Genetic transformation in plants 1.

Introduction
Term genetic transformation refers the ability to move DNA into an organism and thereby alter its genotype or genetic makeup. This technique play central role to both basic and applied molecular biology. The application these conventional or classical techniques has produced significant achievements of major food crops and also overproduction of valuable plant secondary metabolites In nature, many strains of bacteria exchange genetic information during a process known as conjugation, and the new genetic information is passed on to subsequent generations. The advantage of using bacteria relates to the single-celled nature of the organisms. Only one cell needs to be changed in order to integrate the new genetic information and allow for its transmission to the next generation. This natural exchange of genetic information allows the organism an increased capacity to adapt to its environment. Transformation is usually more difficult with multicellular organisms, however, and can be particularly challenging with some important plant species like cereals. The multicellular nature of most plants introduces the complication of transforming each cell of the plant in order to fully integrate the new information. Often the approach taken in the transformation of higher organisms, such as plants, is to transform an individual plant cell and then regenerate it into a whole organism. This step calls upon the pluripotent nature of plant cells. That is, a whole plant can grow from a single cell. Genetic transformation of plants and other organisms occurs naturally. Bacteria carry this out routinely. It has been observed that viruses are also able to move DNA (or RNA) into an organism and cause significant changes in genetic organization in plant cells. Soil bacteria, such as Agrobacterium tumefaciens (tumor inducing bacterial sp.) and Agrobacterium rhizogenes, (root inducing bacteria) are great examples of natural transformation systems, causing crown gall disease and ‘hairy root syndrome’ respectively. Agrobacterium can transfer a section of its own DNA, known as the T-DNA or ‘transfer DNA’ into plant cells. For A. tumefaciens this normally occurs at wound sites and the transferred DNA causes the plant to develop crown gall disease. The story does not end there however. Along with causing a gall, the Agrobacterium harnesses the plant’s machinery to produce unique sugars called opines that the bacterium uses as a nutrient supply.

2. Common plant transformation techniques
Plant Genetic Transformation is carried out by Physical, Chemical & Biological agents. Out of these some important plant Genetic Transformation techniques are listed below.

Agrobacterium mediated gene transfer - transfer of DNA from bacteria to plants. Biolistics - rapidly propelled tungsten or gold micro projectiles coated with DNA are blasted into cells. Electroporation - Electrical impulses are used to increase membrane and cell wall permeability to DNA contained in the surrounding solution. Microinjection - Injection of DNA directly into the cell nucleus using an ultrafine needle. Poly-ethylene glycol mediated gene transfer- plant cell protoplasts treated with PEG are momentarily permeable, allowing uptake of DNA from the surrounding solution. 2.1 Agrobacterium mediated gene transfer The newest and most promising method technology for transformation of Bt corn is Agrobacterium tumefaciens mediated transformation using a bacterial plasmid as a vector for the Bt gene. This method of transformation is the most widely used to introduce foreign genes into plant cells. A. tumefaciens contains a Ti plasmid (tumor-inducing) which normally infects dicotyledonous plant cells, making the bacteria an excellent vector for the transfer of foreign DNA. By removing the tumor inducing genes and replacing them with the genes of interest, efficient transformation can occu Plant transformation mediated by the soil bacteria Agrobacterium tumefaciens has become the most used method for plant transformation. A. tumefaciens naturally infects the wound sites in dicotyledonous plant causing the formation of the crown gall tumors. Since the discovery of the bacterial origin of these

ovule and meristematic cells or a defined compartment of a cell.3 Biolistics Some cells. While direct introduction of DNA into monocots by electroporation has proven successful. the genes involved in the process of T-DNA transfer from the bacterium to the plant cell and for the bacterium-bacterium plasmid conjugative transfer genes. is a commonly used method for genetic transformation of plants and other organisms. 2. The cell membrane of the host cell is penetrable thereby allowing foreign DNA to enter the host cell.e. encoding for enzymes involved in the synthesis of auxins and cytokines and responsible for tumor formation. are located the genes for the opine catabolism. biolistics. In this method. including particle bombardment. Some of these cells will incorporate the new DNA and express the desired gene. To resolve this problem in gene transfer. especially plant cells. a product resulted from condensation between amino acids and sugars. the gene gun machine is used for the transfer of desired gene The gene gun is part of the gene transfer method called the biolistic method. and the genes encoding for the synthesis of opines. By this method. firstly with the hope to understand the mechanisms of oncogenesis in general and applied it to study of cancer disease in animals and humans. DNA-particle complex is put on the top location of target tissue in a vacuum condition and accelerated by powerful shot to the tissue.4 Microinjection Microinjection is the direct mechanical introduction of DNA under microscopical control .A target can be a defined cell within a multicellular structure such as embryo. This technique is effective with plant protoplasts and tissues. causing the crown gall disease. Uncoated metal particles could also be shot through a solution containing DNA surrounding the cell thus picking up the genetic material and proceeding into the living cells. Electroporation Electroporation is the process where cells are mixed with a DNA construct and then briefly exposed to pulses of high electrical voltage. A fine pipet is then used to insert the DNA into the cytoplasm or nucleus. then DNA will be effectively introduce into the target cells. tumefaciens as carbon and nitrogen sources. a cell is held in place with gentle suction while being manipulated with the use of a blunt capillary. 2. tumefaciens is capable to transfer a particular DNA segment (T-DNA) of the tumor-inducing (Ti) plasmid into the nucleus of infected cells where it is subsequently stable integrated into the host genome and transcribed. a large number of researches have focused on the study of this process. which are produced and excreted by the crown gall cells and consume by A. the electroporation technique is used in a relatively small proportion of cases in comparison to other methods. By examination with a microscope. The T-DNA contains two types of genes: the oncogenic genes. tissues and intracellular organelles are impermeable to foreign DNA. Outside the T-DNA. DNA or RNA adheres to biological inert particles i. Later this hypothesis was discarded and the interest on crown gall disease largely decreased until it was evident that A.neoplastic diseases. . gold or tungsten.

