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9 Plant Transormation Methods RNT

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21 views19 pages

9 Plant Transormation Methods RNT

Uploaded by

Sarita Choudhary
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Plant transformation methods

INTRODUCTON

 The production of transgenic plants is considered as a


valuable tool in plant research and the technology is extensively
applied in phytomedicines and agricultural research.

Gene transformation in plants is normally carried out by


Agrobacterium species, application of some chemicals and
physical techniques (electroporation, microprojectile, etc.).

 Despite a variety of available DNA delivery methods,


agrobacterium and biolistic-mediated transformation remain the
two predominantly employed approaches.
Production of transgenic plants

Isolate and clone gene of interest

Add DNA segments to initiate or


enhance gene expression

Add selectable markers

Introduce gene construct into plant cells


(transformation)

Select transformed cells or tissues

Regenerate whole plants


PLANT TRANSFORMATION METHODS

DIRECT INDIRECT
Physical Chemical Biological In Planta
• Microinjection •PEG • A.Tumefaciens •Meristem transf.
•Biolistics – •DEAE- dextran • A. Rhizogenes •Floral
genegun/particle •Calcium phosphate • Virus-mediated dip method
Bombardment • Artificial lipids • Pollen
•Electroporation • Proteins transformation
•Microinjection •Dendrimers
•Silica/carbon fibers
Transformation vector requirements

 Origin of replication
 Bacterial selectable marker
 Gene constructs of interest
 T-DNA borders and other Agrobacterium genes if
using Agro bacterium
 Compatible with helper plasmid if using
Agrobacterium
Agrobacterium mediated genetransfer

Have ability transfer bacterial genes to plant genome

 Contains Ti plasmid which can transfer its T-DNA


region into genome of host plants
Ti-plasmid features
 Two strains of Ti-plasmid:-
1.Octopine strains- contains two T-DNA region: TL
(14kb) and TR ( 7 kb)
2.Nopaline strains- contain one T-DNA region(20 kb)
 Size is about 200 kb
 central role in Crown-gall formation
 Contains one or more T-DNA region that is integrated
into the genome of host plants
 Contain a vir region ~ 40 kb at least 8~11 vir genes
 Has origin of replication
 Contains a region enabling conjugative transfer
 Has genes for the catabolism of opines
Nopaline OctopineAcetosyringone
Forms of T-DNA that are found in Agrobacterium

ds circles - found only in induced bacteria,


not(apparently) in plant cells.

ds linear T-DNA - found only in induced bacteria ,not


(apparently) in plant cells.

 ss linear T-DNA - found in bacteria and plant cells.


ADVANTAGE

Precise integration of DNA sequences with defined end,


 Transfer of desired DNA along with the marker gene,
 High frequency of stable and intact gene transfer
Low rate of transgene silencing, and
 The ability to transfer long stretches of T-DNA (> 150 kb)

DRAWBACKS
 Limitation to carry limited size base pair (<500kb)
 Chances of transgene silencing,
 Poor gene transfer efficiency,

Mehrnaz S. Ohadi Rafsanjani, Amene Alvari, M. Samim, M. Amin Hejazi and


M.Z. Abdin 2012. Application of Novel Nanotechnology Strategies in Plant
Biotransformation: A Contemporary Overview: Recent Patents on Biotechnology
2012, 6, 69-79
Direct Methods

1) Particle bombardment (biolistics)


2) Microprojectile gun method
3) Electroporation
4) Silicon carbide fibres
5) Polyethylene glycol (PEG)/protoplastfusion
6) Liposome mediated gene transfer
Biolistic/Particle Bombardment

Forced bombardment of small gold particles directly


introduce gene into the cells through reversible
penetration.
Formation of passive diffusion pores is also probably
the reason.
Advantages
DNA coated gold particles are bombarded over the cells, tissues
or whole plants.
More stable transformation possibility.
Vectors are not required
Applicable to wide range of plants.
Applicable with other methods that are needed for tissue culture.
Highly efficient.

LIMITATIONS
The transformed DNA is highly rearranged leading to mitotic and
meiotic instability.

The transformed cells survival rate is decreased because of high


membrane damage.
Chemical Method
Fusion of protoplast/cells under the influence of
their charge interaction.
Surfactant like behavior enhances the cellular
permeation.
Advantage:
 Providing stability to gene,
Diminish or reduce the deletion in the DNA, while
used long with the physical techniques,
 Control release pattern of gene delivery,
 Surface modification of liposome (positively
charged)
Potentiate the penetration within a cell,
Show higher degree of reproducibility,
Applicable to a wide range of cell types, and
 Free from cellular toxicity.
THANK YOU…

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