Decisions on GM crops should be taken on the basis of sound science only after incorporating known advances in plant transformation technology.

The recent restraint on commercial release of Bt brinjal despite approval of the Genetic Engineering Approval Committee has become controversial. The step was taken after consultations with diverse stakeholders in several states. The majority view in the consultations was that commercial release of Bt brinjal could wait. Reasons included cautionary advice of European scientists about inadequate bio-safety assessment by seed producer company Mahyco and possible threats to indigenous brinjal biodiversity. Some scientists and politicians consider the decision a setback to advances in agriculture biotechnology and therefore to attainment of food security in the long-term. Many biotechnology researchers have taken it as a blow to their efforts. So we must carefully chart the way ahead for introduction of genetically modified (GM) crops and for relevant avenues of biotechnology research, especially in genetic engineering.

Bt brinjal is a genetically engineered brinjal containing the Bt toxin gene from the soil bacterium Bacillus thuringiensis (therefore, Bt). The Bt toxin confers resistance to two pests — fruit and shoot borer (FSB, Leucinodes orbonalis) and fruit borer (Helicoverpa armigera). Genetic engineering (GE) or recombinant DNA technology (r-DNA) is a path-breaking technique compared to conventional plant breeding, as it allows genes to be transferred across species, from animals to plants, microbes to higher organisms and vice versa. Like other GM crops, commercial production of agriculturally suitable Bt brinjal involves two steps. First is the production of the primary transformant by GE. The gene to be transferred (transgene), for instance, the Bt gene, is inserted into a chromosome of a target crop variety, which is most amenable for its cellular acceptance and integration by a particular r-DNA protocol. The host variety for this primary event has high acceptance for the transgene, but is usually not agriculturally suitable and therefore, we need a second step, namely, the production of the commercially viable and agriculturally suitable GM hybrid or variety. This is done by transferring the Bt gene from the primary transformant to a hybrid or variety by a conventional plant breeding technique based on cross-pollination.

In the first step, GE is used to ensure that an alien gene of a desired trait can be inserted and integrated into a crop of interest. In nature a gene, on accidental entry into an alien cell, is immediately destroyed. The exceptions are the DNA of parasitic bacteria and viruses. They have some DNA of unique mobility and protective armour which confer on them the property of crossing species barriers and infecting and surviving in alien host cells. Genetic engineers have taken advantage of this phenomenon. The transgene is attached to such mobile microbial DNA, which acts as a carrier and then by suitable protocols this recombinant DNA is transferred to host cells. From these transformed host cells, whole plants or primary transformants are developed using tissue culture techniques. Seeds of these are collected for future use.

In the second step, the primary transformant is crossed with suitable hybrids or varieties to produce the usable GM crop. The favoured method for this is backcrossing, to obtain homozygous plants which have uniform expression of the transgene, reliable performance and which breed true with regard to the inheritance of the transgene.

Thus, Mahyco produced Bt brinjal primary transformant by incorporating the Bt gene into a bacterial plasmid DNA (pMON10518) and transferring this r-DNA by the common agrobacterium-mediated transformation technology to a brinjal variety. This primary transformant was crossed with several brinjal hybrids (MHB 4, 9, 10, 80, 99 etc) to produce the Bt MHB lines for commercial release.

What is not so well-known, however, is that for most commercially released GM crops the protocols still date from as far back as the mid-1990s, overlooking the many advances in GE technology since then. For instance, Mahyco uses a slightly modified technique enunciated by M. Fari et al in 1995 (Fari et al, Plant Cell Report, pp.82-86, 1995). The plasmid used continues to have, besides the Bt transgene, antibiotic resistance markers (npt II and aad) and the 35S CaMV promoter. Over the last two decades, however, plant transformation technology has moved ahead rapidly as scientists around the world have endeavoured to make the technology bio-safe. They recognise the danger that since the transgene-vector recombinant DNA had the capacity of ‘jumping into' alien species, it could also ‘jump out' of a transgenic crop and ‘jump into' another species causing gene contamination. In this context, a major apprehension was that the antibiotic resistance marker DNA fragments could spread to other species from the GM crop. To take care of these dangers, genetic engineers developed safe markers and protocols for obtaining marker-free transgenic crops.

