HUMAN GENOME PROJECT

Human Genome project (HGP) is an international project whose principal goal was to sequence the entire human genome. The National Centre for Human Genome Research(NCHGR) was established in the United States in 1989 by James D. Watson, and subsequently by Francis Collins. It was a 13 years project coordinated by the U.S. Department of energy and the national institutes of health.

HGP researchers have deciphered the human genome in following ways:

• To develop a high resolution genetic map of the human genome,
• To produce a variety of physical maps of all human chromosomes and of the DNA of selected model organisms (ie, Escherichia coli, Saccharomyces cerevisiae, nematode (Caenorhabditis elegans), Drosophila and the laboratory mouse.
• To determine a complete sequence of human DNA and the DNA of model organimsms.
• To Develop capabilities for collecting, storing, distributing, and analysing  the data produced
• To create the appropriate innovative and high risk techniques necessary to achieve these objectives.
• To develop programs to address the understanding of the ethical, legal, and social implications (ELSI) of the HGP.

Genome is the entire DNA in an organism, including its genes. Human genome is made up of 23 chromosomes pairs with a total of 2.9 billion base pairs encoding approximately 20,000 to 25,000 genes. All human cells except for mature red blood cells contain a complete genome.

Genes are specific sequences of bases that encode instructions for making proteins. Genes comprises of only 2% of the human genome, the remainder consists of non coding regions. An average gene consists of 3000 bases, but sizes vary greatly with the largest known human gene being dystrophin at 2.4 million bases.

In 2002 some facts about the human genome project was found as:

Ø  The number of genes is almost one third of the expected, about as many as lower organisms and fewer than some

Ø  Human genes are able to encode more proteins than the genes of lower organisms.

Ø  Human proteins are more complex than the protein of similar functions in lower organisms.

Ø  Human genes are not arranged evenly; the ends of chromosomes are more tightly packed.

Ø  About 200 human genes arrived in the mammalian genome directly from bacteria

Ø  Only about 2.5 % of human DNA encodes proteins. Some of the DNA that does not encode genes probably has important functions

Ø  The mutation rate in male is twice than in female

Ø  All human are 99.9% identical in terms of their nucleotide sequence. All the ethnic groups share most genetic differences.

The objective of HGP is helpful in providing scientists with a powerful tool to help them understand the molecular essence of tragic and devastating illness such as schizophrenia, alcoholism, Alzheimer disease, and manic depression. This has lead to improved approaches to predict increased risk, provide early detection, and promote more effective treatment strategies.

Single Nucleotide Polymorphisms (SNPs)

The Human Genome Project has helped to inform us about how remarkably similar all human beings are—99.9% at the DNA level. About 99.9 % of nucleotide bases are exactly the same in all the people. However, this 0.1 % variation of genome within population is currently a focus of intense research, because the data will provide information about increased susceptibility or resistances to diseases. The most common variations are single nucleotide polymorphisms (SNPs). When two haploid genomes are compared, SNPs occur every kilobase, on average, other kinds of sequence variation such as copy number changes, insertions, deletions, duplications, and rearrangements also exist, but at low frequency and their distribution is poorly understood.

SNPs are abundant, stable, widely distributed across the genome. SNPs offer the highest resolution for tracking disease genes and population history. SNPs have potential for mapping complex traits such as cancer, diabetes, and mental illness. Therefore, SNP maps are desirable for identifying genes that make a small contribution to disease risk. In some instances, such maps will also permit predict of individual differences in drug response.

Proteomics:

The constellation of all proteins in a cell is called its proteome. Unlike the relatively unchanging genome, the dynamic proteome changes from minute to minute in response to tens of thousands of intra and extra cellular environmental signals. A protein’s chemistry and behaviour are specified by the gene sequence and by the number and identities of other proteins made in the same cell at the same time. This explains how over a million of modified proteins can exist when the entire human genome codes for less than 30,000 genes.

Human Genome Project in Clinical medicine

Technology and resources promoted by the Human Genome Project are starting to have profound impacts on biomedical research and promise to revolutionize the wider spectrum of biological research and clinical medicine.

