transgenic plants new

S.KARTHIKUMAR,M.Sc.,M.Phil.,M.Tech.,(PhD)ASSISTANT PROFESSORDEPARTMENT OF BIOTECHNOLOGYKAMARAJ COLLEGE OF ENGINEERING AND TECHNOLOGYVIRUDHUNAGAR-626001TAMILNADU, INDIA

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Transgenic Plants

Why do we need transgenic plants ?

• improvement of agricultural value of plant (resistance to herbicides,

resistance to insect attack -> Bacillus thuringiensis toxin)

• living bioreactor -> produce specific proteins

• studying action of genes during development or other biological

processes (knock-out plants, expression down-regulated)

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Transgenic Plants

• Advantages:- Plant cells are totipotent -> whole plant can be regenerated from

a single cell (engineered cells -> engineered plants)- Plants have many offspring -> rare combinations and mutations

can be found- Transposons used as vectors

• Disadvantages:- Large genomes (polypoid -> presence of many genomes in one

cell) - plants regenerating from single cells are not genetically

homogenous (genetically instable)

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Plants

Conventional breeding

Tissue culture

Genetic engineering

Gene – transfer methods

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Gene – transfer methods

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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 plantskarthikumarbt@kcetvnr.org 6

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

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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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Electroporation• Use on cells without walls

(plant protoplasts or animal cells )

• High-voltage pulses cause pores to form transiently in cell membrane; DNA pulled in by electrophoresis or diffusion (?)

• Drawback

Regeneration is difficult

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This electroporator is for low-current applications such as those using small electrodes

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Particle Bombardment

• Less limitations than electroporation• Can use on cells with walls, essentially any

tissue• Can transform organelles!• Method:

1. Precipitate DNA onto small tungsten or gold particles.

2. Accelerate particles to high speeds at cells or tissues.

3. Selective growth and regeneration of transgenic plants as described for Agro-mediated transformation.

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DNA is bound to the microprojectiles, which impact the tissue or immobilized cells at high speeds.

J. Sanford & T. Klein, 1988

Original biolistic gun. A modified 22 caliber.

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Biolistic / Gene Gun

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An Air Rifle for a DNA Gun – Circa 1990

A.Thompson, Bob ?, and D. Herrinkarthikumarbt@kcetvnr.org 15

The Helium Gas Gun – Circa 2000

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The Hand-Held Gas Gun

Purpose:Introduce DNA into cells that are below the top surface layer of tissues (penetrate into lower layers of a tissue)

One interesting use:Making DNA Vaccines in whole animals.

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Agrobacterium - mediated Gene Transfer• Most common method of engineering dicots, but also

used for monocots• Pioneered by J. Schell (Max-Planck Inst., Cologne)

• Agrobacteria– soil bacteria, gram-negative, related to Rhizobia– species:

tumefaciens- causes crown galls on many dicotsrubi- causes small galls on a few dicotsrhizogenes- hairy root disease radiobacter- avirulent

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Crown galls caused by A. tumefaciens on nightshade.

More about Galls: http://waynesword.palomar.edu/pljuly99.htmhttp://kaweahoaks.com/html/galls_ofthe_voaks.html

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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., 1984karthikumarbt@kcetvnr.org 21

Infection and tumorigenesis

• Infection occurs at wound sites• Involves recognition and chemotaxis of the

bacterium toward wounded cells• galls are “real tumors”, can be removed and

will grow indefinitely without hormones• genetic information must be transferred to

plant cells

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Tumor characteristics

1. Synthesize a unique amino acid, called “opine”– octopine and nopaline - derived from

arginine– agropine - derived from glutamate

1. Opine depends on the strain of A. tumefaciens

2. Opines are catabolized by the bacteria, which can use only the specific opine that it causes the plant to produce.

3. Has obvious advantages for the bacteria, what about the plant?

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Ti Plasmid

1. Large (200-kb)2. Conjugative3. ~10% of plasmid transferred to plant cell

after infection4. Transferred DNA (called T-DNA) integrates

semi-randomly into nuclear DNA 5. Ti plasmid also encodes:

– enzymes involved in opine metabolism– proteins involved in mobilizing T-DNA (Vir

genes)

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auxA auxB cyt ocsLB RB

LB, RB – left and right borders (direct repeat)auxA + auxB – enzymes that produce auxincyt – enzyme that produces cytokininOcs – octopine synthase, produces octopine

T-DNA

These genes have typical eukaryotic expression signals!

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auxA auxBTryptophan indoleacetamide indoleacetic acid

(auxin)

cytAMP + isopentenylpyrophosphate isopentyl-AMP

(a cytokinin)

• Increased levels of these hormones stimulate cell division.

• Explains uncontrolled growth of tumor.

