The following points highlight the top six examples of transgenic plants. The examples are: 1. “High Lysine” Corn 2. Enhanced Nitrogen Fixation 3. Herbicide-Tolerant Plants 4. Disease-Insect-Resistant Varieties 5. Male Sterility 6. Transgenic Plants as Bioreactors (Molecular Farming).
Example # 1. “High Lysine” Corn:
The proteins stored in plant seeds function as reserves of amino acids used during seed germination and pre- emergence growth of the young seedling. Plant seed storage proteins also provide the major source of proteins in the diets of most humans and herbivorous higher animals.
Worldwide, the seeds of legumes and cereal grains are estimated to provide humans with 70 per cent of their dietary requirements. Unfortunately, the major seed storage proteins of cereals, called prolamines (Zeins in com or maize), are virtually lacking in the amino acid lysine.
Since prolamines account for about half of the total protein content of cereal seeds, diets based largely on cereal grains will be deficient in lysine (an essential amino acid). In the case of com, the seed proteins are also deficient in tryptophan (another essential amino acid) and to a lesser extend, methionine (an essential amino acid).
Because of the importance of cereal seeds as human and animal foods, plant breeders have attempted for several decades to develop varieties with increased lysine, tryptophan and methionine content.
In com, mutants such as opaque-2, sugary-1 and floury-2 have increased amounts of lysine and/or methionine in seeds, but these mutant strains have undesirable soft kernels and produce lower yields. These “high lysine” mutant strains all result from mutations that alter the relative proportions of different seed storage proteins.
In general, they lower the prolamine (zein) content so that other seed proteins account for a larger proportion of the total seeds proteins. This, in turn, increases the relative amounts of lysine and/or methionine in the seeds.
Several genes of corn encoding zeins have now been cloned and sequenced. With this information in hand, researchers have suggested that it might be possible to produce “high lysine” corn by genetic engineering. Since the zeins have no known enzymatic functions, one might be able to modify zein genes by mutagenesis without inflicting any deleterious effects on function(s).
Specifically site-specific mutagenesis could be used to introduce more lysine codons into zein sequences. Then, these “high lysine” zein coding sequences could be joined to strong promoters such as the CaMV35S promoter and reintroduced into com plants by transformation by means of electroporation or a microjectile gun.
However, a possible difficulty in engineering “high lysine” com by this method is that the modified zein proteins might not package properly in seed storage structures.
The zein proteins are synthesized on rough endoplasmic reticulum and they aggregate within this membranous structure into dense deposits called protein bodies. The formation of protein bodies is thought to involve hydrophobic and weak polar interactions between the zein monomers.
If so, charged amino acids such as lysine might interfere with proper packaging of zeins during protein body formation. In 1988, B.A. Larkin and colleagues have introduced new lysine and tryptophan codon into a zein cDNA by oligonucleotide-directed site-specific mutagenesis.
When BNA transcripts of these modified cDNAs were translated efficiently and the “high lysine” zein products were found to self-aggregate into dense structures similar to those present during polar body formation in com. These results offer encouragements that “high lysine” com might indeed be produced by means of genetic engineering.
Example # 2. Enhanced Nitrogen Fixation:
Plants are only able to utilize nitrogen that has been incorporated into chemical compounds such as ammonia, urea, or nitrates. No green plant is capable of extracting diatomic nitrogen (N2) molecules directly from the atmosphere. Although plants use only a small fraction of the total nitrogen pool, they are dependent on a continuous supply of nitrogen in usable form (most often called “fixed nitrogen”).
On-going fixation of atmospheric nitrogen is required because the fixed nitrogen in soil is constantly being depleted by leaching, by utilization for the growth of plants and microorganisms and by denitrifying bacteria that converts fixed nitrogen back to N2. As a result, millions of dollars/rupees is spent each year on nitrogen fertilizers in order to obtain optimal yields of major crops such as com and the cereal grains.
Biological nitrogen fixation is the alternative to the use of the industrially fixed nitrogen provided in fertilizers. Several species of bacteria and lower algae are capable of converting N2 to fixed forms of nitrogen that can be utilized by plants.
Because the purchase of nitrogen fertilizers represent one of the major expenses incurred with current agricultural production methods, a major effort has been made and continues to be devoted to the development of enhanced methods of biological nitrogen fixation.
