IN THE NOT SO distant future, winter wheat may be able to sustain extended cold temperatures without crop damage — or tomatoes may be able to maintain their quality when stored in the refrigerator.

Molecular geneticists with the Ohio Agricultural Research and Development Center (OARDC) are using a combination of molecular genetic, genomic, bioinformatic and biotechnology techniques to compare genes between cold-tolerant and chilling-sensitive plants in order to understand how plants adapt to varying levels of cold temperatures.

"For each plant, it's quite different in how we might hope to improve low-temperature tolerance," said Eric Stockinger, an OARDC researcher and head of the project. "In wheat, for example, tremendous losses can occur in an extremely cold winter, particularly if there is no snow cover to protect the plants. Some other plants, like the chilling-sensitive tomato, are damaged just by placing them in the refrigerator. By understanding the genes that are turned on at low temperatures and how those genes respond in cold-sensitive crops, we might be able to prevent the economic losses that occur in wheat or might actually end up with tomatoes that are not affected by putting them in the refrigerator."

For the researchers, the road to a more cold-tolerant crop begins with the search to find the genes that protect cold-tolerant plants against freezing temperatures. Stockinger and colleagues have extracted DNA from cold-tolerant varieties of barley, alfalfa, potato and soybean. They are comparing them to similar genes from cold-sensitive counterpart plants by creating genomic libraries — a procedure whereby the DNA is spliced into segments and replicated, and the genes responsible for cold-tolerance are sought.

"Researchers discovered in the mid-1990s that a group of proteins, known as CBFs (C-repeat binding factors), was an important regulator that induced a pathway of genes to turn on and protect a plant against cold damage," said Stockinger. "We are focusing on this group, because we think it might be a critical player in the difference between a chilling-sensitive and a cold-tolerant plant."

Once the CBF gene is identified, the ultimate test to determine whether it alone can confer enhanced cold-tolerance is to then insert it into taxonomically related plants using a bacterial vector — that is, a bacterium that can carry a gene and transfer it into the DNA of a plant. The cold-tolerant gene of wild potato will be inserted into cultivated potato and tomato, the gene of winter barley will be inserted into spring barley and rice, the gene of cold-tolerant wild soybean will be inserted into cultivated soybean, and the gene from Siberian alfalfa will be inserted into related species of cold-sensitive alfalfa.

"Soon we'll have a picture that describes the structure of these genes in crop plants, and we hope to learn more about how these genes are regulated in response to low temperature through other types of studies," said Stockinger.

The research is supported by the OARDC Research Enhancement Competitive Grants program and a five-year, $1.4-million grant from the National Science Foundation Plant Genome Project.

OARDC research associate Kip Gardner prepares an assay to test the genetic material of various plants in the cold-tolerance study.	Graduate student Zhibn Wang prepares cloned CBF constructs for long-term storage. The material will be stored at -80°C to be used for future research.

Assaying RNA gel for quality is a key step in testing genetic material for the cold-tolerance gene. "RNA is extremely labile. We have to know that we recovered RNA molecules before we proceed with the research," said Stockinger.

Plant material is shown above being prepared for genetic testing. Through a series of laboratory tests, RNA molecules will separate from the rest of the plant material (represented by the darker substrate at the bottom of the test tubes). The RNA is then tested for the cold-tolerance gene, also known as the CBF gene.