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Vir (virulent) genes

1. On the Ti plasmid

2. Transfer the T-DNA to plant cell

3. Acetosyringone (AS) (a flavonoid) released by wounded plant cells activates vir genes.

4. virA,B,C,D,E,F,G (7 complementation groups, but some have multiple ORFs), span about 30 kb of Ti plasmid.

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Vir gene functions (cont.)

• virA - transports AS into bacterium, activates virG post-translationally (by phosphoryl.)

• virG - promotes transcription of other vir genes • virD2 - endonuclease/integrase that cuts T-

DNA at the borders but only on one strand; attaches to the 5' end of the SS

• virE2 - binds SS of T-DNA & can form channels in artificial membranes

• virE1 - chaperone for virE2• virD2 & virE2 also have NLSs, gets T-DNA to

the nucleus of plant cell• virB - operon of 11 proteins, gets T-DNA

through bacterial membranes karthikumarbt@kcetvnr.org 28

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

Cellular process of Agrobacterium–host interaction

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Gauthier, A. et al. (2003) J. Biol. Chem. 278:25273-25276

Type IV Secretion Sys.

• many pathogens, also used in conjugation

• promiscuous • forms T-Pilus

• B7-B10 span OM & IM • B7-B9 in OM interacts w/B8 & B10 of IM to form channel

• 3 ATPases

• D4 promotes specific transport

• B2 can form filamentskarthikumarbt@kcetvnr.org 33

VirE2 may get DNA-protein complex across host PM

Dumas et al., (2001), Proc. Natl. Acad. Sci. USA, 98:485karthikumarbt@kcetvnr.org 34

• Monocots don't produce AS in response to wounding.

• Important: Put any DNA between the LB and RB of T-DNA it will be transferred to plant cell!

Engineering plants with Agrobacterium:

Two problems had to be overcome:(1) Ti plasmids large, difficult to manipulate(2) couldn't regenerate plants from tumors

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Binary vector system

Strategy:

1. Move T-DNA onto a separate, small plasmid.

2. Remove aux and cyt genes.

3. Insert selectable marker (kanamycin resistance) gene in T-DNA.

4. Vir genes are retained on a separate plasmid.5. Put foreign gene between T-DNA borders. 6. Co-transform Agrobacterium with both plasmids.7. Infect plant with the transformed bacteria.

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Binary vector system

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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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Viral Vectors

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Transfer into protoplasts

Gene transfer across a protoplast membrane is promoted by some chemicals such as polyethylene glycol

Vector + polyethylene glycol

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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 media

component.

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Selectable Markers

– NPTII - kanamycin (antibiotic)

– Hpt - hygromycin

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Novel Selection Genes

• Luciferase - gene from fireflies – substrate

• Green Fluorescent Protein - from jellyfish - under lights GFP will glow

• GUS - glucuronidase gene will convert added substrate (color less) to blue color.

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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)

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substrate for GUS

GUS oxidative dimerization X-glu → colourless soluble → Blue precipitate of intermediate diX-indigo

5-Bromo-4-chloro-3-indolyl β-D-glucuronide (sodium salt)

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Frequently used promoter: -> 35S promoter from cauliflower mosaic virus

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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 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

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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) μg/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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Delivery of a corn-based edible vaccine

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

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Tearless Onion

Dr EadyCrop & Food Research in New Zealand and his collaborators in Japan

As onions are sliced, cells are broken, - 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 irritantkarthikumarbt@kcetvnr.org 52

COLORED FRIUTS/FLOWERS/VEGETABLES

The-orange-purple-green-cauliflowers

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Purple tomatoes high in anthocyanins

High anthocyanin purple tomato and red wild-type tomato

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World's First Blue Roses On Display In Japan

Tokyo, Japan –

World's first blue roses have been unveiled to the publicfor 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 beenimplanted 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 Blue Rose was developed by Suntory Flowers

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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

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Biosensor

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Researcher grows roots on upper part of plant

(http://www.uu.nl/EN/Current/Pages/Researchergrowsrootsonupperpartofplant.aspx)

Pankaj Dhonukshe discovered a

molecular switch to alter the auxin transport.

By turning on the switch, it is possible 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.

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Herbicide Resistant Plants

A herbicide, commonly known as a weedkiller, is a type of pesticide used to kill unwanted plants. Selective herbicides kill specific targets while leaving the desired crop

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EPSP enzyme

Aromatic a.a.

Glyphosate ‘kills’ EPSP enzyme; so no aromatic a.a.

Not toxic to animals?

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Glyphosate Toxicity to Plants

•Slow down the growth, 3 weeks+

•Depletes aromatic a.a.

•no protein synthesis

•NH3+ accumulates (toxic)

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Herbicide Resistant Crops

•How?•Insert constitutive EPSP gene

(5-enolpyruvylshikimate-3-phosphate synthase)

•Examples•“Round-up Ready” Corn & Soybeans

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Development of Virus- resistant plants

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Human Proteins in Plants

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THANK YOU

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