Plant Genetic Transformation

All stable transformation methods consist of three steps:

• Delivery of DNA into a single plant cell.

• Integration of the DNA into the plant cell genome.

• Conversion of the transformed cell into a whole plant.

Agrobacterium-mediated Transformation

Only known natural example of DNA transport between Kingdoms

1. (Virulent) strains of A. tumefaciens contain a 200-kb tumor inducing (Ti) plasmid

2. Bacteria transfer a portion of the plasmid DNA into the plant host (T-DNA).

T-DNA

•Infects at root crown or just below the soil line.

•Can survive independent of plant host in the soil.

•Infects plants through breaks or wounds.

•Common disease of woody shrubs, herbaceous plants, dicots.

•Galls are spherical wart-like structures similar to tumors.

The T-DNA is transferred from the Bacteria into the Nucleus of the

Plant1. Stably integrates (randomly) into the plant genome.

2. Expression of genes in wild-type T-DNA results in dramatic physiological changes to the plant cell.

3. Synthesis of plant growth hormones (auxins and cytokinins) neoplastic growth (tumor formation)

Genes required to breakdown Genes required to breakdown opinesopines for use for use as a as a nutrient source nutrient source are harbored on the are harbored on the Ti Ti plasmidplasmid in addition to in addition to virvir genes essential for genes essential for the the excisionexcision and and transporttransport of the of the T-DNA T-DNA to to the the wounded plant cellwounded plant cell..

T-DNA

vir genes

opine catabolism

pTipTi~200 kb~200 kb

tra

for transfer to the plant

bacterial conjugation

23 kb23 kb

vir genes

opine catabolism

pTipTitra

for transfer to the plant

bacterial conjugation

Agrobacterium chromosomal DNAAgrobacterium chromosomal DNA

chvA chvB

pscA

oriV

T-DNA-inserts into plant genome

Ti Plasmid

Ti Plasmid

Agrobacterium can be used to transfer DNA into plants

1. Agrobacterium tumefaciens chromosomal genes: chvA, chvB, pscA required for initial binding of the bacterium to the plant cell and code for polysaccharide on bacterial cell surface.

2. Virulence region (vir) carried on pTi, but not in the transferred region (T-DNA). Genes code for proteins that prepare the T-DNA and the bacterium for transfer.

Ti plasmids and the bacterial Ti plasmids and the bacterial chromosome act in concert to chromosome act in concert to

transform the planttransform the plant

3. T-DNA encodes genes for opine synthesis and for tumor production.

4. occ (opine catabolism) genes carried on the pTi allow the bacterium to utilize opines as nutrient.

Generation of the T-strandGeneration of the T-strand

overdrive

Right Border

Left Border T-DNA

virD/virC

VirD nicks the lower strand (T-strand) at the right border sequence and binds to the 5’ end.

5’

Generation of the T-strandGeneration of the T-strand

Right border

Left border

DvirD/virC

gap filled in

T-strand

T-DNA

virE

1. Helicases unwind the T-strand which is then coated by the virE protein.

2. ~one T-strand produced per cell.

1. Transfer to plant cell.2. Second strand synthesis3. Integration into plant chromosome

Right border

Left border

D

T-strand coated with virE

T-DNA

virD nicks at Left Border sequence

Overview of the Infection Process

pTi-based vectors for plant pTi-based vectors for plant transformation:transformation:

2. Early shuttle vectors integrated into the T-DNA; still produced tumors.

1. Shuttle vector is a small E. coli plasmid using for cloning the foreign gene and transferring to Agrobacterium.

E. coli Agrobacterium

pTiShuttle plasmid

conjugation

Transformation of Arabidopsis plants

Dip floral buds in 1 ml of Agrobacterium culture for 5 to 15 min.

Detergent added to allow bacteria to infiltrate the floral meristem.

Transformation of Arabidopsis plants

700 to 900 seeds per plant.

Germinate on kanamycin plates to select transformants.

10 to 20 transformed plants per plant.

10 day old seedlings

MiniTi T-DNA based vector for plants

1. 1. Binary vectorBinary vector: the : the virvir genes required for genes required for mobilization and transfer to mobilization and transfer to the plant reside on a the plant reside on a modified pTimodified pTi..

2. consists of the 2. consists of the right and right and left border sequencesleft border sequences, a , a selectable marker selectable marker (kanomycin resistance) and (kanomycin resistance) and a a polylinkerpolylinker for insertion of for insertion of a foreign gene.a foreign gene.

Disarmed vectors: do not produce tumors; can be used to regenerate normal plants containing the foreign gene.

miniTi

MiniTi T-DNA based vector for plants

modified Ti plasmid

a binary vector system

oriVoriV

virvir

T-DNA deleted

2

LBRB

oriori

kanr polylinker

miniTiminiTi

bombom1

bom = basis of mobilization

Alternate Methods of Transforming Plants: Particle Bombardment

One way of physically introducing DNA into cells is with a particlegun.              

•Very tiny DNA-coated metal particles are suspended in a drop on a macroprojectile.

•A discharge (from a gunpowder explosion or from breakage of a membrane enclosing a pressurized chamber) impels the macroprojectile.

•The macroprojectile is stopped by a stopping plate, but the microprojectiles continue into the tissue below.

•The DNA introduced with the particles is expressed

1. DNA- or RNA-coated gold/tungsten particles are loaded into the gun and you pull the trigger.

Particle Bombardment using the Gene Gun