DNA from the species of interest and a single short oligonucleotide
(usually 10 base sequences) primer. The DNA amplification product is generated from a
region, that is flanked by a part of 0 bp priming sites in the appropriate orientation. Genomic
DNA from different individuals often produces different amplification patterns i.e. RAPDs. A
particular fragment generated for one individual but not for the other represents DNA
polymorphism and can be used as a genetic marker. A DNA sequence different between
individuals in a primer binding site may result in the failure of primer to bind, and hence in
the absence of a particular band among the amplification product. The reaction products are
conveniently analysed on agarose gels stained with Ethidium bromide and seen under UV
light.
In inheritance studies, the amplification products are transmitted as dominant markers.
Thus presence of a RAPD band corresponding to a PAPD detminant allele against absence of
a band that corresponds to a recessive allele. Thus heterozygous and homozugous dominant
individuals cannot be differentiated with RAPD markers.
In F2, the segregating population may be scored as follows. Therefore in F 2
segregating population may be scored a follows :
Band present AA (hemozygtote dominant)
Aa (heterozygous)
Band absent aa (recessive homozygote) therefore in F 2 these two classes would be
expected to segregate in 3:1 ratio.
This causes a loss of information relative to RFLP markers which show co-
dominance.
Applications:-
 Construction of genetic maps
 Mapping of traits
 Ana lysis of genetic structure of populations
 Finger printing of individuals
 Targeting markers to specific region of the genome
 Identification of somatic hybrids
 A useful system for evolution and characterization of genetic resources.
Advantages of RAPD
1. Simple quick to perform
2. Require relatively very small amounts of DNA
3. Involves no radio activity
 Limitations
Unable to distinguish belong homozygous and heterozygous

Character RFLP RAPD

Principle Restriction digest ion DNA amplification
Inheritance Codominont Dominant
Detection Southern blotting DNA staining
DNA quality Relatively pure Crude
DNA amount High (2 to 10 mg) Small (15 to 25 mg)
Primer require None Yes (require random primer)
Use of radio isotopes Yes (in probes) for labeling No
Types of probes Sps specific probes Random base
Used primers Probes Sequence
Time sequence 5 times than RAPD Low

DNA finger printing / DNA profiling / Genetic finger printing

It is a technique in which an individual DNA is analysed to reveal the pattern of
repetition of particular nucleotide sequence through out the genome. The unique pattern of
DNA fragment is identified by southern hybridization or PCR. DNA typing methods have
been used widely in criminal investigation and for determining the pedigree and parentage of
an individual.
A DNA finger print of an individual is prepared by digesting its DNA with restriction
enzymes, subjecting the DNA digest to electrophorosis and southern hybridization with a
probe specific for a highly variable region so that large amount of polymorphism is generated.
In case of human beings many satellite DNA’S are used as probes. But such probes are not
available in plants. RFLPS are commonly used for identification of commercial varieties
characterization of germplasm lines etc.

Lecture No. 27
Quantitative trait loci mapping, marker assisted selection and its
applications in crop improvement – DNA finger printing applications.

 Quantitative Trait is governed by polygenes and is affected by the environment as a

result shows a continuous variation.
 Polygenes have small but cumulative effect on the concerned trait, and several
polygenes affect a single trait.
 Quantitative Trait Loci is a position in a chromosome that contains one or more
polygenes involved in the determination of a quantitative trait
 The genes cannot be mapped using the conventional approaches because it is
impossible to fallow the inheritance pattern of individual poly genes.
 The theory of quantitative trait loci mapping was first described in 1923 by Sax. It was
noted that seed size in bean (a complex trait) was associated with seed coat colour (a
monogenic trait).
 Thoday in 1961 suggested that if the segregation of simply inherited oligogenes could
be used to detect linked QTLs and it should be possible to map and characterize all the
QTLs involved in the control of complex traits.
 QTL mapping involves testing DNA markers through out the genome for the
likelihood that they are associated with a Quantitative Trait Loci.
 The QTL mapping is explained using the example of RFLP markers.
 Two strains are selected A and B
 Crossed to produce – F1 – selfed – F 2 population.
 The F 2 plants are evaluated for the various RFLP markers and the concerned
Quantitative Traits.
 F 2 plants homozygous for the two alleles (HH / hh) of each RFLP marker are
identified and grouped into two separate classes. The mean values of the two groups
for the Quantitative Trait are now compared a significant difference between the two
groups will reveal a linkage between the RFLP marker and the Quantitative Trait Loci
affecting the concerned Quantitative Trait.

 Let us consider the hypothetical example.

