Recent advancements in the area of genetic engineering, molecular biology and plant tissue culture have made it possible to mobilise genes from across the biological world into plants. Development of new techniques of gene cloning, gene transfer to plants and success in regeneration of crop species led to this rapid progress. By using various methods of gene transfer discovered till date, almost all plant species including those which were recalcitrant to regeneration and genetic transformation such as cotton, sugarcane, wheat, rice, etc, have been genetically engineered. Of the various methods of gene transfer, the most notable ones, such as the agrobacterium-mediated and the particle bombardment-based gene delivery, have been deployed for generating transgenics.

Starting from 1986, when the first transgenic crop was tested in a field trial, and until 1997, approximately 25,000 field trials covering 60 transgenic crops and 31.5 million acres of land have been conducted in 45 countries around the world. These trials were carried out mostly with the transgenics of maize, tomato, soybean, canola, potato and cotton with engineered traits which include herbicide tolerance, insect resistance and virus resistance. Some of these genetically modified crop plants with improved traits have already been commercialised (Table 1). Besides, a number of genetically modified plants have been developed with improved quality of oil, protein and starch for food use. The commercial application of these transgenics is expected to increase productivity of crop plants and also contribute significantly to nutritional security.

Table 1: Genetically Modified Crops Already Commercialised in the USA

Genetic Modification

Productivity enhancement: The increasing demand on food has necessitated the application of all the tools available for increasing crop productivity. Our foremost challenge is to increase photosynthetic efficiency through genetic engineering. One of the time tested strategies available for improving the production potential of crops is through the exploitation of heterosis. So far, it has been possible to harvest hybrid vigour in those crops where controlled pollination or cytoplasmic male sterility systems were available. Genetic engineering provides a means of imparting reversible male sterility, thereby ensuring economically viable hybrid seed production. One such technique based on the “barnase barstar” male sterility system has been very effective in Indian mustard. Since this approach is species neutral, attempts are being made to genetically modify other crop plants while looking for new and more efficient alternatives. Ensuring at least 20-30 per cent gains in productivity through hybrid vigour can go a long way in providing food and nutritional security.

Engineering biotic stress tolerance: In India, it has been estimated that biotic stresses caused by insects, nematodes, fungal, bacterial and viral pathogens and weeds, collectively result in approximately 45 per cent yield loss annually. By any standard this is a staggering loss and even a part of it that can be saved will add substantially to the total food basket of the country. So far management of these pests has been through deploying resistant varieties bred by conventional means and by chemicals which are often used injudiciously. Ironically for some of these pests such as Helicoverpa, genes imparting tolerance are not available in the germplasm. Such insects are being managed through chemical sprays, which escalate the cost of cultivation and also result in environmental pollution.

Genetic engineering provides the means of managing these pests in a more sustainable way. A variety of genes have been used for imparting biotic stress tolerance to different crop plants. The most significant development in the area of biotic stress management has been the use of the insecticidal crystal protein genes of the soil bacterium Baccillus thuringiensis (Bt) for engineering varieties of cotton resistant to the most devastating pest, the boll worm.

Significantly, more than 30 plant species have been transformed with a variety of Bt genes. These transgenic crops are being commercially cultivated in a number of countries including the USA, Australia, China and Mexico. Within the ICAR system, Bt transgenics of rice, brinjal and tomato have been successfully developed for resistance to stem or fruit borers, at the National Research Centre on Plant Biotechnology, IARI, New Delhi. Once these transgenics become available for commercial cultivation, they will go a long way in reducing the burden of the extra management cost of chemicals on the part of poor marginal farmers of the country.

Abiotic stresses: In India, in spite of all the developmental efforts in the past, more than 64 per cent of the cultivated land is rainfed. The crops in these areas often have to face situations of short to long periods of drought. Also, more than 8 million hectares of land suffers from salinity and alkalinity. These abiotic stresses result in significant yield losses. Classical plant breeding methods and in vitro induced variation have been applied to improve the stress tolerance of various crop plants but without much success. It has been difficult to manipulate precisely the individual components of stress-tolerance. Research over the past two decades has provided a better understanding of the molecular basis of stress responses in plants. Many genes and gene products have been identified which get induced upon exposure of plants to various abiotic stresses – salinity, drought, low and high temperature, etc. Consequently, biotechnological tools have been applied to transfer some of these useful genes, implicated in stress tolerance, to plants. In addition to these stress-induced proteins, genes encoding enzymes of the biosynthetic pathways of different osmolytes such as proline, glycine betaine, sorbitol, pinitol, etc, have been cloned and exploited in improving abiotic stress-tolerance in plants through genetic engineering.

