How recombinant DNA technology is developed, history, gene cloning, gene transfer methods, its application and drawbacks in plants

2. INTRODUCTION
 DNA is very large molecule made up of small units
called nucleotides which contain sugar(ribose),
phosphate molecule and nitrogenous base.
 The process of introducing a foreign piece of DNA
into the genome of another organism, which
contains our gene of interest.
 This gene, which is introduced is the recombinant
gene and the technique is called the recombinant
DNA technology.
 Recombinant DNA molecules are sometimes
called chimeric DNA.

3. HISTORY
 The idea of recombinant DNA was first proposed
by Peter Lobban of Stanford University Medical
School.
 The first recombinant DNA (rDNA) molecules
were generated in 1973 by Paul Berg,
HerbertBoyer, Annie Chang, and Stanley Cohen
of Stanford University and University of California
San Francisco.
 The first licensed drug generated using
recombinant DNA technology was human insulin
and the first GM food was the Flavr Savr tomato
in 1994.

4. STEPS FOR RECOMBINANT
DNA
 Selection of the desired gene.
 Cutting the desired DNA by restriction
enzymes.
 Selection of the perfect vector.
 Inserting the genes into the vectors.
 Recombinant DNA formed.
 Obtaining the products of recombinant genes.

5. RESTRICTION
ENDONUCLEASES
 Enzymes for the manipulation of DNA.
 Are bacterial enzymes that cut/split DNA at
specific sites called as restriction site.
 Produce sticky as well as blunt ends in
the DNA sequence that will help it bind
specifically to the desired gene.
 Three types of restriction endonuclease-
 Type 1, Type 2,Type 3.

6. •RESTRICTION
ENDONUCLEASES
 When RES acts at the centre of the symmetry, two
complimentary strands of DNA of equal length are
produced, this forms blunt or non-overlapping ends.
 Some RES breaks the DNA on either side of the centre
of the symmetry which forms unequal fragments which
are called as sticky or overlapping ends

7. TYPES OF
RESTRICTIONENDONUCLEASE
S
TYPE 1 TYPE 2 TYPE 3
CLEAVAGE SITE Cut DNA on both
strands at non
specific
location(around at
1000 bp away
from the particular
sequence that is
recognized by
restriction enzyme
Cut DNA on both
strands with
particular
sequence
recognized by
restriction enzyme
Cut DNA on both
strands at 24-
26bp away from
recognition site.
RECOGNITION
SITE
Asymmetric 4-6bp sequence,
Palindromes
5-7bp sequence,
Asymmetric

8. RECOMBINANT DNA
FORMATION
 Every DNA has got number of restriction site
for a number of restriction enzymes.
 DNA fragments produced by restriction
enzymes are known as restriction fragments
 Recombinant DNA can be formed by joining
restriction fragments obtained from different
sources.
 It will have base sequence different from that
of original DNA.

9. AMPLIFICATION
 Amplification is required to make multiple
copies of the recombinant DNA.
 It can be done :
Either by cloning the DNA
Or by polymerase chain reaction

10. GENE CLONING
 Gene is a stretch of DNA that holds the
information to build and maintain organism’s
cells and pass on genetic traits to offspring.
 Gene cloning is the production of exact copy of
a particular gene or DNA sequence using
genetic engineering techniques.
 A vector is required to transfer recombinant
DNA into the cells

11. STEPS OF GENE ClONING
 STEP 1
• The DNA containing the target gene(s) is split
into fragments using restriction enzymes.
• A fragment of that DNA, containing the gene to
be cloned is inserted into the circular DNA
molecule called a vector, to produce recombinant
DNA.
 STEP 2
• The vector act as a vehicle that transport the
gene into a host cell . This process is called
transformation.

12. •STEPS OF GENE ClONING
 STEP 3
• Within the host, vector multiplies producing
numerous identical copies not only of itself
but also of gene it carries.

13. •STEPS OF GENE ClONING
 STEP 4
• When the host cell
divides, copies of
rDNA molecule are
passed to progeny
and further vector
replication takes
place.

14. •STEPS OF GENE CONING
 STEP 5
• After large no. of cell divisions, a copy of
identical host cells is produce. Each cell in the
clone contains one or more copies of the rDNA
molecule.
 STEP 6
• Then, the host cells are lysed and rDNA can
be seperated.