Strain Strain
A B differ for 2 RFLP markers
Protein 4% 8% protein
F1
 F 2 population raised individual plants evaluated for RFLP
markers G and H and for protein content.
 For RFLP marker G the F2 plants are classified into two groups those having G allele
(GG) and g allele (gg) heterozygote are not considered.
 Similarly they are classified for RFLP marker HH and hh
 The mean protein contents of the two groups for each RFLP marker are now
compared.
 A significant difference is detected between the two groups for marker H. This
indicates linkage below RFLP marker H and the Quantitative Trait Loci governing
protein content in rice.
 Hypothetical data on mean protein content of F2 plants from the cross between strains
A and B (rice).
Mean protein content %
RFLP marker Slow moving allele Fast moving allele
G GG 5.2 gg 5.7
H HH 4.9 hh 6.2
Significantly different from the mean protein content of the slow moving group i.e.
from 4.9
 One of the most powerful application of Quantitative Trait Loci mapping is to analyse
gene x gene, gene x environment int eractions, but this requires many large time
consuming experiments.

Molecular Breeding (MAS)

Conventional plant breeding is primarily based on phenotypic selection of superior
individuals among segregating progenies resulting from hybridization. Although significant
strides have been made in crop improvement through phenotypic selections for agronomically
important traits, considerable difficulties are often encountered during this process, primarily
due to genotype – environment interactions. Besides, testing procedures may be many times
difficult, unreliable or expensive due to the nature of the target traits (e.g. abiotic stresses) or
the target environment. Molecular marker-assisted selection, often simply referred to as
marker-assisted selection (MAS) involves selection of plants carrying genomic regions that
are involved in the expression of traits of interest through molecular markers. With the
development and availability of an array of molecular markers and dense molecular genetic
maps in crop plants, MAS has become possible for traits both governed by major genes as
well as quantitative trait loci (QTLs).
If the individual gene(s) or QTLs with significant influence on specific target trait (s)
can be identified based on their linkage to molecular markers, the efficiency of incorporating
the desired traits in elite germplasm could be greatly enhanced.
In general, the success of a marker-based breeding system depends on three main
factors:
(i) A genetic map with an adequate number of uniformly-spaced polymorphic
markers to accurately locate desired QTLs or major gene(s):
(ii) Close linkage between the QTL or a major gene of interest and adjacent markers:
(iii) Adequate recombination between the markers and rest of the genome: and
(iv) Ability to analyze a larger number of plants in a time-and cost-effective manner.
Molecular marker analysis allows to identify genome segments, so-called quantitative
trait loci (QTL), contributing to the genetic variance of a trait and thus to select superior
genotypes at the se loci without uncertainties due to genotype by environment interaction and
experimental error. Selecting for favorable QTL effects based on marker data (marker assisted
selection, MAS) therefore has great potential for improving quantitative traits. In evaluating
the possible impact of MAS it is important to know that in general a quantitative trait is
controlled by quite a large number of genes.
DNA markers are highly reliable selection tools as they are stable, not influenced by
environmental conditions and relatively easy to score in an experienced laboratory.
 Molecular Breeding is used to describe plant breeding programmes that are supported
by the use of DNA – based markers.
 MAS is the breeding strategy in which selection for a gene is based on molecular
markers closely linked to the gene of interest rather than the gene it self, and the
markers are used to monitor the incorporation of the desirable allele from the donor
source.
MAS requires the following Technology.
 Genetic maps, molecular markers linked to agronomic traits, high through put,
automated diagnostic technique.
 The predictive value of molecular markers used in MAS depends on their inherent
repeatability, map position and linkage with economically important quantitative and
qualitative traits.
 The presence of tight linkage between Quantitative Trait and a molecular marker
maybe useful in MAS to increase gain from selection.
 MAS may have potential in population and inbred line development.
 The effective of any MAS will depends on the accuracy of the phenotypic
classification of trait expression and the degree of linkage between the marker(s) and
traits of interest.
DNA Fingerprinting:
Every year in court cases all over the world the ability to establish a person’s identity
is essential for a just decision. Genetics has come to the rescue of the courts and now the
following new questions are routinely asked in the courts: (1) Is the drop of blood found at the
crime scene from suspect on trait? Who is the child’s father? Until recently, there was no
foolproof test. In a criminal case, if there was no identifiable fingerprint left behind at the
crime scene, there was no case. Blood tests can determine who is not the parent, not who is. A
test has now been developed that provides hundred percent positive identification. The test is
called DNA fingerprinting. The test of DNA fingerprinting can show conclusively whether
the genetic material in a drop of blood matches that of the suspect, or it can be used to solve
paternity case.
The technique of DNA fingerprinting relies on developments from recombinant DNA
technology and allows an examination of each individual’s unique genetic blueprint – DNA.
The technique was discovered in England by Alec Jeffreys. It is based on the fact that the
DNA of each individual is interrupted by a series of identical DNA sequences called
repetitive DNA or tandem repeats. The pattern, length, and number of these repeats are
unique for each individual. Jeffreys developed a series of DNA probes, which are short
pieces of DNA that seek out any specific sequence they match, and base pair with that
sequence. Such molecular probes are used to detect the unique repetitive DNA patterns
characteristic of each individual.
The procedure of DNA fingerprinting has the following steps:
1. DNA is purified from a small sample of blood, semen, or other DNA-bearing cells,
and digested into smaller fragments with restriction endonucleases.
2. The fragments are separated by agarose gel electrophoresis.
3. The separated fragments are transferred to a nylon membrane by the technique of
Southern blotting.
4. The DNA probes labeled with radioactive material are added to a solution containing
the nylon membrane.
5. Wherever the probes fit a band containing repetitive DNA sequences, they attach.
6. The X-ray film is pressed against the nylon filter and exposed at bands carrying the
radioactive probes attached to the fragments.
7. The patterns of bands obtained on the film is 100 per cent unique for each person,
except for identical twins who would have the same pattern.
 The forensic application of the DNA fingerprinting technique involves a comparison
between the DNA fingerprint obtained from cells at a crime scene with a DNA fingerprint
from cells provided by the suspect. If the DNA pattern matches exactly, certain identification
is made. For paternity determination, DNA fingerprints of the mother, child and alleged
father are compared. In this case, one -half of the bands in the child comes from the mother
and the other half from the father. All the parental ba nds in child’s DNA fingerprint must
match with the alleged father for positive paternity identification.
In India, DNA fingerprinting tests are carried out at the Centre for Cell and
Molecular Biology (CCMB), Hyderabad. For this purpose, a test with the BKM -DNA probe
(= banded krait minor satellite DNA) earlier used for identification of sex chromosome by
(Dr. Lalji Singh) has been found to cost-one tenth of the cost of tests used in Europe and
U.S.A. Paternity dispute cases are much more common in India and most of them are referred
to CCMB for DNA evidence. The first such test on DNA fingerprinting was used in June,
1989 to settle a drawn-out paternity case in Madras.