This bacterial species has also been used for plant transformation. tumefaciens uses leaf disks. Through selection methods. pea. Agrobacterium tumefaciens was the first vector used for introduction of foreign DNA in plant cells. The basis of this transformation method is the bacterial plasmid. and this area of DNA is key to the tumor growth in infected plants. tumefaciens with plasmids containing the transgene. The region is located between the right border and left border (RB and LB) of the plasmid. four have yielded the best results: Agrobacterium species-mediated transformation. a soil bacterium that infects some plants because of a wound on the plant. After approximately a month of incubation in the tissue culture medium. where tumors are caused by bacteria that causes uncontrolled growth on the stem of the infected plants. With the use of the restriction enzymes.mong the several methods of plant transformation. such as soybean. The plasmids are capable of integrating into the DNA of the host plant. Agrobacterium rhizogenes. two important plant hormones involved in tumor formation. Plasmids also contain other important DNA sequences. and cotton. which contains the genetic sequence that is integrated into the host genome. microinjection. tumefaciens to efficiently transfer plasmid DNA into the host has made it important in early studies in genetic transformation. tomato. Another soil bacteria. seedlings start to develop on the leaf disks. microprojectile bombardment. One of the most important parts of a plasmid is the region responsible for the translocation of its DNA into the host plant genome. causes the growth of secondary roots after infection. transgenic seedlings are identified for whole . Agrobacterium Mediated Transfer Tumors and uncontrolled cellular growth in plants can occur due to genetic factors or bacterial and viral infections. This problem is caused by Agrobacterium tumefaciens. The ability of A. a transgene can be introduced between the right border and left border of the plasmid. At this time there is no single technique that is suitable for all species. Leaf disks of about 6 mm in diameter are cultured on a tissue-culture media containing A. Plasmids present in the bacteria are responsible for tumor growth after infection by A. and this induces the transfer of the bacterial plasmid into the plant. allowing the bacteria to transfer novel genes into the recipient plant. An example is crown gall in plants. some of them control the production of auxin and cytokinin. the protocol has been modified to allow the bacteria to infect some monocot (grass) species as well. and direct transformation. Although Agrobacterium has only been used to infect dicot plant species. The bacteria are able to recognize wounds on the plant. Many research groups working with plants have found this to be the preferred transformation approach. causing uncontrolled plant growth and the formation of tumors. tumefaciens. One of the techniques used for transformation mediated by A. Each of these methods has merits and limitations and is used in specific situations. This is called transfer DNA (T-DNA).

was designated biolistic (biologic + ballistics = biolistic). This method. Each cell to be transformed must be manipulated individually. but it is retained. or DNA of the gene of interest. The assimilation rate can be increased with the addition of polyethylene glycol (PEG) or the use of an electric discharge (electroporation). on a steel mesh so that the microparticles continue in the direction of the target cells. No barrier to direct transformation has been detected. developed at Cornell University. wheat. Many of the commercial transgenic crop varieties on the market today were developed using the gene gun. microcapillary needles are used to introduce DNA directly into cells. This transformation method consists of the acceleration of a macroprojectile loaded with millions of tungsten or gold microspheres about 1 µm in diameter (microparticle). allowing them to acquire the needed momentum to penetrate the target cells. Helium gas at high pressure is used to propel the macroparticle. This is a simple method that consists of adding great amounts of transgenic plasmids to a protoplast culture. cattle. However. after a small distance. Microparticle Bombardment This technique has also been called microprojectile acceleration or biolistics. soybean. which guarantees that a small proportion of the protoplasts will be taken up (assimilated) by the plasmids. and the acceleration chamber operates under a partial vacuum. The problem with this method lies in the difficulty of regenerating a whole . Although very difficult and laborious. and in animals like salmon. and rice. because high-speed microscopic projectiles (microprojectiles) are accelerated into the cells to be transformed. indicating that this method can be used with virtually any species. In this technique. These methods use protoplasts (cells after the removal of the cellular wall) as targets for transformation. Positive results have already been obtained in several crop species such as corn. Microinjection This method was developed for animal transformation but has also been extended to plants. the DNA coating the microspheres is released and can be integrated into the plant's genome. Once inside the target cells. due to its cost and the complex integration patterns resulting from this method. which allows for improved microsphere movement. Direct Transformation Transformation using direct methods was accomplished soon after the first Agrobacterium-mediated transformation. and swine. One of the advantages of this method is that the optimum amount of DNA can be injected into the target cells.plantregeneration. DNA microinjection has yielded positive results and has been used in several laboratories. but microparticle bombardment is the formal name for the machine called a gene gun. several research groups are reducing its use. tobacco. The microspheres are coated with the transgene. The macroparticle is propelled in the direction of the cells at high speed. which helps to ensure optimal integration. Microspheres have a high specific mass.

it has not been used as widely as the other methods.plant starting from protoplasts. . Therefore.

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