Transgenic plants

 

Transgenic plants

Transgenic plants are those plants which carry additional, stably integrated and expressed, foreign gene(s) from Trans species. The whole process of transf=genic plants development involving introduction, integration, and expression of foreign genes in to the host is called genetic transformation. The combined use of recombinant DNA technology, gene transfer methods and tissue culture techniques has led to the efficient transformation and production of transgenics in a wide variety of crop plants. Unlike conventional breeding, only the cloned gene(s) of agronomic importance is being introduced without the co transfer of other undesirable genes from the donor. The recipient genotype is least disturbed and there is no need for repeated back crosses. This will serve as an effective means of removing certain specific defects of otherwise well adopted cultivars.

General procedure used to make a transgenic plant agrobacterium mediated transformation .The first step in the process of development of a transgenic plant involves the formation of the recombinant plasmid and its transfer to plant cells. For the recombinant formation, T-DNA  needs to be disarmed. To do this, the genes encoding the proteins for the production of auxin and cytokinin are simply removed from the T-DNA fragment. New DNA can, then, be inserted between the left and right border repeats. Recombinant T-DNA plasmid vectors contain a number of functional units in addition to the right border and left border elements. These include:

1.      A broad host range origin of replication,

2.      An antibiotic-resistance gene for plasmid selection in bacteria,

3.      A multiple cloning site located between right and left border elements for insertion of the target gene,

4.      A dominant selectable/screenable marker gene for selection of transformed plant cells.

Most such genes used in plants are dominant selectable markers that confer resistance to antibiotics or herbicides. The commonly used selectable marker genes include those conferring resistance to the antibiotics kanamycin and hygromycin; and herbicides glyphosate, phosphinothricin, etc.

Agrobacteria that are carrying a recombinant plasmid with both a selectable marker and a desired transgene are incubated in culture with plant cells. The wounded plant cells at the edge release substances that attract the Agrobacteria and cause them to inject DNA into these cells. Only those plant cells that take up the appropriate DNA and express the selectable marker gene survive to proliferate and form a callus. The growth factors supplied to the callus induce it to form shoots and roots, and grow into adult plants carrying the transgene.



TRANSGENIC PLANTS:APPLICATION

1- Development of insect, virus and herbicide resistant plant Insecticidal resistance Through genetic engineering it is possible to develop crops that are intrinsically resistant to insect. Several different strategies have been used to confer resistance against insect predators. One approach involves a gene for an insecticidal protoxin produced by one of several sub-species of the bacterium Bacillus thuringiensis. Crop plants have been engineered to express the insecticidal toxin gene of B. thuringiensis so that insects attempting to eat these plants are killed. Be thuringiensis, during sporulaltion, forms intracellular crystalline bodies that contain an insecticidal protein called the Seendotoxin. The 6--endotoxin protein (cry If cry II, cry III and others) accumulates in the bacterium as an inactive precursor. After ingestion by the insect, this protoxin is cleaved by proteases, resulting in shorter versions of the protein that display the toxic activity, by bindina to the inside of the insect's gut and damaging the surface epithelium.

Virus resistant plants several approaches have been used to engineer plants for virus resistance, such as the introduction of coat protein gene, antisense RNA approach, and ribozyme-mediated protection. Of these strategies, use of coat protein gene has been the most successful. Transgenic plants having a virus coat protein gene linked to a strong promoter have been produced in many crop plants such as tobacco, tomato, alfalfa, potato, etc. The first transgenic plant of this type was tobacco produced in 1986. It contained the coat protein gene of Tobacco Mosaic Virus (TMV). When these plants were inoculated with TMV, symptoms either failed to develop or were considerably delayed. The effectiveness of coat protein (CP) gene in conferring virus resistance can be affected by both the amount of coat protein produced in transgenic plants and by the concentration of virus inoculum. Most likely, the resistance generated by CP is due to the blocking of the process of uncoating of virus particles, which is necessary for viral genome replication as well as expression. However, other effects seem to be involved in producing coat protein mediated virus resistance; one such mechanism appears to be the prevention or delay of systemic spread of the viruses. In another approach, the transgenic expression of dysfunctional viral movement proteins (MP) is used to make the plant virus resistant. Resistance conferred by transgenic expression of a dysfunctional MP is likely due to competition for plasmodesmatal binding sites between the mutant MP and the wild-type MP of the inoculated virus. An interesting attribute of MP-mediated protection is the broad spectrum efficacy of the resistance mechanism. The protection conferred by the mutant MP of TMV, for example, mediates resistance to other viruses also.