Lecture No. 28*

Genetic transformation - Gene transfer methods
Indirect method of gene transfer
Agro-bacterium mediated gene transfer method

The process of transfer, intigration and expression of transgene in the host cells is known
as genetic transformation. Various genetic transfer techniques are grouped into two main
categories.
1) Vector mediated and Indirect gene transfer.
2) Vectorless and Direct gene transfer
Vector mediated and indirect gene transfer.
In this approach the transgene is combined with a vector which takes it to the target
cells for inteigration. The term plant gene vector applies to potential vectors both for transfer
of genetic information between plants and the transfer of genetic information from other
organisms (bactieria fungi and animals) to plants. The vector mediated transfer is strongly
linked to regeneration capabilities of the host plant.
The plant gene vectors being exploited for transfer of genes are plasmids of
Agrobacterium viruses and transposable elements.
Agrobactreium mediated transformation
 The Agrobacterium system was historically the first successful plant transformation
system, marking the break through in plant Genetic engineering in 1983. The Agrobacterium
is naturally occurring gram negative soil bacterium with two common species A Tumifacience
and A rhizogenes There are known as natural gene engineers for their ability to transform
plants. A tumifacience induces tubers called crown galls, where as A rhizogenes causes hairy
root diseases. Large plasmids in these bacteria are called tumer inducing (Ti plasmil) and root
inducing (Ri plasmid) respectively. The Ti plasmid has two major segments of interest in
transformation that is T DNA and virus region. The T DNA region of the Ti plasmid is the
part which is transferred to plant cell and incorporated into nuclear genome of cells. The
transfer of T DNA is mediated by genes in the another region of Ti plasmid called virs genes
(virulence genes). Modified Ti plasmid are constructed that lack of undesirable Ti genes but
contain a foreign gene (resistant to a disease) and a closely linked selectable marker gene
(Eg:- for antibiotic resistance). With in the T DNA region any gene put in T DNA region of
plasmid cysts transferred to the plant genome. The T DNA is generally integrated in low copy
number per cell. Transfer of gene through to wounded plant organs A. tumifacience has
limited range of host. It can infest about 60% gymnosperms and Angiosperm. Hence
Agrobacterium mediated transformation is the method of choice in dicotyledonous plant
species, where plant regeneration system are well established, However, Monocotyledons
could not be successfully utilized for Agrobacterium mediated gene transfer.
Advantages
 It is a natural means of gene transfer
 Agrobacterium is capable of infecting infact plant cells and tissue and organs.
 Agrobacterium is capable of transfer of large fragments of DNA very efficiently
 Integration of T DNA is a relative precise process.
 The stability of gene transferred in excellent.
Limitations
 Host specificity
 Somaclonal variation
 Slow regeneration
 Inability to transfer multiple genes
Lecture No. 28

