Gene Transfer and its applications; Plant tissue culture and its application for production of secondary metabolites, Modern methods of gene transfer, Modern techniques of gene transfer in plants, How gene is transferrred,Crispr Cas, Latest methods of gene introduction, Gene transfer,

2. Gene transformation is a process in molecular
biology and biotechnology where genetic material
(such as DNA) is introduced into an organism's
cells, resulting in a change in the organism's
genetic makeup.
This process also involve the introduction of
foreign genes.
Introduction
Gene Transformation

3. Agrobacterium tumefaciens
Polyethylene Glycol
Silicon-carbide whisker-mediated transformation
Sonoporation
CRISPR/Cas-Mediated Targeted DNA Insertion
Methods of Gene Transfer in Plants

4. T-DNA Region in Agrobacterium:
◦ Agrobacterium tumefaciens contains a segment of DNA known as
the T-DNA (Transfer-DNA) region within its Ti (Tumor-Inducing)
plasmid. This T-DNA region contains genes that code for the
transfer and integration of the DNA into the host plant's genome.
Recognition and Attachment:
◦ Agrobacterium recognizes wound sites in the plant, where plant
cells are damaged or injured. This is often caused by physical
damage, such as cutting or wounding.
Integration into Plant Genome:
◦ The transferred T-DNA integrates into the host plant's genome.
The specific location of integration may vary, and it can lead to the
expression of the genes contained within the T-DNA.
Agrobacterium tumefaciens

5. Virulence Proteins:
◦ When Agrobacterium encounters a
wounded plant cell, it secretes virulence
proteins and signals that facilitate the
transfer of the T-DNA into the plant cell.
T-DNA Transfer:
◦ The T-DNA region is then transferred into
the plant cell's nucleus. This transfer is
facilitated by both Agrobacterium's
virulence proteins and the plant cell's own
DNA repair mechanisms.
Contin…

6. Expression of Transferred Genes:
◦ Once integrated into the plant genome, the transferred genes become part of the plant's DNA. They are
transcribed and translated, leading to the expression of the genes in the host plant.
Phenotypic Changes:
◦ The expression of the transferred genes can lead to changes in the plant's characteristics or traits. This
can include enhanced resistance to pests, improved tolerance to environmental stress, or altered
biochemical pathways.
Selection and Regeneration:
◦ Transgenic plant cells that have successfully integrated the desired genes are selected and grown on
selective media containing antibiotics or other markers. These cells are then regenerated into whole
plants.
Contin…

7. Advantages:
1. Agrobacterium is capable of transferring large fragments of DNA very efficiently without
substantial rearrangements.
1. Integration of T-DNA is a relatively precise process.
2. Stability of transferred gene is excellent.
Disadvantages:
1. It has limitation of host range, some important crops cannot be infected with agrobacterium.
2. Sometimes cells in a tissue that are able to regenerate are difficult to transform.
Contin…

8. Polyethylene glycol (PEG)-mediated transfer:
The method leads to the physicochemical uptake of DNA via endocytosis. The
protoplast are kept in polyethylene glycol (PEG) solution.
 The concentration of PEG used is 15% having 8000 Dalton molecular weight. After
exposure of protoplasts to exogenous DNA in presence of PEG and divalent cations.
PEG is removed and intact protoplast are then cultured to form cells with walls and
colonies in turn.
Due to the simplest transformation protocol, the method is most widely used among
all. Without removing their cell walls, E. coli and Saccharomyces cerevisiae are both
susceptible to the genetic change caused by polyethylene glycol (PEG).
Polyethylene glycol (PEG)-mediated transfer

9. Advantages of PEG-mediated transformation:
i. A large number of protoplasts can be simultaneously transformed.
ii. This technique can be successfully used for a wide range of plant species.
Limitations of PEG-mediated transformation:
i. The DNA is susceptible for degradation and rearrangement.
ii. Random integration of foreign DNA into genome may result in undesirable traits.
Contin…