Recently, for instance, a bacterial gene Cod A isolated from a soil bacterium Arthrobacter globiformis was introduced into Arabidopsis and rice which normally do not accumulate glycine-betaine in response to stress. There was improvement in the overall ability of the transformed plants to germinate and grow under salt-stress conditions.

The Cod A gene is being deployed currently to improve abiotic stress-tolerance in some of the Indian crops as well. It has been reported that compared to many other compatible solutes such as proline, pinitol, sorbitol, etc, glycine-betaine is most powerful for the enhancement of salinity and drought-tolerance in higher plants. In addition, it has the ability to impart improved tolerance to freezing cold and high temperature stresses.

However, the transgenics produced so far with one of these genes alone have shown only marginally improved tolerance to specific or collective abiotic stress factors. It is being realised that any of these genes alone might not provide the required tolerance levels in the plants, while, pyramiding these genes is one of the alternatives which is being explored for engineering tolerance to abiotic stresses.

Reducing post-harvest losses: In a vast country such as India, transportation of fruits and vegetables across long distances requires extensive periods of storage. Unfortunately, most fruits and vegetables have a very short shelf life that leads to extensive losses during storage as well as transportation due to damages through over ripening. With the advent of biotechnological techniques resulting in transformation of crop plants with novel genes or gene constructs, avenues have been opened for reducing post-harvest losses and for improving the quality of horticultural crops. The main focus here is to prevent and minimise losses due to over-ripening, physical damage, chemical injury and pathological decay.

For this purpose, several genes have been identified and used in sense or antisense orientation in different crops. Currently two approaches are being used to engineer plants with a longer shelf life of fruits:

• Engineering genes controlling ethylene biosynthesis and ethylene sensitivity, and
• Reduction in polygalacturonase activity by antisense gene expression.

Antisense RNA technology has been used as a tool of choice to specifically inactivate the expression of ACC synthase and ACC oxidase genes encoding the key enzymes required for ethylene biosynthesis and to curtail its production in ripening fruits. Expression of antisense RNA derived from the cDNA of the ACC synthase gene has resulted in an almost complete inhibition of mRNA accumulation of both ripening induced ACC synthase genes. This has led to very low levels of ethylene production in transgenic plants. Transgenic fruits still attached to the plant neither turned red nor softened.

At IARI, transgenic tomatoes are being developed with improved post- harvest characteristics using ACC synthase and annexin genes. In a similar approach, transgenic apples and cabbages have also been developed elsewhere with delayed ripening and prolonged shelf-life. Similarly, antisense constructs based on a gene for polygalacturonase (PG) have also been used to reduce softening and ripening in tomatoes. Low PG activity has conferred improved firmness on fruits throughout the ripening process. Bruise resistant tomatoes have been developed which express antisense RNA against the endogenous PG gene. The transgenic tomato developed by Calgene, Inc, (USA) using the antisense PG gene was the first to be commercialised in 1994 and was called ‘Flavr Savr’.

Increase in shelf life of fruits and vegetables will facilitate the safe transportation of these commodities across the country, it will also ensure remunerative prices to farmers who, at present, are unfortunately compelled to make distress sales due to the perishable nature of farm produce.

Development of value-added food products: In addition to traits related to storage and shelf life, harvested products can be endowed with better characteristics such as protein quality, oil quantity and quality, higher starch content and other nutrients. Some of these examples are described below:

Altering the fatty acid composition in vegetable oil: Edible vegetable oils, except coconut and palm oil, possess relatively less saturated fatty acids. The composition of the fatty acids in these oils varies from crop to crop. Genetic engineering provides an easy way for selectively altering the fatty acid composition such that the modified oil is better suited to human health. With the help of antisense technology Calgene, Inc, (USA) has succeeded in developing rape seed with high stearate levels by blocking the stearoyl ACP-desaturase (SAD) activity in seeds. The seed stearate content was increased from a mere 2 per cent to as high as 40 per cent.

The high stearate rape offers a novel and safer alternative to industrially hydrogenated oils for human consumption. Oils stored for a relatively longer period undergo spontaneous changes making them unacceptable for human consumption. An increase in oleic acid content in oil reduces these changes and improves the stability and shelf-life of edible oils. Antisense repression of 1-12 oleoyl desaturase in the transgenic rape seed resulted in an increase in oleic acid content up to 83 per cent.