Certain free-living soil bacteria such as Azotobacter vinelandii and Klebsiella pneumoniae directly convert atmospheric nitrogen to ammonia. These bacteria are an important source of fixed nitrogen and in addition, have proven to be extremely valuable subjects for studies on the mechanism of nitrogen fixation.
In Klebsiella, there are 17 nif (nitrogen fixation) genes organised in seven operons. The complexity of the nitrogen fixation metabolic machinery in these bacteria has important implications for anyone who might aspire to engineer nitrogen-fixing plants.
The situation with nitrogen fixation is very different from that of herbicide tolerance. It is one thing to construct a single chimeric gene and transfer that gene to plants, but it is far more difficult to engineer 17 different chimeric genes, to transfer all of them to the same recipient plant, and to coordinate their expression in the plant so that all the components of the complex nitrogen-fixing enzymatic machinery are synthesized in proper amounts and in the appropriate cells of the plant.
At present, the possibility of engineering nitrogen-fixing plants is largely fantasy, but remember that travelling to the moon was pure science fiction not too many years ago.
Some facts about nitrogen fixation:
The phenomenon of fixation of atmospheric nitrogen by biological means is known as diazotrophy or biological nitrogen fixation and these prokaryotes as diazotrophs or nitrogen fixers. For the first time, Beijerinck (1888) isolated Rhizobium from root nodules of leguminous plants. Thereafter, S .Winogradsky discovered a free-living nitrogen fixing bacterium, Clostridium pasteurianum.
Then a large number of nitrogen fixers were discovered from different sources and associations. For example, Frankia from nodules of non-legumes (e.g., alder, Casuarina, etc.), Nostoc from lichens, Anabaena from Azolla leaves, and coralloid roots of Cycas. The diazotrophs may be free living or in symbiotic form.
Heterocysts are the sites of nitrogen fixation in some cyanobacteria, e.g., Anabaena, Nostoc, etc. Heterocysts are formed in the absence of utilizable combined nitrogen, such as ammonia because it inhibits heterocyst differentiation and N2-fixing enzyme, the nitrogenase. Heterocysts lack oxygen evolving photosystem II, ribulose biphosphate and may lock photosynthetic biliproteins.
Chlorophyll-a is present in heterocysts. Wall of heterocyst contains O2 binding glycolipids which together with respiratory consumption maintain the anaerobic conditions (i.e., highly reduced atmosphere) necessary for N2 fixation. In contrast, vegetative cells adjacent to heterocysts, both photosystem I and II are present; therefore, oxygen evolution takes place by these cells.
Other cyanobacteria, that lack heterocyst also do N2-fixation, e.g., Oscillatoria (see Dubey 2006).
Another very important source of biologically fixed nitrogen is the symbiotic relationship between bacteria of the genus Rhizobium and plants of family Leguminosae (the alfalfa, clovers, soyabeans, peanuts, peas, etc.).
This symbiotic nitrogen fixation occurs in highly differentiated root nodules that develop when Rhizobium bacteria interacts with the roots of legumes. Nodule formation is dependent on genetic information of both the plants and the bacterium. The nitrogenase that catalyzes N2 reduction is encoded by the bacterial genome, but the fixed nitrogen is utilised for growth of both bacteria and the host plants.
Once the mechanisms responsible for establishing this symbiotic relationship and for nodule formation are known, and the genes that control these processes have been identified, it might be possible to use genetic engineering to modify non-legume plants (e.g., com, rice and wheat, such that they will participate in similar symbiotic relationships with nitrogen-fixing bacteria.
However, once again, this will undoubtedly be a challenging task because the genetic control of nodule formation is clearly complex. Nevertheless, experiments are in progress with goals of modifying bacteria so as to enhance their nitrogen-fixing capacity and to broaden their host range to include additional plant species.
Example # 3. Herbicide-Tolerant Plants:
The development of herbicide-tolerant varieties of agronomically important plants such as com, soybeans and the cereas promises to have a major impact on agriculture, both economically and on production practices. Weeds compete with crops for soil nutrients and routinely lead to significant losses in yield.
Modern agriculture makes use of herbcides to control weeds and minimize the losses. Unfortunately, the available herbicides seldom provide the degree of specifications that is desired, and most herbicides will control only certain classes and not others.
Broad-spectrum herbicides may give good weed control, but in doing, so usually have deleterious effects on the growth of crop plants as well. As a result, scientists are now considering alternate approaches to weed control.
