Genetic engineering as a topic is fiercely discussed. It belongs to applied molecular biology. Its principle of action is rather simple:

1. The discovery that bacteria may contain plasmids was the starting point. Plasmids are small, circular DNA molecules occurring in bacterial cells in addition to the original bacterial chromosome. The chromosome of the bacterium is circular, too, but far larger. The plasmid DNA makes up just a few percent of the total bacterial DNA.

2. Bacteria contain a number of highly specific endonucleases (restriction endonucleases, restriction enzymes) that cut the DNA at specific nucleotide sequences. Many restriction enzymes have been isolated, their action spectra are known. Most of them are commercially available.

3. ‘Foreign’ DNA can, too, be cut with restriction enzymes.

4. DNA sequences (nucleotide sequences, fragments) are easily separated from each other due to their differing molecular weights. The method of choice is gel electrophoresis.

5. DNA fragments can be connected and circularized by certain enzymes, so-called ligases, to form hybrid molecules. In other words: any DNA segment can be incorporated into a bacterial plasmid.

6. The bacterial plasmid (including the foreign DNA) can be incorporated (transformation) and multiplied by bacteria provided that a suitable plasmid has been used. Suitability is dependent on the specific experiment. Today, a wide spectrum of very specific plasmids is available. Some of them serve just very limited purposes and survive only in certain strains of bacteria.

The mostly technical problems have largely been solved, but the real problems start with gene expression, since it requires specific signals for transcription (promoters) and translation. The signals recognized by procaryotic cells differ fundamentally from those needed by eucaryotic cells. The transfer of a bacterial plasmid into a eucaryotic cell and its expression there is thus not possible directly. The T_i-plasmid is an exception. Eucaryotic cells understand the signals preceding the synthetase genes of octopine and nopaline. Therefore, a foreign DNA coupled in the right reading frame behind such a signal makes sense for a eucaryotic cell.

Cloning is, in practice, a process consisting of several steps that leads finally to the wanted DNA-hybrid. Only those parts of the T_i-plasmid that are absolutely essential for transfer into the plant and integration into the plant’s genome are used. It is important that the tumor regenerates into a complete plant containing the foreign gene and that the latter is not lost during differentiation. The next challenge is meiosis, but it was principally solved.

Many working groups use genes whose products cause resistances against antibiotics like the gene causing resistance against kanamycin. It retains its ability to be fully active within the plant cell, i.e. the plant cells are now resistant against kanamycin. Untreated cells are sensitive towards kanamycin.

The transcription rate of genes is not only dependent on the efficiency of the promoters preceding them, but is equally strongly controlled by cellular factors whose concentration is dependent on the state of development and/or the respective tissue. P.ECKES, S. ROSAHL, J. SCHELL, and L. WILLMITZER (1986) analyzed the organ-specific expression rate of several potato genes. It turned out that one of the genes could be transferred to tobacco and that its expression is induced by light. The search for suitable promoters lead, among others, to the cauliflower-mosaic virus. It was considered as an alternative to the T_i-plasmid. The genome of this virus integrates into the host cell’s genome, but integration seems to be too unstable for reproducible results. The promoters occurring in the DNA of the cauliflower-mosaic virus, though, proved to be suitable for the activation of subsequent genes (M. B. BEVAN, S. E. MASON, P. GOELET, 1985).

Transfer and expression of many genes tried so far remained without success. Plant cells do neither transcribe nor translate the ADH gene of yeast, for example. The attempt to integrate the phaseolin gene (phaseolin is a legumin of bean) was informative, though. The respective DNA segment is transcribed in transformed tumor cells as could be easily shown, but the translation products were not detected at first. Finally, T. C. HALL and his collaborators (University of Wisconsin, Madison, 1983 (a publication of 11 authors!)) proved that the tumor cells contain polypeptides recognized by antibodies against phaseolin. It does therefore look as if the protein is indeed produced, but is degraded immediately after production.

In 1986, an especially spectacular case of gene transfer was reported. An American working group (D. W. OW and colleagues) succeeded in transferring the luciferase gene of a luminous beetle (Photinus pyralis) to tobacco plants using the Agrobacterium-plasmid together with a cauliflower-mosaic promoter as a vector. Addition of luciferin to the nutrient medium lead to a bright, tissue-specific luminescence. It was especially strong in the vascular tissue of shoots and roots. Nowadays, almost every laboratory has isolated and cloned one or even several genes in this way. Information about the analyzed and isolated genes can be found in relevant data bases. See: http://srs.ebi.ac.uk/

Besides transforming plant cells with foreign genetic material using Agrobacterium tumefaciens, the fusion of protoplasts of different origins turned out to be a suitable method for bringing different genomes together or for transforming a cell with DNA (I. POTRYKUS et al., 1985; H. LÖRZ et al., 1985; B. KRAUTWIG and H. LÖRZ, 1995).

Independent of these studies, it emerged that certain plant cells like egg cells and germinating pollen are capable of taking DNA up directly. It was thus often sufficient to inject a DNA-containing solution into a developing inflorescence (of cereals, for example) or to incubate germinating pollen with a solution of DNA in order to obtain transformed plant cells. The foreign DNA was integrated into the plant genomes, and expressed within the cells. The choice whether to work with whole plants, in vitro-cultures or protoplasts depends on the respective problem. The genetic manipulations of the test material can occur at different states of development and under different conditions of cultivation.

During the last years, the release of genetically engineered plants has been discussed. This topic is covered thoroughly by the essays cited at the beginning. The first release experiment in Germany was performed by the Max-Plank-Institut für Züchtungsforschung at Cologne. The results are given in an essay of W. SCHUCHERT (in German only): :

Further experiments using genetically engineered plants concern virus- and fungus-resistances. (in German only)