As the functional component of DNA, a gene can be reckoned as what constitutes the hereditary factor in any organism. Genetic engineering is the re-configurement of the genome of an organism and transgenics is only a part of genetic engineering. When a gene of interest is transferred from one organism to another it is called transgenics. In other words, transgenic is the means by which genes are transferred from one organism to another---and these organisms could be of either plant or of animal origin.
So today you have plants of different species that have received genes from other species----prominent examples being sunflower, cotton and tomato. Initially application of transgenics in plants was confined to dicotyledonous plants (mostly flowering plants such as rose and magnolias that have leaves or cotyledons in seed embryo), but now crop varieties like maize and rice (monocots) have also been recipients of gene transfer. The key factor in any transfer of genes in crop varieties is of course the possibility of getting better yields.
In Transgenic plants the genome has been significantly altered by the introduction of a foreign gene. In ornamental plants, transgenics has helped to improve flower color and scent. In food crops it has been used for combating disease, drought, and cold wintry conditions and to enhance nutritional value. It has been used for making functional foods and bioactive compounds as well. So in a way transgenics offers additional economic value.
Conventional versus transgenics
But do we really need transgenics in crop plants, especially when farmers down the ages have always been using conventional breeding methods to increase genetic diversity in plants and thereby increase crop harvest? The answer is in the affirmative because conventional crop breeding methods takes too much of time, and to get a superior plant that would yield higher crop yields might take several years often entailing a trial and error process. Besides, there could be problems of the pollen not germinating, or the seed may show abnormal growth or sometimes the entire endeavor could result in transfer of unwanted characteristics. On the contrary, transgenics can avoid the possibility of any of these problems occurring.
Using conventional means a tomato species that ripens early could be crossed with a tomato species of enhanced genetic background to get a tomato that would ripen fast and therefore lessen the time required to bring it into the market. If transgenics were used, although the end result would be similar, scientists would focus more on the genes that are responsible for the required trait (whether it has to do with early ripening or some other trait) and the process itself would be done at the molecular level to transform the plant's genome for getting the desired trait. So today there are transgenic tomatoes with more lycopene content and other tomato varieties whose cell membranes degrade very slowly after ripening. But importantly in transgenics everything is done by reengineering the gene through molecular tools.
In this article our aim is to elucidate how transgenic plants are actually evolved and describe the three part process that includes:
1) Making of recombinant DNA; 2) Transformation of the gene of interest into the plant 3) and how Reproduction and Regeneration of Transgenic Plant Cells happens
1) Making of recombinant DNA
To begin with, the donor plant's DNA has to be isolated by either T-DNA tagging, transpoon tagging (transposons are 2-element mobile genetic systems in which one element encodes a transposase while the other element transposes on account of activity of transposase), or by looking up suitable library for genes of interest (GOI). From nucleotide, passenger DNA can be synthesized but in that case gene of interest would need a vehicle for getting transferred to plant tissues.
Recombinant DNA can be made by cloning the GOI with vectors (as for example plasmids and bacteriophages) and introducing appropriate promoter sequences. Restriction endonucleases and ligases are used for these processes that entail splicing and joining DNA molecules.
2) Transformation of the gene of interest into the plant
There are predominantly two dominant methods to transform the GOI into a particular plant. They are a) By using Agrobacterium or by b) direct gene transfer
a) By using Agrobacterium
The soil bacterium Agrobacterium tumefaciens's cell has tumor inducing (Ti) plasmid (that contains T-DNA) which clings on to the host and causes tumor. The GOI is put into T-DNA after removing tumor causing genes. A. tumefaciens gets the DNA transferred into the plant where it gets aligned with plant genome. In other words, when plant cells are infected with A.tumefaciens plus recombinant DNA the DNA transforms into plant genome for later expression.
b) direct gene transfer
Here chemical (using calcium phosphate or polyethylene glycol), physical means (electropolation) or biolistic approach (using particle gun bombardment) is used to transfer recombinant DNA into plant protoplasts. Electropolation involves the use of high energy field electrical impulses to reversibly permeabilize cell membranes to facilitate uptake of foreign DNA.
Developed by Sanford in 1983, in particle gun bombardment, either microscopic tungsten or gold particles are used to carry the recombinant DNA at high speeds so that they can penetrate cell walls of calli.
3) How Reproduction and Regeneration of Transgenic Plant Cells happens
On being cultured in an artificial media, the cells that get transformed will grow into callus. For proper stimulation of cytokinesis or cell division the ratio of auxin and cytokinin should be appropriate and in this way from plant calli whole plants can be regenerated. This process is called totipotency. In other words plant cells using their genes can transform to tissue and subsequently to whole plants. This ability to generate any cells from starting tissue is called totipotency.
To sum up, the step by step transformation of gene into plant occurs as follows. Firstly both passenger DNA and Vector DNA are digested separately with restriction enzymes. Then it is connected with DNA ligase to make recombinant DNA. Then using either Agrobacterium mediated transformation or by transformation using chemical or physical means plant cells are made. These cells are then converted to calli. Finally calli is regenerated into whole plant.
There are some limitations to transgenics. These are:
• Transgenic expression depends on transgene copy number and site of integration and so there is a certain amount of unpredictability. Transgenic plants with single copy number are preferred because gene silencing could occur with multiple copies.
• In the heterologous system transgene inactivation and silencing by the host is a problem.
• Sometimes co-suppression takes place because the transgene that is introduced could suppress endogenous gene expression.
• Sometimes when transgene is expressed it might inhibit host metabolism by becoming cytotoxic.
• Currently available techniques are found wanting for transferring multiple genes by transformation.
• Some plant species exhibit recalcitrance and this limits transgene expression and regeneration of the plant species
• In transgenics, on account of stresses caused due to transformation techniques somaclonal variation (either genotypic or phenotypic) occurs. This is reckoned as a disadvantage in horticulture and forestry where clonal uniformity is required for quickly propogating elite genotypes.
When transformation techniques are used it is important to study the progress of lignin biosynthesis in such plants as also how cell wall polysaccharides are synthesized and remodulated. The use of transgenics and transformation techniques, have also enhanced our knowledge of other important aspects of plant bioscience such as expression of reporter genes, gene activity, design of transgenes, the use of PCR for finding out transgene copy number, and the techniques for isolating marker genes from transgenic plants.
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