A plant which bears a foreign gene of desired function of other organism is called transgenic plant. During the last 20 years considerable progress has been made on isolation, characterisation and introduction of novel genes into plants.
As per estimate made in 2002, transgenic crops are cultivated world-wide on about 148 million acres (587 million hectares) lands by about 5.5 million farmers.
Transgenic crop plants have many beneficial traits like insect- resistance, herbicide tolerance, delayed fruit ripening, improved oil quality, weed control, etc. Some examples of transgenic crops approved by Food and Drug Administration (FDA) (USA).
Currently, India is importing both grain legumes and edible oils to meet people’s demand. By 2050, India’s population is expected to reach about 1.5 billion. It is hoped that 30% India’s population will be suffering from malnutrition.
Therefore, nutritional security for everyone would require the extensive availability of grain legumes, edible oil, fruits and vegetables, milk and poultry products. These challenges can be met by better resource management and producing more nutritious and more productive crops.
A new institute, the National Centre for Plant Genome Research (NCPGR), has been established in New Delhi to strengthen plant biotechnology research in India. Department of Biotechnology (DBT) (Ministry of Science & Technology) has made enough fund for promotion of crop biotechnology.
Bt-cotton was released for commercial cultivation in March 2003, following bio-safety procedures laid down by the Government of India.
These cotton hybrids have been granted permission for field sowing and are currently in the fields in the six states namely, Maharastra, Gujarat, Madhya Pradesh, Andhra Pradesh, Kamataka and Tamil Nadu. Transgenic mustard hybrids after trials have been found suitable and released for cultivation.
There are several beneficial traits which have been transferred and transgenic plants have been produced for example, stress tolerance, delayed fruit ripening, male sterility, molecular farming, improvement in nutritional quality, etc.
1. Stress Tolerance :
There exists a variety of stresses in natural environment growing anywhere. This is why production and yield are declined. In nature ideal conditions rarely exist. Crop plants always produce maximum under ideal conditions.
All types of stresses are broadly categorised into two major groups: abiotic stress (e.g. salinity, herbicide exposure, draught, low temperature, high temperature, nutrient deficiency, etc.), and biotic stresses (e.g. viral pathogens, bacterial pathogens, fungal pathogens, insect pests, nematode pests, weeds, etc.).
Under these conditions plants respond poorly. Consequently stress causes loss of crop yield and quality. Using biotechnological approaches stress tolerant plants can be produced. These aspects have briefly been discussed below:
(a) Abiotic Stress Tolerance:
As described earlier there are many kinds of stresses which severely affect the growth and yield of plants. In contrast, plants respond against abiotic stresses. They secrete stress-related osmolytes such as sugars (fructans and trehalose), sugar alcohols (mannitol), amino acids (betaine, glycine and proline) and other proteins.
For example, betaine is a highly effective osmolyte that accumulates in some plants during water-stress or high salinity. Betaine is synthesised both in bacteria and plants. A transgenic tobacco was prepared by transferring E. coli bet A gene through Ti-plasmid. The transgenic tobacco was 80% salt tolerant (i.e. 300 mM) than the normal tobacco.
Following bio-technological strategies, several abiotic-streess tolerant transgenic plants can be produced. The transgenes over-express one or more proteins as mentioned above. Therefore, stress-tolerant transgenic plants can tolerate stresses imposed by the environment.
(i) Production of Drought Resistance:
The level of water is gradually going down due to low rains and high temperature. Moreover, the plants requiring substantial amount of water suffer from drought conditions.
This leads to the development of severe drought condition in that area. Consequently the herbivores such as humans and other animals suffer a lot resulting in famine condition. Such situation is spreading in several third world countries.
The recent catastrophic crop failure, in many countries such as southern Africa due to drought, has brought on famine conditions. It also raised the question, what could genetic modification (GM) technology offer to poor farmers working in marginal lands vulnerable to drought, including many of those in sub-Saharan Africa?
