Agrobacterium. A Memoir
This little memoir is not a review; the reader is directed to current authoritative Agrobacterium reviews with genetic (23) or cell biology emphasis (24). Likewise, this is not an update on recent advances in plant genetic engineering, which are the subject of a recent book (13). Rather, I invite you to join me on a foray through the story of Agrobacterium transformation of plant cells. Our journey will take us back in time about 30 years, and we will note early contributions from laboratories
... round the globe, including Belgium, the Netherlands, France, Australia, and several in the United States. The scientists in our story represented many disciplines, from traditional ones such as plant pathology, microbiology, and chemistry to younger fields such as molecular biology, plant tissue culture, and plant metabolic chemistry. Many in the course of investigating Agrobacterium found intellectual haven in the newly emerging field of plant molecular biology. Beginning at a time when bulk DNA was analyzed as a macromolecule, our story spans the birthing and growth of recombinant DNA technology. Lest the experiments we revisit seem simple when viewed from the 21st century, our first stop will be a museum of molecular biology research in the time about which I will write, circa 1970. The catalog of restriction endonucleases was unrecognizably thin. What few enzymes were available often were tainted. Kits were unknown. Procedures often did not work. We sized DNA and determined its percentage of G and C in the model E ultracentrifuge. We measured small volumes with 5-, 20-, 50-, or 100-L glass capillaries. We cultured our plant calli in jelly jars and fleakers. Instead of laminar flow hoods we worked in still air hoods. A few years later when the plasmid came into our lives, we taught ourselves how to do gel electrophoresis, and we designed and built our own gel rigs. (The one with the agarose wicks was known, of course, as the wicked gel.) We made combs from square aluminum rod, using doublestick tape to mount teeth that were pieces of glass cut with great difficulty from microscope slides. Each of us hoarded his or her own collection of glass teeth, and it was not uncommon to hear an anguished voice cry "Who took my teeth?" Research in this period presented unique challenges. The first cloning of DNA was out of sight, just over the horizon, and of course PCR was not yet conceived. With this setting in mind, then, let us turn our attention to the crown gall problem and consider what was known at the beginning of the 1970s. AN IDEA BORN BEFORE ITS TIME Dr. Armin Braun of the Rockefeller University (New York), whom many regard as the godfather of the crown gall story, first demonstrated that tumor cells are transformed, i.e. they can be freed from Agrobacteria and grown in vitro without the supplemental auxin and cytokinin required by normal plant cells in vitro (5). Braun kept tumor lines growing on hormone-free medium quite literally for decades. He reasoned that Agrobacterium must give these cells something, and he proposed that this gift must replicate because it is never lost by dilution. He proposed for it the term TIP (tumor inducing principle). Georges Morel of the Institut National Recherche Agronomique on the grounds of the Palais de Versailles in France discovered copious amounts of new metabolites-octopine and nopaline-in cultured crown gall tumor cells that were free from bacteria (18). Morel's group showed that the Agrobacterium strain, not the plant, determines the opine made by the tumor. Furthermore, each Agrobacterium strain can grow on its own particular opine but not on a different one. He thought Braun's TIP must be or include a gene responsible for opine synthesis in the plant. He proposed that a single enzyme catalyzed opine synthesis in the plant and opine breakdown in Agrobacterium, in order to account for the strain specificity of opine catabolism. We now know that part of Morel's model was not correct (the bacteria use a different enzyme for catabolism), but he was certainly on the right track about opine synthesis in tumors. However, the scientific community in 1970 was far from ready to accept the notion of a bacterial gene getting into a plant cell and functioning there. More direct evidence would be needed to support such a radical idea.