Transgenic plants as bioreactors (molecular farming)

Plants can be used as cheap chemical factories that require only water, minerals, sun light and carbon dioxide to produce thousands of sophisticated chemical molecules with different structures. By transferring the right genes, plants can serve as bioreactors to modified or new compounds such as amino acids, proteins, vitamins, plastics, pharmaceuticals (peptides and proteins), drugs, enzymes for food industry and so on. The transgenic plants as bioreactors have some advantages such as the cost of production is low, there is an unlimited supply, safe and environmental friendly and there is no scare of spread of animal borne diseases.
Tobacco is the most preferred plant as a transgenic bioreactor because it can be easily transformed and engineered. Tobacco is an excellent biomass producer with about 40 tons of fresh leaf production as against e.g. rice with 4 tons. The seed production is very high (approx. one million seeds per plant) and it can be harvested several times in a year.
Some of the uses of transgenic plants are:

Improvement of Nutrient quality

Transgenic crops with improved nutritional quality have already been produced by introducing genes involved in the metabolism of vitamins, minerals and amino acids.
A transgenic Arabidopsis thaliana that can produce ten-fold higher vitamin E (alpha-tocopherol) than the native plant has been developed. The biochemical machinery to produce a compound close in structure to alpha-tocopherol is present in A. thaliana. A gene that can finally produce alpha-tocopherol is also present, but is not expressed. This dormant gene was activated by inserting a regulatory gene from a bacterium which resulted in an efficient production of vitamin E.
Glycinin is a lysine-rich protein of soybean and the gene encoding glycinin has been introduced into rice and successfully expressed. The transgenic rice plants produced glycinin with high contents of lysine.
Using genetic engineering Prof Potrykus and Dr. Peter Beyer have developed rice which is enriched in pro-vitamin A by introducing three genes involved in the biosynthetic pathway for carotenoid, the precursor for vitamin A. The aim was to help millions of people who suffer from night blindness due to Vitamin A deficiency, especially whose staple diet is rice. The presence of beta-carotene in the rice gives a characteristic yellow/orange colour, hence this pro-vitamin A enriched rice is named as Golden Rice.
The genetic engineering is also being used to improve the taste of food e.g. a protein ‘monellin’ isolated from an African plant (Dioscorephyllum cumminsii) is about 100,000 sweeter than sucrose on molar basis. Monellin gene has been introduced into tomato and lettuce plants to improve their taste.

Improvement of seed protein quality

The nutritional quality of cereals and legumes has been improved by using biotechnological methods. Two genetic engineering approaches have been used to improve the seed protein quality. In the first case, a transgene (e.g. gene for protein containing sulphur rich amino acids) was introduced into pea plant (which is deficient in methionine and cysteine, but rich in lysine) under the control of seed-specific promoter. In the second approach, the endogenous genes are modified so as to increase the essential amino acids like lysine in the seed proteins of cereals.
These transgenic routes have helped to improve the essential amino acids contents in the seed storage proteins of a number of crop plants. E.g. overproduction of lysine by de-regulation. The four essential amino acids namely lysine, methionine, threonine, and isoleucine are produced from a non-essential amino acid aspartic acid. The formation of lysine is regulated by feed back inhibition of the enzymes aspartokinase (AK) and dihydrodipicolinate synthase (DHDPS). The lysine feedback- insensitive genes encoding the enzymes AK and DHDPS have been respectively isolated from E. Coli and Cornynebacterium. After doing appropriate genetic manipulations, these genes were introduced into soybean and canola plants. The transgenic plants so produced had high quantities of lysine.

Diagnostic and therapeutic proteins

Experiments are going on to use transgenic plants in diagnostics for detecting human diseases and therapeutics for curing human and animal diseases. Several metabolites and compounds are already being produced in transgenic plants e.g. the monoclonal antibodies, blood plasma proteins, peptide hormones, cytokinins etc. The use of plants for commercial production of antibodies, referred to as plantbodies, is a novel approach in biotechnology. The first successful production of a functional antibody, namely a mouse immunoglobulin IgGI in plants, was reported in 1989. This was achieved by developing two transgenic tobacco plants-one synthesizing heavy chain gamma- chain and other light kappa- chain, and crossing them to generate progeny that can produce an assembled functional antibody. In 1992, C.J. Amtzen and co-workers expressed hepatitis B surface antigen in tobacco to produce immunologically active ingredients via genetic engineering of plants.
Several other therapeutic proteins have also been produced like haemoglobin and erythropoietin in tobacco plants, lactoferrin in potato, trypsin inhivitor in maize etc. The first proteins/enzymes that were produced in transgenic plants (maize) are avidin and beta-glucuronidase and are used in diagnostic kits.

