Download Transgenic Plants: Experiences and Challenges

Transgenic Plants:
Experiences and
Challenges
Dr Anil Kumar
Associate Professor & Incharge
Deptt. Of Molecular Biology &
Genetic Engineering, CBSH
GBPUAT Pantnagar Uttranchal
Pin-263245
Agriculture : a scenario
• Main stay of our economy
• Productivity enhancement by combined
efforts of agricultural scientists
• Green revolution in mid 60’s and 70’s in
20th century
• Alleviation of world hunger, poverty and
malnutrition
Food security
Nutritional security
Enhanced Agricultural
Productivity: Why ?
• Burgeoning and upsurging population
(160 persons per minute)
• Plateuing of productivity due to adopting
traditional farming practices
• Sinking of cultivable land
• Decaying of natural agricultural resource
base
• Catering need of over 11 billion people by
doubling of grain yield by 2050
POPULATION GROWTH :
URGENT NEED TO INCREASE AGRICULTURAL PRODUCTIVITY
1600
1400
POPULATION
(MILLION)
1200
1000
800
600
400
200
0
1980
1990
2000
2010
2020
INDIAN POPULATION IS EXPONENTIALLY GROWING
AND EXPECTED TO REACH 1.38 BILLION BY 2020.
Biotechnology : An answer
• Crop improvement programme through new
found wisdom of Biotechnology
• Remedy of stagnation of crop productivity
• Impact on food production system through
balanced approach and knowledge of
• Plant tissue culture
• Molecular Biology
• Cell Biology
• Biochemical Engineering
• Gene technology
• Plant breeding
Plant Breeding
Biotechnology
Improved
Genotypes/
Cultivars
Integrated
Farm
Management
Enhanced crop
Productivity
And Production
Gene technology
• An additional tool for crop improvement programme
• Two keys of plant improvement
• Creation of variation
• Selection for positive attribute
The attributes :
Improvement of important agricultural traits
Food and nutritional security
Producing specialty foods, biochemicals,
pharamaceuticals and smart designer crops
Protecting plants against the ever increasing threats of
biotic stress
Alleviating the hazards of abiotic stress
IS IT POSSIBLE ?
GENE TECHNOLOGY MAKES IT
POSSIBLE
Bacteria
P
PRO--PRO
P
PRO--EUK
P
EUK--EUK
EUK--PRO
GENE
Plant
Animal
Transgenic Technology : Art and
science of creating designer crops
• Identification of useful gene (s)
• Creation of a suitable gene construct
• Transfer of this construct in to plant cells/tissues in
vitro ( maintained as organized explants such as
immature embryos, stem sections, cotyledons etc.
Using tissue culture techniques, through a process
called transformation.
• Selection of transformed cell lines or seedling using
a suitable marker system and regeneration of fertile
plants from the transformed cells.
• Analysis of transformed plants for several aspects
including stable integration, expression and genetic
behaviour of transgene(s)
Procedure to develop transgenic biotech crop.
How are Transgenic crops made ?
Locating Genes for Plant Traits
Identifying & locating genes for agriculturally important
traits : Most limiting step in transgenic process
Little is Known about specific genes
to enhance yield potential
improve stress tolerance
modify chemical properties of harvested products
otherwise affect plant characters
Identifying a single gene for a trait is not sufficient
Must understand how the gene is regulated
What other effects it might have on the plant
How it interacts with other genes active in the
same biochemical pathway
Designing Genes for Insertion
Once a gene has been isolated & cloned : it must be suitably
modified for effective insertion into a plant
A promoter sequence must be added for the gene to be correctly
expressed
Sometimes, the cloned gene is modified to achieve greater
expression in a plant
The termination sequence signals to the cellular machinery that
the end of the gene sequence has been reached
A selectable marker gene is added to the gene "construct" in order
to identify plant cells or tissues that have successfully integrated the
transgene.
Simplified representation of a constructed transgene, containing
necessary components for successful integration and expression
Promoters used for Transgene expression
It is the ON/OFF switch : controls when & where in the plant the
gene will be expressed
Most promoters used are CONSTITUTIVE : cause gene
expression throughout the life cycle of plant in most tissues.
