The r-DNA debate is too clever by half

GENETIC MODIFICATION of crops through recombinant DNA (r-DNA) technology has been a remarkable outcome of technological innovations in plant breeding, a facet of human development ardently practised since the days prehistoric man turned a settler from the nomadic hunter-gather-scavenger that he was. Without a doubt, r-DNA technology has improved the quality of human and animal life, has a positive impact on the environment and helps sustain the biodiversity. Yet, as with any technological innovation it has its share of detractors and sceptics who prefer to cling to things they are familiar with perhaps because they are ignorant of the new developments in science and technology or are just plainly scared of venturing out of the beaten path. Often such reservations give forth in the form of doomsday predictions, scare mongering and undue haste in bundling every innovation and discovery as unworthy. It leads to many myths and canards, which have a tendency of self- perpetuation, based on non-science, half-truths and misrepresentations.

When a plant breeder wants to introduce resistance to a particular fungal pathogen to a crop species, he would scout for a variety that inherently carried the resistance to the pathogen. Usually such donors are found in the wild or from distant relatives that do not carry any other intrinsic qualities of yield attributes about them. Then he would go through the arduous process of transferring this desired trait onto the cultivated species through crossing once with the donor and then repeated back-crossing of the progeny generations with the cultivated species in an effort to capture the desired trait without dragging down the other favourable attributes of the cultivated species already present in it. This translates into a numbers game: the more the crosses made and the more the progeny screened the better the opportunity of striking the desired combination of disease resistance plus favourable attributes already present in the cultivated species.

What is happening here is that the genes of the two species are mixed up during the process of sexual reproduction and they get reassorted in the progeny in a myriad of permutations and combinations. The trick is to be able to pick one winner among a million or more! Recombinant DNA technology assists in identifying the specific gene(s) conferring the resistance trait and helps splice it onto the genome of the recipient with clinical precision and without having to rehash the whole genome of the recipient. What is more, unlike classical breeding which circumscribes to barriers to gene transfer, r-DNA circumvents it and facilitates transfer of genes across kingdoms. In either event, genes have been shuffled into genomes of cultivated species, save that in classical breeding there are many operational constraints, besides being a very drawn out process and progress is slow whereas the modern method is more precise, obviates the familiar barriers but is more expensive besides being now hemmed in by restrictions imposed by intellectual property rights.

A natural phenomenon

In nature genes have been transferred from and between organisms without discrimination and this has been happening over epochs. There are innumerable studies that show similarities between natural horizontal gene transfer (HGT) and natural DNA rearrangements and those used in laboratory experiments. It is common knowledge that genes move around many microbes. Microbial gene transfer is a well-documented means of exchange of loci among many prokaryotes and some eukaryotes (Paul, JH, 1999, J. Mol Microbiol Biotechnol). A transduction-like mechanism of transfer from viral-like particles produced by marine bacteria and thermal spring bacteria to Escherichia coli has been documented indicating that broad host range transduction may be occurring in aquatic environments. The sequencing of complete microbial genomes has further shown them to be a mosaic tapestry comprising ancestral chromosomal genes interspersed with recently transferred operons that encode for peripheral functions. Genomes of ancient species include genes for replication, transcription and translation that are eukaryotic in complexity while the genes for intermediary metabolism are bacterial in nature. Moreover, in eukaryotes, bacterial genes, believed to have been derived from food sources, have replaced many ancestral eukaryotic genes. Together, these results indicate that microbial sex results in the dispersal of loci in contemporary microbial populations.

Gene movements in insects has been comprehensively reviewed documenting extensive similarities of nature to lab genetic engineering by Robertson and Lampe (1995, Ann Rev Ent).

