r dna

1. Welcome

2. ROLE OF
R-DNA TECHNOLOGY
IN AGRICULTURE
SUBMITTED TO: SUBMITTED BY:
Dr. A.K.SHARMA NAVREET KAUR RAI
(M.Sc. (Ag.) Pre. Yr. Sem-II)
DEPTT. OF GENETICS AND PLANT BREEDING
Swami Keshwanand Rajasthan Agriculture University,Bikaner

3. CONTENT
 Introduction
 Discovery
 Goals and Principles
 Requirements
◦ Enzymes
◦ Host cells
◦ Vectors
 rDNA technology procedure
 Techniques
 Applications in Agriculture

4. Recombinant DNA Technology
 DNA molecules that are extracted from
different sources and chemically joined
together; for example DNA comprising
an animal gene may be recombined with
DNA from a bacterium.
 DNA molecules constructed outside of
living cells by joining natural or synthetic
DNA segments to DNA molecules that
can replicate in a living cell

6.  Discovery of DNA structure: Watson & Crick in 1953
 Isolation of DNA ligase in 1967
 Isolation of REase in 1970
 Paul Berg generated rDNA technology in 1972
 Cohen & Boyer in 1973 produced first plasmid
vector capable of being replicated within a bacterial
host

7. GOALS
 To isolate and characterize a gene
 To make desired alterations in one or more
isolated genes
 To return altered genes to living cells
 Artificially synthesize new gene
 Altering the genome of an organism
 Understanding the hereditary diseases and
their cure
 Improving human genome

8.  Generation of DNA fragments & selection
of the desired piece of DNA.
 Insertion of the selected DNA into a cloning
vector to create a rDNA or chimeric DNA.
 Introduction of the recombinant vectors into
host cells.
 Multiplication & selection of clones
containing the recombinant molecules.
 Expression of the gene to produce the
desired product.

10. REQUIREMENTS
Restriction enzymes
DNA ligase enzyme
Host cells
Vectors

11. RESTRICTION ENDONUCLEASES
 Enzymes for the manipulation of DNA.
 Are bacterial enzymes that can cut/split
DNA at specific sites.
 These were first discovered in E.coli
restricting the replication of
bacteriophages,by cutting the viral
DNA(The host E.coli DNA is protected
from cleavage by addition of methyl
groups).Thus,the enzymes that restrict the
viral replication are known as restriction
enzymes or restriction endonucleases.

12. Recognition sequences:
 Recognition sequence is the site where the
DNA is cut by a restriction endonuclease.
 Restriction endonucleases can specifically
recognise DNA with a particular sequence of
4-8 nucleotides & cleave.
Cleavage patterns:
 The cut DNA fragments by restriction
endonucleases may have mostly sticky ends
or blunt ends.
 DNA fragments with sticky ends are
particularly useful for rDNA experiments,since
single stranded sticky DNA ends can easily
pair with any other DNA fragment having
complementary

17. HOST CELLS
 The hosts are the living systems or cells in
which the carrier of rDNA molecule or
vector can be propagated.
 Host cells can be prokaryotic or
eukaryotic.
 Microorganisms are preferred as host
cells, since they multiply faster compared
to cells of higher organisms.

18. E.coli
 This was the first organism used in the
DNA technology experiments.
 The major drawback is that it cannot
perform post translational modifications.

19. Eukaryotic Hosts
 These are preferred to produce human
proteins, since these have complex
structure suitable to synthesise complex
proteins.
 Mammalian cells possess the machinery
to modify the protein to the active
form.(post translational modifications)
E.g., Tissue plasminogen activator

20. VECTORS
 Are the DNA molecules, which can carry a
foreign DNA fragment to be cloned.
 These are self replicating in an appropriate
host cell.
 Most important vectors are plasmids,
bacteriophages, cosmids & artificial
chromosome vectors.

21. Plasmids
 Are extrachromosomal, double stranded,
circular, self-replicating DNA molecules.
 Usually plasmids contribute to about 0.5%-
5.0% of the total DNA of bacteria.
 A few bacteria contain linear plasmids E.g.,
Streptomyces sp, Borelia burgdorferi.
 E.g., pBR322,pUC
 The plasmids carries genes resistance for
ampicillin & tetracycline that serve as
markers for the identification of clones
carrying plasmids.

23. Bacteriophages
 Are the viruses that replicate within the
bacteria.
 In case of certain phages, their DNA gets
incorporated into the bacterial chromosome
& remains there permanently.
 Can take up larger DNA segments than
plasmids,hence preferred for working with
genomes of human cells.
 E.g., phage λ, phageM13.

24. Cosmids
 Are the vectors possessing the
characteristics of both plasmid &
bacteriophage λ.
 These carry larger fragments of foreign DNA
compared to plasmids.

