Plant transformation methods

Dr. Annamalai MuthusamyManipal Life Sciences Centre

Manipal UniversityManipal – 576 104Karnataka, India

amsamy20@gmail.com

Agricultural Production

Different goods

Plants & Animals

Why Plant Transformation

Conventional & modern practice

Improve the agricultural productivity

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

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.

Plants

Conventional breeding

Tissue culture

Genetic engineering

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 Chemical Biological

MicroinjectionPressureBiolistics - gene gun/particle bombardmentElectroporationMicroinjection Silica/carbon fibersLazer mediated SAT

PEGDEAE-dextranCalcium phosphateArtificial lipidsProteinsDendrimers

A. TumefaciensA. Rhizogenes

Virus-mediated

In planta

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 tumefaciensAgrobacterium tumefaciens & &

♣ ♣ Agrobacterium rhizogenesAgrobacterium 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 diseaseA. rhizogenes - hairy root disease

A. rubi - cane gall disease

A.vitis - galls on grape and a few

other plant speciesOtten et al., 1984

Tzvi Tzfira and Vitaly Citovsky, 2002, Trends in Cell Biol. 12(3), 121-129

Cellular process of Agrobacterium–host interaction

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 FormulaC14H12BrClNNaO7

Molecular Weight444.59

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 oxidative dimerization X-glu → colourless soluble → Blue precipitate of intermediate 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 yellowcolor (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

Tomatoes comes in many varieties, colors and shapesTransgenic tomatoes - expressing different malarial antigens

Medical hypothesis, 2006

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, 10:283–28

Delivery of a corn-based edible vaccine

Transgenic corn kernels (a) Corn snack (b) or Embryo or germ cells (c)

Tearless Onion

Dr EadyCrop & 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

http://www.dailymail.co.uk/news/article-514799/The-orange-purple-green-cauliflowers-scientists-claim-healthier-you.html

Purple tomatoes high in anthocyanins

High anthocyanin purple tomato and red wild-type tomato

http://news.bbc.co.uk/2/hi/health/7688310.stm

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/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 was developed by Suntory Flowers

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.

Myco-diesel

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 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.

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.

1st generation of tg plants

2nd generation of tg plants