Several international agricultural research organizations have already devoted considerable effort to improve drought resistance in the staple cereals that feed most of the world’s poor.
Plant breeders and farmers are well aware that some plants cope with drought conditions much better than the others. GM technology makes it possible to transfer genes conferring this drought tolerance to important food crops.
The introduction of such crops has become the target of attacks by environmentalist groups. Because such crops have the potential to significantly enhance the food production in drought- stricken parts of the world.
The result may be to prevent the whole communities from gaining access to a technological development that could literally make the difference between life and death.
1. A hope from the new technology:
The promising GM technology includes the use of techniques for increasing drought tolerance. Research works on these aspects are being investigated at the International Maize and Wheat Improvement Centre, Mexico and individual countries itself.
Wheat plants, that have been genetically modified to withstand drought, are now being tested in bio-safety greenhouses at the International Maize and Wheat Improvement Centre. Most of the plants produced have shown high tolerance to extreme low-water conditions.
This research illustrates how recent advances in both molecular genetics and genetic engineering can be applied to enhance drought tolerance in plants. Progress has been slow and difficult due to the complex effects of drought on plants.
2. The plant pathway:
At least four independent signalling pathways act in plants to switch on an array of genes in response to dehydration. Some of these genes code for proteins that help to protect various parts of the plant cell during water loss, while the others detoxify the harmful substances. Understanding how it would be best to utilize these genes is a lengthy process.
The researchers at the International Maize and Wheat Improvement Centre have initially focused on incorporating a type of DREB gene (encoding a dehydration-responsive element binding protein), which enables the wheat plants to withstand extreme water loss.
Unfortunately, when this gene is continually switched on, plants are smaller and produce much lower yields than unmodified varieties. This causes a significant disadvantage when it comes to plant breeding.
But the scientists then found that by fusing the DREB gene with the promoter region of another gene (rd29A), it is switched on only under the stress conditions of dehydration or cold temperatures. This results in a normal growth pattern and yield of plants in good conditions.
But it is much more resistant to drought, freezing, and high salinity. More work is now needed to fully characterize the function of the additional gene, and to dissect the complex process by which this gene is expressed.
3. A promising future:
The researchers at the International Maize and Wheat Improvement Centre are optimistic that their technique offers a promising way to deal with the challenges of drought. Other approaches have also been investigated such as:
(i) Over expression of a gene related to drought tolerance.
(ii) Accumulation of sugars and salts to protect against water loss.
(iii) Further investigation, at a molecular level, of the physiological mechanisms by which plants adapt to extreme environments. Such research will lead to a much more complete understanding of drought tolerance in plants.
With the help of genetic engineering it will be possible to create plants with these traits, without the need for long and tedious breeding programmes.
But taking the next critical step (namely moving these plants from the laboratory to the fields of resource-poor farmers in developing countries) will require a supportive public and the well-founded assent and collaboration of developing nations.
A concerted effort is now required to convince both decision-makers and environmentalist critics that the value of crops produced in this way (and the capability to alleviate to some extent the suffering faced by rural people in drought conditions) strongly outweighs any perceived health and environmental dangers.
(ii) Herbicide Tolerance:
Any unwanted herbaceous plants occurring in a crop field is called weed. Weeds compete with crops for light, water and nutrients and thereby cause a decline in crop yield and quality. There are certain herbicides which are used as pre-emergence stage to kill herbaceous weeds before planting the crop.
If crop plants are tolerant to these herbicides, they can be sown in field. All plants are not tolerant to herbicides. Herbicide-tolerance is a genetic trait. The herbicide-tolerance gene expresses enzyme which detoxify the herbicide and tolerate the effects.
Using biotechnological approaches transgenic plants have been produced by introducin herbicide tolerant gene in chloroplast degrading enzyme and detoxify herbicides.
