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1. Techniques in Biotechnology
M.PHOOL BADSHAH

2. Lecture-12
Transformation
In
Bacteria & Plant

3. 1- Transformation in Bacteria
 Introduction:
 It is a process in which foreign genetic material typically DNA is inserted
into a bacterial cell and integrated into its genome is usually known as
transformation in bacteria.
 This process is for horizontal gene transfer between bacteria, giving them
the capacity to acquire new characteristics like antibiotics resistance or the
capability to metabolize diverse chemicals.
 Normally, naked DNA from the surroundings is taken up by the
transformation process. While it happens naturally in some bacterial
species, scientists can carefully trigger transformation in lab settings. The
following are the general stages of bacterial transformation:

4. 1- Competent Cell Preparation:
 To make bacterial cells "competent," or more permeable to outside DNA,
they undergo treatment.
 This is frequently accomplished by applying calcium chloride or using other
techniques that damage the cell membrane to the cells.
2- Foreign DNA Isolation:
 The foreign DNA is separated, frequently as linear pieces or plasmids.
Circular DNA molecules known as plasmids are capable of self-replication
apart from the chromosomal DNA of bacteria.
3- Cells Mixing with DNA:
 The separated foreign DNA is mixed with the capable bacterial cells. The
DNA is absorbed by the cells from their environment.

5. 4- Incubation:
 The bacterial cells are allowed to recuperate and express the new genetic
material during the incubation of the cell-DNA mixture.
5- New Traits Expression:
 The transformed bacteria will express specific qualities if the foreign DNA
contains genes that code for them, such as the ability to make a particular
protein or resistance to antibiotics.
6- Selection:
 To find and separate the successfully changed cells, selective pressure is
frequently used, such as exposing them to an antibiotic that only
transformed cells can withstand.

7.  Note:
• One of the most important methods in genetic engineering and
biotechnology has been bacterial transformation.
• It is widely used in research labs to insert desired genes into bacterial cells
in order to produce proteins, investigate gene function, and create
genetically modified organisms (GMOs).
• The study of bacterial evolution, antibiotic resistance, and the
dissemination of genetic features throughout microbial populations can all
benefit from an understanding of bacterial transformation.

8. 2- Transformation in Plant
• Genetic transformation is the process of introducing an specific gene into a
plant and changing it.
• Transformation in bacteria was first demonstrated in 1928 by the British
bacteriologist Frederick Griffith.
• This can be done in many ways, and which one is used will depend on the
kind of plant, the gene to be added, and other elements.
• Transformation in Plant is proceed through various processes
which discussed down:

9. 1-Selection of Specie of Plant:
 Select the species of plant you want to
transform. Certain plants are more
capable to genetic manipulation than
others, and various plants transform in
different ways.
2- Selection of Gene of Interest:
 Determine which gene of interest to
introduce into the plant and isolate it.
This gene may confer a desired
characteristic, including increased
nutritional content, enhanced yield,
or insect resistance.

10. 3- Construct Vector:
 The target gene should be cloned into a vector. Circular DNA molecules, like
plasmids or viral vectors, are commonly known as vectors and are capable of
transferring foreign genes. Regulatory elements, including as terminators and
promoters, are also usually present in the vector to regulate the plant's expression
of the gene.

11. 5- New gene introduction in Plant Cell:
• Several methods are involved which are given down:
 Agrobacterium-mediated Transformation:
 This widely used technique involves inserting the desired gene into a plasmid,
which is subsequently injected into the bacterium Agrobacterium tumefaciens,
which naturally transfers genes into plant cells. After that, plant tissues are infected
by the bacteria.

12.  Particle Bombardment (Biolistics):
 This technique uses a gene gun to fire microprojectiles coated with the
target gene into plant cells.

13.  Electroporation:
 Through the process of electroporation, transient holes in the plant cell
membrane are made, allowing foreign DNA to enter.
 Protoplast Transformation:
 Using plant cells that have had their cell walls removed (called
protoplasts), foreign DNA is inserted into these cells.

14. 6- Selection & Regeneration:
 Following the introduction of the foreign gene into plant cells, the cells that
have successfully absorbed the gene are identified using a selection
procedure.
 This is frequently accomplished by combining the gene of interest with a
selectable marker gene (such as one that confers resistance to an antibiotic).
 Using tissue culture procedures, altered cells that have been chosen are
encouraged to regenerate into entire plants.
7- Validation:
 Using molecular methods like PCR and gene expression analysis, confirm
the inserted gene's presence and expression in the regenerated plants.
8- Field Examining:
If required, carry out field tests to assess the GM plants' performance in
uncontrolled environments.

15.  Note:
• It is important to remember that genetic transformation is a
complicated process, with different details depending on the type of
plant and the desired results.
• When working with genetically modified organisms, ethical
considerations and local restrictions should also be taken into
account.
• Prior to performing any genetic transformation studies, be sure you
have all the required permissions and are following the right rules.