The agricultural biotech industry has yet to supply a genetically modified (GM) product that is viewed as entirely beneficial by a largely skeptical public. Sections of the community perceive the industry as profit-driven, largely unaccountable, and interested in solving the problems faced by large-scale agricultural industrialists rather than those of the public at large. The ability to bring together traits from different plants, even from animals and microorganisms, concerns many people, and their belief is that such power should be wielded responsibly or not at all.

Traditional crop biotechnology typically involves the transformation of a human food plant with a foreign gene(s) coupled to plant virus DNA and a herbicide/antibiotic resistance gene. The whole process is rightly seen as an extraordinary undertaking, requiring extraordinarily good justifications. The biotech industry has yet to convince skeptics that there are sufficiently extraordinary reasons for most of the products in or approaching the market. The lack of perfect knowledge and consequent unpredictability of the outcome of genetic manipulations further increase the anxiety and concerns about safety and the long-term consequences of GM products.

The first phase of plant GM technology has involved the random insertion of whole genes controlled by viral promoters into plants, and the widespread use of antibiotic/herbicide resistance genes to allow transgene selection. In the near future, these methods will likely be seen as primitive, clumsy, and perceivably risky approaches for creating novel crops with beneficial properties. To date, these techniques have provided very powerful research and development tools and are currently the only way to create truly novel products, such as plants that make biodegradable plastics in cotton fiber for a warmer product(1). The emerging next phase of the technology addresses some of the first phase problems by, for example, using efficient and precise insertion of transgenes into the maternally inherited (at least in most plant species) plastid genome to allow a more controlled effect on plant metabolism. In most crop species, positioning the transgene in the plastid genome prevents its inclusion in pollen and therefore ensures genetic containment and avoids distribution of transgenes to honey products and pollen feeders, another public concern. It is now possible to also remove marker genes once they have been used in the initial identification of transgenic plants(2), a very positive development in the technology. (See "Engineered Chloroplasts Snip Out Antibiotic Resistance Genes," ISB News Report, this issue.)

In addition to the introduction of novel or altered genes into a plant, many metabolic advantages and new products can be gained by switching off plant genes using antisense or partial sense technology. This first phase technology uses a manipulated version of the target gene to control the expression of the plant's own version. This technique brings with it all of the problems already discussed. With the flood of information coming from functional genomics(3), proteomics, and protein structural studies, it is clear that genetic engineering can now incorporate rational design strategies and precision manipulation of heritable material to both knockout and change the characteristics of encoded enzymes.

The task is now to identify ways to create novel plant traits with a minimum amount of change since, armed with this new knowledge, a handful of carefully chosen tiny alterations (in many cases just of one base pair) can often have the required effect(4). Minimizing the degree of genetic alterations is much more palatable to the public than the insertion or deletion of large amounts of coding DNA, with the same end result. This approach would benefit the biotech industry and may increase public support for more ambitious genetic engineering projects with widely beneficial goals. Some research effort should therefore be directed toward the development of tools that allow such effective but minimal genetic manipulations. Surprisingly, until recently the biotech industry has largely tended to reject the exploitation of single point mutations or polymorphisms.

A recent publication by Berns and co-workers(5) demonstrates a method that may one day replace the large-scale genetic tampering currently employed. The DNA of dividing animal cells in culture was made photosensitive by the addition of a dye, and a laser was aimed at a single visually identified region of a chromosome. The laser beam then knocked out, in a specific manner, the cluster of genes known to be located in that region. A heritable genetic modification had occurred without the use of any recombinant DNA. The problem is one of specificity; how does one direct the laser energy to only the specific gene or regulatory element one wishes to change? One possible answer is mentioned in the report's conclusion: utilizing the plants own homologous recombination system to precisely deliver a sequence-specific molecular probe conjugated to a photon-absorbing molecule. The delivery of such probes to the genes of cultured cells is entirely feasible, and this negates the requirement for microscopic aiming of the laser. Such an approach would allow the delivery of sufficient energy to a precise location in the genome, perhaps disrupting a specific regulatory element and causing a cascade of effects predicted from genomic/proteomic information. An example use might be the knocking out of a crucial region of the promoter for a gene encoding a transcription factor required for senescence, postponing flower decay.

The first use of another powerful site-specific mutagenesis technique in plants was reported last year(6). Unlike laser mutagenesis, chimeraplasty is capable of introducing precise single point mutations into the genomes of cultured cells. In this case, however, recombinant heritable material is used. DNA/RNA hybrid oligonucleotides are introduced using standard techniques (electroporation, biolistics) into cultured cells where they bind specifically to the DNA region of interest. A single `error' in the oligonucleotide causes the cell's own DNA repair mechanisms to rewrite the DNA sequence to incorporate the single change. In the report, maize plants gained resistance to a common herbicide via a single change in the gene encoding for a protein involved in amino acid synthesis. The change was predicted to have no other effect on the metabolism of the plant. This is another example of a single precisely delineated alteration having a dramatic and useful effect. Herbicide resistance was used merely to identify altered plants easily, but information from functional genomics studies could identify many other targets with more beneficial outcomes.

In summary, techniques currently in development, when coupled with the enormous amounts of information coming from genomic and proteomic efforts in industry and academia, will stimulate a change in the way targeted genetic modification is employed. The precision of the new methods should address at least some of the public's concerns and help them to comprehend the benefits of agricultural biotechnology.

Sources:

1. Chowdhury B and John ME. 1998. Thermal evaluation of transgenic cotton containing polyhydroxybutyrate. Thermochimica Acta 313(1): 43-53.

2. Iamtham S and Day A. 2000. Removal of antibiotic resistance genes from transgenic tobacco plastids. Nature Biotechnology 18: 1172-1176.

3. Detailed introductory material is available on functional genomics and proteomics in the June 15, 2000 edition of Nature.
4. Many examples of the power of point mutations are in the literature. See for example Cahoon EB, Shah S, Shanklin J and Browse J. 1998. A determinant of substrate specificity predicted from the acyl-acyl carrier protein desaturase of developing cat's claw seed. Plant Physiology 117: 593-598.

5. Berns MW, Wang Z, Dunn A, Wallace V, and Venugopalan V. 2000. Gene inactivation by multiphoton-targeted photochemistry. Proceedings of the National Academy of Sciences 97: 9504-9507.

6. Zhu T, Mettenburg K, Peterson DJ, Tagliani L and Baszczynski CL. 2000. Engineering herbicide-resistant maize using chimeric RNA/DNA oligonucleotides. Nature Biotechnology 18: 555-558.

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