For example initial Monsanto (U.S.A.) produced glyphosate under the trade name Roundup® which is a widely use non-selective herbicide. Transgenic plant Roundup Ready has been produced and commercialisec. It is tolerant to the herbicide Roundup®.
Similarly, Hoechst AG (Germany) produced phosphinothricin under the trade name Bastad which is also a non-selective herbicide. The scientists isolated the bar gene from Streptomyce; hygroscopicus which can degrade phosphinothricin. The bar gene was transferred into tobacco anc potato. The transgenic tobacco and potato showed herbicide tolerance without change in yield performance.
(b) Biotic Stress Tolerance:
The abiotic stresses have also been given above. Biotic stress tolerance has been discussed giving some examples.
(i) Insect Resistance:
There are a large number of mites, and insects that attack crop plants and cause great loss in quality and yield. To reduce the loss by the way of killing insect’s farmers apply insecticides (i.e. synthetic pesticides) on the crop plants.
However, the synthetic insecticides pose a serious threat to the health of plants, animals and humans. We cannot forget the Bhopal Tragedy of MIC (methyl isocyanate) gas leakage in the mid night of 2/3rd December 1984 where thousands of people died. Besides, thousands are still suffering from several side effects.
The alternative and novel ways of rescue from damages of insects are the use of transgenic technology. It is eco-friendly, cost-effective, sustainable and effective way of insect control.
The cry genes of Bacillus thuringiensis (commonly called Bt gene) was found to express proteinaceous toxin inside the bacterial cells. When specific insects (species of Lepidoptera, Diptera, Coleoptera, etc.) ingest the toxin, they are killed. Toxin denatures the epithelium of gut by creating many holes at alkaline pH (7.5 to 8).
The insecticidal toxin of B. thuringiensis has been classified into the four major classes: cry I, cry II, cry III and cry IV based on insecticidal activities against many insects. These toxins affect the specific group of insects. They do not harm the silkworm and butterflies or other beneficial insects.
Using biotechnological approaches many transgenic crops having cry gene i.e. Bt-genes have been developed and commercialised. Some examples of Bt- crops are brinjal, cauliflower, cabbage, canola, corn, cotton, eggplant, maize, potato, tobacco, tomato, rice, soybean, etc.
In India, Bt-cotton was permitted to sow at large scale in field. It contains crylA (c) gene that provides resistance against bollworm (Helicoperpa armigera) which is crylA a notorious pest of cotton. Several transgenic crop plants have been developed and commercialised at national and International levels. However, many transgenic plants are under field trials.
(ii) Virus Resistance:
Plant viruses causes severe disease on crop plants and result in yield loss in several economically important plants.
There are two approaches for developing genetically engineered resistance in plants: pathogen-derived resistance (PDR) and non-pathogen-derived resistance (non-PDR). For PDR, complete or part of viral gene is introduced into the plant which interferes the essential steps in the life cycle of the virus.
For the first time Roger Beachy and co-workers introduced coat protein (CP) gene of tobacco mosaic virus (TMV) into the tobacco. They observed the development of TMV-resistance in transgenic plants.
Now, there are several host-virus systems in which it has been fully established. In many crops virus-resistance transgenics have been developed by introducing either CP gene or replicase gene encoding sequences. Coat protein-mediated resistance (CPMR) is the most favoured strategy to make virus-resistant plants.
Several important crops have been engineered for virus resistance using CPMR approach and released for commercial cultivation. Transgenic plants can yield more than non-transgenic plants. Normal papaya and transgenic papaya resistant to papaya ring-spot virus are shown in.
TMV-tobacco mosaic virus; CMV-cucumber mosaic virus; PVX-potato virus X; PVY- potato virus Y; ZYMV- Zucchini yellow mosaic virus; WMV2- watermelon mosaic virus 2; PRSV- papaya ring spot virus; CP- coat protein.