CaMV 35S Promoter : Most commonly used & gives high degree
of expression in plants
Other promoters : More Specific : respond to cues in the plant’s
internal & external environment
Promoter from cab gene (encoding chlorophyll a/b
binding proteins) : Light – inducible
Promoters from Arabidopsis : Specifically & rapidly
induced by natural plant stress/wounding related
semiochemical cis-jasmone
Selectable Marker Genes
Gene that facilitates the detection of genetically
modified plant tissue during development
Two major types of genes
Conferring resistance to antibiotics
Conferring tolerance to herbicides
Other genes coming up
Drug (Antibiotic)
resistance Marker
Substrate for
selection
npt II (Neomycin
Phosphotransferase)
G418, Kanamycin,
Neomycin
hpt (Hygromycin
Phosphotransferase)
Hygromycin
dhfr (Dihydrofolate reductase)
Methotrexate,
Trimethoprim
ble
Bleomycin
gat (Gentamycin Acetyltransferase)
Gentamycin
Herbicide resistance marker
Substrate for
Selection
bar & pat (Phosphinothricin
acetyltransferase)
Phosphenothricin
(Bialaphos, Basta)
epsps (5-enolpyruvylshikhimate-3phosphate synthase)
Glyphosate
Risks & Concerns of using Antibiotic Resistance Genes
Horizontal Gene Transfer
Transfer of these genes from the transgenic plants to the
microbes
Make the bacteria in the guts of animals and humans
resistant to antibiotics
Make antibiotic medicines less effective
Transfer to soil microbes : A field study shows that
transgenic DNA persists for 2 years after the GM crop has
been harvested
Other genes used as Selectable Marker Genes
man A gene from E coli
encodes mannose phosphate transferase and confer
upon transformed cells ability to use mannose as a sole carbon
source
Ipt (isopentenyl transferase) gene from Agrobacterium
located on T-DNA and induces cytokinin synthesis. Plants
selected on the basis of their ability to produce shoots from
callus on medium lacking cytokinins.
Betaine aldehyde dehydrogenase gene (BADH) from Spinach
Used with chloroplast genome.Converts toxic betaine
aldehyde to non-toxic glycine betaine, which also serves as an
osmoprotectant and confer drought/salt tolerance
Selection of successfully transformed tissues
Following the gene insertion process, plant tissues are transferred
to a selective medium containing an antibiotic or herbicide,
depending on which selectable marker was used. Only plants
expressing the selectable marker gene will survive, and it is
assumed that these plants will also possess the transgene of
interest. Thus, subsequent steps in the process will only use
these surviving plants.
Regeneration of whole plants
To obtain whole plants from transgenic tissues such as immature
embryos, they are grown under controlled environmental
conditions in a series of media containing nutrients and
hormones, a process known as tissue culture. Once whole
plants are generated and produce seed, evaluation of the progeny
begins. This regeneration step has been a stumbling block in
producing transgenic plants in many species, but specific
varieties of most crops can now be transformed and regenerated.
TRANSGENIC CROPS DEVELOPED
SO FAR AT PANTNAGAR
PRODUCTS
GENE
INTROGR
-ESSED
GENETICALLY ALTERED TRAITS
WHEAT
PAT
HERBICIDE RESISTANCE (BASTA)
BRASSICA
OSMOTIN
TOLERANCE TO ALTERNARIA BLIGHT AND
SALINITY
BRASSICA
ANNEXIN
MODULATION OF HYPERSENSITIVE RESPONSE
AGAINST ALTERNARIA BLIGHT AND POWDERY
MILDEW
TOMATO
CARROT
Pr M E
Pr M E
GUS
PRODUCTION OF EDIBLE VACCINE AGAINST
JAPANESE ENCEPHALITIS VIRUS
TRANSGENIC TECHNOLOGY
AN APPROACH FOR ENGINEERING RESISTANCE
CELL DEATH
CELL PROLIFERATION
?
?
PLANT DEFENSE
SYSTEMIC
RESISTANT GENE
OSMOTIN & ANNEXIN
?
?
DIFFERENTIATION
& DEVELOPMENT
ƒ AT LEAST FOUR DIFFERENT SIGNALLING PATHWAYS ARE KNOWN TO
EXIST IN PLANT CELL. ONE OR MORE OF SIGNALLING COMPONENTS CAN
BE MODULATED BY TRANSFERRING STRESS RELATED USEFUL GENE.
THIS MAY ENGINEER RESISTANCE.
Different stages of hardening of transformed plants.
(a) Rooted PCR positive shoots transferred in plastic pots covered with
polybags kept for hardening at transgenic glass house.
(b) A PCR positive plant growing in the pot.
(c) A hardened PCR positive plant growing in plantation pot after 2 months.
(d) Hardened PCR positive plants growing at transgenic glass house.
Alternaria blight tolerance in
Brassica
Five Brassica transformants harboring Annexin(T0 plants)
were scored for various diseases , no. of pods , no. of seeds etc.
PLANT NO.