15. VECTORS
 Vectors-They are considered as the final
vehicles that carry genes of interest into the
host organism.
VECTORS
PLASMIDS
COSMIDS
BACTERIOPHAG
ES

16. CHARACTE
RS
PLASMID BACTERIOPHA
GE
COSMID
Extra
chromosomal
and Self
replicating
DNA
molecules.
Viruses that
replicate within
bacteria.
Possess both the characters
of plasmid and
bacteriophage
Double
stranded
Circular
Can take up
more larger DNA
segments than
Plasmid
Carry larger DNA fragments
than plasmid

17. WORKING OF PLASMID

18. WORKING OF
BACTERIOPHAGE

19. •LYTIC CYCLE OF
BACTERIOPHAGE

20. •LYSOGENIC CYCLE OF
BACTERIOPHAGE

21. WORKING OF A COSMID
 Cosmids are hybrid of plasmids and lambda
phages.

22. OTHER VECTORS
 Ti and Ri plasmids for higher plants.
 Lambda phage vectors.
 Plant viruses as vectors (Cauliflower
Mosaic Virus, Tobacco Mosaic Virus,
Gemini Virus).

23. POLYMERASE CHAIN
REACTION
 Another way of making many
copies of a specific section of
DNA, without the need for vectors
or host cells, is through a
polymerase chain reaction
(PCR).
 It involves repetition of 3 steps
1. Denaturation, which separates
the two nucleotide strands of
the DNA molecule
2. Primer annealing, in which the
primers bind to the single-
stranded DNA
3. Extension, in which
nucleotides are added to the
primers - in the 5' to 3' direction
- to form a double-stranded
copy of the target DNA.

24. GENE TRANSFER
METHODS
CHEMICAL PHYSICAL
BIOLOGICAL
Calcium
phosphate
DEAE-
dextran
Lipofection
Electroporati
on
MICROPROJEC
TILE
Particle
bombardmen
t
Virus
mediated
Agrobacterium
mediated

25.  AGROBACTERIUM-
MEDIATED TRANSFER
 Agrobacteria is a soil borne, Gram -Negative bacteria.
 It is rod shaped, motile and belongs to bacterial family
Rhizobiacea.
 Agrobacteria species:
• tumefaciens- causes crown galls
on many dicots
• rhizogenes- hairy root disease
 Large plasmids were found in A. tumefaciens called
tumor-inducing or Ti plasmids which are large (200-kb),
conjugative and around10% of plasmid transferred to
plant cell after infection.

26. •T-DNA
 It was observed that the disease in host plant was
caused by the transfer of a DNA segment from the
bacterium to plant nuclear genome.
 The DNA fragment is called as T-DNA and is a part of Ti
plasmid of A.tumefaciens.
 Increased levels of these hormones stimulate cell
division and explains uncontrolled growth of tumor.

27. •PROCESS

28. VIRUS-MEDIATED
TRANSFER
 In virus mediated gene delivery DNA is injected
inside a host cell and it offers advantage of the
virus' own ability to replicate and implement their
own genetic material.
 They are more likely to induce an immune
response.
 DNA based:
• Cauliflower Mosaic Virus
• Gemini Virus
 RNA BASED
• Tobacco Mosaic Virus

29. CHEMICAL METHODS
 Chemical methods of gene transfer can use
natural or synthetic compounds to form
particles that facilitate the transfer of genes
into cells.
 These synthetic complexes either interact with
the cell membrane and promote uptake by
endocytosis, or fuse with the membrane and
deliver the DNA directly into the cytoplasm.

30. CALCIUM-PHOSPHATE
TRANSFECTION
 The principle of the technique is that DNA in a
buffered phosphate solution is mixed gently
with calcium chloride, which causes the
formation of a fine DNA-calcium phosphate
coprecipitate.
 The precipitate settles onto the cells and some
of the particles are taken up by endocytosis.
 The reagents are inexpensive.

32. DEAE-dextran
 It is a soluble polycationic carbohydrate that
forms aggregates with DNA through
electrostatic interactions.
 It provides the entire complex with a net
positive charge, which allows it to interact with
the negatively charged cell membrane and
promotes uptake by endocytosis.
 The reagents are inexpensive and the
procedure is simple and efficient
 Less DNA can be used in each experiment.

33. LIPOFECTION
 Liposomes are sphere of lipids used to
transport molecules into cell.
 They are artificial vesicles which can act as
delivery agents for exogenous material such
as transgene.
 They are hydrophobic, phospholipid vesicles.
Such vesicles when mixed with cells in culture,
fuse with the cell membrane and deliver DNA
directly into the cytoplasm.
 Liposomes have low toxicity and long term
stability.

35. PHYSICAL METHODS
 The DNA is delivered directly into either the
cytoplasm or the nucleus using some kind of
physical force.
 There is no requirement for interaction with the
plasma membrane.
 These methods are usually expensive as they
employ some sort of apparatus, which is
required to administer the physical force.

36. ELECTROPORATION
 Introduction of DNA into plant cell by exposing
them for very brief periods into high voltage
electric pulses which induce transient pores in
the plasma lemma for up to 30 minutes, allowing
the uptake of free DNA from the surrounding
medium.
 Two systems-
1. Low voltage and long pulse method:300-
400V/cm for 10-15 milliseconds
2. High voltage and short pulse method:1000-
1500V/cm for 10 micro seconds (more stable)

37. •ELECTROPORATION
MECHANISM
Electroporati
on
Mechanism
of a cell
membrane:
(a) At zero
potential, (b)
Osmotic
imbalance,
(c) Swelling,
(d)
Membrane
ruptures.