Some current and potential applications of genome research include

• Molecular medicine
• Risk assessment
• DNA forensics (identification)

The main aim of human genome project is its applications in molecular medicine. Genomic information helps researches to design drugs targeted at specific pathways involved in the disease. With correct genomic information, doctors can deliver correct drugs and drug dosages that are most likely to work well. These drugs are believed to work better and cause fewer side effects than current treatments. Gene based drugs in the treatment of leukaemia and other cancers have already started to pay off.

Understanding the human genome will have an enormous impact on the ability to assess risks posed to individuals by exposure to toxic agents. Scientists know that genetic differences make some people more susceptible and others more resistant to such agents. Therefore genomic information are valuable source of predicting disease so that more attention is paid for preventing that disease. For instance: if the genome contains variations that raise the risk of heart disease, people might exercise more and take drugs that lower cholesterol.

Understanding genomics will help us understand human evolution and the common biology we share with all of life. Comparative genomics between humans and other organisms such as mice already has led to similar genes associated with diseases and traits. Further comparative studies will help determine the yet-unknown function of thousands of other genes.

Understanding genomics provides valuable source is DNA forensics. It can be used to identify potential suspects whose DNA may match evidence left at crime scenes and to exonerate persons wrongly accused of crimes.

 It can also be used in establishing paternity and other family relationships.

 Another advantage of studying human genome is to match organ donors with recipients in transplant programs.

Insect Resistant Bt.Cotton

Agrobacterium tumefaciens is a soil borne, gram negative bacterium. It causes the crown gall disease in dicots via a unique mechanism of DNA transfer and integration of the transferred DNA (T-DNA) into the host-plant genome. This unique feature has established Agrobacterium tumefaciens as a Nature’s own genetic engineer.

Crown gall formation depends on the presence of a plasmid known as Ti (Tumour inducing) plasmid. Ti plasmids contain one or multiple T-region which is defined by T-DNA border sequences. The border sequences serve as the target for the VirD1/ VirD2 endonuclease and also serve as the attachment site for VirD2 protein. T-DNA is a part of Ti plasmid which is actually transferred from the bacterium into the plant cell where is becomes integrated into the genome of the plant. T-DNA carries genes that encode proteins involved in hormone (auxin and cytokinin) biosynthesis and biosynthesis of novel plant metabolites called opines and agropines. The production of auxin and cytokinin causes the plant cell to proliferate and so form the gall. These proliferating cells also produce opine and agropines which are used by Agrobacterium as its sole carbon and energy source.

T-DNA transfer and Integration

Signal recognition by Agrobacterium

The transformation process begins following the wounding of the plant which causes release of signals such as phenolics and sugars and is perceived by Agrobacterium. These signals activate the bacterial virulence (vir) genes located at the Ti plasmid. 

Attachment to Plant cells

Agrobacterim are chemically attracted to the site of wounding and colonise the wound site. Chromosomal virulence genes chv genes) are involved in the attachment of the bacterial cells to the plant cells. Attachment involves initial attachment via a polysaccharide and then by a mesh of cellulose fibre produced by the bacterium.

Generation of T-DNA transfer complex

A DNA segment (25-bp) is excised from the lower strand of Ti-plasmid by the VirD2/VirD1 endonucleae complex, producing a single stranded T-DNA. VirD2 remains attached to the 5’ end of the T-DNA and guide the T-complex through the bacterial export complex. This immature polar T-complex and several other Vir proteins (like VirE2 VirE3, VirF, and VirD5) are exported into the plant cell cytoplasm via a Type IV secretion system (T4SS), composed of VirD4 proteins and 11 VirB proteins.