Natural infection in plants
growing in glass house
under high humidity and
high temp
No. of pods
obtained
No. of seeds obtained
Control
Alternaria blight (++) and
powdery mildew(++)
72
196
D38
Only powdery mildew (+++)
15
61
D14
Only powdery mildew (+++)
52
186
D11 (1)
Alternaria blight (++) and
powdery mildew (++)
96
361
D62
Died early (only powdery
mildew)
6
35
D17
Alternaria blight (+) and
powdery mildew (++)
76
208
INHERITANCE OF ANNEXIN TRANSGENE IN T1 PLANTS
M
1
2
3
4
5
M
6
7
8
9
10
1.5 kb
1.0 kb
Annexin PCR of T1 progeny of T0 plant
Lane M = 100 bp ladder
Lanes 1-10 = T1 progeny plants of T0 plant
ƒ THIS EXPERIMENT HAS FURTHER REVEALED THAT A TRANSFORMANT PLANT HAS MOSAICS
OF GENE WHEN GROWN IN VITRO FROM CALLUS.
• THE GENE MOSAICS COULD OCCUR AT BOTH ORGAN AND TISSUE OR CELLULAR LEVEL.
Edible vaccine against Japanese Encephalitis virus
Shoot initiation
Callusing
Different stages of regeneration in tomato
Rooting in solid
medium
Rooting in liquid
medium
Presence of Transgene in tomato
by PCR amplification
Transformed plant
kept for hardening
Problems Associated with
production of Transgenic plants
• Low regeneration frequency associated with
albinism and anthocyanin pigmentation
which varied from explants to explants and
crop to crop and resolved by the use of
additives
• Low transformation frequency after cocultivation, varied from 0.0001 to 5% in our
experiments due to induction of
hypersensitive response by Agrobacterium
• Large number of escapes when Kanamycin
is used as a selectable marker
•Production of gene mosaics especially in the
transgenic plants developed through
organogenesis during callus culture
•Pleiotropic effects of transgene insertions like
dwarfism, seed germination, developmental and
differentiation changes and alteration of other
important processes and traits
•Segregation problems in subsequent
generations if homozygosity is not obtained
•Problems of stability in subsequent generations
due to gene silencing
Transgene silencing
Reduced/abolished expression of foreign gene
Loss of expression : Not due to loss of transgene but due
to their inactivation
Concept of gene Space :
Genomes are made of isochores (long stretches of
DNA with high compositional homogeneity)
If a GC rich transgene is integrated into a GC isochore
or an AT rich transgene is integrated into an AT
isochore : It is Transcribed
If a GC rich transgene is integrated into the AT rich
gene space or vice versa : It is Inactivated, as there is
no compositional homogeneity with the neighboring
sequences
Position Dependent & Sequence Dependent Gene silencing
Transgene integrates into a genomic region containing
heterochromatin.
The repressive chromatin structure & DNA methylation can
spread into the transgenic locus from the flanking
genomic DNA
Homology Dependent Gene Silencing
Caused by Multiple copies of transgene (Repeat induced gene
silencing)
Affect not only the stability of transgene but also alter the
activity of endogenous gene (Cosuppression)
Transcriptional Gene Silencing
No mRNA is produced from silenced gene
Affected loci : nucleation points for heterochromatin
formation & DNA methylation
Post – Transcriptional Gene Silencing
Transcription is required for silencing to take place
Induce degradation of mRNA : very little accumulate
in cytoplasm
Genetically Modified Organisms
Pitfalls
Possible
cause(s)
Possible
outcome(s)
Multiple transgene copies.
Loss of proper feedback
control.
Bad expression:
Organism ‘out of harmony’
Low viability or death.
Risk
minimization
Use progressive methods
Should be detectable
during development.
Level, tissue, time.
Disturbance at insertion
site
Danger is with occasional
need for normal function,
such as resistance to a
rare pathogen.
Insertion in appropriately
“benign” region.
Genetically Modified Organisms
Pitfalls
Possible
cause(s)
Possible
outcome(s)
Risk
minimization
Resulting organism
conflicts with environment
and/or interacting
organisms.
Threatened insect
populations. Resistant
pests. GMOs could
spread out of control,
either directly or via their
gametes.
Understand the species,
its modes of propagation,
and its interactions with
other species and the
environment.
Resulting organism
generates inappropriate
food product.
Hormones, pesticides,
residues, allergens etc. in
product.
Understand risks and test
widely for safety.
Genetically Modified Organisms
Pitfalls
Possible
cause(s)
Possible
outcome(s)
Risk
minimization
Public perception on
safety, ethics, welfare.
Market failure
Generate arguably safe
GMOs and educate public
–maybe difficult.
Other unknown causes
Other unknown outcomes
Keep an open and critical
mind.