Transgenic Plants

5/25/2018 Transgenic Plants

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BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Transgenic Plants

Further Readings:

Recombinant DNA (2nd ed.)

by Watson et al., Ch.15http://www.uoguelph.ca/~jdberg/plantran.htm

http://www.isaaa.org


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leaf

protoplast

Clonal

cell line

Selected plant cultivar

Stages in

embryoidformation

plantlet

Tissue

explant

Callus

culture

Shoot

formation

Root

formation

Meristem

explant

plantletplantlet

Shoot

formation

Rootformation

plants

Micromanipulation of Plant Tissue Culture


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Increasing auxin concentration

Inc

reasing

cytokinin

concentration

Regeneration is Induced by Plant Hormones


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A biological method

Agrobacteria and crown gall tumors(www.ppws.vt.edu/~sforza/agro/agro2002b.mov)

Agrobacteria --> Ti-plamsid --> T-DNA

--> integrate into plants Can adopt for plant gene transfer

(www.ppws.vt.edu/~sforza/agro/agro_lab2002b.mov)

Agrobacteria-Mediated Transformation


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Transformation Methods

Explants (Leave discs or roots) Vacuum infiltration (flowering buds and

seeds)


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Agrobacteria: genes for plant-

Agrobacteria interaction (chvA, chvB,

exoC, att: stable binding to plant cells;

ros: efficiency of expression of some of

the genes in the virregion of the Ti-plasmid)

Three Genetic Components of T-DNA Transfer


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Ti-plasmid (outside T-DNA region):

genes for octopine and nopaline catabolism

vir(virulence) genes, switched on by

chemicals from wounded plant cells, help to

excised T-DNA from the plasmid

plant wound --> phenolics --> sense by VirA

--> signal passed to VirG --> VirG activates

expression of other virgenes --> theproducts of virgenes help to excised T-DNA

from the Ti-plamsid



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T-DNA

genes for biosynthesis of nopaline andoctopine

genes for phytohormone biosynthesis

(tms: auxins and tmr: cytokinins) -->unregulated growth of plant cells



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Ti Plasmids as Vectors for

Plant Gene Transfer

Key: any DNA flanked by the two

borders of T-DNA can be

integrated into the plant genome


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Cointegration

Vector

Intermediate shuttle vector

Cloning site

T-DNA

E. coli

plasmid

fragment

Selectable marker forAgrobacterium

(e.g. KanR

)

Marker for selection in plants (e.g. NTPII

gene for KanR)

Gene of interest

Ligate into cloning site

Transform into E. coli

Select for marker on E. coli

plasmid fragment (e.g. AmpR

colonies)


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Infect plant cells

or via electroporation

Mate with

Agrobacterium

T-DNA

Ti plasmid

Agrobacterium

E. coli

Agrobacterium

RecombinantAgrobacterium

T-DNA plasmid integrates into Ti plasmid by

homologous recombination

Cointegration

Vector


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LB Cloning site

RBBacterial origin of

replication

Marker for selection in plants (e.g. NPTII gene for KanR)Marker

for

selection

in

bacteria

Ligate into cloning site

Gene of interest

LB

RB

NPTII

Transform into E. coli and select transformants

E. coli

Transfer intoAgrobacterium plasmid by

mating or electroporation

VirgenesAgrobacterium containing

recombinant plasmid and vir

helper plasmid

Binary

Vectors


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Infect callus generated from explants

T-DNA is transferred to plant cell

Virhelper plasmid supplies

proteins needed for DNA

transfer

Select KanR

plant cells

Regenerateplant

Integrated T-DNA

Binary

Vectors

Agrobacterium containing

recombinant plasmid and virhelper plasmid


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Gene Gun Microprojectile-mediated transformation,

a physical method Good for plants that cannot be

transformed byAgrobacterium, e.g.

most monocot

Good for transient and rapid assays

Low frequency of stable and inheritableintegration of DNA into plant genome;

may get multiple insertions


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Gene Gun


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Promoter for plant expression

Cloned gene

Bacterial sequences

Precipitate DNA

onto particles

Load into particle gun

Tungstenmicroparticles

1m


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Biolistic PDS-1000/He System


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Biolistic PDS-1000/He System


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Helios Gene Gun System


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Other Methods for Plant Gene Transfer

PEG-Mediated Transformation

Digest cells with cellulase to getprotoplasts

PEG induces reversible

permeabilization of the plasma

membrane

Low transformation efficiency (1-2%)Difficult to regenerate

Modify from http://www.uoguelph.ca/~jdberg/plantran.htm


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Preparation of Protoplasts


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Preparation of Protoplasts


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Liposome

Targeted DNA encapsulated in aspherical lipid bilayer termed a

liposome

In the presence of PEG, endocytosis

occurs.