The most promising of the alternate approaches is the development of herbicide-tolerant plant varieties for use with broad-spectrum or totally nonspecific herbicides. Obviously, the potential economic value of herbicide-tolerant plant varieties is significant.
Herbicides are simple chemical compounds that kill or inhibit the growth of plants without deleterious effects on animals. Herbicides usually inhibit the processes that are unique to plants, for example, photosynthesis.
Most frequently, herbicides act as inhibitors of essential enzyme reactions. Thus, anything that diminishes the level of inhibition will provide increased herbicide tolerance.
The two most common sources of herbicide tolerance are:
(1) Over-production of the target enzyme and
(2) Mutations resulting in enzymes that are less sensitive to the inhibitor (usually due to a lower affinity of the enzyme for the inhibitor).
It seems likely that the most successful strategy for developing herbicide- tolerant plants will be to combine both sources of tolerance, that is, to engineer plants that overproduce herbicide-tolerant mutant enzymes. We can consider, here, the example of glyphosate.
Glyphosate is one of the most potent broad-spectrum herbicide known; it is marketed under the trade name Roundup Glyphosate acts by inhibiting the enzyme 5-enolpyruvylshikimate 3-phosphate synthase (EPSP synthase), an essential compound in the biosynthesis of the aromatic amino acids tyrosine, phenylalanine and tryptophan.
These aromatic amino acids are essential components in the diets of higher animals since the enzymes that catalyse the biosynthesis of these amino acids are not present in higher animals. Therefore, since higher animals contain EPSP synthase, glyphosate has no toxic effects on animal systems. In this respect, glyphosate is an ideal herbicide.
Glyphosate of herbicide, does inhibit the EPSP synthases of microorganisms as well as those of plants. By selecting for growth in the presence of glyphosate concentrations that inhibit the growth of wild-type bacteria, researchers have been able to isolate glyphosate-tolerant mutants of Salmonella typhimurium, Aerobacter aerogenes and Escherichia coli.
In bacteria, EPSP synthase is encoded by the aroA gene. When the mutant bacterial aroA genes were provided with plant promoters and polyadenylation signals (producing, chimeric genes) and were introduced into plants, the transgenic plants exhibited increased tolerance to glyphosate (herbicide).
In plants, synthesis of aromatic amino acids takes place in chloroplasts, but the genes encoding the biosynthetic enzymes such as EPSP synthase are nuclear genes. The translation products contain a transit peptide that targets the protein to the chloroplasts.
This transit peptide is then cleaved off proteolytically upon entering the chloroplasts to yield the active enzyme. Experiments have now shown that the petunia transit peptide will target the E. coli aroA gene product into tobacco chloroplasts and will produce glyphosate tolerance in the recipient cell lines.
Example # 4. Disease-Insect-Resistant Varieties:
Several microorganisms and certain native plants produce proteins that are toxic to specific plant pathogens, both microbial pathogens and insects that feed on plants. One goal of plant genetic engineering is to transfer the genes encoding these protein toxins to agronomically important plants with the hope that expressing the toxin genes in these plants will provide biological control Disease-insect of at least some plant diseases and insect pests.
Currently, plant diseases resistant plants and insect pests are controlled almost exclusively by the use of broad- spectrum chemical bacteriocides, fungicides and insecticides. However, there is reason for concern about the potential damage to ecosystems and pollution of groundwater that might result from the widespread use of these chemicals on agricultural crops. Thus, scientists are searching for alternate methods for controlling these pathogens.
The best-known example of the use of natural gene products to control plant pests are the insect toxins of Bacillus thuringiensis. Each of the toxin genes of B. thuringiensis encodes a large protein that aggregate, to form protein crystals in spores and these protein crystals are highly toxic to certain insects.
Some of the insects that are killed by these protein toxins are plant pests of major economic importance. Different subspecies of B. thuringiensis produce toxins that kill different insects. For example, the toxin produced by B. thuringiensis subspecies kurstaki kills lepidopteran larvae such as the tobacco hornworm.
The gene that encodes this toxin has been isolated and shown to synthesize a functional toxin in E. coli. A chimeric gene with the structure CaMV35S promoter/B. thuringiensis subspecies kurstaki toxin coding sequence/Ti nos 3′ termination sequence was constructed.
