Genetic engineering in plants

Because of their economic significance in agriculture, many plants have been the subject of genetic analysis aimed at developing improved varieties. Recombinant DNA technology has introduced a new dimension to this effort because the genome modifications made possible by this technology are almost limitless. No longer is genetic diversity achieved solely by selecting variants within a given species. DNA can now be introduced from other species of plants, animals, or even bacteria. In response to new possibilities, a sector of the public has expressed concern that the introduction of genetically modified organisms (GMOs) into the food supply may produce unanticipated health problems. The concern about GMOs is one facet of an ongoing public debate about complex public health, safety, ethical, and educational issues raised by the new genetic technologies.

THE TI PLASMID SYSTEM A vector routinely used to produce transgenic plants is the Ti plasmid, a natural plasmid derived from a soil bacterium called Agrobac-terium tumefaciens. This bacterium causes what is known as crown gall disease, in which the infected plant produces uncontrolled growths (tumors, or galls), normally at the base (crown) of the stem of the plant. The key to tumor production is a large (200-kb) circular DNA plasmid— the Ti (tumor-inducing) plasmid. When the bacterium infects a plant cell, a part of the Ti plasmid — a region called T-DNA for transfer DNA — is transferred and inserted, apparently more or less at random, into the genome of the host plant (Figure 11-28). The structure of a Ti plasmid is shown in Figure 11-29. The genes whose products catalyze this T-DNA transfer reside in a region of the Ti plasmid separate from the T-DNA region itself. The T-DNA region encodes several interesting functions that contribute to the bacterium's ability to grow and divide inside the plant cell. These functions include enzymes that contribute to the production of the tumor and other proteins that direct the synthesis of compounds called opines, which are important substrates for the bacterium's growth. One important opine is nopaline. Opines are actually synthesized by the infected plant cells, which express the opine-synthesizing genes located in the transferred T-DNA region. The opines are imported into the bacterial cells of the growing tumor and metabolized by enzymes encoded by the bacterium's opine-utilizing genes on the Ti plasmid.

The natural behavior of the Ti plasmid makes it well suited to the role of a vector for plant genetic engineering. If the DNA of interest could be spliced into the T-DNA, then the whole package would be inserted in a stable state into a plant chromosome. This system has indeed been made to work essentially in this way but with some necessary modifications. Let us examine one protocol.

Dna Agrobacterium Transfer Into Plants
Figure 11-28 Infection by Ti plasmid. In the process of causing crown gall disease, the bacterium Agrobacterium tumefaciens inserts a part of its Ti plasmid—a region called T-DNA — into a chromosome of the host plant.

Ti plasmids are too large to be easily manipulated and cannot be readily made smaller, because they contain few unique restriction sites and because much of the plasmid is necessary for either its replication or for the infection and transfer process. Therefore, a properly engineered Ti

Plasmid Vector

Figure 11-29 Simplified representation of the major regions of the Ti plasmid of A. tumefaciens. The T-DNA, when inserted into the chromosomal DNA of the host plant, directs the synthesis of nopaline, which is then utilized by the bacterium for its own purposes. T-DNA also directs the plant cell to divide in an uncontrolled manner, producing a tumor.

Figure 11-29 Simplified representation of the major regions of the Ti plasmid of A. tumefaciens. The T-DNA, when inserted into the chromosomal DNA of the host plant, directs the synthesis of nopaline, which is then utilized by the bacterium for its own purposes. T-DNA also directs the plant cell to divide in an uncontrolled manner, producing a tumor.

plasmid is created in steps. The first cloning steps take place in E. coli, using an intermediate vector considerably smaller than Ti. The intermediate vector carries the transgene into the T-DNA. This intermediate vector can then be recombined with a "disaxmed" Ti plasmid, forming a cointegrate plasmid that can be introduced into a plant cell by Agrobacterium infection and transformation. An important element on the cointegrate plasmid is a selectable marker that can be used for detecting transformed cells. Kanamycin resistance is one such marker.

As Figure 11-30 shows, bacteria containing the coin-tegrate plasmid are used to infect cut segments of plant tissue, such as punched-out leaf disks. In infected cells, any genetic material between flanking T-DNA sequences can be inserted into a plant chromosome. If the leaf disks are placed on a medium containing kanamycin, the only plant cells that will undergo cell division are those that have acquired the kanR gene engineered into the cointegrate plasmid. The transformed cells grow into a clump, or callus, that can be induced to form shoots and roots. These calli are transferred to soil, where they develop into transgenic plants (see Figure 11-30). Typically, only a single copy of the T-DNA region inserts into a given plant genome, where it segregates at meiosis like a regular Mendelian allele (Figure 11-31). The presence of the insert can be verified by screening the transgenic tissue for transgenic genetic markers or the presence of nopaline or by screening purified DNA with a T-DNA probe in a Southern hybridization.

Transgenic Plants With Foreign Genes
Figure 11-30 The generation of a transgenic plant through the growth of a cell transformed by T-DNA.

Transgenic plants carrying any one of a variety of foreign genes are in current use, including crop plants carrying genes that confer resistance to certain bacterial or fungal pests, and many more are in development. Not only are the qualities of plants themselves being manipulated, but, like microorganisms, plants are also being used as convenient "factories" to produce proteins encoded by foreign genes.

Chromosome pair in transgenic plant

T-DNA borders

Progeny

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  • serafino
    How is tdna inserted into a plant?
    8 years ago
  • lily
    How to transfer agrobacterium into plants?
    8 years ago
  • luca
    What is the role of Ti plasmid as a vector used to genetically engineer new genes in plant?
    3 years ago
  • nestore
    What is transgenic plant?
    3 years ago

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