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Genetic Engineering of plants
For Improving agricultural, horticultural and ornamental value of a crop plant
Transgenic plant can act as bioreactor for inexpensive production of imp. Proteins or metabolite
Transgenic plant provides a powerful tools for studying of action of genes during development and
other biological production
Infection of a plant with A. tumefaciens and formation of crown gall
infect only dicotyledonous plants
Schematic representation of Ti plasmid : T –DNA is defined by left and right boarder and includes genes
For biosynthesis of auxin, cytokinin and an opine; these genes are only transcribed and translated in plant cells
Outside of T- DNA region there is cluster of vir genes a gene that codes for enzyme for opine catabolism and
an origin of replication (ori) which permits the plasmid to be stably maintained in A. tumefaciens.
Opine are an unique and unusual condensation
Product of keto and amino acid ot amino acid
and sugar.
These product are used as carbon source
by Agarobacterium to sustain their growth
Limitations of Ti plasmid vector to be used for transformation
Production of phytohormones from transformed cells in culture medium inhibits regeneration into mature plant
Opine synthesis gene in transgenic plant is not useful as it may lower the yield of final product
Size is large 200-800 kb . For recombinant experiments smaller version is preferred
Ti plasmid doe not replicate in Escherichia coli so following requirements are made:A selectable marker gene as neomycine phosphotransferase that confers kanamycine resistance to transformed cell
because this gene is prokaryotic in origin. It is necessary to put it under the control of plant transcriptional
regulational signals including both a promotor and a termination-plyadenylation sequence, to ensure that it is
efficiently expressed in transformed plant cells
Two types of vector used (a) E.coli-A. tumefaciens
shuttle vector with disarmed Ti plasmid
(b) Both vectors are combined to make cointegrate vector
Mechanism of integration of T-DNA into plant (host) DNA
CurrentOpinioninBiotechnology 2006, 17:147–154
Cont…
Leaf disc transformation by A. tumefaciens
Transformation in monocots with agrobacterium
Initially monocots were recalcitrant to agro bacterium mediated transformation
Because wounded monocot tissue do not produce phenolics such as acetosyringone at
sufficient level to induce vir gene expression
In dicot, cell division is often induced by wounding , whereas wounded sites in monocot
tend to be lignified
Transformation efficiency is increased by use of vectors with enhanced
virulence function
Hiei et al showed that co cultivation of agrobacterium and rice embryo in presence
of 100 mM acetosyringone was a critical factor for genetic transformation
The modification of agrobacterium can be done by increasing the expression of vir G
resulting in so called supervirulent bacterial strains such as AGL-1
Vector less or Direct DNA transfer to Plants
Micro projectile Bombardment
Direct DNA transfer into plant protoplast
Electroporation
Liposome fusion
In Planta Transformation
Particle Bombardment method for gene transfer
Gold or tungsten spherical particles (0.4-1.2 µm) are coated with DNA that has
been precipitated with CaCl2, spermidine or polyethylene glycol
The coated particles are accelerated at a velocity sufficient to penetrate the cell wall ( 250 m/s)
Once inside cell, the DNA by some unknown mechanism integrates into plant genome
Many different types of plant materials have been used as transformation targets including
callus, cell suspension culture and organized tissues such as immature embryos, meristem
and leaves
Rice (1991), wheat (1992), Oat (1992), sugarcane and barley (1994)
A. Mature embryo-derived callus of T.
durum showing histochemical
localization of the gus gene activity two
days after bombardment with
pAct1-F/pDM302.
B & C. Treated and control explants,
respectively, on regeneration medium
Genetic transformation of T. aestivum and T. durum by particle bombardment employing mature embryo-derived
calli as the target tissue. The calli were bombarded with either pDM302/pAct1-F or pBY520 or pBI101::Act1
Plant regeneration from
Mesophyll protoplast of Capsicum
Annuum L.
1 fresh isolated protoplast
2 early division of protoplast
3 microcalli from protoplat
4 Macrocalli
5 shoot on regenerating medium
6 Regenerated plant
Chemical mediated gene transfer e.g. chemicals like polyethylene glycol
(PEG) and dextran sulphate induce DNA uptake into plant
protoplasts.Calcium phosphate is also used to transfer DNA into cultured
cells
Microinjection where the DNA is directly injected into plant protoplasts or
cells (specifically into the nucleus or cytoplasm) using fine tipped (0.5 - 1.0
micrometerdiameter) glass needle or micropipette.
Electroporation involves a pulse of high voltage applied to make
transient (temporary) pores in the plasma membrane which facilitates
the
uptake
of
foreign
DNA.
The cells are placed in a solution containing DNA and subjected to
electrical shocks to cause holes in the membranes. The foreign DNA
fragments enter through the holes into the cytoplasm and then to
nucleus
Liposome mediated gene transfer - Liposomes are circular lipid molecules
with an aqueous interior that can carry nucleic acids. Liposomes encapsulate
the DNA fragments and then adher to the cell membranes and fuse with them
to transfer DNA fragments. Thus, the DNA enters the cell and then to the
nucleus. Lipofection is a very efficient technique used to transfer genes in
bacterial, animal and plant cells.
