Tanaman Dan Hewan Transgenik

Genetic Engineering of Plants:
Methodology
Plant transformation with the Ti plasmid of
Agrobacterium tumefaciens
Ti plasmid derived vector systems
Physical methods of transferring genes to plants
(microprojectile bombardment)
Chloroplast engineering
Use of reporter genes in transformed plant cells
Manipulation of gene expression in plants
Production of marker-free transgenic plants
Why genetically engineer plants?

To improve the agricultural or horticultural value of
plants
To serve as living bioreactors for the production of
economically important proteins or metabolites
To provide a renewable source of energy (biofuels)
To provide a powerful means for studying the
biological action of genes and gene products
Plant transformation with the Ti plasmid of

Agrobacterium tumefaciens
A. tumefaciens is a gram-negative soil bacterium
which naturally transforms plant cells, resulting in
crown gall (cancer) tumors
Tumor formation is the result of the transfer,
integration and expression of genes on a specific
segment of A. tumefaciens plasmid DNA called the
T-DNA (transferred DNA)
The T-DNA resides on a large plasmid called the Ti
(tumor inducing) plasmid found in A. tumefaciens
The Ti plasmid of Agrobacterium tumafaciens and the

transfer of its T-DNA to the plant nuclear genome
Fig. 18.3 The Ti plasmid of Agrobacterium tumafaciens and

its T-DNA region containing eukaryotic genes for auxin,
cytokinin, and opine production.
Chapter 18
Genetic Engineering of Plants: Methodology
Figure 18.3
Ti plasmid structure
Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition

Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten
Copyright 2010 ASM Press

American Society for Microbiology
1752 N St. NW, Washington, DC 20036-2904
Chapter 18
Figure 18.1
Infection of a plant with A. tumefaciens

and formation of a crown gall tumor.


Fig. 28-27
Crown Gall on
Tobacco
Fig. 18.1 Infection of a

plant with A. tumefaciens and
formation of crown galls
Figure 18.2 and 18.3

Ti plasmid structure and
function. Note the woundinduced 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.
Chapter 18
Figure 18.4
Conserved nucleotides at the right and left borders of the Ti plasmid are imperfect
direct repeats.


Chapter 18
Figure 18.6
Chemical structures of three
opines produced by plants.


Fig. 18.7 The binary Ti plasmid system involves using a small

T-DNA plasmid (shown below) and a disarmed (i.e., no TDNA) Ti plasmid in A. tumefaciens
Clone YFG (your favorite gene) or

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.
(disarmed)
Disarmed
Ti plasmid
Plant genetic engineering with

the binary Ti plasmid system
Transgenic
plant
Chapter 18
Table 18.1


Chapter 18
Table 18.2


Fig. 18.10
Microprojectile
bombardment or
biolistic-mediated
DNA transfection
equipment
(a) lab version
(b) portable version
When the helium pressure

builds to a certain point, the
plastic rupture disk bursts, and
the released gas accelerates the
flying disk* with the DNAcoated gold particles on its
lower side. The gold particles
pass the stopping screen, which
holds back the flying disk, and
penetrate the cells of the plant.
Chapter 18
Figure 18.10
Microprojectile bombardment
(biolistics) apparatus


Chapter 18
Chloroplasts can be genetically engineered using microparticle bombardment.
Figure 18.12
Figure 18.13


Chapter 18
Table 18.5


Table 18.5 Some plant cell reporter and

selectable marker gene systems
Enzyme activity
Selectable
marker
Reporter
gene
Neomycin phosphotransferase (kanr)
Yes
Yes
Hygromycin phosphotransferase (hygr)
Yes
Yes
Nopaline synthase
No
Yes
Octopine synthase
No
Yes
b-glucuronidase (GUS)
No
Yes
Firefly luciferase
No
Yes
b-galactosidase
No
Yes
Bromoxynil nitrilase
Yes
No
Green fluorescent protein (GFP)
No
Yes
Reporter Genes
For how reporter genes work, see:
http://bcs.whfreeman.com/lodish7e/#800911__811966__
GFP Researchers Win Nobel Prize (October 8, 2008)
Osamu Shimomura, Martin Chalfie, and Roger Tsien won
the Nobel Prize in chemistry for their work on green
flourescent protein, a tool that has become ubiquitous in
modern biology as a tag and molecular highlighter, vastly
improving our ability to understand what goes on inside
cells.
Perhaps you may even want to see a 10 minute YouTube
video on GFP; if so please see
http://www.youtube.com/watch?v=Sl2PRHGpYuU
Manipulation of gene expression in plants