(iii) Resistance against Fungi and Bacteria:
The fungal and bacterial pathogens attack host plants. There occurs plant-pathogen interactions. Consequently plants respond through several defence responses such as pathogenesis-related proteins (PR proteins).
The PR proteins include chitinase (cell wall degrading enzyme), (3-1,3- glucanase, small cystein-rich proteins, lipid transfer proteins, polygalacturonase inhibitor proteins, phytoalexins) and resistance genes from plants, etc.
After introducing desired genes into plants several fungal and bacterial transgenic plants have been produced. Some pathogen-resistant plants have been commercialised.
In 1991, Broglie and co-workers expressed bean chitinase gene in tobacco and Brassica napus. Such transgenic plants showed enhanced resistance to a fungal pathogen Rhizoctonia solani. A soil bacterium Envinia carotovora causes a serious loss to potato growers worldwide.
One group of researchers developed transgenic potato by transferring T4 lysozyme gene into potato. Transgenic potatoes were not infected by E. carotovora. Lysozyme can lyse a wide range of both Gram-positive and Gram-negative bacteria.
2. Delayed Fruit Ripening :
A major problem in fruit marketing is the pre-mature ripening and softening during transport of fruits. Consequently shelf-life of fruit remains short in the market. During ripening genes encode the enzyme cellulase and polygalacturonase.
Therefore, ripening process can be delayed by interfering the expression of these genes. In the U.S.A. a transgenic tomato named FlavrSavr (flavour saver) was produced where ripening is delayed by lowering polygalacturonase activity.
In 1994, the U.S. Food and Drug Administration granted permission to release and commercialise the FlavrSavr as it is safe.
A plant growth hormone ethylene is produced during fruit ripening and senescence. It is synthesised from S-adenosylmethionine through an intermediate compound 1-aminocyclopropane- 1-carboxylic acid (ACC). There is a large number of bacteria that can degrade ACC.
Therefore, bacterial gene (for ACC) deaminase associated with ACC degradation was isolated and introduced into tomato. In transgenic tomato fruit ripening was delayed because it synthesised lower amount of ethylene (due to inhibition in ACC synthesis) than the normal tomatoes. Such tomatoes and other fruits can be transported to a longer distance without spoilage.
3. Male Sterility and Fertility Restoration :
Production of male sterile plant is an important event. Because in such plants hybrid seeds can be produced through control of pollination and manual emasculation. These methods are often practised during breeding programmes.
Male sterile plants can be produced by introducing a gene which encodes RNA hydrolysing enzyme that inhibits pollen formation.
In 1990, C. Macriani and co-workers (Belgium) used a gene construct that contained an anther specific promoter (from TA29 gene of tobacco) and barnase gene (from Bacillus amyloliquifaciens that encodes ribonuclease).
They produced transgenic Brassica napus by introducing gene construct (1 A29-KNase) into tapetal cells of anther.
Using male-sterile barnase gene construct several breeding season starts, male sterile plants have been produced for example, cauliflower, maize, cotton, tobacco, tomato, etc.
4. Transgenic Plants as Bioreactor (Molecular Farming) :
Animal life is possible due to plants. Plants are the natural producers. Plant cells act as the Nature’s cheapest ‘factory’. The cell uses CO2, water, minerals and sun light to synthesise thousands of valuable and complex products which are the basis of animal’s life.
In recent years transgenic plants are used by biotechnology industries as ‘bioreactor’ for manufacturing special chemicals and pharmaceutical compounds. Normally, these chemicals are produced in low amount or not produced by the plants.
In successful trials transgenic plants have been found to produce monoclonal antibodies, functional antibody fragments, proteins, vitamins and the polymer polyhydroxybutyrate (PHB). The PBH can be used to prepare biodegradable plastics. Some of the examples have been discussed in this section.