DNA delivery method
Feature
Agrobacteriummediated
< 50 kb
Chemicalelectrical
5-and20 kb
Microinjection
Particle gun
16 kb
-
Range of plants
Limited
All plants
All plants
All plants
Efficiency
High
High
-
-
-
High degree of
rearrangements
Yes
Yes
Size of DNA
construct
DNA insert
integrity
Not affected by High degree of
rearrangements rearrangements
Need for special
equipment
No
No (chemical)
Yes (eletrical)
Regeneration
protocol
Needed
Needed
Protoplast
culture
Not needed
Necessary
May be needed
Not needed
Applicable to
organised
meristems
Generally,No
No
No
Yes
Applicable to
cereals
Applicable
Frequently used
Possible
High
applicability
Needed(often) Not necessary
Comparative table for different method of genetic transformation
In planta transformation
This method involves the introduction of DNA either by agrobacterium or direct
transfer into plant. The procedure is carried out at an appropriate time in the
plant life cycle so that DNA becomes incorporated in into cells that contribute
to germline, directly into germ cells themselves or into very early plant embryo
This procedure minimizes or eliminates the tissue culture steps usually needed
for generation of transgenic plant
The first in planta transformation system involved imbibing Arabidopsis seeds overnight in
an Agrobacterium culture followed by germination of seeds on antibiotic containing media
In another approach Arabidopsis plants at the early stage of flowering and placed en masse
into a bell jar in a solution of Agrobacterium . A vacuum was applied and then released ,
causing air trapped within plant to bubble off and be replaced with Agrobacterium solution
In ‘floral dip’ method transformation can occur by mere dipping of flowers in Agrobacterium
solution
Virus mediated gene transfer
Viruses provide natural examples of genetic engineering since viral infection of a cell result in addition
Of new genetic material which is expressed in the host. Additional genetic material incorporated in
plant viral genome may be replicated and expressed along with viral gene in plant cell
Virus vector should possess following chactersticks:1. It should have broad host range, virulence, ease of mechanical transmission and rate of seed
transmission.
2. It should have potential to carry additional genetic information ,since there are strict packaging limit
3. Virus suitability as a vector depends on the fact that genetic material must be able to be manipulated
and be infectious
At present three group of virus are being suited for vector development and tranformation
DNA group virus : caulimoviruses : Cauliflower mosaic virus (CaMV)
gemini virus
: African cassava mosaic virus (ACMV), Tomato golden mosaic
virus (TGMV) both are transferred by insect
RNA group virus:
Tobacco mosaic virus (TMV)
(i): Beta vulgaris plants systemically infected with fully optimized
viral vector carrying theGFP reportergene;photographed
Under normal(left) or UV(right)light,10 days post-inoculation
Application of plant genetic engineering
Insect resistant plant :
Bacillus thuringiensis, commonly known as Bt, is a bacterium that occurs naturally in the soil. For
years, bacteriologists have known that some strains of Bt produce proteins that kill certain insects
with alkaline digestive tracts. When these insects ingest the protein produced by Bt, the function of
their digestive systems is disrupted, producing slow growth and, ultimately, death
Today, plants can be genetically engineered to produce their own Bt. Another way of imparting
insect resistance to susceptible plants entails using a gene that encodes an alpha amylase
inhibitor, use of bacterial cholesterol oxidase
Herbicide resistant crops are changing weed management
Several crops have been genetically modified to be resistance to non selective herbicide
These transgenic crops contain genes that enable them to degrade the active ingredient in
an herbicide, rendering it harmless. Farmers can thereby easily control weeds during the
entire growing season and have more flexibility in choosing times for spraying.
Virus resistant genetically modified (GM) plants
In some cases, biotechnology can be used to make virus resistant crops. The most common way of
doing this is by giving a plant a viral gene encoding the virus' 'coat protein'. The plant can then
produce this viral protein before the virus infects the plant. If the virus arrives, it is not able to
reproduce.
All genetically modifiedvirus resistant plants on the market (e.g. papayas and squash) have coat
protein mediated resistance. Similarly fungus and bacterium resistance plant can be prepared
Development of stress and Senescence-Tolerant plants
Transgenic plant expressing high level of Cu/Zn superoxide dismutase are : resistant to
oxidative stress, have increased resistance to herbicide methyl viologen (paraquat), and a
higher tolerance to light stress.
Salt Tolernace
Many plants live in environment where growth can be severely impaired by either drought or high
salinity. To proliferate under these conditions many plants synthesize low molecular weight non
toxic compound osmoprotectants eg betaine . These compound facilitate both water uptake and
retention and also protect and stabilize cellular molecules from damage by high salt.
Several important crops (potato, rice and tomato) do not accumulate betaine. However by
adding betaine biosynthetic enzymes into these plant might enable them to withstand water
stress/salt stress.
Modification of Plant Nutritional Content
A process for the production of plants with increased content of appropriate amino
acids having high nutritional properties through the modification of plant genes
encoding plant storage proteins
eg increased lysine content by deregulating lysine biosynthetic pathway, introduction
of phaseolin gene which encode a seed storage protein of broad spectrum of amino
acids.
By genetic engineering it Is possible to change the degree of unsaturation (no. of C-C
double bonds) and modify the chain length of fatty acids in plant. In canola transgenic
plant with stearic acid variety contains an antisense copy of a Brassica stearate
desaturase gene , which inhibit the expression of normal canola gene and leads to
accumulation of stearic acid rather than the desaturation of stearic acid to oleic acid.
Plants as Bioreactor
Plants are easy to grow and can generate considerable biomass. With these features in
mind ,research has been carried out to determine whether transgenic plant can be used for
production commercial proteins and chemicals
In small scale trials plants have been used to produce monoclonal antibody, functional
antibody fragments and the polymer polyhydroxybutyrate, which can be used to make a
biodegradable plastic like material.
In bacteria such as Alcaligenes eutrophus 3- hydroxybutric acid is synthesized from acetyl
coenzyme A in three steps by three enzymes in three different steps whose genes are
organized on a single operon.
Since plants are unable to process a transcript of an operon with more than one gene,
each of these three genes was cloned and targeted to chloroplast of the plant A. thalliana.