Strong, constitutive promoters (35S Cauliflower mosaic virus
promoter or 35S CaMV or 35S)
Organ and tissue specific promoter (e.g., the leaf-specific
promoter for the small subunit of the photosynthetic enzyme
ribulosebisphosphate carboxylase or rbc)
Promoterless reporter gene constructs to find new organ- and
tissue-specific promoter (see Fig. 18.15)
Inducible promoters
Secretion of transgene products by inclusion of a signal peptide
sequence between a root promoter and YFG and growing the
transgenic plant hydroponically (YFG product will be secreted)
Chapter 18
Figure 18.21
Rhizosecretion using a
plant promoter active in
roots and a signal peptide
sequence.


Chapter 18
Figure 18.26
Marker genes may be a safety issue, so it is best to remove themhere is one strategy.


Genetic Engineering of Plants:

Applications
Insect-, pathogen-, and herbicide-resistant plants
Stress
Genetic manipulation of flower pigmentation
Modification of plant nutritional content
Modification of plant food taste and appearance
Plant as bioreactors
Edible vaccines
Renewable energy crops
Plant yield
Genetically engineered crops/foods allowed in the US food supply
Insect-resistant plants
Bt toxin
Cowpea trypsin inhibitor
Proteinase inhibitor II
a-amylase inhibitor
Bacterial cholesterol oxidase
Combinations of the above (e.g., Bt toxin and
proteinase inhibitor II)
Genetic engineering of Bt-plants

Expression of truncated Bt genes encoding the Nterminal portion of Bt increase effectiveness
Effectiveness enhanced by site-directed mutagenesis
increasing transcription/translation
Effectiveness further enhanced by making codon bias
changes (bacterial to plant)
35S CaMV and rbcS promoters used
Integration and expression of the Bt gene directly in
chloroplasts
Note that Lepidopteran insects like corn rootworm,
cotton bollworm, tobacco budworm, etc., cause
combined damages of over $7 Billion dollars yearly in
the US
Fig. 18.7/19.3 A binary T-DNA plasmid for delivering

the Bt gene to plants (not a cointegrate vector)
(NPT or kanr)
(35S-Bt gene-tNOS)
(Spcr)
Effectiveness of insecticide and Bt-tomato plants

in resisting insect damage
% of plants or fruits damaged
Insect
wt tomato
-insecticide
wt tomato
+insecticide
Bt-tomato
-insecticide
Bt-tomato
+insecticide
Tobacco
hornworm
48
Tomato
fruitworm
20
nd
nd
Tomato
pinworm
100
95
94
80
nd, not determined
Strategies to avoid Bt resistant insects

Use of inducible promoters (that can be turned on
only when there is an insect problem)
Construction of hybrid Bt toxins
Introducing more than one Bt gene (stacking)
Introduction of the Bt gene in combination with
another insecticidal gene
Spraying low levels of insecticide on Bt plants
Use of spatial refuge strategies
Genetically engineered Bt-plants in the field