Genetic transformation - Gene transfer methods – Indirect method of gene

transfer – Agro -bacterium mediated gene transfer method

Genetic transformation of plants or Transgenic Techniques

Genetic engineering over comes the limitations of traditional Breeding and allows
scientists to use new traits from many kinds of plants and other living things such as fish,
insects, bacterium and even humans. If a new gene is introduced to another organisms cell,
the cell begin to produce that genes particular protein and will display the new character and
trait directed by the gene.
Steps for developing new crop varieties:-
Step-I:- Finding a plant or other organism showing the desired characteristic.
Step-II:- Identify the gene controlling that trait and its location on a chromosome (By using
genome mapping)
Step-III:- Marking the gene, so its presence can be detected quickly through “marker
assisted selection”. Specific genes or segments of DNA, called markers are used to mark the
location of the genes to be transferred.
Step-IV:- Isolating the gene from the rest of the DNA so that only the desired information
will be transferred. (By endonuclease enzymes).
Step-V:- Producing large number of the gene and introducing the desired gene into cells of
the organism to be enhanced (Agrobacterium gene transfer or by particle gun method).
Step-7:- Identify which plant cells now contain the desired gene, through the use of markers
as described in step-3
Step-8:- Growing the altered cells into full plants, using a process called tissue culture, to
confirm that the desired trait is expressed properly.
Step-9:- Using traditional plant breeding techniques to transfer the trait into a usable variety.
Transgenic Techniques
The common means of transferring genes into plants is by employing the natural
ability of some bacteria to alter the DNA of organisms, which they infect.
These bacteria have the ability to inject a segment of their DNA into that of an
infected cell, there by affecting its behavior.
For example, the common soil bacterium, A. tumifaciens causes a disease called crown
gall is susceptible plants by inserting its genes into a plants DNA
The bacterial genes disrupt the genetic code of the plants cells in the infected area
causing them to grow into an abnormally shaped mass called a gall.
 The bacterial species produce small, circular pieces of DNA called plasmids, which
function independently of other Bacterial DNA and can enter cells of other organisms,
carrying genetic information with them that then merges with that of the host cell.
To transfer genes from one cell to another, researchers use an enzyme to cut a gap in a
bacterial plasmid then they insert a gene from the donor DNA strand in to the plasmid.
Because the cut ends of both the plasmid and the donor gene segment are chemically ‘sticky’
they attach to each other or recombine to form a plasmid containing the new gene (hence the
name, recombinant DNA (rDNA) Technology). The recombined plasmid now carries the gene
that can result in the expression of the new trait when transferred into a plant cell by the
plasmid. Once a gene is inserted into a plasmid, it can be introduced into a bacterium that is
easily reproduced in the lab when the altered bacteria multiply, the plasmid containing the
desired gene is also copied or cloned in every cell. Because bacteria grow quickly, this
method makes it possible to make millions of copies of the desired gene in a short period.
Using the appropriate enzymes, the plant genes are extracted again from the bacteria
and inserted into the plasmids of A. tumefaciens. Finally, the altered A. tumefaciens is mixed
with the target plant cells. The foreign gene is then incorporated into DNA of some of the
target plant cells.

Lecture No.29*
Direct methods of transformation – Particle bombardment / gene gun
method – Electroporation, Microinjection, Macroinjection, Liposome,
Mediated etc.

Introduction of DNA into plant cells without the involvement of biological agents
such as Agrobacterium and leading to stable transformation is called direct gene transfer.
The various methods of direct gene transfers are
1) Chemical methods
2) Electroporation
3) Particle bombardment
4) Lypofection
5) Micro injection
6) Macro injection
7) Pollen transformation
8) Delivery via growing pollen tubes
9) Laser induced transformation
 10) Fibre mediated transformation etc
Some of these like involve lipofection, electroporation delivery of DNA to protoplast
and thus require regeneration of plants from transformed protoplast

1) Chemical methods:-
It is based on ability of protoplast to uptake the foreign DNA from surrounding
solution. An isolated plasmid DNA is mixed with protoplast in the presence of the poly
ethylene glycol (PEG), PVA and Ca (PO4) polyvinyl alcohol which enhance the uptake of
DNA by protoplast. After 15-20 min of incubation the protoplasts are cultured. On the
presence of appropriate selective agents, the protoplast are regenerated and the transgenic
plants are further characterized for conformation.
2) Electroporaton
Induction of DNA into cell by exposing them for a very brief period to high voltage
electrical pulses to induce transiant pores in the plasma lemma is called Electroporation.
Generally protoplasts are used since they have expand plasma membrane. A
suspension of protoplast with a desired DNA is prepared. Then a high voltage current is
applied through the protoplast DNA suspension.

