Nh

I"

Mannopine

FIGURE 9-27 Metabolites produced in Agrobacterium-infected plant cells. Auxins and cytokinins are plant growth hormones. The most common auxin, indoleacetate, is derived from tryptophan. Cytokinins are adenine derivatives. Opines generally are derived from amino acid precursors; at least 14 different opines are produced by enzymes encoded by the Ti plasmids of different Agrobacterium species.

This rare example of DNA transfer from a prokary-ote to a eukaryotic cell is a natural genetic engineering process—one that researchers can harness to transfer recombinant DNA (instead of T DNA) to the plant genome. A common cloning strategy employs an Agrobacterium with two different recombinant plas-mids. The first is a Ti plasmid from which the T DNA segment has been removed in the laboratory (Fig. 9-28a). The second is an Agrobacterium-E. coli shuttle vector in which the 25 bp repeats of the T DNA flank a foreign gene that the researcher wants to introduce into the plant cell, along with a selectable marker such as resistance to the antibiotic kanamycin (Fig. 9-28b). The engineered Agrobacterium is used to infect a leaf, but crown galls are not formed because the T DNA genes for the auxin, cytokinin, and opine biosynthetic enzymes are absent from both plasmids. Instead, the vir gene products from the altered Ti plasmid direct the transformation of the plant cells by the foreign gene—the gene flanked by the T DNA 25 bp repeats in the second plasmid. The transformed plant cells can be selected by growth on agar plates that contain kanamycin, and addition of growth hormones induces the formation of new plants that contain the foreign gene in every cell.

The successful transfer of recombinant DNA into plants was vividly illustrated by an experiment in which the luciferase gene from fireflies was introduced into the cells of a tobacco plant (Fig. 9-29)—a favorite plant for transformation experiments because its cells are particularly easy to transform with Agrobacterium. The potential of this technology is not limited to the production of glow-in-the-dark plants, of course. The same approach has been used to produce crop plants that are resistant to herbicides, plant viruses, and insect pests (Fig. 9-30). Potential benefits include increased yields and less need for environmentally harmful agricultural chemicals.

Biotechnology can introduce new traits into a plant much faster than traditional methods of plant breeding. A prominent example is the development of soybeans that are resistant to the general herbicide glyphosate (the active ingredient in the product RoundUp). Glyphosate breaks down rapidly in the environment (glyphosate-sensitive plants can be planted in a treated area after as little as 48 hours), and its use does not generally lead to contamination of groundwater or carryover from one year to the next. A field of glyphosate-resistant soybeans can be treated once with glyphosate during a summer growing season to eliminate essentially all weeds in the field, while leaving the soybeans unaffected (Fig. 9-31). Potential pitfalls of the technology, such as the evolution of glyphosate-resistant weeds or the escape of difficult-to-control recombinant plants, remain a concern of researchers and the public.

FIGURE 9-28 A two-plasmid strategy to create a recombinant plant.

(a) One plasmid is a modified Ti plasmid that contains the vir genes but lacks T DNA. (b) The other plasmid contains a segment of DNA that bears both a foreign gene (the gene of interest, e.g., the gene for the insecticidal protein described in Fig. 9-30) and an antibiotic-resistance element (here, kanamycin resistance), flanked by the two 25 bp repeats of T DNA that are required for transfer of the plasmid genes to the plant chromosome. This plasmid also contains the replication origin needed for propagation in Agrobacterium.

When bacteria invade at the site of a wound (the edge of the cut leaf), the vir genes on the first plasmid mediate transfer into the plant genome of the segment of the second plasmid that is flanked by the 25 bp repeats. Leaf segments are placed on an agar dish that contains both kanamycin and appropriate levels of plant growth hormones, and new plants are generated from segments with the transformed cells. Nontransformed cells are killed by the kanamycin. The foreign gene and the antibiotic-resistance element are normally transferred together, so plant cells that grow in this medium generally contain the foreign gene.

Agrobacterium cell

Ti plasmid without T DNA / (b)

Recombinant plasmid with foreign gene and kanamycin-resistance

Diabetes 2

Diabetes 2

Diabetes is a disease that affects the way your body uses food. Normally, your body converts sugars, starches and other foods into a form of sugar called glucose. Your body uses glucose for fuel. The cells receive the glucose through the bloodstream. They then use insulin a hormone made by the pancreas to absorb the glucose, convert it into energy, and either use it or store it for later use. Learn more...

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