Improving protein quantity and quality: Plant proteins are an important source of essential amino acids in human diet. Indians, primarily being vegetarians, depend to a large extent for their protein on plants and plant-derived products. Conventional breeding methods have had limited success in both quantity and quality improvement of proteins because of tedious and time consuming selection procedures. Genetic engineering techniques provide a way out to bring in directional changes in both quantity and quality of proteins. For example, over expression of glutenin gene has resulted in increasing the glutenin protein content in wheat leading to an overall increase in the total protein. Likewise, attempts are being made to engineer protein quality by mobilising heterologous genes such as 2S storage protein from Amaranthus into potato and rice (JNU, New Delhi). It has been demonstrated that this storage protein from Amaranthus has higher levels of some essential amino acids than those recommended by World Health Organization (WHO) (Table 2).

Table 2: Essential Amino Acids (per cent) of AmA1 Protein vs WHO Recommended Values

Amaranthus is a minor millet widely cultivated and consumed both for leafy vegetable and puffed grains, and, therefore, this protein has been a component of the human diet for a long time. Mobilisation of this gene from Amaranthus into other target crops through genetic engineering is expected to improve the protein quality without any deterimental effects on human or environmental health. The protein quality is also being improved by manipulating the enzymes involved in aspartate pathways such as homoserine dehydrogenase, dihydrodipicolinate synthase and threonine dehydratase, thereby increasing the quantity of certain essential amino acids such as lysine, threonine and methionine.

Increasing the starch content: Genetic engineering techniques have been effectively utilised in modification of the starch content in a number of crop plants. In potato, the over expression of the enzyme ADP-glucose pyrophosphorylase (AGPase) resulted in increased amount of starch in potato (Monsanto, USA). These transgenic potatoes were found to be suitable for finger-chips. Generally, the chips produced from normal potatoes show browning of the margin on frying, primarily because the peripheral tissues contain relatively less starch. The finger-chips from the transgenic potatoes result in uniform frying besides the added advantages of less oil being used during frying and reduction in oil intake in human diet. There are several other possible strategies of modifying natural starches for food use through genetic engineering (Table 3).

Table 3: Potential Strategies for Obtaining Modified Natural Starches

Increasing the vitamin content in plants: Naturally occurring tocopherols are lipid-soluble antioxidants. Of the different tocopherol species present in foods, alpha-tocopherol is most important to human health. It possesses the highest vitamin E activity. However, plant oils, the main dietary source of tocopherols, typically contain alpha-tocopherol as a minor component whereas gamma-tocopherol is more abundant. Using the genetic engineering approach overexpression of a gene encoding gamma-tocopherol methyl transferase (gamma-TMT) resulted in an increased level of alpha-tocopherol in Arabidopsis seed oil by converting gamma-tocopherol to alpha-tocopherol. It is thus possible to increase the vitamin E activity by increasing the proportion of alpha-tocopherol through genetic engineering in a number of oils and crops such as Indian mustard (Brassica juncea) and groundnut. It is estimated that a vitamin E-rich diet can reduce the risk of cardiovascular disease and some cancers, improve immune function and slow down the progression of a number of degenerative human diseases. Similarly, vitamin A content has been increased by increasing the beta-carotene content in rape seed oil by genetic engineering by Monsanto, Inc, USA.

Edible vaccines: Transgenic plants that express foreign proteins with industrial or pharmaceutical value represent an economical alternative to fermentation-based production systems. Specific vaccines have been produced in plants as a result of the transient or stable expression of foreign genes. It has recently been shown that genes encoding antigens of bacterial and viral pathogens can be expressed in plants in a form in which they retain native immunogenic properties. Transgenic potato tubers expressing a bacterial antigen stimulated humoral and mucosal immune responses when they were provided as food. These results provide ‘proof of the concept’ for the use of plants as a vehicle to produce vaccines.

Some examples of edible vaccines that have been developed by genetic engineering of plants are: Hepatitis B surface antigen and E coli heat labile enterotoxin in tobacco, cholera toxin – B subunit in potato and Streptococcus surface protein in tobacco.

Relevance of Genetic Modification

Plant breeding methods used for crop improvement ensure bringing together a constellation of genes responsible for traits of economic importance, be they productive traits, quality traits or novel traits. In genetic engineering, individual genes affecting the traits of economic importance are mobilised in superior cultivars through in vitro techniques. If the gene happened to be the one which affects the quality of the food such as modification of starch or oils or protein, etc, then the genetically engineered product is expected to have better nutritive value. Such modifications are possible through conventional mechanisms.

