May 12, 2003

Frog Genes May Be Key To
Curbing Rhododendron Root Rot
By Sherry Fisher

frog from Africa may be just what a popular Connecticut plant needs to thrive, according to Mark Brand, a professor of plant science who specializes in ornamental horticulture. Brand is inserting the genes from an African clawed frog into rhododendrons, to create a plant that is resistant to a common soil fungus that causes root rot, Phytophthora cinnamomi.

"It's a significant disease in nurseries as well as in the landscape," says Brand, who has been conducting the transgenic research for four years. Jane Knapp, a post-doctoral fellow, is working with him on the project.

Mark Brand, professor of plant science, with a health transgenic rhododendron plant, left, that is resistant to the fungus that causes root rot, and an untreated plant that has succumbed to the fungus. Photo by Dollie Harvey

"Basically, the fungus clogs up the vascular tissue in the roots so they can't take up water or nutrients," Brand says. "The plants wilt and die."

His research has economic significance for the state, because ornamental plant production is the largest agricultural industry in Connecticut, with rhododendrons being one of the main crops.

"Several million dollars of rhododendrons are lost to the disease before they even get to garden centers," Brand says.

So why did Brand chose this slippery amphibian from across the world? Because it had the right gene.

"We looked at other organisms, like the silkworm, but thought this frog would be the most effective in killing the fungus," Brand says.

This particular frog, which lives in stagnant ponds and other bodies of water, is constantly defending itself against pathogens. The cells in its skin produce an anti-fungal protein that acts as a defense mechanism.

What's nice about this gene is that it's not harmful to plants or animals, he says.

"The frog gene makes a protein that damages the cell membranes of bacteria and fungi, leaving the cells unable to function properly," Brand says. "It only affects bacteria and fungal membranes."

Although the thought of a frog gene inserted into a plant may conjure up images of rhododendrons hopping around a yard, or leaves with bulging eyes, think again. "You won't come up with a frogadendron," Brand jokes. "You can't tell the difference between the transgenic plants and the others."

In fact, Brand never gets to see the frog. The original frog DNA was provided by a local biotechnology company. "It comes in a little vial as a DNA plasmid inside bacterial cells," he says. "If you're going to try to put DNA into some species, you need a lot of it. The E. coli is used as a factory to manufacture your DNA. You start out with a few cells of the bacteria, and overnight you have millions of them."

The gene is inserted into the plant with a blast from a gene gun.

"The original ones were based on a .22 caliber rifle," Brand says. The metal and plastic box sitting on the lab counter hardly resembles a weapon, however. It consists of a small stainless steel chamber connected to a vacuum pump.

The gene gun uses a burst of helium, traveling downward at the speed of sound, to "shoot" gold dust coated with DNA onto a petri dish containing the rhododendron plant leaves. Gold dust is used because it's inert, very dense, and tends to puncture into the cells, Brand says.

The plant leaves are induced with hormones to form shoots, which are then tested to see whether they contain the gene, and those that do are put into test tubes to take root. They are then inoculated with the fungus to see if they show resistance. Eventually, these shoots grow into ordinary-looking rhododendron plants.

Brand opens the door of a growth chamber lined with pots of plants. Some contain transgenic rhododendrons and others are ordinary. Both sets of specimens have been inoculated with the fungus that has colonized rice grains. "That's how we'll tell whether the plants are resistant or not," Brand says. Rice grains are used because they are a food for the fungus, and provide a means for adding a uniform amount of fungus to each plant.

Brand points to several plants with yellow, wilted leaves. "These both have the fungus," he says. "They're not picking up any nutrients." The plants that don't have the fungus are a healthy-looking dark green.

Brand says it will take at least five years before these transgenic plants will be introduced to the marketplace. First, he says, "we have to generate data and information that proves it's going to be safe for the environment."