With the discovery of the secrets of gene manipulation, it has been possible to manipulate and transfer genes from one organism (plant or animal) to another, whether there is any relationship between the two or not.

What to speak about the gene transfer between two animals or two plants; it has been successfully carried out between an animal and a plant and vice versa.

The basic purpose underlying this transfer is to have something good for the human society, either increase productivity or express a new character for aesthetic pleasure or express and harvest a new beneficial product at a fairly high level. Now there exists no reproductive barrier for transferring genes to heterologous cells.

All these gene transfers across the reproductive barrier have opened up a new branch of genetic engineering known as transgenics.

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The gene that is transferred is known as a transgene and the organism carrying and expressing the gene as transgenic. Care is taken to see that the transgene is integrated and maintained in the host genome in a stable manner and expresses at a high level continuously.

The entire process of gene transfer is carried out in vitro. It involves a sequence of steps as outlined below:

1. The identification of the beneficial character, its encoding gene and its cell location in the donor organism.

2. The isolation of the cell carrying the beneficial gene.

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3. The isolation of the beneficial gene of interest.

4. Ligation of the gene to a suitable vector (carrier) and the formation of a recombinant vector.

5. Isolation of the target cell for the transfer of the beneficial gene.

6. Transformation of the target cell in vitro with the recombinant vector (vector-gene combine).

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7. Transfer of the transformed cell into its normal location.

8. Monitoring the expression of the transgene in the transformed cells.

Transfer of the transgene into the target cell

The isolation of the beneficial gene from the donor organism is carried out, following a standard protocol. However, the transfer of the gene to the target cell in vitro has been the main issue of the process. There are two types of practice: (l) transfer by transformation and (2) direct transfer.

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Transfer by transformation

Some plant bacteria have plasmids as extrachromosomal DNA circles. These plasmids are genetically engineered and used as vectors (carriers) for the transgene. Bacteria carrying the recombinant plasmid (plasmid + transgene) are used to transform host plant cells.

This will result in the integration of the recombinant plasmid into the host cell genome. Alternately, some plant viruses also can be used as vectors for the transformation of plant host cells.

a) Plasmid mediated transformation

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The vector is the carrier of the transgene to the target cell. The vector of choice in most plant cell transformations is the Ti (tumor inducing) plasmid, present in the Gram-negative soil bacterium, Agrobacterium tumefaciens This bacterium infects most dicotyledonous plants through a wound, often at the crown separating the stem from the root, causing a tumor (uncontrolled cell growth) known as the crown gall tumor.

It has been found that the actual infectious agent is the Ti plasmid. A 30 kb part of this plasmid is known as T (transforming) DNA. The transfer and integration of the T DNA is dependent on two 25 bp directly repeated sequences, present on both sides.

The T DNA is excised from the plasmid by the interaction between the bacterium and host cell. It forms a circle and then integrates into the host cell genome. Its excision, transfer and integration are controlled by a vir (virulence) gene, also present on the plasmid.

The part of the T DNA between two direct repeats is not necessary for excision, transfer and integration. Therefore, it may be replaced by a transgene a tumor inducing (Ti) plasmid of Agrobacterium tumefaciens used for gene transfers in plants.

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Luciferase gene of bacteria or the firefly is used as a common reporter gene. In the presence of the substrate luciferin, the luciferase gene expresses and exhibits a detectable bioluminescence. A tobacco plant with an incorporated luciferase gene has been successfully created [see (Unit IV) chapter-5 in the Part-I of this book for vectors used for transformation of plant cells under the section “Eukaryotic hosts and vectors (Plants)”] The Ti plasmid is genetically engineered in vitro.

A large part of the Ti plasmid (excluding the T DNA, direct repeats and the vir gene) is removed. A linear pBR 322 is annealed to it. A constitutive promoter such as Ca MV 35S (for continual expression of the transgene) and a reporter gene (for selecting a stable integration) are also engineered into the plasmid. The resultant is circularized.

The T DNA excluding the direct repeats is removed and in its place the foreign gene (transgene) is inserted and ligated. A recombinant plasmid is formed. An A. tumcfaciens is transformed with this recombinant plasmid. The bacteria carrying the recombinant plasmids are allowed to infect plant cells in culture. The Ri (root inducing) plasmid of A. rliizogenes is also engineered in the same way as that of the Ti plasmid,

(b) Virus mediated transformation

Some viruses infect plant cells and produce large number of viral particles inside the host cell leading to gene amplification. Most viral genomes do not integrate into plant cell genome. Therefore, these cannot be used as vectors for transformation of plant cells. However, DNA viruses, such as cauliflower mosaic virus (Ca MV) and Gemini virus are sometimes used for transforming plant cells.

Direct transfer

Transfer of the transgene directly without the mediation of bacteria or viruses is known as direct transfer. The transgene may be delivered directly as such or via a vector.

Several methods like electroporation and microprojectile bombardment etc are used for the purpose. Isolated protoplasts are the best targets for this type of gene transfer, since these are devoid of cell walls, [see (Unit IV) chapter-5 in the Part-I of this book for transformation of plant cells under the section “Alternate methods of gene transfer”]

Economically important plants through transgenics

Transgenics have been applied to the generation of agriculturally important plants. Novel plants have been generated through this procedure, which otherwise would not have been possible through traditional breeding method. A few of the applications are discussed below.

Virus coat protein and viral infection resistance

Most plant viruses are pathogenic and pose a serious problem to agricultural crops one such virus is tobacco mosaic virus (TMV). It infects tobacco plant and causes serious damage to the plant. Its genetic material is RNA.

