Transcript Lecture 3
Plant transformation based on direct DNA delivery 1. Polyethylene glycol (PEG)-mediated protoplast transformation. 2. Particle bombardment 3. Electroporation 4. Microinjection 5. Ultrasound mediated 6. Pollen-mediated 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 Advantage: there is no need to establish callus or suspension culture first. Explants (embryos) were directly electroporated. electroporation Pollen Pollinate flowers Transgenic plant Electroporated pollens can be 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 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 the potential contamination. The transformed tissue was not regenerated into 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 npt gene. But they didn’t carry out 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 plant was 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 cell but not to the nucleus. Further, it does not present much advantage over other methods and therefore it hasn’t been explored much. 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 (drug) resistant. This method has been very useful and applied to several plant species. But it is a tedious procedure! Protoplasts are treated with DNA in the presence of PEG and Ca++ Particle bombardment PDS-1000 gene gun Particle inflow gun ACCELLTM technology PDS-1000 First 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. 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 Design of Particle Inflow gun 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. Gold or tungsten particle are used gold tungsten Factors affecting particle bombardment-mediated transformation Selectable marker genes: choosing a proper selection marker is critical. Markers npt II (Tn5) hpt Gentamycin acetyl transferase SPT (Streptomycin phosphotransferase) dhfr mutant form bar (PAT) aroA (EPSP synthase) als (Acetolactate synthase mutant form) Bromoxynil nitrilase (bxn) selection agent G418 (geneticin), kanamycin, neomycin, paromycin hygromycin B gentamycin streptomycin, spectinomycin methotraxate PPT, bialaphos glyphosate (roundup) sulphonyl urea 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. negative positive positive positive positive positive GFP expression in rice callus LUC+ tobacco 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” All major monocots were transformed by biolistic method by 1992. Agrobacterium produces simple integration patterns Biolistic produces complex patterns LB T-DNA Binary vector RB Co-combardment of binary vector with VirD1 and VirD2 expression cassettes: produced integration patterns just like the ones generated by Agrobacterium.