Increasing evidence suggests that the VirD2-conjugated T-strand (immature T-complex) and VirE2, along with other effector proteins are exported independently to the host plant cell’s cytoplasm. Once inside the plant cell cytoplasm, mature T-complex formation begins with the association of the VirD2-conjugated T-strand with the VirE2 molecules. The T complex is then imported into the host cell nucleus. The nuclear pore imposes several physical and molecular restrictions on the nuclear import of the T-complex, and the bacteria overcomes these barriers by using the host protein karyopherin α, which interacts with the bacterial VirD2 protein, and leads to directed translocation of the T-complex through the nuclear pore complex(NPC) into the karyoplasm. Though VirE2 is essential for the translocation, it doesnot interact directly with the host nuclear-import machinery. However, it interacts with VirE2-interacting protein 1 (VIP1), which functions as an adaptor between the host nuclear import machinery and the host cell chromatin and VirE2. A recent report shows that VIP1 is a target protein for an Agrobacterium induced MAPK, a possible mechanism by which Agrobacterium induces and uses the defense signaling of the cell to deliver its T-complex into the host cel nucleus.

Inside the nucleus, VirF associates with the T-complex via its binding to VIP1 and recruits the host cell ASK1 and cullin proteins, forming the SCFVirF complex, which activates proteasomal degradation of VIP1 and VirE2, leading to uncoating of the T-strand.

The uncoated T-strand can undergo partial degradation in the nucleus and then becomes converted to a double stranded form. Integration of T-DNA occurs by non-homologous recombination (NHR). In the NHR pathway DNA-protein complex binds to the free double stranded ends of the T-DNA and is directed to DSBs formed in the host genome. The integrating double stranded T-DNA molecules are ligated to the ends of the DSB via the activity of plant DNA ligases.

In this way Agrobacterium uses host cellular mechanisms and pathways for infection. More specifically, the transfer and integration of T-DNA into the host genome requires interaction with the host nucleus at different levels.

Agrobacterium Mediated Transformation

Agrobacterium tumefaciens is a soil borne, gram negative bacterium. It causes the crown gall disease in dicots via a unique mechanism of DNA transfer and integration of the transferred DNA (T-DNA) into the host-plant genome. This unique feature has established Agrobacterium tumefaciens as a Nature’s own genetic engineer.

Crown gall formation depends on the presence of a plasmid known as Ti (Tumour inducing) plasmid. Ti plasmids contain one or multiple T-region which is defined by T-DNA border sequences. The border sequences serve as the target for the VirD1/ VirD2 endonuclease and also serve as the attachment site for VirD2 protein. T-DNA is a part of Ti plasmid which is actually transferred from the bacterium into the plant cell where is becomes integrated into the genome of the plant. T-DNA carries genes that encode proteins involved in hormone (auxin and cytokinin) biosynthesis and biosynthesis of novel plant metabolites called opines and agropines. The production of auxin and cytokinin causes the plant cell to proliferate and so form the gall. These proliferating cells also produce opine and agropines which are used by Agrobacterium as its sole carbon and energy source.

T-DNA transfer and Integration

Signal recognition by Agrobacterium

The transformation process begins following the wounding of the plant which causes release of signals such as phenolics and sugars and is perceived by Agrobacterium. These signals activate the bacterial virulence (vir) genes located at the Ti plasmid.  

Attachment to Plant cells

Agrobacterim are chemically attracted to the site of wounding and colonise the wound site. Chromosomal virulence genes chv genes) are involved in the attachment of the bacterial cells to the plant cells. Attachment involves initial attachment via a polysaccharide and then by a mesh of cellulose fibre produced by the bacterium.

Generation of T-DNA transfer complex

A DNA segment (25-bp) is excised from the lower strand of Ti-plasmid by the VirD2/VirD1 endonucleae complex, producing a single stranded T-DNA. VirD2 remains attached to the 5’ end of the T-DNA and guide the T-complex through the bacterial export complex. This immature polar T-complex and several other Vir proteins (like VirE2 VirE3, VirF, and VirD5) are exported into the plant cell cytoplasm via a Type IV secretion system (T4SS), composed of VirD4 proteins and 11 VirB proteins.