10. Silicon-carbide whisker-mediated transformation:
Silicon carbide fibers are added to a suspension of plant
cell and plasmid DNA using a vortex, shaker or blender.
The fibres are used which are single crystals of silica
organic minerals like silicon carbide which possess an
elongated shape, having a diameter of 0.6mm
DNA coated fibers will penetrate the cell membrane
through small holes created by collisions between the
plant cells and fibres.
The method being done has been reported in case of
transformation of cotton (Gossypium hirsutum)
Silicon-carbide whisker-mediated transformation

11. Sonoporation ( Ultrasound mediated gene
transfer )
The method involves rupturing the cellular membranes
by acoustic waves which leads to the possibility of
introduction of molecules like DNA into the interior of
cell.
The membrane permeability increases and thereby, the
entrance of molecules into the cells is facilitated.
This phenomena leads to generate the microscopic
channels.
DNA has been introduced to the protoplasts of beetroot
and tobacco by 20kHz ultrasound at 0.5 to 1.5 W/cm2
Sonoporation

13. Step 1: Design the gRNA
The first step is to design a guide RNA (gRNA) that specifically targets the site in the plant's DNA
where you want to insert the gene. The gRNA contains a 20-nucleotide sequence that matches
the target DNA site.
Step 2: Prepare the Cas9 Protein
The Cas9 protein is a nuclease enzyme that acts as molecular scissors to cut the DNA. It must be
present in the form of a Cas9 protein-gRNA complex to make precise cuts at the target site.
Step 3: Deliver the CRISPR-Cas9 Components
The Cas9 protein and the gRNA are introduced into plant cells. There are several methods for
delivering these components, including:
Crispr Cas9

14. a. Agrobacterium-Mediated Transformation: Using
Agrobacterium as a vector to deliver the CRISPR-Cas9
components to plant cells. b. Particle Bombardment:
Using particle guns to introduce DNA-coated particles
into plant cells. c. Protoplast Transformation: Directly
introducing the components into isolated plant
protoplasts.
Step 4: Cas9-gRNA Complex Formation
In the plant cell, the Cas9 protein and the gRNA
combine to form a Cas9-gRNA complex. This complex
searches for the target DNA site based on the gRNA's
20-nucleotide sequence.
Contin…

15. Step 5: DNA Cleavage
The Cas9-gRNA complex binds to the target
DNA site and induces a double-strand break
(DSB) in the plant's DNA at that specific
location. This break triggers the plant cell's
natural DNA repair mechanisms.
Contin…

16. Step 6: DNA Repair
The plant cell can repair the DSB in one of two ways:
a. Non-Homologous End Joining (NHEJ): This repair mechanism is prone to errors and may
result in the insertion or deletion of DNA bases. If an insertional DNA is provided along with the
CRISPR components, it may be integrated at the break site, leading to gene insertion.
b. Homology-Directed Repair (HDR): HDR is a more precise repair mechanism. It involves using a
donor DNA template that contains the desired gene sequence flanked by regions homologous to
the target site. The cell incorporates this template to repair the DSB, leading to the insertion of
the desired gene.
Contin…

17. PsLOX2 Gene in Yellow Pea

21. CRISPR/Cas: a Nobel Prize award-winning precise genome editing technology for gene therapy and
crop improve
Gene Transfer in Crop Improvement | Scienceement - PMC (nih.gov)
(PDF) Genetic Transformation Methods for Crop Improvement- A Brief Review (researchgate.net)
What is CRISPR gene editing, and how does it work? (theconversation.com)
Efficient Polyethylene Glycol (PEG) Mediated Transformation of the Moss Physcomitrella patens - PMC
(nih.gov)
(PDF) DNA-Delivery Methods to Produce Transgenic Plants (researchgate.net)
Polyethylene Glycol (PEG)-Mediated Transient Gene Expression in a Red Alga, Cyanidioschyzon
merolae 10D | Plant and Cell Physiology | Oxford Academic (oup.com)
014-0012(2023).pdf (iaras.org)
REFERENCES