5/19/2016

GENETIC MODIFIED
ORGANISM

PLANT GENETIC
ENGINEERING

Agustina Setiawati

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Why Change a Plant’s DNA?

Can change plant so that it has new or different
characteristics
Stress resistance (cold, drought, disease...)
Insect resistance (Bt toxin)
Herbicide resistance (Round-up)

GM crops by country...

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Common GM Crops in the U.S.

% of Total US Acres

http://blog.wire
d.com/wiredsci
ence/2007/09/
monsanto-is-
hap.html

Example: What are uses GM Plant?

Nutraceutical
Golden rice
Vitamin A enriched

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The Golden Rice Story

• Vitamin A deficiency is a major health problem

• Causes blindness
• Influences severity of diarrhea, measles

• >100 million children suffer from the problem

• For many countries, the infrastructure doesn’t exist

to deliver vitamin pills

• Improved vitamin A content in widely consumed

crops an attractive alternative

β-Carotene Pathway Problem in Plants

IPP

Geranylgeranyl diphosphate

Phytoene synthase

Phytoene
Problem: Phytoene desaturase
Rice lacks
these enzymes ξ-carotene desaturase

Lycopene
Lycopene-beta-cyclase
Normal
Vitamin A β -carotene
“Deficient” (vitamin A precursor)
Rice

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The Golden Rice Solution

β-Carotene Pathway Genes Added

IPP

Geranylgeranyl diphosphate

Daffodil gene Phytoene synthase

Phytoene
Vitamin A
Phytoene desaturase
Pathway Single bacterial gene;
is complete performs both functions
and functional ξ-carotene desaturase

Lycopene
Daffodil gene Lycopene-beta-cyclase

Golden β -carotene
Rice (vitamin A precursor)

What are the Uses of GM Plants?

Bioreactors / Molecular farming

Therapeutic proteins
Human lactoferrin to treat iron deficiencies
Antibodies
Vaccine production
Antigen expression
HepC, HIV
Antibody-producing tomato plant
Nicholas Ewing
California State University,
Sacramento

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Agrobacterium sp
Gram negative soil borne bacterium
Causes crown gall tumours
Mempunyai plasmid Ti yang bisa dipindahkan ke sel
tanaman

Crown-gall Hairy-root
disease disease
A.tumefaciens A.rhizogenes

Agrobacterium infect plants, inserting some of their DNA

into the plants genome and forming a gall.

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Fig. 28-27

Crown Gall on
Tobacco

Fig. 18.1 Infection of a

plant with A. tumefaciens and
formation of crown galls

Agrobacterium infect plants, inserting some of their DNA

into the plants genome.

Fig 20.25

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After inserting a gene into the Agrobacteria, they will insert

that gene into the plant's DNA.

Fig 20.25

Plasmid Ti

The production of phytohormon prevent being regenerated into

mature plants
Ti-plasmid are large (200 to 800 kb)
Need to developed new strategy

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Figure 18.2 and 18.3

Ti plasmid structure and
function. Note the wound-
induced plant phenolics
induce the vir genes on
the Ti plasmid.

The infection process:

1. Wounded plant cell releases phenolics and nutrients.
2. Phenolics and nutrients cause chemotaxic response of A. tumefaciens
3. Attachment of the bacteria to the plant cell.
4. Certain phenolics (e.g., acetosyringone, hydroxyacetosyringone) induce vir gene
transcription and allow for T-DNA transfer and integration into plant chromosomal DNA.
5. Transcription and translation of the T-DNA in the plant cell to produce opines (food) and
tumors (housing) for the bacteria.
6. The opine permease/catabolism genes on the Ti plasmid allow A. tumefaciens to use
opines as a C, H, O, and N source.

Chemical structures of three

opines produced by plants.

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1. pTi-based vectors for plant transformation

(Co-integrative Strategy)
1. Shuttle vector is a small E. coli plasmid using for
cloning the foreign gene and transferring to
Agrobacterium.
2. Shuttle vector integrated to T-DNA

Shuttle plasmid pTi

conjugation
E. coli Agrobacterium

2. Binary Strategy

Clone the target gene in the small T-DNA

plasmid in E. coli, isolate the plasmid
and use it to transform the disarmed
A. tumefaciens as shown.

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Selectable Marker & Reporter Gene

1. Selectable Marker : a gene used to determine if a nucleic acid sequence
has been successfully inserted into an organism's DNA eq: antibiotic
resistance gene
2. Reporter gene : a gene that is used to test the strength and/or
regulation of a regulatory sequence that they are fused to eq: GFP

Some plant cell reporter and selectable

marker gene systems
Enzyme activity Selectable Reporter
marker gene
Neomycin phosphotransferase (kanr) Yes Yes
Hygromycin phosphotransferase (hygr) Yes Yes
Nopaline synthase No Yes
Octopine synthase No Yes
β-glucuronidase (GUS) No Yes
Firefly luciferase No Yes
β-galactosidase No Yes
Bromoxynil nitrilase Yes No
Green fluorescent protein (GFP) No Yes

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TRANSFORMATION

Metode transformasi pada GM plant:

1. Co-cultivation
2. Electroporation
3. Biolistic transformation – “Gene gun”

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Co-cultivation
Bacterial
Ti Plasmid chromosome
Agrobacterium
contains Ti plasmid
recombinant

Co-cultivation of the
Agrobacterium with
plant pieces transfers
the DNA Petri dish
with leaf pieces
plus Agrobacterium

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Electroporation

Prinsip: pembukaan membran pembentukan pori sel

tanaman dengan muatan listrik
DNA in the surrounding solution can enter the cell
through these pores and become incorporated into the
cell’s nuclear genome through illegitimate recombination

Biolistic transformation – “Gene gun”

DNA is precipitated on the

surface of heavy metal (gold;
tungsten) particles
Loaded particles are driven
into plant cells by high velocity
gas propulsion (originally
gunpowder; now helium)
Distance between discharge
nozzle and tissue can be
optimized, as can particle
velocity
Target tissue must be
regenerable

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Microprojectile bombardment
(biolistics) apparatus

EDIBLE VACCINE
VIRUS-RESISTENT PLANT

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This was the first “genetically modified” food approved by the

FDA in 1994. It was eventually pulled off the market in 1997
because of the controversy surrounding it. Questions arose
about it’s effects on human health, the environment, potential
gene transfer, and the creation of “Frankenfood”.

Traditional

The traditional tomato must be

harvested while it is still green
and firm so that it is not crushed
on the way to the supermarket.

The traditional tomato is

sprayed with ethylene
after shipping to induce
ripening.

Ripe but decreased Flavour.

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Antisense Technology
• Flavr SavrTM tomato
introduced in 1994
• Ripe tomatoes normally
produce the enzyme,
polyglacturonase (PG)
which digests pectin
• Scientists isolated the PG
gene, produced a
complementary gene which
produces a complementary
mRNA that binds to the
normal mRNA inactivating
the normal mRNA for this
enzyme

Flavr Savr

The Flavr Savr tomato ripens

on the vine – resulting in fuller
flavour. It is modified so that it
remains firm after harvesting.

Ripe and Increased Flavour.

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Methods Used in Plant Transgenesis

RNA Interference (RNAi)

• Inhibits gene expression by
interfering with transcription or
translation of RNA molecules

Practical Applications in the Field

Genetic Pesticides
• Bacillus thuringiensis (Bt)
produces a protein that is
toxic to plant pests
• Transgenic plants contain
the gene for the Bt toxin
and have a built-in defense
against these plant pests

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What exactly are

“edible vaccines?”
• Biopharmaceuticals
• Plants or crops that produce human vaccines
• The next generation of vaccines

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VIRUS RESISTANT PLANT

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Transgenic Animals

SOMATIC NUCLEAR CELL TRANSFER

Transgenic animal was

constructed based on
SNCT by Robert et al
(1952)

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DOLLY SHEEP, first succeed cloning

Uses for transgenic animals

Gene pharming

Food/Feed
Xenotransplantation

Industrial

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Transgenic Animal

The foreign gene is constructed using recombinant

DNA technology.
In addition to a structural gene, the DNA usually
includes other sequences to enable it to be
incorporated into the DNA of the host, and to be
expressed correctly by the cells of the host.

Recombinant protein production in the milk of

a transgenic sheep

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Knock-in

A new gene is added (knocked in) by random

insertion into the genome of the host organism.
Your goal is to express that gene, but you don’t
care where it ends up in the genome.
Circular plasmid construct DNA can break anywhere in its
sequence and insert anywhere into the recipient cell genome.
Usually performed by microinjection into one of
the two pronuclei of a newly fertilized egg.
Example:
Knock in a β-galactosidase gene driven by a promoter being
tested for tissue specificity.
Watch where and when the β-galactosidase is expressed.

TRANSGENIC ANIMALS

“A little bit of this,

and a little bit of
that?”

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Methods of producing transgenic animal

1. DNA microinjection

2. Embryonic stem (ES) cell transfer

3. Retroviral infection

Source: A.Yuswanto, Fac. of Pharmacy, UGM

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Microinjection

Male pronuclei

Pregnant mare serum gonadotropin follicular

50 Human chorionic gonadotropin ovulation
50

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51 A.Yuswanto, Fac. of Pharmacy, UGM

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Retroviral infection

Drawback ?

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Embryonic stem (ES) cell transfer

53 Source: A.Yuswanto, Fac. of Pharmacy, UGM 53

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Pronuclear injection.

High efficiency for

random knock-ins.

How to analyse transgenic mice

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Knock-out transgenic

A gene of the host organism is inactivated (knocked

out) by insertion of a foreign sequence.
A mutant allele replaces the normal one by
homologous recombination.
This is known as “targeted” insertion of a gene.
Targeting involves incorporating sequence identical to the
target gene in the vector.
Successful homologous recombination is rare and must be
Selected and
Screened

KNOCKOUT MICE

Normal (+) gene X

Isolate gene X
Genome and insert it into vector.
Inactivate the gene
by inserting a marker gene
that make cell resistent
to antibiotic (e.g. puromycin)
Defective Transfer vector with (-) gene X
(-)
Gene X into ES cells
(embryonic stem)
VECTOR

MARKER GENE e.g.(NeoR)

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TERIMA KASIH

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