Similarly, scientists had reservations about the gene switch (promoter) derived from the Cauliflower Mosaic Virus (35 S CaMV) as parts of its base sequence resembled some sections of the HIV virus. To avoid it, safe promoters have been designed. Innovations on promoters have also focused on tissue-specific promoters for tissue-specific transgene expression in plants. For instance, at present in the commercially released Bt crops, Bt gene expression being non-specific, Bt toxin is formed in all organs of the plant. It would be better if it were expressed only in the susceptible tissues and not everywhere including the roots. With new tools it is now possible to have expression of a gene, only where it is needed and by using controls of temporal expression, when it is needed. Random unpredictable insertion of the transgene into the genomic DNA has been another concern of the researchers. Such random insertion, even in non-genic segments of the genome, could have unintended negative consequences. Therefore, attempts for site-directed non-random insertion of a transgene have been a thrust area of research with some success in the case of plants. A rapidly developing area constitutes attempts to insert multiple genes in a crop or gene stacking. One such example, besides Golden Rice, is the production of GM cotton with the Bt gene along with a gene for resistance to sucking pests developed at the National Botanical Research Institute, Lucknow under the leadership of Rakesh Tuli.

Many such advances have been made in plant transformation technology to make it more efficient, relevant and bio-safe and this is a continuing effort around the world, including India. However, it is disconcerting to note that in India the GM crops released or waiting to be released have been produced through an underdeveloped technology dating from the mid-1990s.

A key concern regarding the second step of transfer of the transgene from the primary transformant to a suitable hybrid or variety through repeated backcrossing is the choice of the acceptor host hybrid or variety. In India with diverse agro-climatic zones a preferable strategy would be to use acceptor lines which are best adapted to particular zones of cultivation of a crop. This approach is being followed for Bt cotton being developed by the Central Institute for Cotton Reseach (CICR), Nagpur, under the stewardship of K.R. Kranthi. However, commercial release of GM crops tends to take a short-run view and to attract farmers, companies use very high yielding hybrid lines as acceptors. These very high yielding hybrids and their Bt counterparts require much higher inputs of fertilizers and irrigation than even the Green Revolution hybrids. The even more important point is that world-over the higher yields of GM crops are not because of the inserted transgenes but due to the use of very high yielding hybrids or varieties as the acceptor host.

In view of the above considerations, it would be wiser to take precautionary measures and not rush into commercialisation of a technology that is currently still being perfected by our scientists. One should wait till truly bio-safe GM crops, especially bio-safe food crops have been produced using advances in plant transformation technology. Researchers are already working towards producing marker-free GM crops, with safe promoters, site-directed insertion of single or stacked genes, genes expressing in specific tissues, and other necessary attributes for bio-safety. A major endeavour of genetic engineers is the production of transplastomic GM crops through chloroplast transformation rather than nuclear transformation. In such transgenic crops there is enhanced formation of the transgene product, as a plant cell contains only one nucleus but many chloroplasts. Further, with transplastomics there is little chance of gene contamination by pollen flow. This should become a thrust area of plant transformation initiatives. In this context, the proposal that GM research should mainly be in the public sector is of great relevance. There could also be public-private partnered GM crop production.

As Prof. M.S. Swaminathan has said, “Unless R&D efforts on GM foods are based on principles of bio-ethics, bio-safety, bio-diversity conservation and bio-partnerships, there will be serious public concern in India, as well as many developing countries, about their ultimate nutritional, social, ecological and economic consequences.”

(The writer is Director, Bio-Science, Samaj Pragati Sahayog, Madhya Pradesh. Former Professor of Botany at CCS University, Meerut, he was associated with pioneering work on agrobacterium-tobacco DNA combination at the Roswell Park Memorial Cancer Research Institute, New York.)

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