Increasing evidence suggests that the VirD2-conjugated T-strand (immature T-complex) and VirE2, along with other effector proteins are exported independently to the host plant cell’s cytoplasm. Once inside the plant cell cytoplasm, mature T-complex formation begins with the association of the VirD2-conjugated T-strand with the VirE2 molecules. The T complex is then imported into the host cell nucleus. The nuclear pore imposes several physical and molecular restrictions on the nuclear import of the T-complex, and the bacteria overcomes these barriers by using the host protein karyopherin α, which interacts with the bacterial VirD2 protein, and leads to directed translocation of the T-complex through the nuclear pore complex(NPC) into the karyoplasm. Though VirE2 is essential for the translocation, it doesnot interact directly with the host nuclear-import machinery. However, it interacts with VirE2-interacting protein 1 (VIP1), which functions as an adaptor between the host nuclear import machinery and the host cell chromatin and VirE2. A recent report shows that VIP1 is a target protein for an Agrobacterium induced MAPK, a possible mechanism by which Agrobacterium induces and uses the defense signaling of the cell to deliver its T-complex into the host cel nucleus.

Inside the nucleus, VirF associates with the T-complex via its binding to VIP1 and recruits the host cell ASK1 and cullin proteins, forming the SCFVirF complex, which activates proteasomal degradation of VIP1 and VirE2, leading to uncoating of the T-strand.

The uncoated T-strand can undergo partial degradation in the nucleus and then becomes converted to a double stranded form. Integration of T-DNA occurs by non-homologous recombination (NHR). In the NHR pathway DNA-protein complex binds to the free double stranded ends of the T-DNA and is directed to DSBs formed in the host genome. The integrating double stranded T-DNA molecules are ligated to the ends of the DSB via the activity of plant DNA ligases.

In this way Agrobacterium uses host cellular mechanisms and pathways for infection. More specifically, the transfer and integration of T-DNA into the host genome requires interaction with the host nucleus at different levels.

Plant Derived Vaccines

Since prehistory, mankind has used plants for medicinal purpose. Thousands of plants have been employed for the treatment and therapies of human and animals. Still today, the search for new phytochemicals is still going on. In recent years, the development of genetically engineered plants has paved the way for using plants to produce vaccines and other pharmaceuticals. This technique is relatively new and its gaining momentum. The traditional method of producing proteins and vaccine using fermentation technique is limited due to high production cost, sophisticated purification technique and construction, and long validation period of manufacturing products (Mei et al, 2006).  However, modern plant biotechnology has used plants in providing an alternative way of producing vaccine which has the potential to overcome the constraints faced by traditional method, ie using fermentation technique. Modern method of vaccine production is the use of principles of genetic engineering to express foreign gene in plants. Plants can be genetically transformed to express any recombinant protein in them from which they can be purified or can be consumed directly. Moreover, when these recombinant proteins are expressed in the edible plant tissue like seeds, fruits, there is no need of purification, cold chain delivery of vaccine, and injection for the administration of vaccine.

 Plants are used as a biofactories for manufacturing large quanitities of recombinant protein in short period of time. Plant made vaccine and therapeutics refer to protein products with clinical applications for both humans and animals. There are numerous benefits of employing plants for the production of recombinant proteins. Plants are inexpensive to grow as they have very simplistic requirement of sunlight, soil water and cheap fertilizers and when they are engineered to express recombinant protein in edible tissues, overall production cost will significantly get lowered. Also, the need of expensive fermentation, purification, cold storage, transportation and sterile delivery is eliminated (Daniell et al, 2009). The products that are currently being produced in plants include bioactive peptides, vaccine antigens, antibodies, diagnostic proteins, nutritional supplements, enzymes and biodegradable plastic (Sharma and Sharma, 2009). These products can be expressed in edible plant tissue like fruits, seeds or leaves and easily processed or consumed directly (Warzecha and Mason, 2003). Therefore the edible plant vaccine is a cost effective means of delivering vaccines. Edible vaccines are like subunit preparations in which the antigens are expressed in them using genetic engineering, but they bear no genes that would enable whole pathogens to form. Also, the risk of contamination of plant vaccine with pathogens is eliminated. Subunit vaccines are just as efficacious as whole pathogen vaccine. In addition to that, they are safer means of vaccination (Streatfield et al, 2001). Plant derived antigenic proteins (vaccine) have been proved as an  efficient means of delaying or preventing the onset of disease in animals and have also been proven to be safe and functional in human clinical trials (Walmsley and Arntzen, 2000).