38. MICROINJECTION
 The direct microinjection of intact DNA into
the cytoplasm or nuclei of cultured cells.
 Very small quantities of DNA are sufficient.
 This method is performed under specialised
optical microscope setup called as
micromanipulator.
 Shearing can also occur in the delivery
needle, and large DNA fragments are often
protected by suspension in a high salt buffer
and/or mixing with polyamines and other
protective agents.
 The procedure is expensive, time
consuming and requires skilled labour.
 Example-tobacco and alfalfa etc.

39.  PARTICLE BOMBARDMENT
 Particle bombardment uses accelerated micro
projectiles to deliver DNA into intact tissues or
cells.
 It involves coating micrometer-sized gold or
tungsten particles with DNA and then
accelerating them into cells or tissues.
 DNA can be delivered to deep cells in tissue
slices, and the depth of penetration can be
adjusted by changing the applied force.
 The gene gun is a device that fires DNA into
target cells. Type of guns used: Helium

40. •PARTICLE BOMBARDMENT
MECHANISM
DNA is
bound to
the micro
projectiles
which
impact the
tissue or
the
immobilise
d cells at
high
speed.

41. SCREENING OF
TRANSGENE
 The presence of transgene or gene of
interest is detected by several methods.
1. Northern Blot Technique
2. Southern Blot Technique
3. Western Blot Technique

43. RECOMBINANT DNA
TECHNOLOGY
• VACCINES
• GROWTH
HORMONES
• ANTIBODIES
• VECTORS
THERAPEUTIC
PRODUCTS
• FRUITS
• GM vegetables
• GM crops
• GM animals
GENETICALLY
MODIFIED
PRODUCTS
• Bioethanol
• Biohydrogen
• Biomethanol
• Biobutanol
ENERGY
APPLICATION

44. GENETICALLY MODIFIED
PLANTS
Crop plants that have been modified in laboratories to
enhance traits such as resistance to pesticides or
improved nutritional content.

45. APPLICATIONS OF
GENETICALLY MODIFIED PLANTS
 Stress tolerance: Tolerance to high and low
temperatures, salinity, and drought
 Pest and Disease resistance: For example, American
chestnut trees resistant to fungal blight
 Biofuels: Plants with altered cell wall composition for
more efficient conversion to ethanol
 Phytoremediation: Plants that extract and
concentrate contaminants like heavy metals from
polluted sites.
 Nutritional enhancement: Higher vitamin content,
enhanced nutrient composition and food quality
 Crop production: increased crop yields, reduced
costs for food or drug production, greater food security

46. •APPLICATIONS
 Crops that mature faster and tolerate
aluminum, boron, salt, drought, frost, and other
environmental stressors, allowing plants to
grow in conditions where they might not
otherwise flourish.
 EXAMPLE:
 Herbicide tolerance (Soybean)
 Insect resistance (Corn)
 Altered fatty acid composition (Canola)
 Virus resistance (Plum)

48. HERBICIDE TOLERANCE
 Herbicides mainly affect processes like
photosynthesis or biosynthesis of essential
amino acids.
 By these 2 methods, we can make Herbicide
resistant plants:
1. Introducing a pathway that will detoxify the
herbicide.
2. Either the target molecule should become
insensitive to herbicide or target protein
should be overproduced.

49. A. Detoxification of Herbicide
 Detoxification of Herbicide means that the herbicide
will kill the weeds and not the crops.
 Transgenic tomato plant with bxn gene from Klebsiella
and bar gene from Streptomyces; oil seed rape
(Brassica napus) and sugar beet using bar gene from
Streptomyces are found to be herbicide resistant.
1.GST (glutathione-S transferase) detoxifies herbicide atrazine in
maize.
2.Nitrilase coded by Klebsiella pneumoniae detoxifies bromoxynil.

50. B. Roundup Ready® crops
 Crops genetically engineered to be
glyphosate-tolerant are called Roundup
Ready crops.
 Glyphosate inactivates a key enzyme
involved in amino acid synthesis that is
present in all green plants; molecules
the plants need to make protein.
 It is an effective broad spectrum
herbicide against nearly all weeds.
 By introducing a gene(EPSPS) into the
plant cells of petunia scientists have
been able to produce plants that can
survive herbicide intolerance.
 This enables farmers to apply
glyphosate as a weed-killer without
worrying about the effect on their crop.