Product
Institution(s)
Engineered Trait(s)
Sources of New
Genes
Name
Corn
Bayer
Resist glufosinate herbicide to control weeds/Bt toxin to control insect pests (European corn borer)
Bacteria, virus
StarLink-1998 (animals
only)
Corn
Dow/Mycogen
Bt toxin to control insect pests (European corn borer)
Corn, bacteria, virus
NatureGard-1995
Corn
Dow/Mycogen
Resist glufosinate herbicide to control weeds/Bt toxin to control insect pests (Lepidopteran)
Herculex I-2001
DuPont/Pioneer
Corn
Monsanto/DeKalb
Bacteria
Bt-Xtra-1997
Corn
Monsanto
Bacteria
YieldGard-1996
Corn
Monsanto
Resist glyphosate herbicide to control weeds/Bt toxin to control insect pests (European corn borer)
Arabidopsis, bacteria,
virus
?-1998
Corn
Syngenta
Bacteria
Bt11-1996
Corn
Syngenta
Knock Out-1995
Corn (pop)
Syngenta
Knock Out-1998
Corn
(sweet)
Syngenta
Bacteria
Bt11-1998
Cotton
Monsanto/Bayer
Resist bromoxynil herbicide to control weeds/Bt toxin to control insect pests (cotton bollworms
Bacteria
?-1998
and tobacco budworm)

Cotton
Monsanto
Bt toxin to control insect pests (cotton bollworms and tobacco budworm)
Bacteria
Bollgard-1995
Potato
Monsanto
Bt toxin to control insect pests (Colorado potato beetle)
Bacteria
NewLeaf-1995
Potato
Monsanto
Bt toxin to control insect pests (Colorado potato beetle)/resist potato virus Y
Bacteria, virus
NewLeaf Y-1999
Potato
Monsanto
Bt toxin to control insect pests (Colorado potato beetle)/resist potato leafroll virus
Bacteria, virus
NewLeaf Plus-1998
Fig. 18.7 Procedure for putting CuMV

coat protein into plants
Virus-resistant plants
Overexpression of the virus
coat protein (e.g. cucumber
mosaic virus in cucumber
and tobacco, papaya ringspot
virus in papaya and tobacco,
tobacco mosiac virus in
tobacco and tomato, etc.)
Expression of a dsRNase
(RNaseIII)
Expression of antiviral
proteins (pokeweed)
Genetically engineered Papaya to resist the Papaya

Ringspot-Virus by overexpression of the virus coat protein
Herbicides and herbicide-resistant plants
Herbicides are generally non-selective (killing both weeds and

crop plants) and must be applied before the crop plants
germinate
Four potential ways to engineer herbicide resistant plants
1. Inhibit uptake of the herbicide
2. Overproduce the herbicide-sensitive target protein
3. Reduce the ability of the herbicide-sensitive target to
bind to the herbicide
4. Give plants the ability to inactivate the herbicide
Chapter 19
Engineering Plants To Overcome Biotic and Abiotic Stress
Table 19.3


Herbicide-resistant plants:
Giving plants the ability to inactivate the herbicide
Herbicide: Bromoxynil
Resistance to bromoxynil (a photosytem II inhibitor) was
obtained by expressing a bacterial (Klebsiella ozaenae)
nitrilase gene that encodes an enzyme that degrades this
herbicide
Herbicide-resistant plants:
Reducing the ability of the herbicide-sensitive target to bind to the
herbicide
Herbicide: Glyphosate (better known as Roundup)

Resistance to Roundup (an inhibitor of the enzyme EPSP
involved in aromatic amino acid biosynthesis) was obtained by
finding a mutant version of EPSP from E. coli that does not bind
Roundup and expressing it in plants (soybean, tobacco,
petunia, tomato, potato, and cotton)
5-enolpyruvylshikimate-3-phosphate synthase (EPSP) is a
chloroplast enzyme in the shikimate pathway and plays a key
role in the synthesis of aromatic amino acids such as tyrosine
and phenylalanine
How to make a Roundup Ready Plant
Fungus- and bacterium-resistant plants
Genetic engineering here is more challenging; however, some

strategies are possible:
Individually or in combination express pathogenesis-related (PR)
proteins, which include b1,3-glucanases, chitinases, thaumatinlike proteins, and protease inhibitors
Overexpression of the NPR1 gene which encodes the master
regulatory protein for turning on the PR protein genes
Overproducing salicylic acid in plants by the addition of two
bacterial genes; SA activates the NPR1 gene and thus results in
production of PR proteins
Development of stress- and senescence-tolerant plants:

genetic engineering of salt-resistant plants
Overexpression of the
gene encoding a Na+/H+
antiport protein which
transports Na+ into the
plant cell vacuole
This has been done in
Arabidopsis and tomato
plants allowing them to
survive on 200 mM salt
(NaCl)
Development of stress- and senescence-tolerant plants:

genetic engineering of flavorful tomatoes
Fruit ripening is a natural aging or senescence process that involves two independent
pathways, flavor development and fruit softening.
Typically, tomatoes are picked when they are not very ripe (i.e., hard and green) to allow
for safe shipping of the fruit.
Polygalacturonase is a plant enzyme that degrades pectins in plant cell walls and
contribute to fruit softening.
In order to allow tomatoes to ripen on the vine and still be hard enough for safe shipping
of the fruit, polygalacturonase gene expression was inhibited by introduction of an
antisense polygalacturonase gene and created the first commercial genetically engineered
plant called the FLAVR SAVR tomato.
Flavor development pathway

Red
Green
Fruit softening pathway

Hard
polygalacturonase
Soft
antisense polygalacturonase
Fig. 20.18 Genetic manipulation of flower pigmentation
Manipulation of the
anthocyanin
biosynthesis pathway
Introduction of maize
dihydroflavonol 4reductase (DFR) into
petunia produces a brick
red-orange transgenic
petunia
Novel flower colors in
the horticultural
industrial are big money
makers!
Note a blue rose would
make millions!
New pathway in
petunia created by
the maize DFR gene
Modification of plant nutritional content
Amino acids (corn is deficient in lysine, while legumes are

deficient in methionine and cysteine)
Lipids (altering the chain length and degree of unsaturation is
now possible since the genes for such enzymes are known)
Increasing the vitamin E (a-tocopherol) content of plants
(Arabidopsis)
Increasing the vitamin A content of plants (rice)
Modification of plant nutritional content: increasing the

vitamin E (a-tocopherol) content of plants
Plants make very little a-tocopherol
but do make g-tocopherol; they do
not produce enough of the
methyltransferase (MT)
The MT gene was identified and
cloned in Synechocystis and then in
Arabidopsis
The Arabidopsis MT gene was
expressed under the control of a
seed-specific carrot promoter and
found to produce 80 times more
vitamin E in the seeds
Dean DellaPenna, Michigan State Univ. Professor

B.S. 1984, Ohio University
Modification of plant nutritional content: increasing the

vitamin A content of plants (Fig. 20.7)
124 million children worldwide are
deficient in vitamin A, which leads
to death and blindness
Mammals make vitamin A from bcarotene, a common carotenoid
pigment normally found in plant
photosynthetic membranes
Here, the idea was to engineer the
b-carotene pathway into rice
The transgenic rice is yellow or
golden in color and is called
golden rice
GGPP
Daffodil phytoene synthase gene
Phytoene
Bacterial phytoene desaturase gene
Lycopene
Daffodil lycopene b-cyclase gene
b-carotene
Endogenous human gene
Vitamin A
Plants as bioreactors
Production of therapeutic agents (proteins)
Production of recombinant vaccines or edible
vaccines
Production of antibodies
Chapter 20
Engineering Plant Quality and Proteins
Table 20.6


Chapter 20
Table 20.7


Chapter 20
Table 20.8


Chapter 20
Table 20.9


Plants are also being genetically engineered for:

Biofuel production (e.g., lower lignin, lower
recalcitrance)
Phytoremediation (i.e., bioremediation using
plants)
Biopolymers (i.e., biodegradable plastics)
Transgenic Animals: Methodology and

Applications
Transgenic mice: methodology (Retrovirus vector, DNA
microinjection, Engineered embryonic stem cell, Cre-loxP
recombination system, High capacity vectors)
Transgenic mice: applications (Alzheimer disease, test systems,
conditional regulation, control of cell death)
Cloning livestock by nuclear transfer
Transgenic cattle, sheep, goats and pigs
Transgenic birds
Transgenic fish
Chapter 21
Transgenic Animals
Figure 21.1
Retroviral vectors can be used to create

transgenic animals


Chapter 21
Transgenic Animals
Figure 21.3
DNA microinjection is the main
method used to create transgenic
animals


