rDNA Technology - Tools and it's application in Agriculture

1. Tools of rDNA Technology and
Application of biotechnology in
Agriculture

2. Recombinant DNA Technology/Genetic Engineering
Technique to alter the chemistry of genetic material (DNA or RNA), to introduce
these into host organisms and thus change the phenotype of the host organism.

3. The tools of recombinant DNA
technology
1. Restriction
enzymes
2. DNA - Desired
gene
3. Ligases
4. Vectors
5. Host organism

4. 1. Restriction Enzymes
• In the year 1963, the two enzymes responsible for restricting the growth of
bacteriophage in Escherichia coli were isolated. One of these added methyl
groups to DNA, while the other cut DNA. The later was called restriction
endonuclease.
• Hamilton smith discovered and isolated HIND II REN from Haemophilius
influenzae in 1968. Hind II was found to identify a specific sequence called the
recognition sequence.
• More than 900 restriction enzymes have been isolated.

5. • REN are enzymes naturally occurring in bacteria as defensive enzyme. It cuts and destroys
bacteriophage DNA that infects bacterial cell.
• It recognize specific palindromic sequences to cut in DNA. Different types of REN are
identified and isolated for different palindromic sequence.
• The RENs are called molecular knives or molecular scissors as they cut DNA
Palindromic sequences:
• Palindromic sequences are ‘invert repeats’ which have the same nucleotide sequences
when read in 5‘to 3' on both the strands.
Eg,. MALAYALAM.

6. Types of nucleases
The naming of restriction enzyme:

7. The fragments of DNA produced are called
restriction fragments. The fragments of DNA
may have ‘blunt ends’ or ‘staggered ends’
(sticky ends).
2. DNA - Desired gene
The functional or normal gene of
our interest taken from donor
cell. It is also known as foreign
gene or trans gene.
3. Ligases
• The enzyme that joins the two
sticky ends of DNA is called
DNA ligase. It is used as
molecular sitchers in genetic
engineering.
• DNA ligase was discovered by
H G Khorna.

8. 4. Vectors
• The carrier DNA that act as a vehicle to carry desired
gene to the host cell is called vector.
• The plasmid and bacteriophages are used as cloning
vectrors. It has the ability to replicate within bacterial
cells independently.
• vector should contain three features for gene cloning.
They are
1) Ori site 2) Cloning sites 3) selectable markers.
ORI – Origin of Replication
• Replication starts
• Replicates any piece of DNA linked to it.
• Controls the copy number.

9. Selectable Marker
• Helps in identifying non transformants and
selectively permitting the growth of
transformants.
• Transformation is a procedure through which a
piece of DNA is introduced in a host bacterium.
Cloning sites
• To link the DNA, the vector needs to have
preferable restriction sites. More than one
restriction site will complicate cloning.
• The ligation of alien DNA is carried out at a
restriction site present in one of the two antibiotic
resistance genes.

10. lacZ - production of β-galactoisdase enzyme that metabolizes the
lactose. (X-gal)

11. Vectors used for cloning genes in plants.
Ti plasmid: Tumour inducing Ti plasmid of `Agrobactrium tumifaciens , used as
cloning vector. It is nonpathiogenic to the plants. It can be used to deliver genes of
our interest in to variety of plants.
Vectors used for cloning genes in animals.
Retroviruses. The trimmed non pathogenic retrovirus are used to deliver desirable
genes into animal cells.
5. Host cell:
The cell to which desired new gene is introduced is called host cell. Any living cell
can be used as host cell.
• Commonly E.coli bacterial cell is used as host cell
in genetic engineering. Because,
1. It is a simple prokaryotic cell and non pathogenic bacteria.
2. It can be cultured easily in laboratory condition. It has short generation time.
3. It contains self replicating plasmid that can be handled as a vector easily.

13. Application of biotechnology in Agriculture
• Rapid multiplication of crop plants, medicinal plants, forest plants and
endangered plants using tissue culture.
• Production of viral and other pathogen resistance plants.
• Production of transgenic plants as nitrogen fixing plants, insect resistance plants
etc.
• The yield of crops has mainly increased due to:
1. Use of improved varieties of crops
2. Use of agrochemicals - fertilizers and pesticides (pollution, expensive)
3. Increase in the yield with conventional methods of breeding.

14. Plants which genome is modified using r-DNA technology by introducing alterative
gene are called GM Plants.
• The Gm plants are useful in the following ways:
1. Gm crops plants more tolerant to abiotic stresses like cold, heat, drought,
salinity, etc.
2. It has reduced the dependence chemical pesticides as they are made pest-
resistant. ex: Bt crops.
4. Food produced from GM crops has enhanced nutritional value. Ex Vitamin ‘A’
enriched rice.
5. GM plants has been used to supply resources to industries such as starch, fuel,
pharmaceuticals, etc.
genetically modified plants (GM Plants):

15. Pest Resistant Plants

16. Meloidegyne incognitia