Slash-Pine Clone Created in a Lab

IN one hand, plant physiologist Ron Newton holds a culture dish of pine-tree cells that have been altered by the insertion of foreign genetic material. His other hand grips a test tube containing a pine "seedling" cloned from an unaltered cell.

Soon he hopes to bring the two processes together by first altering a cell and then cloning it into a slash pine, a commercially valuable tree species in the Southeast. A pine more resistant to fusiform rust disease and more tolerant of drought is the goal of Dr. Newton and his fellow researchers at the Texas Agricultural Experiment Station at Texas A&M University.

Timber is one of the top three cash crops in Texas, where the industry employs 20,000 people. The largest timber companies plant 133 million seedlings annually in the state, but summer drought may kill up to half of them in their first year. If they had lived to harvest, the trees would be worth $50 million.

The procedure Newton's team is attempting has been performed by other scientists on spruces, poplars, and walnut trees. But no one has succeeded with pine.

Old-fashioned cross-breeding, the technique pioneered on his garden peas by Austrian Gregor Johann Mendel, the 19th-century father of heredity research, might eventually get the same results. But that technique requires 20 years to develop a second generation of pines. Also, crossing pines may get not only the desired "good" genes but other "bad" genes as well. For precise gene selection and speed of developing a new species, researchers turn to the laboratory to splice genes into a cell's chromosomes an d then clone the cells into a complete organism.

Chromosomes are composed of deoxyribonucleic acid (DNA) molecules, which look like long twisted ladders, explains John Cairney, a molecular biologist on the project. Each rung of the ladder is a pair of nucleotides, the basic building blocks of genes. Hundreds of adjacent pairs make up an individual gene.

If each pair is like a letter of the alphabet, Dr. Cairney says, then chromosomes are long strings of text in which only 5 percent may be words or sentences. The rest, Cairney says, is meaningless "packing material."

One way to insert new genetic information into a cell's chromosomes is to transmit it via soil-borne Agrobacterium. This kind of bacterium can invade a cell and insert its own genetic material into the chromosome of the cell, which then causes the cell to make food for the bacterium. Scientists can alter the bacterium so that it doesn't harm the cell and will inject genes of their choosing into the cell's chromosomes.

Newton works with a second method: biolistics. This involves loading genetic material into a compressed gas "gun" and blasting it like buckshot onto a culture dish full of target cells.

The genetic ammunition contains several components. One gene causes the cell to turn blue in the presence of the chemical X-gluconate. Newton tests to see if the bombarded cells received the new genetic material by exposing them to X-gluconate.

Another gene makes the cell tolerate the presence of the antibiotic kanamycin. Other genes of choice can be included, such as ones that enhance resistance to drought and disease.

A YEAR ago, Newton was fortunate if 10 or 20 cells turned blue. But last month he achieved a breakthrough that increased his success at altering cells a hundredfold.

Newton had been using the "promoter" region of a gene taken from the cauliflower mosaic virus. The promoter is necessary for a gene to perform its function. Newton made a switch, pairing the "blue" gene with a promoter taken from a carrot. Now 2,000 cells per blast will turn blue.

Unfortunately, that test kills the cells. Thus, none that were altered successfully can be cloned. Newton is working to get around that problem by developing other screening methods.

Right now he is subjecting target cells to kanamycin. Cells that incorporated the transgenic materials should survive, while the others die. Newton can double- check the effectiveness of this screen by exposing some of the surviving cells to X-gluconate. If they turn blue in overwhelming numbers, then he can make clones from other batches, confident that they are indeed transgenic cells.

But that raises the question of finding the desirable genetic material in the first place. Hans van Buijtenen, a geneticist on the project, has "mapped" an ordinary slash pine's chromosomes. But it's a map without the names of places or roads.

"We really don't know anything about the architecture of the pine chromosome," Dr. van Buijtenen says. He hopes to identify the approximate location of drought-resistant and fusiform-resistant genes by comparing his map with maps from pines with those traits. Cairney could then transfer those genes to another plant for evaluation.

He's already doing that with genes from the loblolly pine that seem to be "switched on" by drought. He has inserted them into cotton and tobacco to test their usefulness. And Newton is working with them to make sure he can insert them into slash pine, illustrating the multiple-track approach the team is taking to reach its goal of improving the slash pine.

"This kind of thing takes a team effort" these days, Newton says, noting the variety of disciplines brought to bear on the project.

Once the location of the resistance genes is known, van Buijtenen will be able to tell if a seed has the desired properties. For now, he must plant it and wait five to seven years to conduct tests.

Even when an individual tree is resistant, pollination by a nonresistant tree might cause the offspring to lose that trait. That's why Newton hopes eventually to eliminate the need for seeds and clone new trees directly from the pine needles of individuals known to be resistant.

"I could then produce thousands and thousands of resistant trees," he says. But at this point, cloning will not work on cells from a mature tree.

Cairney adds that, in time, natural selection would achieve the results that the experiment station team hopes for with the slash pine. "We can wait for a million years, or we can make that change now," he says.