Steps in production of plant vaccines

WHO has defined vaccine as any preparation which is able to stimulate the production of antibody conferring immunity against a particular disease. Vaccines may include killed or attenuated microorganisms or purified products derived from them. Traditionally, vaccines have been produced using variety of transgenic systems, including cultured mammalian cells, microorganism like bacteria, viruses and fungi. The use of transgenic plants in the production of vaccine and pharmaceutics is much more recent and promising technique. In transgenic plants, bacterial and viral antigens can be faithfully expressed to form immunogenic proteins. When these proteins are expressed in edible plant tissue, they form edible vaccine.

Sharma and Sharma (2009) has mentioned the following steps for the manufacture of plant vaccine:

1. selection of host plant system,
2. Selection of vector and promoter,
3. Integration of gene constructs into the plant genome and regeneration of plants expressing the desired protein, and
4. Identification and purification of recombinant proteins.

1. Selection of host plant system

Selection of plant system is the key issue in producing plant vaccine. Selection of host depends upon the type of recombinant product to be obtained. Different plants have different ability to express recombinant proteins. Also the life cycle of host, biomass yield, containment and scale up costs are the deciding factors for efficient production of recombinant product. Plant or plant parts should express high level of recombinant proteins and should be suitable for extensive storage and oral delivery. These days, tobacco tomato, banana, rice, maize, wheat, carrot, soybean, pea, potato, lettuce and alfalfa are used in production of plant vaccine (Rybicki, 2009).

2. Selection of Vector and promoter

Mei et al (2006) has mentioned two approaches of transforming plant to express recombinant protein in them. First is the Agrobacterium mediated transformation and second is the use of recombinant viruses to infect non transgenic plants. Agrobacterium is a plant pathogen that in the process of infection transfers a segment of its DNA into the genome of the host plant. This feature can be used in transferring genes from bacteria to plants. After that, the transformed cells are selected and tissue cultured to form transgenic plants (Walmsley and Arntzen, 2000). Another method is the use of recombinant plant virus to deliver recombinant genes into the plant cells. First, the genome of plant virus is engineered and then transduced into host plant. Inside the plant cells, the virus multiplies and many copies of desired DNA are produced which results in expression of transgenes in host plant (Giddings et al, 2000). Later on the entire plant is harvested to extract the recombinant protein.

  

Figure 1. Showing two different approaches of engineering plants for the expression of recombinant protein in them.

The factor involved in controlling the expression of high level antigen coding gene is the selection of suitable promoter. Selection of promoters is desirable to achieve high level of transcription which is needed for optimising expression level of recombinant protein. Promoters can be constitutive, tissue specific or inducible (Sharma and Sharma, 2009). High level expression of protein is essential to develop economically competitive transgenic plant within confined field with controlled envirionmental and contained biosafety conditions (Tiwari et al, 2009). Also, expression of proteins other than the target organ leads to heavy loss.

3. Production and Purification

Transgenic plant vaccine can be obtained or used in two ways: one is plentiful production of antigen proteins and then converting into vaccine by separation and purification; and the other is direct consumption of edible plant tissue (Mei et al, 2006). Purification of recombinant proteins accounts for high cost of pharmaceutical products. Purification of recombinant plant proteins has many problems like influence of polyphenols, alkaloids, quinonoid compounds, and other secondary metabolites (Mei et al, 2006). Therefore, the better approach will be the expression of recombinant proteins in the edible tissues of plant which can be consumed without processing. This eliminates the purification expenses. Also, proteins expressed in edible parts are stable as they can be stored for a longer period and transportation is easier (Tiwari et al, 2009). The most important aspect of recombinant protein expressed in edible tissue is the reduced production cost. Oral delivery of recombinant protein reduced about 90 percent of the production cost.

 Applications of Plant derived vaccine

Vaccination is one of the most effective means of preventing infectious diseases. Since the discovery of vaccine in 1796   by Edward Jenner, many vaccines have been developed and helped in saving life of humans and animals. The traditional method of vaccine production includes the use of cultured mammalian cells, or recombinant microbes. These production methods are limited due to high cost, sophisticated technique, risks of human pathogenic contamination, etc.. Also microbes are not ideal for synthesizing many mammalian proteins because of the differences in metabolic pathway, protein processing, codon usage and the formation of inclusion bodies (Giddings et al, 2000). Also, lack of post translational modification can be taken as a main draw back in bacterial fermentation (Peeters et al, 2001).

