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Plant transformation methods
Dr. Annamalai MuthusamyManipal Life Sciences Centre
Manipal UniversityManipal 576 104Karnataka, India
amsamy20@gmail.com
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Agricultural Production
Different goods
Plants & Animals
Why Plant Transformation
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Conventional & modern practice
Improve the agriculturalproductivity
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ICAR
Union Minster of Agriculture - President
Director General & Secretary to GOI in DARE
8 Deputy Directors-GeneralCrop Sci, Natural Resource Management,
Animal Sci, Agricultural Edu, Agricultural Ext, Fisheries, Horticulture &
Agricultural Eng.
33,000 (7000 Res & Management) in28 SAUs
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Plant Agriculture
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Sustain life on Earth
Oxygen
Food, Fiber & Shelter
Habitats for animals
Preserve soil
Plants beautify
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Pulses macronutrients & minerals, pulses also contain PSMs that are
increasingly being recognized for their potential benefits for human health.
Health potential of pulses - bioactivity of pulses
Isoflavones, phytosterols, resistant starch,
bioactive carbohydrates, alkaloids & saponins.
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Ensure with five food groups
Creals
Vegetables & fruits
Milk & milk products
Pulses / non-veg protein
Fat, oils & sugars
Good breakfast, moderate lunch & light dinner
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Sufficient, nutritionally adequate & culturally acceptablefood for an active, healthy life.
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Plants
Conventionalbreeding
Tissue culture
Genetic engineering
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Conventional breeding
Selection
Tissue culture
Without in vitro selection
With in vitro selection
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Plant Transformation
Plants are the easiest of higher organisms to transform
Both physical and biological methods exist for transformation
Until recently, only transgenic organisms in wide public
release were plants
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Plant Transformation Methods
Physical Chemical Biological
Microinjection
Pressure
Biolistics - gene gun/
particle bombardment
ElectroporationMicroinjection
Silica/carbon fibers
Lazer mediated
SAT
PEG
DEAE-dextran
Calcium
phosphate
Artificial lipidsProteins
Dendrimers
A. Tumefaciens
A. Rhizogenes
Virus-mediated
In planta
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Transformation
Plants - physical methods
Microinjection
Electroporation
Biolistics - gene gun
Silica/carbon fibers
Lazer mediated
SAT
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Microinjection of GOI
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This electroporator is for low-current applications such as those using small
electrodes
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Biolistic / Gene Gun
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Sonication & SAAT
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Biological Transformation
dsDNA vectors - i.e cassava mosaic virus
Agrobacterium tumefaciens &A. rhizogenes
Soil bacterium
Causes diseases in plants called crown gall &
hairy root
SAAT
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Biological Transformation
AgrobacteriumAgrobacterium tumefacienstumefaciens &&
AgrobacteriumAgrobacterium rhizogenesrhizogenes
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Possible plant compounds, that initiateAgrobacteriumto infect plant cells.
Acetosyringone, ferulic acid, gallic acid,
Hydroxybenzoic acid, pyrogallic acid, vanillin etc.
In monocot not efficient
Transformation frequencey very less
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Agrobacterium
Agrobacterium (diseasesymptomologyand hostrange)
A. radiobacter - avirulent species
A. tumefaciens - crown gall diseaseA. rhizogenes - hairy rootdisease
A. rubi - canegall disease
A. vitis - galls on grapeand afewotherplantspecies
Otten et al., 1984
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Tzvi Tzfira and Vitaly Citovsky, 2002, Trends in Cell Biol. 12(3), 121-129
Cellular process of Agrobacteriumhost interaction
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Plant Transformation Methods
Virus-mediated gene transfer(Plant viruses as vectors)
Caulimoviruses ds DNA CaMV
Geminiviruses - 2ss DNA maize streak virus
RNA plant viruses - TMV
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In Planta Transformation
Meristem transformation
Floral dip method Pollen transformation
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Chloroplast transformation
- Horizontal gene transfer
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Selectable Markers
A gene encoding an enzyme
Antibiotic resistance
Herbicide resistance
Positive selection genes
genes that allow use of some necessary mediacomponent.