Herbicidal resistance Many crops have been engineered for resistance to herbicides such as glyphosate. Glyphosate (trade name roundup) is a non-selective herbicide that inhibits 5-enolapyruvylshikimate-3-phosphate (EPSP) synthase, a key enzyme in the biosynthesis of aromatic amino acids in plants. EPSP synthase converts shikimate and phosphoenol pyruvate into 5-enol-pyruvylshikimate-3-phosphate, a precursor for synthesis of aromatic amino acids tryptophan, tyrosine and phenylalanine. Glyphosate competes with phosphoenol pyruvate for binding with EPSP synthase. Two approaches have been used to engineer resistance so that the herbicide can be used for weed control without damaging the crop. In the first approach, the target protein of the herbicide (EPSP synthase) can be over-produced so that resistance occurs as a consequence of having more enzyme available to the cell. A second approach results from the expression of a mutant version of EPSP synthase that is resistant to the herbicide within cells.

2-Developmentvelopment of stress tolerant plant like oxidative stress and salt stress,

3-Modification of plant nutritional content like amino acid, lipid, vitamin and iron.The use of genetic engineering techniques allows scientists to develop the plants with improved nutritional quality. For example, rice is extremely low in vitamin A. Potrykus and Beyer developed genetically engineered rice (popularly known as golden rice), which is enriched in pro-vitamin A by introducing three genes involved in the biosynthetic pathway for carotenoids, the precursor for vitamin A. To obtain a functional provitamin A Macarotene) biosynthetic pathway in rice endosperm, genes coding for phytoene synthase (psy) and lycopene cyclase (lyc) both from Narcissus pseudonarcissus together with a gene coding phytoene desaturase (crtl, from bacteria Erwinia uredovora) were introduced. This combination covers the requirements for pm-carotene synthesis. Additional experiments later revealed that the presence of iyc was not necessary, because psy and crtl alone were able to drive pacarotene synthesis as well as the formation of further downstream xanthophylls.

4-Plants as bioreactor for production of antibodies, polymers etc. Plants are easy to grow and can generate considerable biomass. That is the reason the transgenic plants are used for the production of commercial proteins and chemicals. Plants have been used to produce monoclonal antibodies (plantibodies); the polymer polyhydroxybutyrate, which can be used to make a biodegradable plastic like material; and a number of potential therapeutic agents like, human protein C (anticoagulant), human hirudin variant 2 (anticoagulant), human erythropoietin (anemia), human alpha interferon (hepatitis C and B) and human growth hormone (dwarfism).

The term plantibodies is used for antibodies that are synthesized in transgenic plants. The difference between plantibodies and edible vaccines is that plantibodies are pre-made antibodies that are produced in the transgenic plant; whereas edible vaccines promote the production of specific antibodies by the human immune system once the vaccine is administered to the patient. Plantibodies are advantageous for people who are immunosuppressed and are unable to produce antibodies even after they are vaccinated.

5-Synthesis of edible vaccines Commercial vaccines are expensive to produce and package, and require trained personnel to administer injections. So, it would be advantageous if vaccines could be delivered inexpensively on a broad scale in an edible form, e.g. as part of a fruit or vegetable. An edible vaccine, in contrast to traditional vaccines, would not require elaborate production facilities, purification, sterilization, packaging or specialized delivery system. The gene encoding of the orally active antigenic protein is isolated from the pathogen, and a suitable construct for constitutive or tissue-specific expression of the gene is prepared. The gene is introduced and stably integrated into the genome of selected plant species and expressed to produce antigen.

6-Delayed ripening

Genetic engineering is used to delay ripening in fruits. A number of genes control the ripening process,. One of these, encoding the enzyme polygalacturonase, is involved in the slow breakdown of the polygalacturonic acid component of cell walls in the fruit pericarp. Its effects result in a gradual softening that makes the fruit edible. However, the longer the enzyme is able to act on the cell walls, the softer and more over-ripe fruit will become. Therefore, if the effects of the enzyme can be delayed, then the fruit will ripen more slowly. This approach has been used in tomatoes to delay ripening. Tomatoes have been engineered so that they express less of the polygalacturonase enzyme. This was achieved through the antisense technology. Using antisense technology, Calgene Fresh Inc. (USA) has permanently introduced an antisense copy of the gene for polygalacturonase with the aid of Agrobacterium tumefaciens. Expression of the antisense copy of the gene gives antisense RNA molecules. Antisense RNA has the opposite sense to mRNA (sense RNA). The presence of complementary sense and antisense RNA molecules in the same cell can lead to the formation of a stable duplex, which may interfere RNA processing or possibly translation. Antisense RNA binds to the normal-sense RNA preventing the tomato from making the usual amount of polygalacturonase. This genetically modified tomato—marketed under the trade name Flavr Savr will, therefore, resist softening and have extended self-life.

7-Production of bioplastics

The bacteria alcaligens euthropus produce polyhydroxybutyrate PHB a biodegradable and renewable biopplumer. The gene from A. euthropus that codes for an enzymeres ponsible for biosynthesis of PHB is being transferred to plant for production of biodegradabke plastic.

 

 

 

 

 

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