Likewise in plants, R. A. Emerson was the first to document red- white segments in `Calico' corn (Emerson, 1914, Am Nat) which was later shown by Brink and Nilan (1952, Genetics) to be the phenomenon established by Barbara McClintock as ``Controlling Elements'' (1945, Carnegie Inst Wash Year Book). Since then, ``Controlling Elements'', ``Mobile Elements'' or ``Transposable Elements'' as they are called have been researched extensively for their genetics and characterised at the molecular level by numerous researchers including this author (Natarajan, 1987, Iowa State University). These mobile transposable elements (TEs) have been shown to exist in multiple families with autonomous and non- autonomous members, move within and between chromosomes, disrupt gene function, cause target site duplications and multiply. TEs can exist in a genome in a quiescent state and can be activated by biotic or abiotic stresses that have been collectively termed as ``genomic stress''. What is more, TEs have been found in every plant taxon investigated thus far.

There are two recent publications that have raised the horizon of our understanding of the dynamic role of these TEs in the plasticity of eukaryotic genomes. SanMiguel and his collegues have reported (1998, Nat Genet) evidence of retrotransposon activity in doubling the size of the maize genome within the past 3 million years, demonstrating the active role of such elements in restructing a genome. The other, Kalendar et al (2000, PNAS, USA) illustrate a genome size variation due to retrotransposon amplification and intra-element deletion.

At another plane, horizontal gene transfer (HGT) in nature into plants from a soil bacterium Agrobacterium tumefaciens is well documented (Fraley et al, 1983, PNAS, USA) and is a popular technique adopted by scientists to introduce many desired genes into plants. In fact, 17 human disease genes ranging from hyper- insularism to heredity deafness, fam cardiac myopathy, myotonic dystrophy have high levels of similarity to the genes discovered in Arabidopsis thaliana, a crucifer. In addition, 37 per cent of the human genome is composed of virus-like foreign DNA!

Thus, in all three kingdoms - microbe, plant and animal - a whole range of changes such as additions, duplications, deletions, mutations, modification, activation and silencing of genetic material has been regularly occurring in a random manner and in a ``foreign'' environment over the millennia mediated by and actively engaged in by viruses, retroviruses, bacteria, plasmids, phages, transposable elements and extra-nuclear genomes.

The plasticity of the genome has been established in every organism examined. In fact, it would seem that the genome's integrity is indeed sustained, aided and enhanced by such dynamism in a changing milieu spanning different epochs.

Other myths

One of the many myths floating around is that some of the products of r-DNA technology as the glyphosate-based herbicide is toxic to animals and humans. The science of the matter is that glyphosate is non-toxic to mammals and fishes. In fact it gets bound on contact with soil components and is rapidly degraded by soil microorganisms, leaving little or no residue (Wilkins, 2000, Critical Rev Plant Sci); what is more, there is no known case of reported herbicide resistance to this product. Likewise, that Bt- mediated resistance to insects conferred upon corn and cotton are destructive to monarch butterflies that feed on the pollen of genetically engineered plants. Nothing can be farther from the truth and empirical data in peer-reviewed publications have shown that r-DNA technology does not harm the environment or cause risk to the biodiversity but on the contrary, aids and promotes the reduction of toxic wastes that would otherwise be generated from massive application of pesticides and herbicides to protect the crops.

Marker genes

The use of marker genes of r-DNA work has been marauded by the ignorant with claims that they are antibiotic-resistant genes and that this creates the spread of antibiotic resistance to all organisms that come in contact with the transgene. There is no scientific evidence for the occurrence of direct gene transfer of DNA present in the transgenic crop or food to humans, animals or microbes including those from the gastrointestinal tracts of animals to its microflora. This is so because, the half-life of plant genomic DNA is extremely short. In the case of genetically engineered corn leaf fed to a cow, the low pH and degradative enzymes in the ensilation process would result in rapid DNA degradation. DNA not degraded to single strands prior to consumption would be subject to the harsh degradative environment in the gut and rumen. It has been clearly established (Ausubel, 1987, Wiley and Sons) that plant cells inherently have an abundance of highly active nucleuses that will digest plant DNA upon cell lysis during mastication and the process of digestion. In lab experiments to isolate plant DNA, the integrity of plant DNA can be ensured only through adding protein denaturing agents, without which all DNA will be degraded to fragments of less than 500 base pairs. Neither ruminants nor humans produce such stabilising agents in their stomachs. Few, if any, DNA that escape the above steps of degradation would be subject to digestion by the extra-cellular nucleuses from ruminal and gut bacteria (McAllan, 1980, Brit J of Nutrition). The action of intracellular restriction endonucleases which are common in ruminal bacteria would be a further deterrent to intact DNA (Morrison, 1996).