Production of vaccine in plants has overcomed this problem and has been proved to be an efficient and inexpensive way of producing and delivering vaccines. Following are some of the major advantages of plant based vaccine:

ü  Low production cost

ü  Rapid and easy scale up

ü  Convenient storage

ü  Need of cold chain transport is eliminated

ü  Reduced use of needles and syringes

ü  Contamination of vaccine with human pathogens is eliminated

ü  The need of trained health professionals for the administration of vaccine is minimized

ü  The fear of vaccination via injection, particularly in children is eliminated.

The main advantage of plant vaccine is the reduced manufacturing cost. Sala et al (2003) has mentioned that the production cost will be reduced by 100-1000 times as compared with that of traditional vaccine. The capital investment is low as the existing infrastructures can be used for cultivation, harvesting, storage and processing of transgenic crops (Giddings et al, 2000). The plants can be grown in green houses which eliminate the expensive cost of using fermenters and bioreactors. Plant derived vaccine are stable so that they can be conveniently stored. Also the need of cold chain method of storing and transporting vaccine is eliminated. All these reduce the overall cost of vaccine production in plant.

Plant vaccine has the advantage to scale up the vaccine manufacturing time. Gleba et al (2005) has mentioned that milligrams and grams quantities of recombinant protein are available in 3-4 weeks, and the within less than one year, the scale up to 100 kg is possible. Therefore, plant vaccine can be employed for combating mutating viruses like flu virus and norovirus. Bernstein (2009) has repoted that scientists have already discovered the first vaccine against norovirus also called cruise ship virus in tobacco plants. Viruses like noroviruses are always mutating, making it difficult for developing vaccines in short period of time. However, plant biotechnology has the potential of quicker manufacturing technique suitable to combat mutating viruses like norovirus and the flu. It is mentioned that with plant based vaccine has the advantage to scale up vaccine manufacture within weeks instead of months (Bernstein, 2009).

One of the main advantages of plant derived vaccine is the protein folding mechanism in the endomembrane system of plant that is homologous to those in mammalian cells. This allows the expression of monoclonal antibodies, other types of immunoglobin molecules and multimeric complexes (Ma et al, 2004). Also when recombinant proteins are expressed in plants, there is minimized risk of contamination with human or animal pathogens.

The other advantage of plant vaccine is oral delivery. Plant derived oral vaccines are not digested in stomach as the antigens are bioencapsulated by plant cell wall. When plant derived vaccines are given orally, they are also capable of inducing both mucosal and humoral immunity (Arntzen et al, 2005). Streatfeild et al (2001) has mentioned that vaccines against enterotoxigenic strains of E.Coli and swine transmissible gastroenteritis virus (TGEV) have been expressed in corn seeds and these antigens were able to elicit protective immune responses.  Walmsley and Arntzen (2000) has cited the expression of hepatitis antigen in tobacco and lettuce, rabies antigen in tomato, and cholera antigen in tobacco and potatoes; human cytomegalovirus in tobacco. Also, when vaccines are delivered orally, there is no need of syringes and needles. Therefore, it eliminates the fear of vaccination via injection, particularly in children. Also, the need of trained health professionals for the delivery of vaccine is minimized.

One of the greatest breakthroughs of plant vaccine will be for the people in developing countries where people don’t have access to medicines and vaccines vaccine because of their high cost and complicated delivery system. However, plant derived vaccine has the potential to overcome these problems as it is inexpensive and the need of cold chain storage or delivery is eliminated. So, plant derived vaccines can be accessed by poor and people at remote areas. Therefore vaccines and medicines derived from plants will be a great boon of modern plant biotechnology to these people.

Plant vaccine is regarded as the safer means of vaccine production. Vaccines that are produced in plants are less likely to get contaminated with human or animal pathogens because plant doesn’t act as a host for those infectious agents (Giddings et al, 2000). Also, plants have the ability to carry out post translational modification that is similar to higher eukaryotes (Streatfield and Howard, 2003). These features are beneficial for the safe consumption of plant derived vaccines.