The electric current leads to the formation of small temporary holes in the membrane
of the protoplasts through which the DNA can pass. After entry into the cell, the Foreign
DNA gets incorporated with the host genome, resulting the genetic transformation The
protoplasts are then cultured to regenerate in to whole plants. This method can be used in
those crop species in which regeneration from protoplast is pos sible
3) Particle bambordment / microprojectile / biolistic / gene gun / particle acceleration
The process of partical acceleration (or) biolistics acceleration of DNA into cells with
sufficient force such that a part of it gets integrated in to DNA of target cells. The process of
transformation employes foreign DNA coated with minute 0.2-0.7 µm gold (or) are tungstun
particles to deliver into target plant cells. Two procedures have been used to accelerate the
minute particles
 By using pressurized helium gas
 By electro static energy released by a droplet of water exposed to a high voltage
This method is being widely used because of its ability to deliver foreign DNA into
regenerable cells, tissue (or) organs irrespective of monocots (or) dicots
 Because of the physical nature of process there is no biological limitation to the active
DNA delivery that makes it, genotype independent. This method allows the transport of genes
into many cells of nearly any desired position in an experimental system without too much
manual labour.
The method was first used by Klein et al. in 1987, and Sanford et al 1987.
4) Lypofection
Introduction of DNA into cells via lyposomes is known as lipofection, lioposomes are
small lipid artificial vesicles. The procedure of liposome encapsulation was developed to
protect the foreign DNA during the transfer process
The DNA enclosed in the lipid vesicles when mixed with protoplast under appropriate
condition penetrates into the protoplast where lipase activity of the protoplast dissolves the
lapid vesicles and DNA gets released for integration into the host genome. This method has
not been commonly used as it is difficult to construct the lipid vesicles. The success depends
upon the protoplast regeneration
5) Microinjection
The DNA solution is injected directly inside the cell using capillary glass micropipetts
with the help of micromanipulators of a microinjection assembly. It is easier to use protoplast
than cells since cell wall interferes with the process of microinjection. The protoplast are
usually immobilized in agarose (or) on a glass slides coated with polylysine or by holding
them under suction by a micropipette. The process of microinjection is technically demanding
and time consuming a maximum of 40-50 protoplasts can be microinjected in one hour
6) Macroinjection
The injection of plasmid DNA into the lumen of developing inflorescence using
hypodermic syrange is known as macro injection
An aqueous solution of DNA was introduced into the developing floral tillers 14 days
prior to meiosis. Transformed seeds were obtained from these injected tillers after cross
pollination with other and injected tillers. However the mechanism by which the DNA entered
the zygotic tissue yet unknown.
7) Pollen transformation
Involves the gene transfer by soaking the pollengrains in DNA solution prior to their
use for pollination. The method is highly attractive in view of its simplicity and general
applicability but so far there is no definite evidence for a transgene being transferred by
pollen soaked in DNA solution
8) DNA Delivary via growing pollen tubes
 The stigmas were cut after pollination exposing the pollentubes, the DNA was
introduced onto the cut surface that presumably diffused through the germinating pollentube
into the ovule. This method is simple easy and very promising provided consistent result and
stable transformations are achieved The mechanism of DNA transfer into zygote through this
method is not yet established.
9) Laser induced transformation
It is method of introducing DNA into plant cells with a laser microbeam. Small pores
in the membrane are created by laser microbeam. The DNA from the surrounding solution
may than enter into the cell cytoplasm through the small pores
Lasers have been used to deliver DNA into plant cells But there is no information on
transient expression or stable integration.
10) Fibre Mediated transformation
The DNA is delivered into the cell cytoplasm and nucleus by silicon carbide fibres of
0.6 µm diameter and 10-80 µm length. The fibres mediated delivery of DNA into the
cytoplasm is similar to microinjection. The method was successful with maize and tobacco
suspension cell culture
It is the most rapid and expensive method of DNA delivary provided stable
integrations are achieved
DNA imbition by cells tissue , embryos and seeds
When dry isolated embryos of wheat barly, rye, pea and bean are imbibed in a DNA
solution, they take up the DNA and show the expression of marker gene. Dry seeds, whose
seed coats have been removed also take up DNA when imbibed in a DNA solution. The
imbibed seeds or embryos are germinated on appropriate selective medium to isolate the
transformed embryos. It was throught that the DNA is taken up by the embryos through the
cells injured during their isolation. The DNA then moves through plasmadesmata to other
cells of embryos. Recently two DNA delivary systems Namely 1) Agrobacterium mediated
gene transfer and 2) particle bombardment are widely used for the development of transgenic
embryos.

Lecture No. 30*

Transgenic plants – Applications in crop improvement – Limitations

Transfer of gene from an organism into a plant cell and its integration into the genetic
material of the later usually employing recombinant-DNA technique is known as Genetic
engineering of plants It is the most potent biological approach which constitute the transfer of
specifically constructed gene assembles through various transformation techniques. The
plants obtained through genetic engineering contain a gene (or) genes usually from an
unrelated organisms. Such genes are called transgenes and plants containing transgenes are
known as transgenic plants.
The first transgenic plant was produced in 1983 when a tobacco line expressing
Kanamycin resistant was produced. Soon after transgenic crop varieties resistant to
herbicides, insects (or) viruses (or) expressing male sterility, delayed ripening, and slow fruit
softening were developed.
Flavr savr tomato was first transgenic variety to reach market. Fruits of this variety
remain fresh for a prolonged period.