After endocytosis, the DNA is free torecombine and integrate with the host

genome.



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Electroporation

Intensive electrical field leads topores on plasma membrane, allowing

DNA to enter

Protoplast regeneration is still a

problem



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Silicon Carbide Fibers

Use silicon carbide fibers to punchholes through cultured plant cells

Silicon carbide fibers and cultured

plant cells are added to a tube and

vortexed vigorously

The mechanical force generated bythe vortex drives the fibers into the

cellModify from http://www.uoguelph.ca/~jdberg/plantran.htm


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Microinjection

Uses fine glass needles to inject theforeign DNA directly into the host cell

Developed to inject DNA into

protoplasts, cultured embryonic cell

suspensions and multicellular

structures.Time consuming



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Electrophoresis

Meristematic tissue subtended by two tubes

DNA is mixed with agar, poured into an open-

ended tube containing the cathode and just

agar is poured into the tube containing the

anode.

Under an optimized electric field, the DNA

passes through the agar, onto the tissue,

passes between the cellulose fibers of the

cell wall and into the cell


BIO4320 L t M t i l P d b D H Mi L


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Laser Microbeam

The laser pokes precise tiny holes inthe cells allowing plasmid DNA to be

taken up

Not yet successful in transforming

plants


BIO4320 Lecture Materials Prepared by Dr Hon Ming Lam


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Desiccation

Dried embryos can be mixed with anutrient solution containing the

foreign DNA

The DNA would be taken up as the

embryo rehydrates and seedlings can

be germinated in the presence of aselection medium to assess the

incorporation of the foreign DNA.


BIO4320 Lecture Materials Prepared by Dr Hon Ming Lam


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Possible Applications in Agriculture

Solving agricultural problems

Environmental stress tolerance:

temperature, water, salinity, heavy

metals, etc.Biological stress resistance: weeds,

insects, bacteria, fungi, viruses, etc.

Nutritional value enhancement Shelf life extension

Yield improvement

BIO4320 Lecture Materials Prepared by Dr Hon-Ming Lam


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Minghui63 control

Minghui63/Bt

Insect-Resistant Rice

Courtesy of Prof. Jumin Tu

BIO4320 Lecture Materials Prepared by Dr Hon-Ming Lam


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BIO4320 Lecture Materials, Prepared by Dr. Hon Ming Lam

Insect-Resistant Rice

Courtesy of Prof. Jumin Tu

Transgenic

Contol



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BIO4320 Lecture Materials, Prepared by Dr. Hon Ming Lam

Virus Resistant Papaya

Before InoculationBefore Inoculation

Courtesy of Prof. Robert E. Paull



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, p y g

Virus Resistant Papaya

After InoculationAfter Inoculation

Courtesy of Prof. Robert E. Paull



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p y g

Virus Resistant Tomato

Regular Tomato Genetically Engineered

Virus Resistant Tomato

Courtesy of Prof. Samuel Sun



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Herbicide Tolerant Soybean



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metals, etc.

Biological stress resistance: weeds,



Yield improvement



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Vitamin A Enriched Rice



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Quality Improved Oil



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metals, etc.




Yield improvement



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Delay Ripening Tomato



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metals, etc.




Yield improvement



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Three Maize C4

Photosynthesis GenesWere Introduced into Rice

PEPC: phosphoenolpyruvate carboxylaxse

(cytosolasm)PEP + HCO3- oxaloacetate

PPDK: pyruvate, orthophosphate dikinase(chloroplast)

pyruvate + ATP PEP + AMP + PPi

NADP-ME: NADP-malic enzyme (chloroplast)

malate + NADP pyruvate + CO2 + NADPH

Courtesy of Prof. Maurice Ku



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Photosynthetic Performance in the Field




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Growth

Wild-type PPDK

PEPC NADP-ME




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Grain Yield Field Trials

Transgenic rice expressing both maize PEPC and PPDK producedhigher grain yields (30-90%), especially under adverse conditions.

WT

PC PKC/K

WT

PC

PK

C/K




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Other Applications of Transgenic Plants

Male sterility

Horticulture

Biodegradable plastics

Bioreactors



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Male Sterile Flower



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Altering Flower Color, Shape and Shelf-Life



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Altering Flower Color, Shape and Shelf-Life



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Biodegradable Plastics



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Plant Bioreactors

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Transgenic Plants

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