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Selectable Markers
NPTII- kanamycin (antibiotic)
Hpt - hygromycin
PMI - changes mannose to useable carbohydrate
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Novel Selection Genes
Luciferase - gene from fireflies substrate
Green Fluorescent Protein - from jellyfish - underlights and filter the transgenic plants - GFP
GUS - glucuronidase gene will convert added
substrate to blue color.
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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
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5-Bromo-4-chloro-3-indolyl -D-glucuronide
(sodium salt)
Synonym - BC-Indicator
X-GlcA/
X-Glu
X-glucuronide
Molecular Formula
C14H12BrClNNaO7Molecular Weight
444.59
Activity - quantitative way or through visualization
Beta-glucuronidase E. Coli
Richard Anthony Jefferson (1987)
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X-glcA (X-gluc or X-glc or X-glcU) - substrate for GUS
GUS oxidative
dimerization
X-glu colourless soluble Blue precipitate of
intermediate diX-indigo
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Chloroplast transformation
- Horizontal gene transfer
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Selection & Regeneration
Cells which contain the selectable marker gene can
grow
All plants that develop are transgenic
Plant transformation using physical or biological
methods requires a tissue culture stage
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In Planta Transformation
Meristem transformation
floral dip
Pollen transformation
electroporation
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Analysis of T0 plants
Morphology
Physiology
Yield characters
GUS expression
Gene expression
Confirmation with selectable marker,
Screenable marker, Negative &
Positive control
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Resistance & Stress tolerance in plants:
Resistance:
- able to break-down or
- metabolize foreign molecules or- introduction a new enzyme to metabolize
Tolerance:
- able to grow -foreign molecules- either the target enzyme or- altered form of enzyme
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Golden rice contains increased levels of pro-vitamin A .
Traditional rice is white (a).
The prototype of golden rice was developed in 2000 and is a light yellow
color (b). It contains 1.6 mg/g of carotenoid.
In 2005, new transgenic lines were developed that dramatically increased the amount
of carotenoid synthesized, making the rice a deep golden color (c).
This latest form contains 37 mg/g of carotenoid, of which 84% is b-carotene trial
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Miraculin - taste-modifying protein miracle fruit, the red berries of
Richadella dulcifica - shrub native to West Africa
Active principle - protein miraculin - not sweet
Unusual property - turn a sour taste into a sweet taste
Sour foods - lemons, limes & grapefruit, taste sweet when tasted
together with this protein
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Recombinant miraculin -
tomatos
leaves (102.5) &
Fruits(90.7) g/g fresh weight
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Tomatoes comes in many varieties, colors and shapes
Transgenic tomatoes - expressing different malarial antigens
Medical hypothesis, 2006
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Normal and mutant tomato fruit
high-pigment 1 (hp1/hp1), high-pigment 2 (hp2/hp2), Never-ripe (Nr/Nr),
Green-ripe (Gr/Gr), Colorless non-ripening (Cnr/Cnr) &
ripening-inhibitor (rin/rin) mutations
Current Opinion in Plant
Biology 2007, 10:28328
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Delivery of a corn-based edible
vaccine
Transgenic corn kernels (a)
Corn snack (b) or
Embryo or germ cells (c)
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TearlessO
nion
Dr Eady
Crop & Food Research in New
Zealand and his collaborators in
Japan
As onions are sliced, cells are broken, alliinases - break down aa sulphoxides -
generate sulphenic acids - unstable - rearrange into a volatile gas - syn-propanethial-S-
oxide diffuses by air - reaches the eye - reacts with the water to form a diluted solution
of sulphuric acid - Tear glands produce tears to dilute and flush out the irritant
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http://www.dailymail.co.uk/news/article-514799/The-orange-purple-green-
cauliflowers-scientists-claim-healthier-you.html
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Purple tomatoes high in anthocyanins
High anthocyanin purple tomato and red
wild-type tomato
http://news.bbc.co.uk/2/hi/health/7688310.
stm
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ProfCathie Martin from the John Innes Centre
Anthocyanins offer protection against certain cancers, cardiovascular
disease and age-related degenerative diseases. Anthocyanins also
have anti-inflammatory activity, promote visual acuity and hinder obesity
and diabetes.