 Limitations and potentials of plant derived vaccine

Plant biotechnology is a rapidly emerging technology and is extending from the boundaries of crop improvement to the production of vaccines. Plant derived vaccine holds considerable potential in the vaccination of humans and animals as it is the inexpensive and efficient way of delivering vaccines. Despite having numerous benefits, there is significant public debate regarding the use of genetically modified crops for vaccine production. Doran (2000) has mentioned some of the pitfalls of plant vaccine as follows:

1. There is a possible fear of containment of genetically modified plants in the environment;
2. Recombinant proteins might cause allergic reactions to plant protein glycans and plant antigens;
3. There is a possibility of contamination of recombinant product by mycotoxins, pesticides, herbicides, and endogenous plant secondary metabolites;
4. There is a regulatory uncertainty, particularly for protein requiring approval for using plant vaccine as a human drug.

The major concern against using genetically engineered crops for vaccine production is the cross contamination of wild type plants with genetically altered crops. Amos, J. (2004) has reported that the transgenic plants should be grown pharmaceutical production units on dedicated land, isolated from food crops with regulatory oversights. Also, these plants should be genetically isolated, i.e. sterility in male so that they don’t produce pollen. The crops should be harvested and stored using dedicated equipments. Also, these contained conditions should be properly regulated.

Also, Mei et al (2006) has mentioned that repeated exposure to an oral antigen has the potential to produce immunological tolerance or unresponsiveness. However, the antigen dose to induce protection is generally smaller than the dose for unresponsiveness. Also, all vaccines should go extensive testing to define the correct dosage and the appropriate schedule for boosting. Therefore, tolerance from prescribed doses is highly unlikely (Streatfield and Howard, 2003).

The production of plant vaccine is an economical and efficient means of vaccinating animals. When large herds of farmed animals are to be vaccinated, they require huge amount of vaccines. Therefore, plant derived vaccine can be sucessfully implemented. Also, plant derived vaccine can be used in immunization of wild animals, which may act as a reservoir of disease, like rabies. Giddings et al (2000) reported that feeding pigs with edible maize vaccine protects them from the transmissible gastroenteritis virus.

One concern regarding plant based vaccine is the extent and nature of glcosylation, which is sometimes different from that found in animals. Giddings et al (2000) has acknowledged that some carbohydrates are unique to plants and when they are administered regularly, may present an antigenic challenge to the immune system. Therefore, sufficient knowledge and awareness should be provided to the people about the consumption of plant derived vaccine. It is mentioned that the specific problem of transgene expression in plants like gene silencing, low expression level and altered glycolysation pattern must be overcomed for the efficiency of plant derived vaccines (Warcheza and Mason, 2003).

 Discussion

Plant based vaccine holds considerable promise in future vaccination programmes. Plants have been proven as a virtually unlimited source of inexpensive vaccines for human and animal therapeutics. Plants are proved as ideal bioreactors for the production of vaccines and pharmaceuticals. Plant vaccines are far more advantageous and economical than fermentation technique. The production of vaccine in plants has been taken as a breakthrough for the delivery of vaccine to the people in developing and poor countries. Plant derived vaccine doesn’t need the cold chain process for its delivery and it can be administered without the need of trained health professionals. These vaccines and pharmaceuticals can be stored and distributed as seeds, tubers, or fruits. This makes vaccination cheaper and easier to administer. Therefore, using plant derived vaccine, diseases can be prevented or cured through consumption of food. Another major scope of plant derived vaccine is the vaccination of farmed and wild animals. Since, plant derived vaccines are cheap, edible and with a rapid scale up, it has the potential of protecting large number of animals from infectious diseases. Plant vaccine is a new technology and various challenges are still to be overcomed. So there is a need of adequate regulation, constant monitoring and research for the safe production of vaccines in plants. The use of genetically modified plants should be given utmost emphasis and also developed countries should be supportive to the developing countries for vaccine production in plants by providing advice, education, and monetary support.

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