(a) Nutritional Quality:
Nutritional quality of plants can be improved by introducing genes. Transgenic plants have been produced that are capable of synthesising cyclodextrins, vitamins, amino acids, etc. Consumption of such plant will help in improving the health of malnurished people in poor countries. In this context some examples are given below:
Cyclodextrins (CD) are cyclic oligosaccharides containing 6, 7 or 8 glucose molecules in ex, (3 or y linkage respectively. CDs are synthesised from the starch by the action of cyclodextrin glucosyl transferase (CGTase) enzyme.
It is used in pharmaceutical delivery system, flavour and odour enhancement and removal of undesirable compounds (e.g. coffeine) from food. A CGTase gene isolated from Klebsiella was transferred successfully into potato. The transgenic potato tubers produced CDs.
2. Vitamin A:
Vitamin A is required by all individuals as it is present in retina in eyes. Deficiency of vitamin A causes skin disorder and night blindness. Throughout the world 124 million children are the sufferers of vitamin A. Each year about 20 million new children are victimised due to deficiency of vitamin A.
You know that rice is used as staple food almost in every country. The contents of vitamin A are very low in rice. Vitamin A is synthesised from carotenoid which is precursor of vitamin A. Carotenoid is synthesised by three genes.
Prof. Ingo Potrykus and Peter Beyer produced genetically engineered rice by introducing three genes associated with biosynthesis of carotenoid. The transgenic rice was rich in pro-vitamin A. Since the seeds of transgenic rice are yellow in colour due to pro-vitamin A, the rice is commonly known as golden rice.
Golden rice is an interesting development which could open the way for improving nutritional standards in rice-eating cultures.
Similarly, the work done in India by Ashish Dutta (1992, 2000) on the introduction of amal gene (encoding balanced amino acid-protein) from Amaranthus into potato holds promise for enhancing nutritional value of low protein food. The transgenic potatoes having amal gene are undergoing field trials.
(iii) Quality of Seed Protein:
Seeds are the reservoir of all proteins, amino acids, oils, etc. and used as food throughout the world. However, nutritional quality of legumes and cereals can be affected due to deficiency of certain essential amino acids such as lysine (in cereals like rice, wheat), methionine and tryptophan (in pulses e.g. pea).
Following recombinant DNA technology improvement in quality of seed protein has been done. The two approaches were done for improvement in nutritional quality of seeds.
In the first strategy a gene (encoding protein containing sulphur-rich amino acid) tagged with seed-specific promotor was transferred into cultured tissue of pea plant (rich in lysine but deficient in methionine and cystein).
The transgenic pea produced protein containing sulphur rich amino acids. In the second strategy improvement in endogenous genes is done. The modified gene introduced in cereals produces higher amount of essential amino acids such as lysine.
(iv) Diagnostic and Therapeutic Proteins:
There are some proteins which are used in diagnosis of human diseases. These are called diagnostic proteins. Similarly, proteins used in cure of human diseases are called therapeutic proteins. Examples of these proteins are monoclonal antibodies, blood plasma protein, cytokinins, peptide hormones, human serum albumins.
In recent years transgenic plants have been produced by introducing foreign genes that can be used to produce diagnostic proteins and therapeutic proteins in bioreactor on a large scale at low cost.
Many foreign proteins such as human serum albumin, human interferon-a, human erythropoietin and immunoglobulin A (IgA) and immunoglobulin G (IgG) have been produced successfully in transgenic plants.
In 1999, the Indian Scientists at the International Centre for Genetic Engineering and Biotechnology (ICGEB), New Delhi successfully produced transgenic maize, tobacco, rice, etc. which synthesised interferon gamma (INF-y).
The whole transgenic plant or their specific parts express the above proteins such as tobacco leaves, potato tubers, sugarcane stem, maize endosperm, carrot roots, cabbage leaves, tomato fruits, etc.
(b) Edible Vaccines:
Vaccines are the antigenic proteins that induce B-cells to secrete antibodies. Transgenic crop plants can be constructed which produce vaccines to be eaten i.e. edible vaccines on a large scale at low cost.
