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Tuesday, November 27, 2012

Science in Action: Part I. Engineering Plants Using Crown Gall Disease

Science in Action: Part I. Engineering Plants Using Crown Gall Disease
By Robert Ll. Morris

Ever since Mendel discovered that characteristics in pea plants could be inherited, scientists have been improving plants through hybridization; two related plants were crossed and the resulting offspring had characteristics of both parent plants. Breeders then selected and reproduced the offspring that had the desired traits. These conventional plant breeding techniques were relatively imprecise because they shuffled thousands of genes around and distributed them to the offspring just to get one important change in a plant that was economically worth pursuing.

One challenge encountered in Mendelian breeding is that generally only closely related species of plants could be crossed. If no closely related species with desirable traits existed, breeders had no way of passing on these traits to the other plant. Another problem was that some of the genes were linked to each other. This is seen today in tomatoes that have been bred so that they can be shipped long distances but with a substantial loss in flavor.

Since the early 1980’s scientists have been using the tools of modern biotechnology to insert a single gene, or just two or three genes, into a plant giving it new, advantageous characteristics. With this technology a single gene could be inserted into a plant giving it a desired characteristic instead of the mixing all the genes from two plants through traditional plant breeding and hoping for the best. This technique could develop a new plant with much more control and precision and at a rate much faster than ever before.

The bioengineering of plants emerged from discoveries by researchers in previous years on how bacteria caused plant tumors, how viruses protected plants from other viruses and what enabled some bacteria to kill insects. Some first major step toward biotechnology occurred early in the twentieth century with a plant disease called crown gall. Crown galls are tumor-like plant growths that occur on many woody plants including fruit trees, grape vines, and ornamentals. In 1907 researchers at the USDA discovered that the cause of crown galls was a soil bacterium, Agrobacterium tumefaciens. Other bacteria were known to cause plant diseases but A. tumefaciens had the unusual ability to cause the plant that was hosting it to grow a disfiguring tumor.

Forty years later in 1947 researchers at the Rockefeller Institute for Medical Research (now named Rockefeller University), curious about the crown gall bacterium and using it for insight into how tumors developed, grew crown gall tissue culturally free of any associated bacterium AND free of the plant host as well. They found that these uninfected crown galls could continue to grow, as crown gall tissue, independently of the crown gall bacterium and of the plant host for many years. It was concluded that normal plant growth in some unknown way had been permanently and irretrievably transformed by A. tumefaciens. Understanding how would have to wait nearly another thirty years.

During the 1950s and 1960s, scientists discovered DNA’s role in transmitting information from plant to plant and ultimately controlling plant growth. Armed with this new information, scientists began looking more closely at DNA’s role in the formation of crown gall. The crown gall mystery was attacked again when several investigators began, logically, looking for the tumor-inducing factor in the bacterium's DNA.

Bacterial DNA is relatively simple compared to other types of organisms since it can be normally found on a single chromosome. It wasn’t found there. Instead it was found by Flemish researchers on a smaller, mobile DNA unit called a plasmid that was not part of the bacterium's single chromosome. In a series of experiments at the University of Washington ending in 1977 researchers found that this bacterial plasmid was spliced into the chromosomes of plant cells when the bacteria infected the plants.

This was at the same time that researchers in other fields were just beginning to understand how to manipulate genetic information by a technique called, in lay terms, gene splicing – to cut and splice foreign DNA into the genetic code of an organism.

It became clear now that A. tumefaciens transmitted the genetic information needed to cause a plant to produce tumor-like growth through the transfer of a “packet” of information called a plasmid.  This plasmid altered the genetic makeup of the plant so that the infected cells of the plant were induced to divide continually, developing galls containing the genetic information from A. tumefaciens.

What if scientists could manipulate A. tumefaciens so that it no longer transferred the genetic information for creating crown gall but instead transferred genetic information into plants that produced desirable traits such as resistance to insects or disease?

To convert the A. tumefaciens plasmid into a beneficial plasmid (now called a vector) researchers first had to locate and then replace the tumor-inducing genes. By 1983, plant molecular biologists had developed the first plasmid vector for plants susceptible to crown gall from A. tumefaciens. The crown gall disease had changed plant breeding forever.
A tool for introducing genes into plants is useful only if scientists have found genes that they want to transfer. Enter Monsanto. In the late 1960’s researchers at Monsanto wanted to know what made the nonselective herbicide glyphosate (RoundupTM) a potent killer of so many different kinds of plants; weeds and crop plants as well. It seemed reasonable that if you could alter crop plants so that they were resistant to glyphosate, then spraying an herbicide like glyphosate “over the top” of a mixture of emerging combination of resistant crops and weeds would kill the weeds but not the crop.

Through combined research starting early in the 1970’s glyphosate’s genetic “mode of action’, destruction of an enzyme vital to all plants, was specifically identified by research performed at Monsanto and by German researchers.

In 1983 researchers at Calgene and Monsanto identified the gene, and Monsanto modified the gene, so that the enzyme it produced was no longer sensitive to glyphosate. The A. tumefaciens plasmid vector was used to introduce the modified gene into crop plants. The new tomatoes, petunias and other modified plants were resistant to damage from glyphosate which was reported in the research in1985.

Gene gun. More on this subject at
The USDA requires field testing of genetically modified plants for several seasons before its release and that testing includes potential changes in food safety, nutrient levels, development of potentially toxic substances and safety to the environment. In 1996, the first glyphosate-resistant soybean, cotton, canola, and corn seeds were made available to farmers.

During the1980s and 1990s other ways were developed to introduce beneficial genes into plants. One is called a "gene gun," which literally shoots DNA-covered particles attached to metal “bullets” through plant cell walls and membranes to the cell nucleus. Inside the nucleus the foreign DNA combines with the plant's own DNA and transforms the plant. Other techniques involve electrical or chemical treatments assisting DNA molecules to pass through plant cell walls and membranes barriers and combine with a plant’s genetic information, a high-tech form of plant breeding. Wouldn’t Mendel be impressed?

Previously published in Southwest Trees and Turf

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