Role of transgenic plants in crop improvement:-

Transgenic plants are those which carry additional stability integrated and expressed
foreign genes, usually transferred from unrelated organisms. The combined use of
recombinant-DNA technology, gene transfer methods and tissue culture technique has lead to
efficient productions of transgenics in a variety of crop plants. Transgenic plants or
genetically modified plants (GMP) have both basic and applied uses which are briefly
summerised below
1) Transgenes will be important in increasing the efficiency of crop production systems.
For instance tragenic plants resistant to herbicides, insects, viruses and other biotic and
abiotic stress have already been produced.
2) Transgenic breeding is an effective means of inducing male sterility in crop plants . Eg:-
Barnase and barstar systems in Brassica napus
3) Transgenic plants which are stable for food processing have also been produced Eg:-
Bruise resistant and delay ripening in tomato
4) Several gene transfers have been aimed in improving the produce quality. Eg:- Protein
and lipid quality : improved quality may be achieved by either supression or over
production of endogenous genes.
5) Transgenic plants are aimed to produce novel biochemicals like interferon, Insulin,
Immunoglobin etc useful biopolymers like poly hydroxy butyrate which are not
produced by normal plants. These compounds are extracted from plants can be used as
pharmaceutical (or) industrial substrates
 6) The utilization of transgenic plants as bioreacters (or) factories for the production of
special chemical and pharmaceuticals is known as Molecular forming.
7) Transgenic plants have been produced that express a gene encoding antigenic protein
from a pathogen. Use of transgenic plants as vaccines for immunization against
pathogens fast emerging as an important objective.
8) Transgenic plants have proved to be extremely valuable tools in studies on plant
molecular biology. Regulation of gene action, identification of regulatory, promoters
sequences etc,
Limitations:
1. Transgenic plants some times exhibit instable performance for character under
consideration
2. The transformants had the undesirable side effects of trans genes
3. The position and integration of foreign gene in host genome effects the expression of
transgene in the transformant
4. Inability to transfer polygenic traits
5. Transgenic breeding is a very expensive method of crop improvement
6. Requires high technical skills.
7. Transgenic cells are recovered at a very low frequency in cell culture, more over
regene ration of transgeniccells into whole plants is also difficult and time consuming
task.
8. Transgenic breeding acts against natural evolution.
9. There are chances of developing new weed species through transgenic breeding
10. Sometimes the foreign genes has adverse effects on genome of the recipient parent, in
such cases it may give rise useless gene combinants which may become a problem.