Its genome encodes for few proteins, one of which is the coat protein. Transgenic tobacco plants are produce by introducing the TMV coat protein gene through A.

tumefactions mediated gene transfer. Such transgenic plants exhibited an increased resistance to TMV infection. More recently, effective protection against viral infection has been achieved in crop plants like potato, alfalfa and tomato by increased viral coat protein expression.

Bacillus thuringiensis and biopesticide

Bacillus thuringiensis is a Gram positive soil bacterium, synthesizing a crystalline protein, known as Bt protein. This protein is toxic to larvae of a majority of the lepidopteran pests.

The gene encoding the BT protein is present on a plasmid. It has been introduced into plant cells in culture and Bt- transgenic plants generated. The transfer is mediated by A. tumefaciens. Such plants exhibited an increased resistance to many insect pest infections. The most outstanding examples of the application of this technology are Zfr-cotton, developed by Monsanto, USA and Bt-corn developed by StarLink.

Herbicide tolerant plants

Weeds are undesirable plants present in the field along with the crop plants. These interfere with the normal growth of the crop plants and hence, reduce the harvest. Some chemical agents, weedicides, also known as herbicides are used to kill the weeds.

The crop plants are not resistant to these herbicides. They are also affected by the herbicides. Therefore, the entire purpose is defeated. However, herbicide resistant transgenic plants have been generated by transferring bacterial herbicide resistant genes into plant cells in culture. Glyphosate is the most commonly used herbicide. It is available under the commercial name of Roundup.

This herbicide inhibits the chloroplast enzyme 5-enolpyruvylshikimate 3-phosphate synthetase (EPSPS), involved in the biosynthetic pathways of aromatic amino acids. A mutant EPSPS, present in Escherichia coli is resistant to glyphosate. Its encoding gene is isolated and introduced into crop plant cells in culture and glyphosate-resistant plants are generated. Transgenic glyphosate-resistant tomato, potato, petunia, corn and tobacco have been successfully produced.

Transgenic plants producing bio-plastic

Plastic is a polymer of organic compounds. Natural plastic is isolated from crude petroleum. It is also manufactured by polymerizing organic compounds. Whatever is the source of plastic, this product has become an integral part of modern life.

These are non-biodegradable and hence, caused serious pollution problems through dumping. An alternative to this has been discovered in biotechnology. A group of diverse microorganisms from the genera, Alcaligenes,Azospirillum,Acinetobact’er, Clostridium, Halobacterium, Microcystis, Pseudomonas, Rhizobium, Spirulina, Streptomyces and Vibria synthesizes bio-polymers collectively known as poly-P- hydroxy alkaonate (PHA). These are synthesized and stored in cells for use as a source of carbon under unfavourable conditions.

One of these species, Alkalogenes eutropus produces the PHA, polyhydroxybutyrate (PHB). The gene encoding for the enzyme in the biosynthesis of PHB is isolated and transferred to corn plant cells in culture. The regenerated transgenic corn expresses the transgene and synthesizes the bio-polymer.

This bio-polymer is used as bio-plastic. The advantage of bio-plastic is that it is bio-degradable and hence, does not cause any environmental pollution problem.

Novel transgenic plants

Flower colours are also manipulated by transgenics. Novel flower colours, not naturally found, are also generated. Chalone synthetase (CHS) is an enzyme in the biosynthetic pathway for the synthesis of anthocyanin, a purple pigment, present in many flowers and maize kernel.

The gene encoding for this enzyme is introduced into mutant Petunia protoplasts in culture. The flowers of mutant Petunia were light pink.

The maize kernel CHS gene integrated into the protoplast genome in a stable manner and generated petunia plants, whose flowers were purple to brick red. Another speculation is to introduce a gene encoding for the enzyme involved in the biosynthesis of the blue pigment, delphinidium into rose protoplasts in culture and raise transgenic rose plants, which would give blue flowers. This has not yet become successful. However, experiments are underway to produce rose plants with blue flowers.

Protein producing plants

Plants are already known to produce a variety of chemicals for use in the industry for the production of medicines, dyes and paints. Now, transgenic plants are projected as bioreactors for the synthesis of many therapeutic proteins.

The plant systems stand as an alternative to mammalian cell cultures, which require a high degree of sophistication and hence, the products harvested are very expensive.

The plant system provides a cheap and alternate source for the production of these products. Presently, this is confined to laboratory experiments. Many heterologous proteins such as enkephalin (a neuro-peptide) and human serum albumin have been expressed in plants. Expression of mouse monoclonal antibodies in plants is another application of the technology.

The heavy and light chain genes of the monoclonal antibody were introduced into separate Ti plasmids. A. tumefaciens were transformed by these recombinant plasmids separately and these transformed bacteria were allowed to infect tobacco plant cells in culture. Transgenic plants having the ability to synthesize either heavy chains or light chains were obtained.

These two varieties of plants were sexually crossed to produce progeny, which had the ability to synthesize complete monoclonal antibodies. The production of anticoagulants in the canola plant is another case in point.

The anticoagulant gene of leech is transferred to canola plant cells in culture. The regenerated canola plant synthesized anticoagulant and stored in the seeds. Anticoagulants have an important role in treating coronary diseases.

Golden rice

In 2000, the Swiss Institute of Crop Science struck the media head line by raising Golden Rice. It was a transgenic variety of rice raised by a collaborative research of the scientists from Sweden and Germany.

This rice had elevated levels of P -carotene, a precursor of vitamin A. The biochemical pathway for the synthesis of P-carotene was engineered by inserting genes from daffodil, Narcissus pseudonrcissus and a fungus, Erwinia uredovora. This rice may help combat blindness caused due to the deficiency of vitamin A.

The ethical issues of transgenic plants and animals are discussed in the section of the next chapter.