Plant Cells Transformation

Methods
Vimal Kumar
Subject- Plant and Animal Biotechnology
UID: - 21MBT1180
UIBT
Transformation Basics
• The process by which the genetic material of
the organisms is altered by the integration of
new genes into its genome.
• Historical context: - The transformation was
the first time applied by British Bacteriologist
Frederick Griffith in 1928 in Diplococcus
pneumonia (Streptococcus pneumoniae).

Ref: - www.https://bio.libretexts.org/
 Improve crop yields.

Importance
of Transgenic Improvement of varietal traits.
Plants:
Transgenic plants have protection
against their parasites, pests and harsh
weather conditions.
 Methods of Plant
Transformation.
• Fig: - An overview of plant
transformation methods
enlisting direct (physical and
chemical) and indirect
(biological) methods. An
overview of plant transformation
methods enlisting direct
(physical and chemical) and
indirect (biological) methods.
• Ref:- Saifi. K.S et al., 2020.
Direct Methods or non-biological methods

These methods involve the physical- or chemical-based transfer of the transgene

to a host cell.

Various techniques such as electroporation, lipofection, microinjection,

sonication, particle bombardment/biolistic, silicon carbide fiber-mediated gene
transfer, and laser microbeam-induced gene transfer come under this category.
 • Physical gene transfer methods—
electroporation, microinjection, particle
bombardment etc.
Categorization
of Direct gene
transfer • Chemical gene transfer methods—Poly-
ethylene glycol (PEG)-mediated, diethyl
amino ethyl (DEAE) dextran-mediated,
calcium phosphate precipitation.
Electroporation
• This method involves suspension of plant protoplasts in a
suitable ionic solution containing linearized recombinant
plasmid DNA. This mixture is then exposed to low voltage-
long pulses or high voltage short pulses for the desired
number of cycles.

• The electrical pulses are thought to induce transient pores

in the plasma lemma through which the DNA molecules are
incorporated. Treated protoplasts are then cultured to
obtain cell colonies and plants.
Electroporation

•Fig. Electroporation Cartoon.

•Ref: https://
www.btxonline.com
 Microinjection
• The mechanical insertion of the desirable DNA into a target cell.
• The target cell may be the one identified from intact cells,
protoplasts, callus, embryos, meristems, etc. Microinjection is
used for the transfer of cellular organelles and for the
manipulation of chromosomes.
• The technique of microinjection involves the transfer of the gene
through a micropipette (0.5-10.0 pm tip) into the
cytoplasm/nucleus of a plant cell or protoplast. While the gene
transfer is done, the recipient cells are kept immobilized in
agarose embedding, and held by a suction holding pipette.
 • Fig: - Microinjection insertion.
• Ref: - www.btxonline.com

Microinjection.
 • The micro projectile bombardment method
was initially named as biolistics by its inventor
Sanford (1987).
Particle • Biolistics is a combination of biological and
ballistics. In this method, 1-2 µm gold or
Bombardme tungsten particles coated with DNA are shot
into the plant cells using a helium pressure
nt Gun particle gun device.
• Biolistics, short for “biological ballistics” and
Method also known as particle-mediated gene
transfer, is the method of directly shooting
DNA fragments into cells using a device called
a gene gun.
Gene Gun
• Ref: - Matt Carter, Jennifer Shieh, in
Guide to Research Techniques in Neur
oscience (Second Edition)
, 2015
Gene Gun
 • Liposome-mediated transformation includes
liposome adhesion to the surface of the
Liposome protoplast, its fusion at the attachment site,
and the release of plasmids inside the cell.
mediated • Cationic lipids are those with a positive
transformation charge that are used for nucleic acid
transfer.
• Liposomes can interact more readily with
the negatively charged cell membrane than
uncharged liposomes.
Liposome
Mediated Gene
Transfer
Chemical Gene
Transfer methods:-
• Certain specific chemicals are used which make
incorporate the desired genes or DNA into host
(Plant cells).
• PEG mediated gene transfer.
• Calcium Phosphate co-precipitation.
• DEAE-dextran mediated transfer.
 • Polyethylene glycol (PEG)is a
polymer that disrupts the plasma
membrane of protoplasts in the
presence of divalent cations (using
Ca2+) and makes it permeable to
naked DNA.
• 15-20% PEG is mostly used.
• It promotes the fusion and
PEG mediated endocytosis.

