Plant Transformation Methods

8/6/2019 Plant Transformation Methods

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Plant transformation methods

Dr. Annamalai MuthusamyManipal Life Sciences Centre

Manipal UniversityManipal 576 104Karnataka, India

[email protected]


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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

Plant Transformation Methods

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