April 1, 2011 (Vol. 31, No. 7)

Editor’s Note

The Biotechnology Industry Organization (BIO) reports that over 13 million farmers around the globe now work with agricultural biotechnology techniques. Since first making their appearance on the world stage in the 1990s, agbiotech crops have been planted on more than 2.3 billion acres of farmland. BIO also notes, in particular, that almost 93% of the farmers growing biotech crops are from small and developing countries where feeding their local populations remains a key issue.

This issue’s article commemorating GEN’s 30th Anniversary is reprinted from March/April 1983. It describes one of the first steps taken by biotech researchers to create genetically engineered plants. In this case, a series of experiments was devised to insert a bacterial gene for antibiotic resistance into cultured tobacco cells using a plasmid from Agrobacterium tumefaciens. Significantly, the gene was subsequently expressed. As the article points out, the main goal was not to impart an antibiotic resistant gene into a plant but to develop a model system for genetically engineering plants with important traits such as herbicide resistance, higher yield, nitrogen fixation, and stress tolerance. Looking at the wide range of genetically engineered crops now available in most parts of the world, let’s salute these pioneering researchers for their outstanding work.

—John Sterling, Editor in Chief

“As Seen in GEN–Flashback” Volume 3, Number 2, March/April 1983

Bacterial Gene Is Expressed in Cultured Tobacco Cells

Scientists from three separate laboratories have reported the successful expression of a bacterial gene for antibiotic resistance in plant cells using the tumor-inducing (Ti) plasmid of the soil bacterium Agrobacterium tumefaciens as a gene vector.

Reports of the experiments hilighted the 15th Annual Miami Winer Symposium, held in January, which focused this year on agricultural applications of genetic engineering.

The Monsanto Co., in whose laboratory one of the successful experiments took place, emphasized the long-term significance of the achievement. Dr. Ernest Jaworski, the firm’s director of molecular biology, asserted that the technique eventually will be intended to enable researchers to improve crop plants by inserting useful genes into them in a manner allowing the plants to express them constitutively.

Principal researchers for Monsanto were Drs. Robert Horsch, Stephen Rogers and Robert T. Fraley. Their work took place more or less simultaneously with similar experiments done by Dr. Michael Bevan in the lab of a second Miami Symposium speaker. Dr. Mary-Dell Chilton of Washington University, St. Louis. (Dr. Chilton will leave the university shortly to join the research staff at Ciba-Geigy.) A third Symposium speaker, Dr. Jeff Schell, director of the Max Planck Institute for Plant Breeding, Cologne, West Germany, reported very similar experiments with two antibiotic resistance genes.

Dr. Chilton and Dr. Rogers, in interviews with GEN, emphasized that resistance to the antibiotic is not a particularly useful trait to the plant. The significance of the experiments, they said, is in its role as a model system.

Tumor-Inducing Genes

The tumor-inducing property of the plasmid has been isolated to a particular set of genes on the plasmid. Dr. Schell’s group, in collaboration with Dr. Mark Van Montagu at the State University of Ghent, Belgium, has for several years explored the possibility that the tumor genes could be muted in such a way that the natural genetic engineering capacity of the Ti plasmid might be exploited without causing the transformed cells to lose their differentiation. Tumor cells grow well in culture, but cannot be regenerated into whole plants.

Agrobacteria are close relatives of the nodulating soil bacteria which allow some plants to fix their own nitrogen. However, instead of nodules, Agrobacteria cause a disease called crown gall which manifests tumors. The soil bacterium, on the other hand, has in its Ti plasmid a gene encoding an enzyme called nopaline synthase, which is inert in the bacterium itself. But when inserted onto the plant cell it induces the cell to produce an amino acid derivative called nopaline., which is a good source of carbon and nitrogen for the bacterium.

The nopaline synthase gene is a plant-type gene that functions only in the plant cell. The gene has a promoter, a regulatory sequence which causes the cell to begin transcribing the gene and producing the protein it encodes, which operates in the host plant only.’

The three separate but similar studies announced at the Miami Symposium used essentially the same solution to this problem. Both used an experimental microbial gene that encoded resistance to an antibiotic. The problem was that the promoter sequence attached to this gene was unintelligible to the plant. In each lab, the researchers removed the bacterial promoter from the gene and attached it to a promoter which could be understood by the plant cell—the nopaline synthase promoter. This construction, a “chimeric gene,” was inserted into the Ti plasmid, which in turn was used to transform the plant cells using the available technique for doing so without causing tumorous growth.

Plant Regeneration

The problem of how to regenerate plants from genetically engineered cells was solved by Dr. Andrew Binns of the University of Pennsylvania in collaboration with two post-doctoral students in Dr. Chilton’s lab—Dr. Kenneth Barton (now of Cetus Madison) and Antonius Matzke. Dr. Binns found a class of genetically engineered nopaline Tiplasmids that was avirulent, i.e., not tumor-inducing,” in tobacco, but which nontheless transferred tDNA to plant cellss. Dr. Binns isolated nopaline-positive cells which were not tumorous and which regenerated into complete tobacco plans.

Dr. Barton showed that the plants contained 10-20 copies of the tDNA incorporating the genetically engineered traits. The plants have produced seed and progeny hat also carry multiple copies of the trait.

The usefulness of this process remains to be verified, but Dr. Chilton asserted that the problems associated with creating the vector will be minimal from here on. Monsanto’s Dr. Rogers added that the principle of attaching a chimeric gene to a Ti plasmid might be extended to other kinds of vectors

The technique may lead to geen transfers of obvious usefulness for plant improvemetn, especially to dicotyledonous crop plants such as alfalfa and tobacoo. Agrobacteria infect only dicots. Traits of interst would be herbicide resistance, higher yield, nitrogen-fixation and stress tolerance, said Dr. Chilton.

However, it is not clear with today’s technology how to isolate genes for higher yield.

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