Lecture No. 31*

Engineering for insect, herbicide and disease resistance
The development of transgenic plants is the result of an integrated application of rDNA
technology, gene transfer methods and tissue culture techniques. Remarkable achievements
have been made in the production, characterization and field evaluation of transgenic plants in
several field, fruit and forest plant s pecies and the available statistics reveal that the transgenic
varieties of soybean occupy major global area (more than half) under cultivation of transgenic
corps. The research and developmental efforts on genetic transformation of crop plants has
been targeted towards the generation of transgenic resistant plants for insects herbicides and
diseases etc. transgenic plants have also been developed that possess some novel features or
show modification of one or the other quality trait..
Engineering insect resistance
The genetic Engineering has received maximum attention for the development of
resistance to insects and herbicides. Several insect resistant transgenic varieties have been
tested and released in crops like tomato, potato, cotton, and maize. Two approaches have
been adopted to develop insect resistance transgenic plants by transferring insect control –
protein genes.
1) Introduction of resistant genes from micro organisms
2) Introduction of resistant genes from higher plants
1) Introduction of resistant genes from micro organisms
The cry gene of Bacillus thuringenesis (Bt) produces a protein, which forms
crystalline inclusions in the bacterial spores. These crystal proteins are responsible for the
insecticidal activities of the bacterial strains. On the basis of their insecticidal activity the cry
proteins are classified into four main groups or ranks viz., cry I, cry II, cry III and cry IV cry
protein is a parasporal inclusion (crystal) protein from Bacillus thuringenesis (Bt) that
exhibits heamolytic activity. These proteins are solubilized in the alkaline environment of
insect midget and are proteolytically processed to yield toxic core fragment. The toxic
function is localized in the N -terminal half of the protein, the C-terminal half of these prote ins
is highly conserved and involved in the crystal formation
The cry I proteins are insecticidal to lepidopteron insects.
The cry II A proteins are insecticidal to Lepidopteron and Diptera
The cry III B proteins are insecticidal to Diptera
The cry III proteins are insecticidal to coleopteran
The cry IV proteins are insecticidal to Diptera
The cry V & VI. proteins are insecticidal to nematodes
Toxic action of cry proteins
When cry proteins are ingested by insects they are dissolved in the alka line juices
present in the midget lumen, the gut proteases process them hydrolytically to release the core
toxic fragments
The Bt a lepidopteron specific gene from Bacillus thuringenesis sub sp
kurstaki has been widely and successfully used in tobacco, tomato, potato, cotton, rice and
maize for developing resistance against several lepidopteran insect pests. A lowered level of
expression of Bt gene in plants was experienced that had been intensified through the choice
of suitable promoters or by modifying the coding region of Bt gene. Gene promotor activity
has been accomplished by using two copies of the constitutive 35s promotor from cauliflower
mosaic virus (CaMV). Use of redesigned synthetic Bt gene with increased Guanine, cytosine
content has also been used in some of these crops and in several instances that synthetic
version exhibited upto 500 folds increase in the expression. Several insect resistant transgenic
varieties like Bollgard of cotton maximizer and yield gard of maize, new leaf of potato have
been released in USA. Bt strain contained a great diversity of delta endotoxin coding genes,
each of which has a specific activity spectrum for example Cry 1Ab protein is highly toxic
against European corn borer and is used in Bt corn hybrids. The cry 1Ac protein is highly
toxic to both tobacco bud warm and cotton bollwarm larvae and is expressed in Bt cotton
varieties.
While the Cry 3A protein is expressed in Bt potato varieties and provides protection
against colorado potato beetles.
2) Introduction of resistance genes from higher plants
Several insecticidal proteins of plant origin such as protease in inhibitors, amylase
inhibitors, and lectins can retard insect growth and development when injected in high doses.
Proteiase inhitiors
The protease inhibitors deprive the insects by interfering with the digestive enzymes
of the insects. Eg:- Cowpea trypsin inhibitor (CpTi) gene found in cowpea (V.unguiculata )
produces antimetabolite substances that provide protection against the major storage pest
Bruchid beetle (callosobruchus maculatus)
? - amylase inhibitors
The ? -amylase inhibitor gene (?-A1-Pv) isolated from p vulgaris has been expressed
in tobacco. This ? - amylase inhibitor protein blocks the larval feeding in the mid gut. The
larva generally secreates a gut enzymes called ?-amyla se that digest the starch, By adding a
protein that inhibits insect gut ? -amylase the insect can be starved and it dies.
Lectins
These are plant glycoproteins used as insect toxins. Lectin from snowdrop (Galanthus
n ivalis) is known as GNA because it has shown the activity against aphids.
Engineering plants for herbicide resistance
The use of herbicides to control weeds play a pivotal role in modern Agriculture. In
general, herbicides inhibit either photosynthesis or the biosynthesis of an essential
aminoacids. More progress has been achieved in herbicide resistance as single genes govern
the resistance.
 Following are the two approaches to produce herbicide resistant transgenic plants.
Detoxification
Transfer of gene whose enzyme product detoxifies the herbicide. In this approach the
introduced gene produces the enzymes which degrades the herbicide sprayed on the plant.
Introduction of bar gene cloned from bacteria, streptomyces hygrosopicus into plants makes
them resistant to the herbicide based on phosphinothricin (ppt). The ppt herbicide kills the
plants by inhibiting glutamine synthase. The bar gene produces an enzymes phosphinothricin
acety (transferase (pat) which degrades phosphinothricin into a non toxic acetylated form.
Plants engineered with bar gene were found to grow in ppt at levels 4-10 times higher than
normal field application. Like wise bxn gene from klebsoiella ozanea which produces nitrilase
enzyme imports resistance to plants against the herbicide bromoxynil. Bromaxynil effects
photosys tem II. A transgenic cotton variety, bxn cotton resistant to bromoxynil has been
released in USA.
Streptomyces

Bargene isolated (it codes for phosphinothricin acetyl transferase PAT)

Produces enzyme
PAT Phosphinothricin into Non toxic acetylated form
Detoxifies Plants resistant