Moreover, attention has been paid to produce such antigens that can stimulate mucosal immune system to produce secretary immunoglobulin A (S-IgA).
In 1990, first report of production of edible vaccine in tobacco at 0.02% of total leaf protein was published in the form of a patent application under the International Patent Co-operation Treaty. Thereafter, production of many antigens in several plants was reported.
In 1988, V.S. Reddi at ICGEB produced transgenic tobacco that produced hepatitis B surface antigen (HBsAg) active against hepatitis B virus in uncontrolled way. The recombinant HBsAg expressing leaves gradually lose chlorophyll and turn to yellow/white.
The rHBsAg secreting cells show 5-10 fold increase in size and lack of properly stacked stroma and granum (Fig. 8.12B). The plant derived rHBsAg was similar to yeast derived rHBsAg in providing immunity to mice.
The transgenic plants can be eaten as raw for immunisation or vaccination. The transgene expresses antigenic protein in the cells of transgenic plants. After ingestion antigenic protein activates the immune system to produce antibodies. The antibodies provide immunity against the specific pathogens present in human system.
Due attention has been paid to produce such transgenic plants which can be used even uncooked as raw such as tomato fruits, carrot, sugar beet, banana, etc. Otherwise after cooking the antigenic proteins may be denatured.
There are many advantages associated with edible vaccines such as no problem of storage, easy delivery into system after feeding, low cost of production (as compared to recombinant vaccines produced by bacteria and fungi through fermentation in bioreactors). The edible vaccines provide similar effects as the recombinant vaccines.
(c) Biodegradable Plastic:
You know that plastic materials have created a lot of environmental problems because these are non-biodegradable. Microbiologists have started exploring the possibility for production of bioplastics or biopolymers.
Polyhydroxybutyrate (PHB)-producting bacteria have been searched out to reach the goal. PHB is synthesised from acetyle Co-enzyme in three steps catalysed by three enzymes. These genes are organised in a single operon.
Attempts have also been made to produce transgenic plant containing gene for production of PHB i.e. biodegradable plastic at low cost. Transgenic Arabiopsis plants have been produced by transferring PHB producing gene in the chloroplast without interfering their growth and development.
Each of the three PHB genes was fused to DNA fragment that encodes the chloroplast transit peptide of the sub-unit of pea RuBPcase, and was replaced under transcriptional control of CaMV 35S promotor. Each gene was introduced into Arabidopsis thaliana plant with Ti-plasmid.
A large number of PHB granules are produced in the chloroplasts (Fig. 8.14). Furthermore, commercial production of PHB through transgenic plants like poplar has been done. PHB can be extracted from the leaves of such plants.
(d) Metabolic Engineering and Secondary Products:
There are some secondary metabolites of plants which are very useful in the area of medical sciences. But these are produced in a very low amount. Hence, production of such metabolites cannot meet the demand of people.
For example, anticancerous compounds (e.g. vincristine, taxol, etc.) have much demand but the production is low. Hence these products are costly.
Due to over-exploitation such compound-producing plants (e.g. Taxus, Podophyllum) have become endangered. Therefore, plant biotechnology can help in over-production of such compounds.
The genes which encode the first enzyme involved in metabolic pathway need to be induced so that desired products can be produced in higher amount.
Adopting tissue culture method, a transformed Taxus sp. was produced by transferring taxol overproducing gene into Taxus that produced taxol in higher amount. This approach has given a hope for commercialisation.
On the other hand, there are certain metabolites which are produced in roots. Therefore, the amount of roots governs the product yield. More roots will result in more secondary metabolite production.
It is possible through hairy root culture method. The explants inoculated with suspension of Agrobacterium rhizogenes are grown on nutrient medium (or plant protoplast can be co-cultured with A. rhizogenes).
The plantlets growing on medium enhanced number of hairy roots. The transformed hairy root culture has much importance for commercial exploitation and high level production of secondary metabolites.