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Genomics

Safer biotechnology

Genetic modification technique enables GM products with less environmental risk

The words genetically modified cause shivers down the spine of some people, fearful that man’s intervention in the DNA of other species may spread genes in Nature in an uncontrolled manner and alter the original characteristics of other creatures. But, to judge from the results of the new technique of manipulating the genetic code of plants, this concern might disappear in the case of vegetable species. In working on the cell structure outside the nucleus, the chloroplast, which stops the gene introduced, and consequently, the artificially created element, being transmitted to other life forms, the method paves the way for the production of ecologically safe genetically modified plants.

“More that 95% of vegetable species do not have chloroplasts in their pollen grains (which contain the male sexual cells)”, says Helaine Carrer, of the Luiz de Queiroz College Agriculture (Esalq) of the University of São Paulo (USP), one of the pioneers in the use of the technique. “Therefore, genetic modifications cannot be transmitted to other plants”. There are two exceptions: alfalfa and the geranium (the pollen grains contain chloroplasts).

In the article written jointly with German researchers from the University of Freiburg, published in September’s Nature Biotechnology, Helaine presented the first fertile plant with edible fruit produced by this method: a tomato (Lycopersicon esculentum, Santa Clara variety), whose chloroplast was given the aadA gene, making the plant resistant to certain types of antibiotic, such as spectinomycin and streptomycin. The work showed that, in practice, it is possible to make modifications to the genome of plants used as human food, and this is a new piece of information in this field of study.

Vegetable factories
“With the development of this technique, we will be able to use agricultural crops, in a controlled manner, in about five or ten years’ time, as protein, vitamin or vaccine factories”, predicts the Esalq researcher. Helaine is now engaged in studying the sugarcane chloroplast, which will be used next year in the first experiments with the new technique. Sequencing the genome of this cell structure of the plant, conducted by the Esalq team within the scope of the FAPESP Sugarcane Genome Project, is practically complete – it will be the eighth chloroplast genome to be mapped in the world. Initially the group will repeat the tomato experiment with the sugarcane and, in future, use the leaves of the plant to produce a type of biodegradable plastic.

The organelle responsible for photosynthesis, the chloroplast is one of three compartments of the cells of vegetable species that have DNA fragments – the others are the nucleus and the mitochondrion, the structure responsible for energy production. Biotechnology began in the 80’s with procedures that worked on the genetic material contained in plants’ nuclei. This corresponds to more than 90% of the genome of these species. Since then, companies have announced new varieties of agricultural product genetically modified in the genome of the nucleus – tomato, corn, rice, and cotton, for example. This approach dominates. Research into the mitochondrial genome, on the other hand, is at the toddler stage and research into the chloroplast’s genetic material is gathering pace.

Requirements
Until the publication of Helaine’s work, the only vegetable species successfully manipulated through the chloroplast was an inedible plant, tobacco (Nicotiana tabacum). To be completely successful, a genetically modified plant has to have three features: be able to reproduce in Nature outside laboratory conditions; be able to present clearly and unmistakably the traces deriving from the introduction of a gene originally foreign to its genome; and not be able to transmit the gene introduced to other plants through its pollen. Except for tobacco, and now the tomato, none of the vegetable species altered by the technique has given satisfactory results. The genetically modified versions of rice, potatoes, and Arabidopsis thaliana, a model plant for molecular biology produced by this approach, have all proved sterile.

Besides enabling better control of the spreading out of the genes in Nature, the technique of working with the chloroplast, according to the researcher, offers an additional advantage compared to the traditional method of inserting genes into the nucleus: the modified plant expresses the acquired characteristic to a greater degree after a DNS fragment has been introduced into its organelle. In the tomato, for example, the researchers observed that more than 5% of the total amount of soluble protein found in the cells resulted from the genetic modification. With tobacco, as already seen, this can amount to 40%.

Using the conventional technique, according to the researcher, this figure comes to around 1% and, in some cases, the results can be virtually imperceptible. “Sometimes, the gene inserted into the genome of the nucleus is simply not expressed by the plant and remains silent (inactive)”, explains Helaine. It is as if the organism had not been genetically modified at all. This inconvenience, it seems, does not happen with modifications made in the chloroplast. The high standard of expression provided by the alternative method is probably due to the fact that there are around 10,000 copies of the chloroplast genome in each cell, which expands the results of genetic modification. The genome of the nucleus is unique, with no copies.

Pioneering
It is no exaggeration to say that the researcher from São Paulo is one of the world’s leading experts in the new technique. When doing her doctorate in the United States, Helaine was part of the Pal Maliga’s group, at the Waksman Institute of Rutgers University, which, at the beginning of the 90’s, took the initial steps toward the new technique. In a similar manner to that employed on the tomato, the Maliga team managed to insert genes in tobacco chloroplasts demonstrating the feasibility of the then unprecedented approach. The researcher’s contribution to this project earned her a share in the international patent for commercial use of the technique.

Today, the name of the Esalq researcher is on four patents relating to the method, the most recent of which is associated with the use of genetic engineering in tomato chloroplasts. It was during her stay at the Waksman Institute that Helaine met Ralph Bock, nowadays a researcher at the University of Freiburg and co-author of the article in Nature Biotechnology. In 1997, after both had returned to their native countries, the Brazilian and the German began a partnership that led to the development of the genetically modified tomato through the chloroplast. It involved three years of joint work.

The new technique demands patience and dedication by the researchers to produce results. First, they have to develop an efficient culture medium for regenerating the plant based on a single laboratory cell, in a process that usually takes months. The next step is to analyze the information provided by the sequencing of the genome of the chloroplast of the vegetable species to be modified. When working on this cellular organelle, this type of information is indispensable.

The stages
Because it is small (180,000 circular-shaped base pairs, at most), the chloroplast genome has few genes (around 130), all very close to one another. There is little free space inside the organelle genome where a new gene can be inserted without altering the structure and the workings of the other genes. The solution is to fit the new gene into the tiny space separating two original genes. The introduction of an external fragment of DNA in the chloroplast is done by a technique called biolytic: microscopic particles of gold or tungsten, lined with the gene to be inserted, are bombarded against the leaves of the plant. After penetrating the cell and reaching the chloroplast, the gene lodges in the place provided for the genome. It seems simple and easy, but out of every 100 attempts, five, at most, work.

The final stage is to confirm whether the gene has actually been incorporated into the genome of the chloroplast. When this happens, the altered species begins to show the characteristic deriving from the inserted DNA fragment. In the case of the tomato prepared by Esalq and the University of Freiburg, the bombarded leaves were cut into pieces and these were placed in a selection medium, a solution containing antibiotics. The plant fragments that survive in this medium, have, obviously, incorporated the gene giving them this ability to resist. The others died; consequently, they did not receive the modification. The leaf fragments resistant to the antibiotics were regenerated and cultivated in the laboratory until generating a plant able to reproduce”, explains Helaine.

The Project
SucEST – Sequencing the Sugarcane EST project (nº 99/02860-8); Type
Normal research support line; Coordinator Helaine Carrer – Esalq/USP; Investment R$ 49,954.45 and US$ 207,341.15

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