Target modification
Transfer of a gene whose enzyme product becomes insensitive to herbicide. In this
approach a mutated gene is introduced and produces modified enzyme in the plant that is not
recognized by herbicide as a consequence of which, it cannot kill the plant. For instance a
mutant aroA gene from bacteria salmonella typhimurium has been used for developing
tolerance to herbicide glyphosate. The target site of glyphosate is a chloroplast enzyme 5-enol
pyruvylsikimic acid 3-phosphate synthase (EPSPS) inhibiting the synthesis of aromatic amino
acids. Introduction of mutant aroA gene produces modified EPSPS, not recognizable to
glyphosate. “Round up ready” soyabean and Round up ready cotton varieties resistance to
glyphosate have been released.
Like wise sulphonylurea, imidazolinones herbicides inhibits acetolactate synthase
(ALS) chloroplast protein. Which inhibits the synthesis of leucine, lysine and valine tolerance
to these herbicides has been achieved by engineering the expression of the mutant gene ALS
derived plants. (Aceto lactate synthase)
Engineering plants against disease resistance
Virus resistance: The disease resistance generated by employing pathogen genes is referred
to as “PATHOGEN DERIVED RESISTANCE (PDR)” has been realized in the cases of
bacterial and fungal diseases
i) Coat protein Mediated resistance (CP-MR):
Introduction of viral coat protein gene into plants makes it resistant to virus from which
gene and the coat protein was derived. If susceptible strain of a crop is inoculated with a mild
strain of a virus, there the susceptible strain develops resistance against more virulent strain.
Powel – Abel et al (1986) first demonstrated that transgenic tobacco expressing TMV coat
protein showed resistance similar to that occurring in viral – mediated cross protection.
The accumulation of their protein in unaffected cells results in effective resistance by
uncoating the virus particles before translation and replication. It was first reported by
Dr I. Demonstarted in TMV in tobacco.
ii) Satellite RNA mediated Resistance:
Some RNA viruses have small RNA molecules (300 nucleotides) called satellite
RNA. Satellite RNAs are RNA molecules that are dependent up on helper virus for its
replication and transmission, even though they are unrelated to viral genome. The presence of
SAT RNA leads to reduction in severity of disease symptoms and thus have been used to
develop resistance against specific viruses. Eg: Engineering cucumber using cucumber mosaic
virus. CMV, Satellite RNA leads to transgenic plant resistant to CMV.

Fungal Resistance
GE for fungal resistance has been limited
i) Antifungal Protein Mediated Resistance: Introduction of two genes coding for
chitinase and glucanase makes the plant resistant to fungal infection by degrading
the major constituents of fungal cell wall. (chiten and ß -1,3 – glucan). Co
expression of chintinase and glucanase genes in tobacco and tomato plants confers
higher level of resistance than either gene alone.
ii) Antifungal Compound Mediated Resistance: The low molecule weight
compounds such as Phytoalexins possess antimicrobial properties and play an
important role in plant resistance to fungal and bacterial pathogens. Expression of
stilbene synthase gene from grapevine in tobacco resulted in the production of new
phytoalexin (resveratrol) and enhanced resistance to infection by Botrytis cinerea
and blast resistance in rice. Active oxygen species (AOS) including hydrogen
 peroxide also play important role in plant defence responses to pathogen infection.
Transgenic potato plants expressing an H 2O 2 generating fungal gene for glucose
oxidase were found to have elevated levels of H2O2 and enhanced levels of
resistance both to fungal and bacterial pathogens particularly to Verticillium wilt.
The introduction of H2O2 gene has also improved fungal resistance in rice.
iii) Bacterial Resistance: The expression of a bacterio phage T 4 lysozyme in
transgenic potato tubers led to increased resistance in Erwinia carotovora. the
expression of Barley ? thionin gene significantly enhanced the resistance of
transgenic tobacco to bacteria pscudomonas syringa e

A list of some insect herbicide and virus resistant transgenic varieties approved for
commercial cultivation in U.S.A.

Crop Trait improved by trangene Transgenic Year of Company

variety approval
Cotton Cry gene incorporated plants (resistant to Bollgard* 1995 Monsanto
cotton bollworm, tobacco budworm and
pink bollworm)
Resistance to bromoxynil (Buctril) B X N* 1995 Calgene
Resistant to glyphosate (herbicide Roundup) Roundup 1996 Monsanto
Ready*
Resistant to sulphonoyl area ----- 1996 Dupont
Maize Cry gene incorporated (resistant to corn Maximizer* 1995 Ciba Geigy
borer)
Resistant to glyfosinate -ammonium --- 1996 Decalb
Resistant to glufosinate -ammonium Liberty Link* 1996 Agro-Evo
Cry gene incorporated (resistant to corn Yield Gard* 1996 Monsanto
borer)
Resistant to sethoxydin herbicide (poast) SR* - -
Resistant to imidozolinone herbicides IMI* - -
Cry gene incorporated (resistant to corn - 1996 Northrup King
borer)
Potato Cry gene incorporated (resistant to New Leaf* 1995 Monsanto
Colorado potato beetle)
Insect resistant by cry gene - 1996 Monsanto
Rapeseed / Altered oil composition (high lauric acid) Laurical 1995 Calgene
canola
Soybean Resistant to glyphosate (herbicide) Roundup 1995 Monsanto
Ready
Resistant to glyfosinate – ammonium Liberty Link* - -
(herbicide Liberty)
Resistant to sulphonyl urea herbicides STS* - -
Squarh Resistant to viruses Freedom* 1995 Asgrow

Lecture No. 32 Engineering For Male Sterility, Flavr – Savr Tomato