25. Artificial Chromosome Vectors
 E.g.,Human artificial chromosome, Yeast
artificial chromosomes, Bacterial artificial
chromosome
 These can accept large fragments of
foreign DNA

27. CUTTING OF DNA
 DNA can be cut into large fragments
by mechanical shearing.
 Restriction enzymes are the scissors
of molecular genetics.
 RE are the special class of sequence-
specific enzymes.

28. JOINING OF DNA
DNA ligases
 These were originally isolated from viruses,
also occur in E.coli & eukaryotic cells.
 The cut DNA fragments are covalently
joined together by DNA ligases.
 DNA ligase joins the DNA fragments by
forming a phosphodiester bond b/n the
phosphate group of 5’-carbon of one
deoxyribose with the hydroxyl group of 3’-
carbon of another deoxyribose.

29. Amplifying the recombinant DNA
 Transforming the recombinant DNA into a
bacterial host strain.
 The cells are treated with CaCl2
 DNA is added
 Cells are heat shocked at 42C
 DNA goes into the cell by a mechanism.
 Once in a cell,the recombinant DNA will
be replicated.
 When the cell divides, the replicated
recombinant molecules go to both
daughter cells.

31. TECHNIQUES USED
 Gel electrophoresis
 Cloning libraries
 Restriction enzymes mapping
 PCR
 Nucleic Acid Hybridisation
 DNA Microarrays

32. METHODS OF GENE TRANSFER
1. Transformation
◦ The uptake of plasmid DNA by E.coli is
carried out in ice-cold CaCl2(0-5C) & a
subsequent heat shock (37-45C for about
90sec)
2. Conjugation
◦ Is a natural microbial recombination
process.
◦ Plasmid-insert DNA is transferred from one
cell to another.

33. 3. Electroporation
◦ Is based on the principle that high voltage
electric pulses can induce cell plasma
membranes to fuse.
◦ Liposome-mediated gene
transfer(lipofection) are circular lipid
molecules, having aqueous interior that
can carry nucleic acids.
4. Direct transfer of DNA
◦ DNA is directly transferred into the
nucleus by microinjection & particle
bombardment.

35. Transgenic Plants

36. IMPORTANCE
 Used for production of transgenic
plants with:
◦ Higher yield
◦ Nutritional values
◦ Increased resistance to stress and pests
◦ Disease resistant
◦ Source of biopharmaceuticals
 70-75% of food in supermarket is
genetically modified.

37. Plants With Improved Yield
 Genes are inserted into plants to
increase their yield.
 Researchers at Japan’s National
Institute of Agrobiological Resources
added maize photosynthesis genes to
rice.
 Increased yields by 30 percent

38. Stress Tolerant Plants
 Plant resistance to environmental
stress:
◦ rDNA technology is used to develop crops
that can tolerate abiotic stress.
◦ Genetically modified tomato and canola
plants that tolerate salt levels 300 percent
greater than normal.
◦ Transgenic plants survive , produce fruit,
and remove salt from soil.

39. Herbicide Resistant Plants
 Roundup is an herbicide but it kills almost
all species of plants.
 Using rDNA technology, modified EPSP
synthase gene have been introduced into
crop plants
 Such as cotton and soybean.
 Genes from Salmonella conveys resistance
to glyphosate.
 E.g.,
Alfalfa,Canola,Cotton,Maize,Soybean,etc.

40. Insect Resistant Plants
 Cry genes (popularly known as Bt
genes) from a bacterium Bacillus
thurengiensis are isolated.
 Bt toxin is naturally occurring toxin
harmful only to insects.
 Various crop plants are modified using
this gene.
e.g cotton, rice, maize, potato, brinjal,
cauliflower, cabbage etc.
 Genes for Phytophthora resistance
inserted into potato crop

41. Disease Resistant Plants
 Plants are modified to produce
resistance against diseases.
e.g. Tobacco was first modified to produce
resistance against tobacco mosaic virus.

44. ACHIEVEMENTS
 Human artificial insulin
 Epoetin Alfa ( protein used for treatment
of anemia )
 Fungal disease resistance in plants
 Fertility restoration in plants
 Non-browning phenotype in plants
 Delayed fruit ripening
 Antibiotic resistance

45.  Transgenic plants-
 Cotton
 Eggplant
 Eucalyptus
 Maize
 Melon
 Papaya
 Bt gene plants-
Cotton,Tobacco,Maize,etc.
 Transgenic Carrots have been used to
produce the drugs Taliglucerase alfa which
is used to treat Gaucher’s disease.

46.  Carnation,Rose & Petunia- Modified
flower colour
 Maize- increased lysine, drought
tolerance.
 Papaya and Potato- virus resistant
 Soybean- increased oleic acid
production
 Sugarcane- drought tolerance
 Tobacco - Nicotine reduction

47. THANK YOU