Fig. 21.3 Establishing transgenic

mice by DNA microinjection
Most commonly used method
Only 5% or less of the treated eggs
become transgenic progeny
Need to check mouse pups for DNA
(by PCR or Southerns), RNA (by
northerns or RT-PCR), and protein (by
western or by some specific assay
method)
Expression will vary in transgenic
offspring: due to position effect and
copy number
Chapter 21
Transgenic Animals
Figure 21.4
Less than 5% of the
microinjected
fertilized eggs
become transgenic
progeny


Creating a transgenic mouse using the

DNA microinjection method
See
http://bcs.whfreeman.com/lodish7e/#800911__812052__
Chapter 21
Transgenic Animals
Figure 21.5
Genetically engineered embryonic
stem (ES) cells can be used to create
transgenic animals, but this method is
labor intensive and used to allow for
gene targeting via homologous
recombination.


Establishing
transgenic animals
using engineered
embryonic stem
(ES) cells
But what are ES
cells?
Transgenic animals-Engineered embryonic stem cell

method (used for gene knockouts)
Step 1: Get the ES cells (Fig. 21.5)
Step 2: Genetically engineer the ES cells

(Figs. 21.5 and 21.6)
Step 3: Place
engineered ES cells
into an early embryo
(Fig. 21.5)
see
http://bcs.whfreeman.com/lodish5e/pages/bcs-
main.asp?v=category&s=00020&n=09000&i=09020.01&o=|00510
|00610|00520|00530|00540|00560|00570|00590|00600|0070
0|00710|00010|00020|00030|00040|00050|01000|02000|030
00|04000|05000|06000|07000|08000|09000|10000|11000|12
000|13000|14000|15000|16000|17000|18000|19000|20000|2
1000|22000|23000|99000|&ns=486
Transgenic
animals-Using CreloxP for tissue or
time-specific gene
knockouts
Transgenic mice can be produced with high

capacity vectors
Generally done by microinjection of numerous genes
contained in a YAC
Production of mice that can produce human
antibodies is one notable example
Transgenic mice/animal: applications

Transgenic models for Alzheimer disease, amyotrophic lateral
sclerosis, Huntington disease, arthritis, muscular dystrophy,
tumorigenesis, hypertension, neurodegenerative disorders,
endocrinological dysfunction, coronary disease, etc.
Using transgenic mice as test systems (e.g., protein [CFTR] secretion
into milk, protection against mastitis caused by Staphylococcus
aureus using a modified lysostaphin gene)
Conditional regulation of gene expression (tetracycline-inducible
system in Fig. 21.19)
Conditional control of cell death (used to model and study organ
failure; involves the organ-specific engineering of a toxin receptor
into the mice and then addition of the toxin to kill that organ)
And then there is transgenic art with GFP
Fig. 21.22 Cloning

livestock by nuclear
transfer (e.g., sheep)
Hello Dolly
Transgenic cattle, sheep,

goats, and pigs
Using the mammary gland as a
bioreactor (see adjacent figure)
Increase casein content in milk
Express lactase in milk (to remove
lactose)
Resistance to bacterial, viral, and
parasitic diseases
Reduce phosphorous excretion
Table 21.2 Some human proteins expressed in

the mammary glands of transgenic animals
Erythropoietin
Factor IX
Factor VIII
Fibrinogen
Growth hormone
Hemoglobin
Insulin
Monoclonal antibodies
Tissue plasminogen activator (TPA)
a1-antitrypsin
Antithrombin III (the first transgenic animal drug, an
anticlotting protein, approved by the FDA in 2009)
Enviropigs
Transgenic pigs expressing the
phytase gene in their salivary glands
The phytase gene was introduced via
DNA microinjection and used the
parotid secretory protein promoter
to specifically drive expression in the
salivary glands
Phytate is the predominant storage
form of phosphorus in plant-based
animal feeds (e.g., soybean meal)
Pigs and poultry cannot digest
phytate and consequently excrete
large amounts of phosphorus
Enviro-pigs excrete 75% less
phosphorus
Microinjected an E. coli phytase
gene under the control of a mouse
parotid secretory protein promoter
EnviropigTM an environmentally friendly