However, breeding for them is tedious and time consuming and, therefore, the success rate is limiting as compared to the genetic engineering approach which brings in a precise change in a genotype through the directed transfer and expression of a gene that controls a given trait of economic importance in an otherwise agronomically superior genotype. It is, therefore, evident that irrespective of the methodology used product development is qualitatively similar.

Current Concerns

Since genetic engineering provides an opportunity for mobilising genes across the biological world, there are debates going on as to how a gene implanted in an alien environment will behave. The concern being expressed in the world press on biosafety related issues of genetically engineered plants stems from two major factors:

• Lack of information on the behaviour of a gene transferred from unrelated organisms into an alien atmosphere, and
• Gene products possessing adverse impact on human health and environment. As we know, a gene per se is a stretch of DNA – an organic molecule which primarily acts through protein or enzyme produced by it. Therefore, it can act independently and it might get influenced by the native environment. In many cases, the interactive output cannot be anticipated and, therefore, remains undefined. The debate, therefore, has its origin in a “fear of the unknown” because if the gene product per se turns out to be allergenic or toxic then certainly it’s a matter of concern when such a product forms a component of human or animal food chain.

Added to this is another dimension concerning the genes which are, as such, not important from the economic point of view but form an indispensable part of total genetic engineering approach because of their use as selectable markers. This is an essential requirement because in a multicellular system only a few cells which are transformed need to be selected and allowed to get differentiated into transgenic plants from the bulk of untransformed cells. These selectable marker genes are those which impart tolerance to antibiotics or herbicides.

Apprehensions on their accidental transfer to microbes in the human system in case of antibiotic tolerance and to wild relatives in case of herbicide tolerance have been raised at various fora. The current range of marker genes do not code for any protein with known toxicity and, therefore, are considered safe. Moreover, most antibiotic resistance marker genes used in genetically modified plants are of no clinical importance and are widely spread in microflora.

Scientific scrutiny of the apprehensions voiced across the globe about the adverse effect of genetic engineering products on human health and environment leads to the conclusion that while some of the concerns might be genuine, most come from ignorance and a ‘fear of the unknown’. Genuine could be those cases where the gene product might be allergic or toxic to human health.

Our experience with transgenics, at present, is too meagre and inadequate. World opinion on views of transgenic plants have got distinctly polarised in support of or against the use of the genetically modified plants. The factors for this polarisation are many. The corporate sector which has invested a huge sum of money is eager to quickly start getting returns on investment and, therefore, favours a quick acceptance of this technology by the masses. The public, on the other hand, would like to be convinced about the safety of such foods before their commercialisation. The governments, on their part, have taken the role of regulators of this technology.

Several developing countries, including India, have their biosafety protocols in place, ensuring thereby a critical evaluation of transgenic plants for harmful effects, if any, on human or animal health and environment before permitting large scale cultivation. India, on its part, is quite aware about the benefits of genetic engineering to address the problems related to agricultural productivity in quantitative and qualitative terms. Indian regulators have taken a stand where each transgene must be subjected to a critical evaluation on a case to case basis. Genes found to be innocuous from the angle of biosafety and with proven economic values need to be utilised in generating transgenics.

In order to achieve this, each transgenic product must be evaluated following prescribed parameters for its allergenicity, toxicity, etc, in scientific laboratories of repute. The environmental impact of a transgenic resulting in a shift in ecological balance when a transgene, consciously or unconsciously released in the environment, is an aspect which would require continuous observation over a period of time. Therefore, initial recommendations for the suitability of such a transgenic can be decided only by extrapolation of available related information.

Horizontal gene transfer in the plant kingdom has played a crucial role in evolution in the past and it continues to expand the existing genetic base even today. Transgenics released for commercial cultivation will become an integral part of our total bioresource and, depending upon their growing habits including sexual compatibility, are expected to release/receive genes from other related plants. Nature has always supported the survival of the fittest and, therefore, a gene with higher fitness values will be favoured and retained. Since case to case studies will address this aspect of gene behaviour, a gene found to have higher adaptive negative values will certainly not be permitted to be released. This would require prior testing and verification of the behaviour of the gene in question in the natural environment.

Since testing and verification is highly time consuming, alternative methods are being suggested wherein the transgenes do not get transmitted through pollen grains to the next generation. The genes can be transferred to plastids in the cell which are inherited maternally in almost all crop plants. Once such protocols are fully developed, at least the apprehensions related to the pollen transfer of genes will be completely eliminated, and related environmental issues will stand resolved. Till such time, the country must strengthen the facilities for generating biosafety data at a few institutions of repute so that the advantages emanating from this technology are quickly harnessed.