In this article we will discuss about Transgenic Plants:- 1. Meaning of Transgenic Plants 2. Reasons for making Transgenic Crop Plants 3. Fundamentals of Transgenic Plant Development 4. Steps Involved in the Production of Transgenic Plants 5. Steps Involved in the Production of Transgenic Plants 6. Integration of the Transgene in the Genome of the Target Plant and Other Details.
- Meaning of Transgenic Plants
- Reasons for Making Transgenic Crop Plants
- Fundamentals of Transgenic Plant Development
- Steps Involved in the Production of Transgenic Plants
- Steps Involved in the Production of Transgenic Plants
- Integration of the Transgene in the Genome of the Target Plant
- Analysis of Transgene Integration
- Detection of mRNA Expression
- Inheritance of Transgenes
- Future Development of Transgenic Technology
1. Meaning of Transgenic Plants:
Transgenic plants or genetically modified plants are plants whose DNA is modified using genetic engineering techniques. In most cases the aim is to introduce a new trait to the plant which does not occur naturally in this species. Examples include resistance to certain pests, diseases or environmental conditions, or the production of a certain nutrient or pharmaceutical agent.
A transgenic crop plant contains a gene or genes which have been artificially inserted instead of the plant acquiring them through pollination. The inserted gene sequence, known as the transgene, may come from another unrelated plant or from a completely different species. Plants containing transgenes are often called genetically modified or GM crops.
2. Reasons for making Transgenic Crop Plants:
i. The process of transgenic plant development primarily aims at assembling a combination of genes in a crop plant which will make it as useful and productive as possible. Depending on where and for what purpose the plant is grown, desirable genes may provide features such as higher yield or improved quality, pest or disease resistance, or tolerance to heat, cold and drought.
ii. Combining the best genes in one plant is a long and difficult process, especially as traditional plant breeding has been limited to artificially crossing plants within the same species or with closely related species to bring different genes together.
For example, a gene for protein in soybean could not be transferred to a completely different crop such as corn using traditional techniques. Transgenic technology enables plant breeders to bring together in one plant useful genes from a wide range of living sources, not just from within the crop species or from closely related plants.
iii. This technology provides the means for identifying and isolating genes controlling specific characteristics in one kind of organism, and for moving copies of those genes into another quite different organism, which will then also have those characteristics.
iv. This powerful tool enables plant breeders to do what they have always done-to generate more useful and productive crop varieties containing new combinations of genes-but it expands the possibilities beyond the limitations imposed by traditional cross-pollination and selection techniques.
3. Fundamentals of Transgenic Plant Development:
The underlying reason that transgenic plants can be constructed is the universal presence of DNA (deoxyribonucleic acid) in the cells of all living organisms. This molecule stores the organism’s genetic information and orchestrates the metabolic processes of life. Genes are discrete segments of DNA that encode the information necessary for assembly of a specific protein.
A specific protein (or an enzyme) encodes for a particular trait. In the production of a transgenic plant our primary aim is to transfer a foreign gene, encoding for some novel traits, into the genome of the plant stably.
After transferring we also need the transgene to integrate and express in the plant’s cells. This process as a whole generates a new variety of plant which is new in its own kind and interests us in its massive large scale cultivation.
4. Steps Involved in the Production of Transgenic Plants:
The fundamental steps involved in the transgenic plant production are as follows:
Step 1: Identifying, Isolation and Cloning of Genes for Agriculturally Important Traits:
The very first step in the generation of transgenic plant is to identify and isolate the novel transgene that we want to transfer into the genome of the target plant. Usually, identifying a single gene involved with a trait is not sufficient. We also have to understand how the gene is regulated, what other effects it might have on the plant, and how it interacts with other genes active in the same biochemical pathway.
Step 2: Designing Gene Construct for Insertion:
After entering the plant cell the transgene must inter-grate into the genome of the plant stably and express itself successfully so as to produce higher amount of transgenic protein which will be indirectly reflected in the trait controlled by it.
To achieve this we have to design a “gene construct” or gene-set, having all the DNA segments necessary to achieve the integration and expression of the transgene. Once a gene has been isolated and cloned (amplified in a bacterial vector), it must undergo several modifications before it can be effectively inserted into a plant.
A gene-set which will be transferred to the target plant has following segments:
I. A Promoter Sequence:
This must be added for the gene to be correctly expressed (i.e., translated into a protein product). The promoter is the on/off switch that controls when and where in the plant the gene will be expressed. To date, most promoters in transgenic crop varieties have been “constitutive”, i.e., causing gene expression throughout the life cycle of the plant in most tissues.
The most commonly used constitutive promoter is CaMV 35S, from the cauliflower mosaic virus, which generally results in a high degree of expression in plants. Other promoters are more specific and respond to cues in the plant’s internal or external environment. An example of a light-inducible promoteris the promoter from the cab gene, encoding the major chlorophyll a/b binding protein.
II. The Transgene:
Sometimes, the transgene is modified to achieve greater expression in a plant.
For example, the Bt gene for insect resistance is of bacterial origin and has a higher percentage of A-T nucleotide pairs compared to plants, which prefer G-C nucleotide pairs. In a clever modification, researchers substituted A-T nucleotides with G-C nucleotides in the Bt gene without significantly changing the amino acid sequence. The result was enhanced production of the gene product in plant cells.