Frequently concerns are expressed in the popular press about food security and the propriety in adopting r-DNA technology to address these issues. A brief review of some of the fundamental approaches adopted by researchers that help us feed a hungry world comprising over 6 billion inhabitants today would be pertinent here. The fact is that from among a pool of 250,000 flowering plants, only a hundred or so are intensely cultivated and a limited number among them provide all the energy and nutrients. From prehistoric days until today, plants have been transformed and rendered useful through a process of selection from among the variants. Along the way several transformations took place, prominent ones being determinate growth habit, elimination of shattering of grains/seeds, reduced growing cycle, uniform maturity, enhanced fruit size, increased grain output, resistance to pests, diseases and drought/flooding and so on.

Consider the ancestral marble sized, terribly bitter and poisonous Lycopersicon that has given rise to the now familiar dainty and succulent tomato and the transformations that this species has undergone through human intervention to get a perspective of the processes involved in creating a useful plant product. The narrow pool of native genetic diversity is perpetually augmented in nature by mutations (brought about by horizontal gene transfer), hybridisations and selections. Plant breeders add to these variations by using ionising radiation, mutagenic chemicals or cell culture. The more the variations, the better the prospects of pyramiding useful traits into a cultivated variety. Since no single plant carries all the desirable traits, in traditional plant breeding, crosses are made between two parents to bring about useful traits in the progeny followed by selection. However, the process meant mixing of thousands of genes, as it were, between the two parents. However, modern r-DNA technology achieves the same in a very precise manner by inserting only one or two genes at a time. Thus, the new technology is no different from the classical one save that it is more precise, more accurate and importantly, puts together more traits in a desired plant than was hitherto possible due to restrictions imposed by sexual incompatibility of species or intransigence of cells and tissues when cultured.

It is further well documented that integration of genes and whole genomes have taken place in nature to result in useful plant species, prominent examples being the modern bread wheat, Triticale, nectarine and so on. In agriculture, plant breeders have been moving genes from one species to another for a very a long time through sexual crosses, often using ``bridging'' species. In wheat and rice, for example, many disease resistance traits were introduced from ``alien'' species (Khush and Toenniessen, 1991, Biotechnology in Agriculture, Wallingford). Using modem biotechnology, plants have been made more resistant to insects, bacteria, fungi, and viruses, all of which lead to global production losses of well over 35 per cent estimated at over US$ 200 billions annually (Krattiger 1997, ISAAA Briefs 2). Food quality enhancement by reducing certain enzymes in fruits and perishable vegetables reduces their perishability and significantly cuts post-harvest losses (Neupane et al 1998, in: Acta Horticulturae, Brisbane, Ed. R. A. Drew). Further, certain naturally occurring substances in plants can be increased such as anticancer compounds naturally found in soybeans (Wang and Wixon 1999, INFORM), vitamin A in rice (Burkhardt et al 1997, Pl Journal), iron content in cereals (Theil et al 1997, Eur J Cli Nutr), or more non-saturated fatty acids in canola (Kramer and Sauer 1993, Scan J Nutr), and other oil crops. Plants can also be used to deliver edible vaccines, which would have a tremendous impact in developing countries.

In conclusion

In the past 15 years of intensive governmental, academic and commercial scrutiny, not a single incidence of actual harm to human or animal health, safety or the environment has ever been documented concerning the approved crops or the health-care products on the market today. Does this tantamount to a zero risk situation? Absolutely not. Zero does not exist in terms of risk. But, what is the evidence of adverse effects? Absolutely none.

Like any technology, r-DNA technology carries with it many advantages and some perceived risks. The challenge would be to manage the risks in order to maximise the advantages. A judicious combination of the best of science and due caution, tempered by transparency and enabling systems are key ingredients to harnessing the benefits of this technology for the large good of mankind.

Dr. GURUMURTI NATARAJAN

A plant breeder and molecular biologist

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