gene transfer
 • The DNA is permitted to mix with calcium
chloride solution and isotonic phosphate
buffer to form DNA-calcium phosphate
precipitate.
• Calcium phosphate facilitates the binding of
the condensed DNA in the co-precipitate to
Calcium phosphate the cell surface, and the DNA enters the cell
by endocytosis.
co-precipitation • The principle of calcium phosphate co-
precipitation involves mixing DNA with
calcium chloride in a buffered
saline/phosphate solution to generate a
calcium-phosphate–DNA co-precipitate,
which is then dispersed onto cultured cells.
 • Diethylaminoethyl (DEAE)-dextran is a
polycationic derivative of the carbohydrate
polymer dextran, and it is one of the first
chemical reagents used to transfer nucleic
acids into cultured mammalian cells (Vaheri
and Pagano, 1965).

DEAE-Dextran • The cationic DEAE‑dextran molecule tightly

associates with the negatively charged
Method backbone of the nucleic acid, and the net
positive charge of the resulting nucleic acid-
DEAE‑dextran complex allows it to adhere
to the cell membrane and enter into the
cytoplasm via endocytosis or osmotic shock
induced by DMSO or glycerol.
 Biological • Biological agents are involved in such
gene transfer.

Methods of • Agrobacterium tumefaciens mediated

Gene
gene transfer.
• Virus Mediated gene transfer.

Transfer
Agrobacterium species
Agrobacterium tumefaciens :- Crown gall disease
Agrobacterium rhizogenes :- Hairy root disease.
Agrobacterium radiobacter:- Avirulent strain.

Features:-
Gram-ve Soil bacterium.
Rod shaped.
Infect Dicot plants.
 Agrobacterium-mediated transformation is
highly efficient and hence is most usually
used method for plant genetic engineering.
Agrobacteriu
m tumefaciens The mechanism of Agrobacterium-mediated
transformation is based on the transfer of a
piece of plasmid by the bacteria into the
mediated plant cells during infection.

gene transfer. The plasmid then integrates into the nuclear

genome in order to express its own genes
and affect the hormonal balance in the host
cell.
 •Ref:- Gene cloning and DNA analysis: T.A. Brown 6th
Crown Gall edition

Disease
Ti plasmid.
Ref.
www.microbionotes.com
AMGT Conti.
 • Ref: -Gene cloning and DNA
analysis. T.A. brown 6th edition

TRANSFORMED PLANT BY
AGT
Process of AMT

Ref:- www.microbenotes.com
Novel Approaches of
AMGN.

Binary Vector.
Co-integration Vector
In planta Method of
AMT
• The in-planta method of transformation
is a new and efficient method of
Agrobacterium-mediated
transformation that skips the need for
the tissue culture-based regeneration
of transgenics.
• In this method, Agrobacterium with the
required transgene is allowed to infect
the meristematic tissue of the plant
directly, eliminating the intervening
tissue culture steps.
• This is a cost-effective, fast, and very
efficient method compared to tissue
culture-based transformation and can
open new gates for recalcitrant species.
(Bechtold and Bouchez, 1995)
Comparative
methodology
of callus and
planta method
Virus Mediated Gene transfer

The potential of plant Only two classes of DNA

viruses as cloning vectors virus are known to infect
has been explored for higher plants, the
several years but without Caulimoviruses and
great success. Geminiviruses.
 • Gene cloning and DNA Analysis 6th editiion
• Andrieu, A., Breitler, J., Sire, C., Meynard, D.,
Gantet, P., Guiderdoni, E., 2012. An in planta,
Agrobacterium-mediated transient gene
expression method for inducing gene silencing
in rice (Oryza sativa L.) leaves. Rice 5 (1), 23.
https://doi.org/10.1186/1939-8433-5-23.
References • Saifi K.S., Passricha N., Tuteja R., Kharb P.,
Tuteja N 2020. Chapter: - 21. Advancement in
crop improvement techniques.
• www.bio.libretexts.org.
• www.btxonline.com
• Matt Carter, Jennifer Shieh in guide to
research techniques in neuroscience.
• Cambridge University Press (Expert review).
• www.microbionotes.com.
THANK YOU ANY QUESTIONS.