III. Termination Sequence:
This signals to the cellular machinery that the end of the gene sequence has been reached.
IV. A Selectable Marker Gene:
This is added in order to identify plant cells or tissues that have successfully integrated the transgene. This is necessary because achieving incorporation and expression of transgenes in plant cells is a rare event, occurring in just a few per cent of the targeted tissues or cells.
Selectable marker genes encode proteins that provide resistance to agents that are normally toxic to plants, such as antibiotics or herbicides. Only plant cells that have integrated the selectable marker gene will survive when grown on a medium containing the appropriate antibiotic or herbicide.
The essential features of an ideal reporter gene are:
1. Lack of endogenous activity in plant cells of the concerned enzyme,
2. An efficient and easy detection, and
3. A relatively rapid degradation of the enzyme.
The commonly used selectable marker genes include those conferring resistance to the antibiotics kanamycin (nptII, encoding neomycin phosphotransferase) and hygromycin (hptIV, encoding hygromycin phosphotransferase, isolated from E. coli); and broad range herbicides glyphosate (modified versions of the enzyme EPSPS, 5-enolpyruvate shikimate-3-phosphate synthase, isolated from E. coli or Salmonella typhimurium), phosphinothricin (bar, isolated from Streptomyces hygroscopicus, codes for phophinothricin acetyltransferase), etc.
Step 3: Transforming Target Plants with the Gene Construct:
There are two ways of genetically transforming the target plant:
I. Vector mediated gene transfer, and
II. Vector less or direct gene transfer.
Step 4: Selection of The Transgenic Plant Tissue/Cells:
Following the gene insertion process, plant tissues are transferred to a selective medium containing an antibiotic or herbicide, depending on which selectable marker was used. Only plants expressing the selectable marker gene will survive and it is assumed that these plants will also possess the transgene of interest.
Step 5: Regeneration of the Transgenic Plants:
To obtain whole plants from transgenic tissues, they are grown under controlled environmental conditions in a series of media containing nutrients and hormones by the process of plant tissue culture.
5. Integration of the Transgene in the Genome of the Target Plant:
In general, transgenes integrate at random sites in any of the chromosomes of the genome of host cells. Usually, in a given cell, integration occurs at a single location. As a result, different cells may be expected to show integration of the transgene at different chromosomal locations.
The number of copies integrated per genome ranges from one to several hundred. In general, multiple copies are integrated when large amounts of DNA are used for transfection, while single copies are integrated with smaller amounts.
When multiple copies are integrated, they are mostly integrated at one site joined to each other head-to-tail, i.e., as a concatemer. However, in a small proportion of cases, the multiple copies are located at several sites in the same genome.
The mechanism of random integration is not known. The entire gene construct, including the vector DNA, becomes integrated. When two different gene constructs are mixed and used for transfection, they tend to be integrated together at the same site; this is known as co-transfection. The sequences flanking a gene on either side influence the expression of this gene.
Therefore, the same transgene integrated at different locations in the genome may show different levels of expression; this is known as position effect. Transgene integration frequently leads to various forms of rearrangements, e.g., duplication, deletion, etc., near the site of integration.
If these changes are large enough, the host gene located at the site of integration may become non-functional. A host gene would also become non-functional if the transgene becomes integrated within the coding region of this gene. When integration of a transgene leads to the loss of function of a host gene, it is called insertional mutagenesis; it often produces aberrant phenotypes.
6. Analysis of Transgene Integration:
The integration of transgene into the genome is confirmed by Southern hybridization of genomic DNA extracted from the considered transgenic individuals. The DNA is digested with a suitable restriction enzyme prior to electrophoresis.
By choosing appropriate restriction enzymes for DNA digestion, not only the integration of transgene can be established beyond doubt, but information on the number of copies per cell, the orientations of tandemly arranged copies and the presence of single or multiple integration sites is also obtained from Southern hybridization. All the individuals that give positive result with Southern hybridization are regarded as confirmed transgenic.
7. Detection of mRNA Expression:
The mRNAs produced by transgenes is most readily detected if they are with unique sequences, which have no counterparts among those produced by the host genome. A high purity RNA preparation is obtained from the appropriate tissue of transgenic individuals, and is subjected to RNA dot blot hybridization with a radioactive probe specific for the transgene.
Alternatively, the RNA preparation may be used for northern hybridization, which provides additional information on transcript size as well.
8. Inheritance of Transgenes:
The transgenes which are stably integrated are inherited in a Mendelian fashion. They are usually dominant. Instability may occur due to point mutation, like methylation, or rearrangements of the T-DNA region. In addition, homologous recombination between copies of the transgene inserted in the same nucleus can also lead to instability of the gene.
9. Future Development of Transgenic Technology:
New techniques for producing transgenic plants will improve the efficiency of the process and will help resolve some of the environmental and health concerns.
Among the expected changes are the following:
a. More efficient transformation, that is, a higher percentage of plant cells will successfully incorporate the transgene.
b. Better marker genes to replace the use of antibiotic resistance genes.
c. Better control of gene expression through more specific promoters, so that the inserted gene will be active only when and where needed.
d. Transfer of multi-gene DNA fragments to modify more complex traits.