breed of pigs that utilizes plant
phosphorus efficiently.
Fig. 21.32 Establishing

transgenic chickens by
transfection of isolated
blastoderm cells
Resistance to viral, bacterial,
and coccidial diseases
Better feed efficiency
Lower fat and cholesterol
levels in eggs
Better meat quality
Eggs with pharmaceutical
proteins in them
Transgenic fish
Genes are introduced into fertilized eggs by DNA microinjection or
electroporation
No need to implant the embryo; development is external
Genetically engineered for more rapid growth using the growth hormone
gene (salmon, trout, catfish, tuna, etc.)
Genetically engineered for greater disease resistance
Genetically engineered to serve as a biosensor for water pollution
Genetically engineered for a novel pet (Glofish-see http://glofish.com/)
Transgenic fish (more detail)

Salmon were genetically engineered for more rapid growth using the growth
hormone gene under the control of the ocean pout antifreeze protein gene
promoter and 3 untranslated region (currently under FDA consideration)
Madaka fish were genetically engineered to serve as biosensors for
environmental pollutants (e.g., estrogens) by using an estrogen-inducible
promoter (the vitellogenin promoter) to control expression of the GFP gene
Fig. 21.33
Fig. 21.34

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Genetic Engineering of Plants:

Why genetically engineer plants?

Plant transformation with the Ti plasmid of

The Ti plasmid of Agrobacterium tumafaciens and the

Fig. 18.3 The Ti plasmid of Agrobacterium tumafaciens and

Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition

Copyright 2010 ASM Press

Infection of a plant with A. tumefaciens

Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition

Copyright 2010 ASM Press

Fig. 18.1 Infection of a

Figure 18.2 and 18.3

The infection process:

Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition

Copyright 2010 ASM Press

Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition

Copyright 2010 ASM Press

Fig. 18.7 The binary Ti plasmid system involves using a small

Clone YFG (your favorite gene) or

Plant genetic engineering with

Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition

Copyright 2010 ASM Press

Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition

Copyright 2010 ASM Press

When the helium pressure

Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition

Copyright 2010 ASM Press

Chloroplasts can be genetically engineered using microparticle bombardment.

Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition

Copyright 2010 ASM Press

Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition

Copyright 2010 ASM Press

Table 18.5 Some plant cell reporter and

Neomycin phosphotransferase (kanr)

Hygromycin phosphotransferase (hygr)

Green fluorescent protein (GFP)

Manipulation of gene expression in plants

Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition

Copyright 2010 ASM Press

Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition

Copyright 2010 ASM Press

Genetic Engineering of Plants:

Genetically engineered crops/foods allowed in the US food supply

Genetic engineering of Bt-plants

Fig. 18.7/19.3 A binary T-DNA plasmid for delivering

Effectiveness of insecticide and Bt-tomato plants

nd, not determined

Strategies to avoid Bt resistant insects

Genetically engineered Bt-plants in the field

Bt toxin to control insect pests (European corn borer)

Corn, bacteria, virus

Corn, bacteria, virus

Bt toxin to control insect pests (European corn borer)

Bt toxin to control insect pests (European corn borer)

Bt toxin to control insect pests (European corn borer)

Bt toxin to control insect pests (European corn borer)

Corn, bacteria, virus

Bt toxin to control insect pests (European corn borer)

Corn, bacteria, virus

Bt toxin to control insect pests (European corn borer)

and tobacco budworm)

Bt toxin to control insect pests (cotton bollworms and tobacco budworm)

Bt toxin to control insect pests (Colorado potato beetle)

Bt toxin to control insect pests (Colorado potato beetle)/resist potato virus Y