Transgenic plants new

S.KARTHIKUMAR,M.Sc.,M.Phil.,M.Tech.,(PhD)
ASSISTANT PROFESSOR
DEPARTMENT OF BIOTECHNOLOGY
KAMARAJ COLLEGE OF ENGINEERING AND
TECHNOLOGY
VIRUDHUNAGAR-626001
TAMILNADU, INDIA
karthikumarbt@kcetvnr.org 1

2
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)
karthikumarbt@kcetvnr.org

3
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)

Plants
Conventional
breeding
Tissue culture
Genetic engineering
Gene – transfer methods

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Gene – transfer methods

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

Plant Transformation Methods
Physical Chemical Biological
Microinjection
Pressure
Biolistics - gene gun/
particle bombardment
Electroporation
Microinjection
Silica/carbon fibers
Lazer mediated
SAT
PEG
DEAE-dextran
Calcium phosphate
Artificial lipids
Proteins
Dendrimers
A. Tumefaciens
A. Rhizogenes
Virus-mediated

Transformation
• Plants - physical methods
• Microinjection
• Electroporation
• Biolistics - gene gun
• Silica/carbon fibers
• Lazer mediated
• SAT

Microinjection of GOI

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

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

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.

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.

Biolistic / Gene Gun

An Air Rifle for a DNA Gun –
Circa 1990
A.Thompson, Bob ?, and D. Herrinkarthikumarbt@kcetvnr.org 15

The Helium Gas Gun – Circa 2000

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.

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 dicots
rubi- causes small galls on a few dicots
rhizogenes- hairy root disease
radiobacter- avirulent

Crown galls caused by A.
tumefaciens on
nightshade.
More about Galls:
http://waynesword.palomar.edu/pljuly99.htm
http://kaweahoaks.com/html/galls_ofthe_voaks.
html

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 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

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?

Ti Plasmid
1. Large (200-kb)
2. Conjugative
3. ~10% of plasmid transferred to plant cell
after infection
4. 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)

auxA auxB cyt ocsLB RB
LB, RB – left and right borders (direct repeat)
auxA + auxB – enzymes that produce auxin
cyt – enzyme that produces cytokinin
Ocs – octopine synthase, produces octopine
T-DNA
These genes have typical eukaryotic expression signals!

auxA auxB
Tryptophan indoleacetamide  indoleacetic acid
(auxin)
cyt
AMP + isopentenylpyrophosphate  isopentyl-AMP
(a cytokinin)
• Increased levels of these hormones stimulate cell division.
• Explains uncontrolled growth of tumor.

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.

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

Tzvi Tzfira and Vitaly Citovsky, 2002, Trends in Cell Biol. 12(3), 121-129
Cellular process of Agrobacterium–host interaction

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 filaments

VirE2 may get DNA-protein complex across host PM
Dumas et al., (2001), Proc. Natl. Acad. Sci. USA, 98:485

• 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

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.

Binary vector system

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

39
Viral Vectors

40
Transfer into protoplasts
Gene transfer across a
protoplast membrane is
promoted by some chemicals
such as polyethylene glycol
Vector + polyethylene
glycol

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

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.

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)

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)

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

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

Tomatoes comes in many varieties,
colors and shapes
Transgenic tomatoes –
expressing
different malarial
antigens
Medical hypothesis, 2006

Delivery of a corn-based edible
vaccine
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,
- 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

Purple tomatoes high in anthocyanins
High anthocyanin purple tomato and red
wild-type tomato

World's First Blue Roses On Display In Japan
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 Blue Rose was
developed by Suntory
Flowers

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

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.

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

EPSP
enzyme
Aromatic
a.a.
Glyphosate ‘kills’ EPSP enzyme; so no aromatic a.a.
Not toxic to
animals?

Glyphosate Toxicity to Plants
•Slow down the growth, 3 weeks+
•Depletes aromatic a.a.
•no protein synthesis
•NH3+ accumulates (toxic)

Herbicide Resistant Crops
•How?
•Insert constitutive EPSP gene
(5-enolpyruvylshikimate-3-phosphate synthase)
•Examples
•“Round-up Ready” Corn & Soybeans

63
Development of Virus- resistant plants

Human Proteins in Plants

THANK YOU

Transgenic plants new

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