Edible vaccines

Crop plants offer cost-effective bioreactors to express antigens which can be used as edible vaccines. The approach is to isolate genes encoding antigenic proteins from the pathogens and then expressing them in plants. Such transgenic plants or their tissues producing antigens can be eaten for vaccination/immunization (edible vaccines). The expression of such antigenic proteins in crops like banana and tomato are useful for immunization of humans since banana and tomato fruits can be eaten raw.
Transgenic plants (tomato, potato) have been developed for expressing antigens derived from animal viruses e.g. rabies virus, herpes virus. In 1990, the first report of the production of edible vaccine (a surface protein from Streptococcus) in tobacco at 0.02% of total leaf protein level was published in the form of a patent application under the International Patent Cooperation Treaty (Mason and Arntzen,1995).The first clinical trials in humans, using a plant derived vaccine were conducted in 1997 and were met with limited success. This involved the ingestion of transgenic potatoes with a toxin of E. coli causing diarrhea.

The process of making of edible vaccines involves the incorporation of a plasmid carrying the antigen gene and an antibiotic resistance gene, into the bacterial cells e.g. Agrobacterium tumefaciens. The small pieces of potato leaves are exposed to an antibiotic which can kill the cells that lack the new genes. The surviving cells with altered genes multiply and form a callus. This callus is allowed to grow and subsequently transferred to soil to form a complete plant. In about a few weeks, the plants bear potatoes with antigen vaccines.

The bacteria E.coli, V. cholerae cause acute watery diarrhea by colonizing the small intestine and by producing toxins. Chloera toxin (CT) is very similar to E.Coli toxin. The CT has two subunits, A and B. Attempt was made to produce edible vaccine by expressing heat labile enterotoxin (CT-B) in tobacco and potato.

Another strategy adopted to produce a plant-based vaccine, is to infect the plants with recombinant virus carrying the desired antigen that is fused to viral coat protein. The infected plants are reported to produce the desired fusion protein in large amounts in a short duration. The technique involves either placing the gene downstream a subgenomic promoter, or fusing the gene with capsid protein that coats the virus.

Advantages of edible vaccines

The edible vaccines produced in transgenic plants will sole the storage problems, will ensure easy delivery system by feeding and will have low cost as compared to the recombinant vaccines produced by bacterial fermentation. Vaccinating people against dreadful diseases like cholera and hepatitis B, by feeding them banana, tomato, and vaccinating animals against important diseases will be an interesting development.

Biodegradable plastics

Polythenes and plastics are one of the major environmental hazards. Efforts are on to explore the possibility of using transgenic plants for biodegradable plastics. Transgenic plants can be used as factories to produce biodegradable plastics like polyhydroxy butyrate or PHB. Genetically engineered Arabidopisis plants can produce PHB globules exclusively in their chloroplasts without effecting plant growth and development. The large-scale production of PHB can easily be achieved in plants like Populus, where PHB can be extracted from leaves.

Molecular Breeding
The term molecular breeding is frequently used to represent the breeding methods that are coupled with genetic engineering techniques. Up till now, conventional breeding methods have been used to meet the food demands of the growing world population and the challenges of poverty and improved crop production and yields. However in the years to come, the development in the agriculture yields and techniques is going to be due to the use of molecular breeding programme.
Linkage analysis which deals with the studies to correlate the link between the molecular marker and a desired trait is an important aspect of molecular breeding programme. In the past, linkage analysis was carried out by use of isoenzymes and the associated polymorphisms. Now a days, molecular markers are being used.

Molecular breeding involves breeding using molecular (nucleic acid) markers. A molecular marker is a DNA sequence in the genome which can be located and identified therefore molecular markers can be used to identify particular locations in the genome.

Due to mutations, insertions, deletions, etc. the base composition at a particular location may be different in different plants. These differences, termed polymorphisms, allow DNA markers to be mapped in a genetic linkage group.
Generally, there are three types of markers used in screening/selection:

a) Morphological marker based on visible character (phenotypic expression) e.g. flower color, seed color, height, leaf shapes, etc. Morphological markers could be dominant or recessive. There are certain constraints in using these markers as the morphological markers are easily influenced by environmental factors and thus may not represent the desired genetic variation. Some of the visible markers have not much role to play in the plant breeding programme.

b) Biochemical marker: The proteins produced by gene expression are also used as markers in plant breeding programmes. The most commonly used are isozymes, the different molecular forms of the same enzyme. Each individual variety has its own isozyme variability (profiles) which can be detected by electrophoresis on starch gel.

c) Molecular marker based on DNA polymorphism detected by DNA probes or amplified products of PCR, e.g.Restriction fragment length polymorphism (RFLP), Randomly Amplified polymorphic DNA (RAPD), variable Number Tandom Repeats (VNTR), Microsatellites, etc. Plant breeders always prefer to detect the gene as molecular marker, although it is not always possible. Molecular markers provide a true representation of the genetic make up at the DNA level. They are consistent and free from environmental factors, and can be detected much before the development of plants occur. The advantage with a molecular marker is that a plant breeder can select a suitable marker for the desired trait which can be detected well in advance. A large number of markers can be generated as per the needs. The molecular markers to be used in plant breeding programme should have the following characteristics: (a) the marker should be closely linked with the desired trait, (b) the marker screening methods should be effective, efficient, reproducible and easy to carry out, (C) the entire analysis should be cost effective.