51. Insect Resistance
 Crop varieties can be engineered to express a
bacterial gene that controls certain insect pests,
reducing the need for harmful synthetic pesticides.
 The potentially useful gene is isolated, using a
naturally occurring enzyme, in one plant.
 It is then transferred into the cell of a desirable
crop, by using a naturally occurring bacterium that
infects plants.
 Those with the new gene are identified by means
of a marker.
 The transformed cells are then cultured and
grown into whole plants and tested in
greenhouses to ensure that the transferred gene
functions properly.

52. EXAMPLE
 Insect-resistant crops contain genes from the soil
bacterium Bacillus thuringiensis (Bt)
 It is a safe and effective insecticide used in
farming.
 Insertion of this toxin-producing gene from
bacteria into the DNA of corn and cotton makes
them resistant to some insects and protects them
from disease.
 These crops require less spraying of the
insecticide, as the plants produce the toxin to kill
the insects themselves.
 It protects plants from caterpillars or moths such
as cotton bollworms, also the control of
stemborers for maize and rice.

53. Viral Resistance
 Resistance to viruses
can be achieved by
transferring to plants
certain viral genes that
interfere with the
normal replication of
the virus thereby
inhibiting the spread of
infection.
 The papayas plants
grown in Hawaii are
resistant to attack by
some viruses.
 Example: rice stripe
virus, alfalfa mosaic
virus, and potato leaf

54. MALE STERILITY AND
FERTILITY RESTORATION
 Production of male sterile
(barnase gene) in Brassica
napus
 TA29 gene from
tobacco(anther specific
promoter) and barnase
gene(ribonuclese) from
Bacillus amyloliquefaciens
were taken for production of
transgenic plants in B. napus.
 Normal pollen development
stopped in plant due to
barnase gene product as it is
cytotoxic, killing the tapetal
cells, preventing pollen
development and leading to
 Fertility restorer (barster
gene) in Brassica napus.
 TA29 gene from
tobacco(anther specific
promoter) and barster
gene from Bacillus
amyloliquefaciens were
taken for production of
transgenic plants in B.
napus.
 The product of barstar
gene is ribonuclease-
inhibitor and forms
complex with ribonuclease.
By this it neutraliezes its
cytotoxic properties and
fertility is restored.

55. FLAVR SAVR TOMATO
 Ripening of fruits and vegetables can be
delayed by manipulating the genes through
genetic engineering.
 FLAVR SAVOR tomato is implanted with a
gene from E. coli. It is a transgenic tomato that
will keep tomatoes keep their colour, have
natural flavours and increased shelf life.
 It was the first genetically engineered crop
granted licence for human consumption.

56. •MAKING FLAVR SAVR
TOMATO
 Softening of the fruits is due to degradation of cell
wall by polygalacturonase(PG) gene which
encodes sense mRNA producing PG enzyme
leading to fruit ripening.
 PROCEDURE
1. Isolation of DNA from tomato plant that encodes
the enzyme polygalacturonase(PG).
2. Transfer of the gene to a vector bacteria and
production of complimentary DNA (cDNA)
molecules.
3. Introduction of cDNA into a fresh tomato plant to
produce transgenic plants.

57. •MECHANISM
The cDNA of PG
encodes for
antisense mRNA
which is
complimentary
to sense mRNA.
The
hybridisation
between them
renders sense
mRNA effect
resulting in
delayed
ripening.

58. TRANSGENIC PLANTS
RISKS
 Tampering with nature
 Violation of organism’s natural intrinsic values
 High potential to become invasive plants or weeds
 Better competitors among native plant varieties as
they have high resistance to environmental stress
 Other risks, include effects on
1. soil microorganisms and nutrient cycling
2. nearby insect populations
3. food supply for animal species and agricultural
practices, depending on the plant variety and the
transgenes they bear.

59. •OTHER RISKS
 Exposure to new allergens in
genetically modified foods, as
well as the transfer of
antibiotic-resistant genes to
gut flora.
 Horizontal gene transfer of
pesticide, herbicide, or
antibiotic resistance to other
organisms would not only put
humans at risk, but it would
also cause ecological
imbalances, allowing
previously innocuous plants
to grow uncontrolled, thus
promoting the spread of
disease among both plants
and animals.

63. REFERENCES
 Gupta P K(1997) Elements of biotechnology.Pp.14
-106.Capital Offset Press, Delhi, India.
 Lu CY, Nugent G and Wardley T(1990) Efficient, di
rect plant regeneration from stem segments of chr
ysanthemum (Chrysanthemum morifolium Ramat.
cv. Royal Purple) Plant cell reports 8:733-6.
 Hoshida H, Tanaka Y, Hibino T, Hayashi Y, Tanaka
A, Takabe T and Takabe T(2000)Enhanced toleran
ce to salt stress in transgenic rice that overexpres
ses chloroplast glutamine synthetase. Plant Mol Bi
ol.43:103-11.

64. THANKYOU