transgenic plants

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

Transgenic PlantsFurther Readings:

“Recombinant DNA (2nd ed.)”by Watson et al., Ch.15

http://www.uoguelph.ca/~jdberg/plantran.htmhttp://www.isaaa.org

leaf

protoplast

Clonalcell line

Selected plant cultivar

Stages in embryoidformation

plantlet

Tissue explant

Callus culture

Shoot formation

Root formation

Meristemexplant

plantletplantlet

Shoot formation

Root formation

plants

Micromanipulation of Plant Tissue Culture


Increasing auxin concentration

Incr

easi

ng c

ytok

inin

conc

entra

tion

Regeneration is Induced by Plant Hormones


• 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


Transformation Methods

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

seeds)


• 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 vir region of the Ti-plasmid)

Three Genetic Components of T-DNA Transfer


• 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 vir genes --> the products of vir genes help to excised T-DNA from the Ti-plamsid



• T-DNA– genes for biosynthesis of nopaline and

octopine– genes for phytohormone biosynthesis

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



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


CointegrationVector

Intermediate shuttle vector

Cloning site

T-DNA

E. coliplasmid fragment

Selectable marker for Agrobacterium(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. coliplasmid fragment (e.g. AmpR

colonies)


Infect plant cells

or via electroporation

Mate with Agrobacterium

T-DNA

Ti plasmid

Agrobacterium

E. coli

Agrobacterium

Recombinant Agrobacterium

T-DNA plasmid integrates into Ti plasmid by homologous recombination

CointegrationVector


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 siteGene of interest

LB

RB

NPTII

Transform into E. coli and select transformants

E. coli

Transfer into Agrobacterium plasmid by mating or electroporation

Vir genesAgrobacterium containing recombinant plasmid and virhelper plasmid

Binary Vectors


Infect callus generated from explants

T-DNA is transferred to plant cell

Vir helper plasmid supplies proteins needed for DNA transfer

Select KanR

plant cellsRegenerate plant

Integrated T-DNA

Binary Vectors

Agrobacterium containing recombinant plasmid and virhelper plasmid


Gene Gun• Microprojectile-mediated transformation,

a physical method• Good for plants that cannot be

transformed by Agrobacterium, e.g. most monocot

• Good for transient and rapid assays• Low frequency of stable and inheritable

integration of DNA into plant genome; may get multiple insertions


Gene Gun

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming LamPromoter for plant expression

Cloned gene

Bacterial sequences

Precipitate DNA onto particles

Load into particle gun

Tungsten microparticles

1μm


Biolistic PDS-1000/He System


Helios Gene Gun System


Other Methods for Plant Gene Transfer

• PEG-Mediated Transformation– Digest cells with cellulase to get

protoplasts– 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


Preparation of Protoplasts



• Liposome– Targeted DNA encapsulated in a

spherical lipid bilayer termed a liposome

– In the presence of PEG, endocytosisoccurs.

– After endocytosis, the DNA is free to recombine and integrate with the host genome.




• Electroporation– Intensive electrical field leads to

pores on plasma membrane, allowing DNA to enter

– Protoplast regeneration is still a problem




• Silicon Carbide Fibers– Use silicon carbide fibers to punch

holes through cultured plant cells– Silicon carbide fibers and cultured

plant cells are added to a tube and vortexed vigorously

– The mechanical force generated by the vortex drives the fibers into the cell




• Microinjection– Uses fine glass needles to inject the

foreign DNA directly into the host cell– Developed to inject DNA into

protoplasts, cultured embryonic cell suspensions and multicellularstructures.

– Time consuming



Other Methods for Plant Gene Transfer• 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




• Laser Microbeam– The laser pokes precise tiny holes in

the cells allowing plasmid DNA to be taken up

– Not yet successful in transforming plants




• Desiccation– Dried embryos can be mixed with a

nutrient solution containing the foreign DNA

– The DNA would be taken up as the embryo rehydrates and seedlings can be germinated in the presence of a selection medium to assess the incorporation of the foreign DNA.



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


Minghui63 control

Minghui63/Bt

Insect-Resistant Rice

Courtesy of Prof. Jumin Tu


Insect-Resistant Rice

Courtesy of Prof. Jumin Tu

Transgenic

Contol


Virus Resistant Papaya

Before InoculationBefore Inoculation

Courtesy of Prof. Robert E. Paull


Virus Resistant Papaya

After InoculationAfter Inoculation

Courtesy of Prof. Robert E. Paull


Virus Resistant Tomato

Regular Tomato Genetically EngineeredVirus Resistant Tomato

Courtesy of Prof. Samuel Sun


Herbicide Tolerant Soybean


Vitamin A Enriched Rice


Quality Improved Oil


Delay Ripening Tomato


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


Photosynthetic Performance in the Field



Growth

Wild-type PPDK

PEPC NADP-ME



Grain Yield – Field Trials

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

WTPC PK

C/K

WTPC

PK

C/K



Other Applications of Transgenic Plants• Male sterility• Horticulture• Biodegradable plastics• Bioreactors


Male Sterile Flower


Altering Flower Color, Shape and Shelf-Life


Biodegradable Plastics


Plant Bioreactors

transgenic plants

Documents

honming lam tdna genes

tdna region

seedsbio4320 lecture

honming lam agrobacteria

honming lam tiplasmid

recombinant dna

honming lamti plasmids

plasmid plant wound