Author: Carolyn C. Parrott
Woodrow Wilson Biology Institute
Do you prefer your vegetables canned or frozen? Most people have a preference for one or the other as far as taste is concerned but they realize that little is sacrificed in nutrition by either form of processing vegetables for our tables. Ice, therefore, does not harm processed foods. Why then be concerned about frost formation on crops in the field? The answer lies in living tissue, which is made mostly of water. When the temperature drops to freezing, ice crystals form and the expanding water ruptures the tissues of the plant making them more susceptible to pathogens and to drying up. One has little doubt how serious this is - just look at the face of a Florida citrus grower after a killing frost. Losses can be in the millions of dollars.
Until recently, farmers simply worried about their crops, burned smudge pots, or sprayed the crops with water to minimize the damage when frost hit the fields and groves. Now, a new tool of biotechnology called gene splicing has permitted scientists to produce bacteria that prevent plants from freezing when the temperature hits the freezing mark. How could bacteria help? As the story unfolds, we discover that bacteria were actually involved in two ways in this scientific breakthrough.
The first discovery takes us back to 1961 and a botanist called Dr. Hall Hoppe who worked for the U.S. Department of Agriculture. Dr. Hoppe worked with a corn fungus and each season would grind up the infected leaves to a powder which he applied to test corn the next season to follow the pattern of the disease. When a frost unexpectedly hit the midwest that year, all of the plants dusted with this powder froze in the fields. Strange as it may seem, corn that had not been dusted did not freeze. To add to the mystery, the fungal pathogen alone did not induce freezing, even at temperatures below zero.
Plants with ice-plus P. syringae freeze at higher temperatures than plants without them.
The mystery remained unsolved until the early 70's when graduate student Stephen Lindow at the University of Wisconsin - Madison discovered a bacterium in the dried leaf powder. He found that placing the bacterium on corn leaves that did not already have it made the leaves very susceptible to freezing. Conversely, leaves without the powder did not freeze. The bacterium was Pseudomonas syringae, an organism commonly found in nature. Dr. Lindow identified the next question: How does P. syringae trigger ice to form in cold weather?
Discovering different strains of P. syringae followed. Some caused ice to form and others did not. Dr. Lindow called the ice forming strain the" ice-plus" and the non-ice forming the" ice-minus" P. syringae. It turned out that the ice-minus form was a mutant of the ice-plus form! Under conditions of joint occupancy on plants, the two strains would compete with each other, although the ice-plus bacteria never appeared in large numbers.
A knowledge of everyday physics provided the subsequent clue. When ice crystals form in the atmosphere, moisture and cold are needed but so are dust and other particles. Scientists refer to these particles that "seed" the formation of ice the nucleation centers. Dr. Lindow discovered that the ice-plus strain, the ice forming bacteria, acted as these nucleation centers! They act by having special proteins on their surfaces - proteins that the ice-minus bacteria lack. Another piece was added to the puzzle.
Dr. Lindow asked the question, "Why not use ice-minus bacteria to protect crops from freezing in the fields? It seemed logical.... but there was a catch. The mutagens that produced the ice-minus bacteria in the first place had altered other genes in the bacterium ! Refusing to let an idea die, he questioned whether the ice-plus gene and only the ice-plus gene, could be removed from the genes of the bacterium. Recombinant DNA would provide the technology for this to happen. It couldn't have come at a better time.
A technique was used in which the DNA from P. syringae was cut into pieces. This was done with biological scissors called restriction enzymes. These cut pieces of DNA were then inserted into the plasmids of the bacterium E. coli. (See diagram.). In other words, DNA from one species of bacterium (P. syringae) was inserted into the genetic material of a second bacterium (E. coli.). Since several different recombinants were made, the ice-forming gene still had to be isolated. Fortunately, the researchers found this gene quickly. Now that the gene was isolated, it was cloned so that the researchers would have multiple copies to mutate to the ice-minus, inactive form. The newly reconstructed gene was then inserted into normal P. syringae... and recombination occurred inside the bacterium! The operation was a success.
The first trial runs with the recombinant ice-minus bacteria occurred in a greenhouse under very controlled conditions and raised the hopes that the technique would be applicable to crops raised in the field. Sounds simple enough but there were some hurdles to cross. New questions arose. How does the ice-minus bacteria prevent the growth of the ice-plus variety? What happens when the environment is less controlled and the plants are subjected to the perils of wind and weather? To get permission from the EPA (Environmental Protection Agency) to run a field trial, they must be convinced that the risk from releasing a recombinant organism into society is minimal compared to the benefits gained. Some environmentalists maintained that the risks were too great and sued the government to prevent the field tests. A lesson we have learned in the past tells us that introducing new organisms can upset the ecosystem. Take the situation with the gypsy moth, accidentally introduced from Europe. These moths have no natural predators in the United States and continue to devastate the hardwood forests of the northeastern U.S. Perhaps it would be true in this case. Some feared the ice-minus bacteria might spread throughout the environment and wouldn't be able to be recalled into the laboratory.
Dr. Lindow and his colleagues countered with test results in which the recombinant bacteria were found to be harmless. They also argued that the ice-minus form would always be in the minority in nature. They also pointed out that the ice-minus bacteria would have to be reapplied annually--a definite advantage if you are worried about the life span of the recombinant. Some of the critics pointed to the normal uncertainty of nature and others, the harshest critics, thought changing any organism was too risky.
For four years the heated debate continued until Dr. Lindow and his colleagues were finally given permission to field test the recombinant, an experiment that was successful and showed no indication of the recombinant spreading to the adjacent fields. But the story doesn't end there.....there are more tests to run and more plants on which to try new traits. Ever hear of the flavor sav'r tomato? It's coming to your local grocery store soon!
Some people are still fearful of anything called genetic engineering. Just as in other situations, the more you know about a topic, the better able you will be to "cast your vote." As an educated citizen, you may someday be called upon to pass judgement that requires knowledge of biological and social issues. Read the following statements and discuss them with members of your group. IS THE STATEMENT JUSTIFIED SCIENTIFICALLY? (Refer to the article.) WHY OR WHY NOT ? Be prepared to explain your answer to the whole class.
Other uses exist for this ice-minus bacteria. Can you think of a popular winter sport that might benefit from this technology?
This activity was adapted from The Biotechnology Workbook, Prentice and Hall. All diagrams are from the same source.