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Ice-minus Bacteria ( P. Syringae) - Frost-fighting Superman?

BY: Lakshmi K Sugavanam | Category: Applications | Submitted: 2011-04-12 18:50:15
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Article Summary: "Recombinant DNA techniques have allowed the creating and introduction of Pseudomonas syringae mutants that help avoid crop losses due to frost damage..."


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The nature of agriculture and crop development is such that the weather plays a very important role in the success or failure of a season's harvest. In regions that have very cold climates, frost can be a hindrance to a successful crop. It has been estimated that frost alone accounts for crop losses of about approximately $1 billion in the United States. Pseudomonas syringae, a commonly occurring bacterium, is present on leaves and other plant surfaces. It has a peculiar ice-nucleating character which allows the formation of frost which, in turn, damages the plant. It achieves this by the production of an ice nucleation active protein or ice-plus protein. It is generally found to be produced on the outer cell wall of P. syringae, where it acts as ice nucleating centers. Since it allows ice to be formed, it has derived the name 'ice-plus' P. syringae.

A variant of the Pseudomonas syringae is known as ice-minus bacteria. This variant is cannot produce the protein that is produced by P. syringae as it is a mutant that does not have the gene for ice-nucleation. Hence, it does not allow ice formation and has been named 'ice-minus' bacteria.

Both ice-plus and ice-minus strains can be found in nature. However, genetic engineering has allowed the gene to be altered in an ice-plus strain so that the protein is not produced. This makes the environment unfavorable for the formation of ice. It was hypothesized that introducing ice-minus strain in plants that are infected by ice-plus strains, will induce competition between them. Irrespective of which strain is able to establish itself on the plant, the effect of the ice-plus strain will be greatly reduced, thus improving crop yield.

Hoppe was among the first to show that bacteria were related to frost damage. His study involved applying ground infected corn leaves to healthy corn plants. A sudden frost led to a serendipitous discovery- the plants that were treated with the infected powder were the only ones to suffer from frost damage; the rest were healthy. But, the reason behind this occurrence was not explored in detail. Later, in the early 1970s, Arny and Upper found the bacterium responsible for the frost. Lindow identified it and discovered that the plants exposed to these bacteria suffered frost damage. He also discovered the ice nucleation property and later on, developed the mutant ice-minus strain by genetic engineering.

It was in the year 1987 that the effectiveness of P. syringae in frost damge prevention was field tested. A strawberry field was sprayed with the ice minus strain before frost and the results obtained were almost successful ( a part of the field was burnt down by environmental activists). However, Lindow's experiment on a potato crop was a success. The ice minus strain on P. syringae prevented the frost damage of the potato crop.

The genetic engineering of the P. syringae ice minus strain involves the following steps- The ice nucleating gene is first isolated and amplified. It is then deactivated and introduced back in to the bacteria. The detailed process followed involves the isolation of P. syringae DNA followed by its restriction digestion with restriction endonucleases. Then, random insertion of the DNA fragments is carried out where the fragments may insert at different places in the plasmid, allowing for many variants to be created.

These recombinant plasmids are inserted into E. coli thus transforming them. Then, of these transformed E. coli, the recombinant strains containing the ice nucleating gene are identified. These will possess the property of ice-nucleation and thus be ice-plus phenotype. Then, the ice nucleating gene is identified and amplified by polymerase chain reaction. Then, DNA is subjected to mutagenic agents like UV light to create mutant of the ice-plus gene, thus creating the ice-minus mutant. This gene is then inserted into a plasmid and transformed into E. coli. The recombinant mutant ice-minus strains are identified and the gene is inserted into an ice-plus P. syringae, which creates the mutant ice-minus strain that can be introduced on crops.

Genetic engineering of P. syringae is feared to have a negative effect on the climate. It has been shown that the ice nucleation proteins may have some effect on the formation of ice crystals in clouds. The introduction of genetically modified ice-minus strain may affect the process of rain formation.

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