Download Plant transformation methods

Plant transformation methods
Dr. Annamalai Muthusamy
Manipal Life Sciences Centre
Manipal University
Manipal – 576 104
Karnataka, India
[email protected]
Why Plant Transformation
Agricultural Production
Different goods
Plants & Animals
Conventional & modern practice
Improve the agricultural
productivity
ICAR
Union Minster of Agriculture - President
Director – General & Secretary to GOI in DARE
8 Deputy Directors-General
Crop Sci, Natural Resource Management,
Animal Sci, Agricultural Edu, Agricultural Ext, Fisheries, Horticulture &
Agricultural Eng.
33,000 (7000 – Res & Management) in
28 SAUs
Plant Agriculture
Sustain life on Earth
Oxygen
Food, Fiber & Shelter
Habitats for animals
Preserve soil
Plants beautify
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.
Ensure with five food groups
Creals
Vegetables & fruits
Milk & milk products
Pulses / non-veg protein
Fat, oils & sugars
Good breakfast, moderate lunch & light dinner
Sufficient, nutritionally adequate & culturally acceptable
food for an active, healthy life.
Conventional
breeding
Tissue culture
Genetic engineering
Plants
Conventional breeding
♣
Selection
Tissue culture
♣
Without in vitro selection
♣
With in vitro selection
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
Plant Transformation Methods
Physical
Microinjection
Pressure
Biolistics - gene gun/
particle bombardment
Electroporation
Microinjection
Silica/carbon fibers
Lazer mediated
SAT
Chemical
PEG
DEAE-dextran
Calcium
phosphate
Artificial lipids
Proteins
Dendrimers
Biological In planta
A. Tumefaciens
A. Rhizogenes
Virus-mediated
Transformation
• Plants - physical methods
•
•
•
•
•
•
Microinjection
Electroporation
Biolistics - gene gun
Silica/carbon fibers
Lazer mediated
SAT
Microinjection of GOI
This electroporator is for low-current applications such as those using small
electrodes
Biolistic / Gene Gun
Sonication & SAAT
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
Biological Transformation
♣ Agrobacterium tumefaciens &
♣ Agrobacterium rhizogenes
Possible plant compounds, that initiate Agrobacterium
to infect plant cells.
Acetosyringone, ferulic acid, gallic acid,
Hydroxybenzoic acid, pyrogallic acid, vanillin etc.
In monocot – not efficient
Transformation frequencey – very less
Agrobacterium
Agrobacterium (disease symptomology
and host range)
A. radiobacter - “avirulent” species
A. tumefaciens - crown gall disease
A. rhizogenes - hairy root disease
A. rubi
- cane gall disease
A. vitis
- galls on grape and a few
other plant species
Otten et al., 1984
Cellular process of Agrobacterium–host interaction
Tzvi Tzfira and Vitaly Citovsky, 2002, Trends in Cell Biol. 12(3), 121-129
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
In Planta Transformation
♣
♣
♣
Meristem transformation
Floral dip method
Pollen transformation
Chloroplast transformation
- Horizontal gene transfer
Selectable Markers
• A gene encoding an enzyme
• Antibiotic resistance
• Herbicide resistance
• Positive selection genes
– genes that allow use of some necessary media
component.
Selectable Markers
– NPTII - kanamycin (antibiotic)
– Hpt
- hygromycin
– PMI - changes mannose to useable carbohydrate
Novel Selection Genes
• Luciferase - gene from fireflies – substrate
• Green Fluorescent Protein - from jellyfish - under
lights and filter the transgenic plants - GFP
• GUS - glucuronidase gene will convert added
substrate to blue color.
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
5-Bromo-4-chloro-3-indolyl β-D-glucuronide
(sodium salt)
Synonym - BC-Indicator
X-GlcA/
X-Glu
X-glucuronide
Molecular Formula
C14H12BrClNNaO7
Molecular Weight
444.59
Activity - quantitative way or through visualization
Beta-glucuronidase – E. Coli
Richard Anthony Jefferson (1987)
X-glcA (X-gluc or X-glc or X-glcU) - substrate for GUS
GUS
X-glu
→
colourless soluble
intermediate
oxidative
dimerization
→
Blue precipitate of
diX-indigo
Chloroplast transformation
- Horizontal gene transfer
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
In Planta Transformation
• Meristem transformation
– floral dip
• Pollen transformation
– electroporation
Analysis of T0 plants
Morphology
Physiology
Yield characters
GUS expression
Gene expression
Confirmation with selectable marker,
Screenable marker, Negative &
Positive control
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
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
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
Recombinant miraculin tomatos
leaves (102.5) &
Fruits(90.7) μg/g fresh weight
Medical hypothesis, 2006
Tomatoes comes in many varieties, colors and shapes
Transgenic tomatoes - expressing different malarial antigens
Current Opinion in Plant Biology 2007, 10:283–28
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
Delivery of
vaccine
a corn-based edible
Transgenic corn kernels (a)
Corn snack (b) or
Embryo or germ cells (c)
Tearless Onion
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-Soxide – 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
http://www.dailymail.co.uk/news/article-514799/The-orange-purple-greencauliflowers-scientists-claim-healthier-you.html
Purple tomatoes high in anthocyanins
http://news.bbc.co.uk/2/hi/health/7688310.
stm
High anthocyanin purple tomato and red
wild-type tomato
Prof Cathie 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
Biodegradation of explosives (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
Biosensor
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/3
327043/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
The Blue Rose was
plant pigment that produces a blue hue but is not found
developed by
naturally in roses.
Suntory Flowers
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.
Myco-diesel
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," Strobel
said. "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 fossil
fuels 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.
Researcher grows roots on upper part of plant
Utrecht University (The Netherlands) and Ghent University (Belgium) with help
from scientists in Japan, USA and Switzerland. The results of this research
appeared as an advance online publication of the weekly science journal Nature
on 26 October 2008.
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 is
produced and roots began to emerge here
where normally leaves would grow.
(http://www.uu.nl/EN/Current/Pages/Researchergrowsrootsonupperpartofplant.aspx)
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
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2nd generation of tg plants