This chimeric gene was placed in a Ti vector, and tomato leaf disc cells were transformed by co-cultivation with A .tumefaciens harboring the engineered Ti vector-chimeric gene construct. Transgenic tomato plants were regenerated and shown to express the chimeric gene. The toxicity of the gene-product synthesized in the transgenic plants was tested by allowing tobacco hornworm larvae to feed on the transgenic plants and on control plants.
All the larvae applied to the transgenic plants died within a few days; larvae applied to the control plants remained healthy and eventually consumed the entire plants. These results support the feasibility of using genetic engineering to produce transgenic tomato. Pathogen resistant plant varieties.
BT Cotton:
The transgenic technology provides alternative and innovative method to improve pest control management which are ecofriendly, effective, sustainable and beneficial in terms of yield. The first genes available for genetic engineering of crop plants for pest resistance were cry genes (popularly known as Bt genes) from bacterium Bacillus thuringiensis. These genes are specific to particular group of insect pests and are not harmful to other useful insects such as butterfly, silk worms and honeybee.
Transgenic crops (e.g., cotton, rice, maize, potato, tomato, brinjal, cauliflowers, cabbage, etc.) with Bt genes have been developed and such transgenetic variety proved effective in controlling insect pests and it has been claimed worldwide that it has led to significant increase in yield along with dramatic reduction in pesticide use.
The most notable example is Bt cotton (which contain cry/Ac gene) that is resistant to a notorious insect pest bollworm (Helicoperpa armigera). Bt cotton was adopted for mass cultivation in India in year 2002.
Example # 5. Male Sterility:
Male sterile plants are very important to prevent unnecessary pollination and to eliminate the process of emasculation during the production of hybrid plants. Such sterile male plants are created by introducing a gene coding for an enzyme (barnase), which is an RNA hydrolyzing enzyme) that inhibits pollen formation. This gene is expressed specifically in the tapetal cells of anther using tapetal specific promoter TA29 to restrict its activity only to the cells involved in pollen production.
Example # 6. Transgenic Plants as Bioreactors (Molecular Farming):
Plants are amazing and cheap chemical factories that need only water, minerals, sunlight and carbon dioxide to produce thousand types of chemical molecules (see Dubey 2006). Given the right genes, plants can serve as bioreactors to new compounds such as amino acids, proteins, vitamins, plastics, pharmaceuticals (peptides and proteins), drugs, and enzymes for food industries and so on.
Thus, transgenic plants can be used for the following purposes:
(i) Nutrient quality:
In section 58.2, under the heading of ‘High-lysine com’, we have described how cereals rich in certain essential amino acids such as lysine, methionine and tryptophan can be developed by genetic engineering. Likewise, rice is being modified into Golden rice by Prof. Inge Potrykus and Dr. Peter Beyer.
This is done so that vitamin A potential is maintained even after the husks are removed, a procedure adopted to allow for storage since the husks become rancid. This change may improve health of millions of people throughout the world.
(ii) Diagnostic and therapeutic proteins:
Transgenic plants can also produce a variety of proteins used in diagnosis for detecting human diseases and therapeutics for curing human and animal diseases in large-scale with low-cost. The monoclonal antibodies, blood plasma proteins, peptide hormones and cytokinins are being produced in trangenic plants and their parts such as tobacco (in leaves), potato (in tubers), sugarcane (in stems) and maize (in seed endosperm).
(iii) Edible vaccines:
Crop plants offer cost-effective bioreactors to express antigens which can be used as edible vaccines. The genes encoding antigenic proteins can be isolated from the pathogens and expressed in plants and such transgenic plants or their tissues producing antigens can be eaten for immunization (edible vaccines).
The expression of such antigenic proteins in crops such as banana and tomato are useful for immunization of humans since both of these fruits can be eaten raw. Such edible vaccines of transgenic plants have the following advantages: lessening of their storage problems, their easy delivery system by feeding and low cost as compared to the recombinant vaccines produced by bacteria.
(iv) Biodegradable plastics:
Transgenic plants can be used as factories to produce polyhydroxy butyrate (PHB, biodegradable plastics). Genetically engineered Arabidopsis plants produced PHB globules exclusively in their chloroplasts without effecting plant growth and development. The large-scale production of PHB may be easily achieved in tree plants such as populus, where PHB can be extracted from leaves.