Plant transformation based on direct DNA delivery

1.Polyethylene glycol (PEG)-mediated protoplast transformation.2.Particle bombardment3.Electroporation4.Microinjection5.Ultrasound mediated6.Pollen-mediated7.Cell penetrating peptides

PEG-mediated protoplast transformation

It is the oldest (direct DNA) reliable method for plant transformation. In the first report (Krens et al. 1982 Nature 296:72), Agrobacterium Ti plasmid was introduced into petunia protoplasts. Formation of tumors, opine synthesis and Southern blot provided the verification, which is an extensive and complete analysis to show success of transformation.

The first report of generating transgenic plants using this method was provided by Paszkowski et al. (1984). They regenerated transformed protoplasts into plants that were kanamycin resistant.

Protoplasts are treated with DNA in the presence of PEG and Ca++

This method has been very useful and applied to several plant species.

But it is a tedious procedure!And it is necessary to be able to produce protoplasts and to to regenerate from them.

Electroporation• Use on cells without walls

Use on cells without walls (plant protoplasts or animal cells).

• High-voltage pulses cause pores to form transiently in cell membrane; DNA pulled in by electrophoresis and diffusion.

• Drawback - its more cumbersome to regenerate plants from single protoplasts than from the tissue transformations with Agrobacterium

animal cells).• High-voltage pulses

Electroporation

Use of high voltage electric current (1-1.5 kV) to permeabilize cell membranes to facilitate DNA uptake.

D’Halluin et al. (1992) Plant Cell 4: 1495-1505

Immature Embryos(maize)

electroporation Embryogenic callus Transgenic plant

Pollenelectroporation

Pollinate flowersTransgenic plant

Electroporated pollen can supposedly germinate at 30% efficiency. However, no transgenic plant has so far been reported using this concept, even though it has been shown that pollen grains can be permeated with macromolecules such as DNA.

Electroporation method is very efficient in permeating DNA into cells and protoplasts, therefore this method is very effective for transient expression studies.

Advantage: there is no need to establish callus or suspension culture first. Explants (embryos) were directly electroporated.

Pollen mediated transformation

Nicotiana langsdorfii X Nicotiana glauca = Hybrid (develops genetic tumors)

In 1976, attempts were made to use pollen to take up foreign DNA and then cross fertilize a related species. glauca pollen were incubated in DNA isolated from langsdorfii. The DNA treated pollen were used to pollinate emasculated glauca plants. One group claimed that the sexual progeny thus obtained formed tumors on the stem.

However, these experiments were not reproduced in other labs.

Microinjection

Injection (sometimes it is simply pipetting) of DNA into cavities containing meristems or sexual organs. Some studies claimed success while others reported negative results. The verification was difficult due to contamination. The transformed tissue was not regenerated into a plant.

In 1987 a report was published in Nature that described the generation of transgenic rye plants by injecting DNA into floral tillers. Authors reported kanamycin resistant plants and Southern analysis to prove the integration of the npt gene. But there was no progeny analysis. A large number of labs tried to repeat this method but to no avail.

Two more similar reports (irreproducible) were made in barley in 1990s.

Current status: unreliable, tricky, low (or no) success rate.

Ultrasound-induced (sonication) DNA uptake

Ultrasound treatment causes the formation of bubbles with generation of high pressure and temperature and violent-flow or streaming of fluids. This method has been used to introduce DNA into plant protoplasts. First report described the introduction of DNA into tobacco protoplasts but no transgenic plants were obtained. Subsequent reports described generation of transgenic tobacco and wheat but insufficient molecular evidence was provided.

It appears that this method may be effective in introducing DNA into a cell but not into the nucleus. Further, it does not present much advantage over other methods and therefore has not been much explored.

Particle bombardment

PDS-1000 gene gun

Particle inflow gun

ACCELLTM technology

PDS-1000First particle gun was developed by Sanford and colleagues in 1987. They introduced CAT (chloremphenicol acetyl transferase) gene into onion epidermal cells and detected transient gene activity.

First transgenic plants were produced in 1988 using soybean and tobacco tissue culture. Christou (1988) and Klein (1988) bombarded soybean shoot meristem and tobacco leaf, respectively. Christou recovered chimeric transgenic soybean plants that transmitted the gene into next generation.