Molecular makers are of two types: (a) based on nucleic acid (DNA) hybridization- This involves the cloning of the DNA piece followed by the hybridization with the genomic DNA, which is later detected.
The Restriction fragment length polymorphism (RFLP) was the very first technology employed for the detection of polymorphism, based on the DNA sequence differences. RFLP is mainly based on the altered restriction enzyme sites, as a result of mutations and recombinations of genomic DNA. The procedure involves the isolation of genomic DNA and it’s digestion by restriction enzymes. The fragments are separated by electrophoresis and finally hybridized by incubating with cloned and labeled probes.

(b) Molecular markers based on PCR amplification.
Polymerase chain reaction (PCR) is a novel technique for the amplification of selected regions of DNA. The most important advantage is that even a minute quantity of DNA can be amplified and the PCR- based molecular markers require only a small quantity of DNA to start with. Random amplified polymorphic DNA (RAPD) markers use PCR amplification where the DNA is isolated from the genome and is denatured. The template molecules are annealed with primers and amplified by PCR. The amplified products are separated on electrophoresis and identified. Based on the nucleotide alterations in the genome, the polymorphisms of amplified DNA sequences differ which can be identified as bends on gel electrophoresis.
Amplified fragment length polymorphism (AFLP) is a novel technique involving a combination of RFLP and RAPD. AFLP is based on the principle of generation of DNA fragments using restriction enzymes and oligonucleotide adaptors (or linkers), and their amplification by PCR.

Microsatellites
Microsatellites are the tandemly repeated multiple copies of mono-, di-, tri-, and tetra nucleotide motifs. In some instances, there are unique flanking sequences present in the repeat sequences. Primers are designed for such flanking sequences to detect the sequence tagged microsatellites (STMS) which is done by PCR.

Commercial use of transgenic plants
The main goal of producing transgenic plants is to increase the productivity. In 1995-96, transgenic potato and cotton plants were used commercially for the first time in USA. By the year 1998-99, five other major transgenic crops cotton, maize, canola, soybean, and potato were introduced to the farmers. These accounted for about 75% of the total area planted by crops in USA. There are still a lot of concerns regarding the harmful environmental and hazardous health effects of transgenic plants. The major areas of public concern are- the development of resistance genes in insects, generation of a super weeds by mutation etc. Certain other legal and regulatory hurdles pertaining to commercial use of transgenic plants, needs to be addressed.

Bioethics in Plant genetic Engineering
There are issues and concerns regarding the use of transgenic crops and their effects on the health and the environment in general. The major concerns about GM crops and GM foods are:

a) Effect of GM crops on biodiversity and environment- As the GM crops are created artificially, there is no natural process of evolution in their development. Hence, there is a question of this affecting the biodiversity and overall effect on the environment.

b) The risk of transfer of transgene from GM crops to pathogenic microbes- Antibiotic marker genes are used to identify and select the modified cells. If GM food containing antibiotic resistance marker gene is consumed by animals and humans, there is a risk that the transgene will transfer from GM food to microflora of human and animals. This may lead to the gut microbes to become resistant to antibiotics.

c) The transfer of genes from animals into Gm crops for molecular farming may change the fundamental vegetable nature of plants.

d) The GM crops may bring about changes in evolutionary patterns. The plants adapt to the changing environment in the natural way by changing their genes and developing better races with superior traits which ultimately leads to the development of evolved races and varieties. What will be the evolutionary pattern of the GM crops? There are concerns about the effect of transgene flow from GM crops to other non-GM plants and the alteration of these non-GM crops.

e) There is a risk of transferring allergens (usually glycoproteins) from GM food to human and animals.

f) There is a risk of “gene pollution” i.e. transfer of transgene of GM crop through pollen grains to related plant species and development of super weeds.

g) There are also some religious issues related to the consumption of transgenic plants with animal genes introduced into them, especially, for some strict vegetarian people and some ethnic groups with certain food preferences and restrictions.

h) There is a need to study thoroughly as to how the genetically engineered plants will affect the ecological balance, once they are released in the environment.