Mar 29, 2006

Basic info

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Pollen without foreign genes

Plastid transformation

In genetically modified plants, the new gene is normally inserted into the DNA of the cell nucleus. But there are other parts of the plant cell which contain genes and turn them into proteins: plastids for instance. In most flowering plants plastids do not occur in the pollen cells. By using plastids as the carriers of new genes it is possible to biologically confine the new genetic information in the plant.

Plastids probably originate from formerly free-living bacterium-like organisms that were taken up by plant cells in primitive times. The relationship is mutually beneficial: one example is the chloroplasts, a special group of plastids used by the plant to convert the sun’s energy. For their part, the plastids’ forebears found a protected environment in the plant cells. They have retained some of their own genes, while others have over time become integrated into the cell nucleus of the plant cells.

Plastids have their own system that they use to ‘translate’ their DNA into proteins. The DNA in the plastids is also known as ‘plastome’.

In principle, it is possible to integrate foreign (transgenic) DNA into the plastome. To do this, the target gene is ‘flanked’ by sequences from the plastome. Using the homologous recombination procedure (see diagram) the transgenic DNA is integrated into the plastome. Unlike in the plant’s nucleus genome, this process occurs naturally. The plants modified using this kind of plastid transformation are called ‘transplastomic’.

In general, the pollen cells of flowering plants do not contain plastids. If a foreign gene is present only in the plastome it will not be passed on during propagation.

As well as the security aspect – the desire to confine the foreign genes to the transgenic plant – the increased activity of the foreign genes in the plastids is also interesting. The reason for this lies in their numbers: there are up to 100 plastids in each cell, each carrying up to 100 copies of their genetic material. The genetically modified plants carry the new gene on every copy in all plastids. This means that after successful transfer of a foreign gene, almost half of all proteins in the cell are produced by this gene. This level of productivity cannot currently be achieved using traditional methods. However, implementing the procedure is proving to be a technological challenge.

Introducing foreign genes into plastids: first steps

Established procedures are used to introduce the transgenic DNA into the cell/plastids – e.g. transformation using Agrobacteria, particle gun or microinjection. The important thing is that the tissue used should transform and regenerate easily and contain plastids, e.g. chloroplasts. Suitable tissues that have been used so far are leaves, microspores (unripe pollen with cell walls that are not yet fully formed), immature embryos and hypocotyls of young seedlings.

Plastid transformation is much more complex than cell nucleus transformation because there are up to 10,000 plastid genomes in each cell. Transplastomic lines are genetically stable only if all plastid copies are modified in the same way, i.e. uniformly. This is achieved through repeated regeneration under certain selection conditions. Plastid transformation has already been successfully performed in tobacco, potatoes, oilseed rape, rice, Arabidopsis thaliana (thale cress).

Plastid transformation for the production of genetically modified plants has advantages and disadvantages.

• Plastic transformation reduces the probability of the transgene spreading via pollen, since the plastids are usually inherited from the maternal plant, rather than via the pollen.
• Transformation and regeneration of transplastomic plants is technically more complex. The transformation efficiency rate is lower. Very high expression levels are limited to leaf tissue.

One completed biosafety research project achieved some success with plastid transformation in oilseed rape and maize. Several current biosafety research projects are focusing on plastid transformation in petunias, Arabidopsis, tobacco, maize and oilseed rape.