Particle bombardment is the method of choice for chloroplast transformation.

Method:

1.Precipitate DNA onto small tungsten or gold particles.2. Accelerate particles to high speeds and aim them at

cells or tissues.3. Selective growth and regeneration of transgenic plants

as described for Agro-mediated transformation

Gold or tungsten particle are used

gold tungsten

DNA is bound to the microprojectiles, which impact the tissue or immobilized cells at high speeds.

J. Sanford & T. Klein, 1988

Original 22-caliber biolistic gun

The Helium Gas Gun – Circa 2000

Helium particle gun

ACCELL technology

It uses high voltage electric discharge to vaporize a water droplet which produces a controlled shock wave. Initial shock wave is reflected to produce secondary shock wave, which in turn accelerate a mylar sheet coated with particles. A screen stops the mylar sheet and allows the particles to hit the target.

Particle Inflow gun

Particles are supported by a screen in a syringe filter unit and accelerated in a stream of helium without the need of macrocarrier. The target tissue is held in a chamber under vacuum. A solenoid controlled by a timer relay is used to generate a burst of low pressure helium.

Advantages: Not patented, easy to design, gives good results.

Disadvantages: not commercially available, some safety concerns

DNA coated particles are suspended in ethanol and deposited on a metal sieve plate. Since the particles are not dried up and stuck to plastic like in PDS-1000 system, low pressure He is able to accelerate them.

Design of Particle Inflow gun

Factors affecting particle bombardment-mediated transformation

Selectable marker genes: choosing a proper selection marker is critical.

Markers selection agentnpt II (Tn5) G418 (geneticin), kanamycin, neomycin, paromycin

hpt hygromycin BGentamycin acetyl transferase gentamycinSPT (Streptomycin phosphotransferase) streptomycin, spectinomycindhfr mutant form methotraxatebar (PAT) PPT, bialaphosaroA (EPSP synthase) glyphosate (roundup)als (Acetolactate synthase mutant form) sulphonyl ureaBromoxynil nitrilase (bxn) bromoxynil

A mutant form of dhfr isolated from mouse is resistant to methotrexate drug, which eventually interferes with DNA synthesis. Thus this gene serves as a selectable marker.

Glyphosate inhibits photosynthesis by competitive inhibition of EPSP synthase, which is involved in shikimate pathway for amino acid synthesis. EPSP synthase is encoded by nucleus and localized in chloroplast. aroA gene was isolated from Salmonella typhimurium strain resistant to glyphosate.

Reporter genes

First reporter gene:CAT: chloremphenicol acetyl transferase (chromatography and autoradiography)

nos or ocs: nopaline or octopine synthase (chromatography)

uidA or gusA (GUS): MUG or X-gluc (color or fluorescence) Jefferson (1987)

luc (firefly luciferase) luciferin (bioluminiscence) Ow et al. (1986)

gfp (green fluorescent protein) no substrate, UV irradiation.

LUC+ tobaccoGFP expression in rice callus

negative positive positive

positivepositivepositive

Gene copy number

Number of copies of the introduced DNA, the pattern of integration, and the chromosomal location of integration have profound effect on the expression of the introduced genes.

Direct DNA transfer methods generally produce more complex integration patterns than those produced by Agrobacterium-mediated transformation. Several people are studying the parameters that can simplify integration patterns such as amount of DNA to be introduced. It has been reported that by reducing the amount of introduced DNA, one can produce higher number of transgenic lines containing simple integration pattern.

Since Agrobacterium generates more single copy integrations than say biolistics, a novel approach called Agrolistics was developed by Novartis Co. to facilitate production of single copy transgenic plants of maize, wheat and other such crops that are not amenable to Agrobacterium transformation.

“Agrolistic”

T-DNA RBLB

All major monocots were transformed by biolistic method by 1992.Agrobacterium produces simple integration patternsBiolistic produces complex patterns

Co-combardment of binary vector with VirD1 and VirD2 expression cassettes:produced integration patterns just like the onesgenerated by Agrobacterium.

Binary vector