Tomatoes already contain high levels of the antioxidant lycopene. Highly
processed tomatoes are the best source, or tomatoes cooked in a little
oil, which helps to release the lycopene from cells.
Flavonoids meanwhile are soluble in water, and foods containing both
water soluble and fat-dissolved antioxidants are considered to offer the
best protection against disease.
In this study the scientists expressed two genes from snapdragon that
induce the production of anthocyanins in snapdragon flowers. The
genes were turned on in tomato fruit.
Anthocyanins accumulated in tomatoes at higher levels than anything
previously reported for metabolic engineering in both the peel and flesh
of the fruit. The fruit are an intense purple colour.
http://www.seedquest.com/News/releases/2008/october/24091.htm
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Biodegradation of ex
plosives (TNT, RDX)
Aresa Danish biotech company- planting tg tabacco plant to detect
- Permission from Serbian authorities
- Enzymatic detection & destruction
19 strains of Rhodoccus use RDX as N2 source
Cytochrome p450 system - breakdown
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Biosensor
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World's First Blue Roses On Display In Japan
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World s First Blue Roses On Display In Japan
- Danielle Demetriou, Daily Telegraph, October 31, 2008,
See the rose at
http://www.telegraph.co.uk/news/worldnews/asia/japan/3327043/Worlds-first-blue-roses-on-display-in-Japan.html
Tokyo, Japan - World's first blue roses have been
unveiled to the public for the first time at an international
flower fair in Japan, following nearly two decades of
scientific research.
The blue-hued blooms are genetically modified and have
been implanted with a gene that simulates the synthesis
of blue pigment in pansies.
Its scientists successfully pioneered implanting into the
flowers the gene that produces Delphinidin, the primary
plant pigment that produces a blue hue but is not found
naturally in roses.
The world's first genetically modified blue roses were
unveiled in the laboratory four years ago, although further
research was required to make them safe to grow in
nature.
The Blue Rose wasdeveloped by
Suntory Flowers
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Bozeman, Mont. -- U.S. scientists say a FUNGUS in the Patagonian rainforest might be
a new source of biofuels since it produces a number of diesel compounds from cellulose.
"This is the only organism that has ever been shown to produce such an important
combination of fuel substances," said Montana State University Professor Gary Strobel,
making it a better source of biofuels than anything used now.
The fungus, Gliocladium roseum, produces various molecules made of hydrogen and
carbon that are found in diesel, the researchers said. Because of that, the fuel it produces
is called "myco-diesel."
"Gliocladium roseum lives inside the Ulmo tree in the Patagonian rainforest," Strobelsaid. "We were trying to discover totally novel fungi in this tree by exposing its tissues
to the volatile ANTIBIOTICS of the fungus Muscodor albus. Quite unexpectedly, G.
roseum grew in the presence of these gases when almost all other fungi were killed.
"It was also making volatile ANTIBIOTICS. Then when we examined the gas
composition of G. roseum, we were totally surprised to learn it was making a plethora of
hydrocarbons and hydrocarbon derivatives," Strobel said."
Strobel said the discovery brings into question scientists' knowledge of the way fossilfuels are made.
The discovery is reported in the journal Microbiology.
The fungus grows inside the Ulmo tree in the temperate Patagonian rainforest of
Chile and Argentina.
Myco-diesel
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Researcher grows roots on upper part of plant
(http://www.uu.nl/EN/Current/Pages/Researchergrowsrootsonupperpartofplant.aspx)
Utrecht University (The Netherlands) and Ghent University (Belgium) with help
from scientists in Japan, USA and Switzerland. The results of this researchappeared as an advance online publication of the weekly science journal Nature
on 26 October2008.
Pankaj Dhonukshe discovered a molecular
switch to alter the auxin transport.
By turning on the switch, Dhonukshe was
able to reduce the extent of auxin transport
towards the roots.
The hormone then began to accumulate at
the places in the young leaves where it isproduced and roots began to emerge here
where normally leaves would grow.
The photo on the left shows a normal plant with normal leaves and a root and the photo on the right shows a plant on which root has started to
grow at the place of young leaf. The shoot part is shown in orange and the roots in green.
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1st generation of tg plants
2nd generation of tg plants