So how do we do this? We usually do this with the help of a bacteria called agrobacterium tumefaciens. To understand how this happens, I'm gonna just do a little review of basic biology, and talk about the cell dogma. The cell dogma says that all cells have their genetic information in DNA. The DNA transfers this information to RNA. And the RNA encodes this information, which is decoded into proteins. In all organisms, the DNA is made up of four chemicals. Cytosine, Guanine, Adenine and Thymine. In all organisms, the RNA is made up of four chemicals, cytosine, guanine, adesine and uracine. And in all organisms, proteins are made up the exact same 21 amino acids. They key point is, in all organisms, DNA is DNA is DNA. RNA, is RNA, is RNA. And then amino acid, is an amino acid, is an amino acid. Actually because of this, we get proteins, that is amino acids, in our diet from many different plants or animals. Because of this, genes can be exchanged and actually are exchanged all the time between different organisms and remain active. So now let's go back to agrobacterium. Agrobacterium is both a pathogen and a useful tool. It's a pathogen, because it causes a disease in plants that's called a gall. A crown gall, very similar to a tumor. If you've seen trees or plants that have a tumor like growth on them, like in these pictures, this is caused by agrobacterium. It causes cells to grow, which forms an environment for the bacteria to live in. But it's this tumor inducing property which also makes it a wonderful tool for us. This tumor is induced by a bacteria, and this has been known for over 100 years. Actually, it was an Italian scientist named Fridiano Cavara, who was the director of the botanical garden in Sicily that found that a bacteria causes galls in grapes. At the turn of the century, a scientist of the United States Department of Agriculture found that he could isolate the bacteria from a gall of a plant, grow this bacteria on a petri dish, and then reinoculate a healthy plant. And the same gall would start to grow. But what's interesting, is that the maintained growth of the gall, the maintained growth of the tumor is not dependent on the bacteria, and we can see that in this following experiment. If you take a stem of a periwinkle, and inoculate it with agrobacterium, within four days, five days, you start seeing small galls being formed. Now let's go back and redo this experiment in several ways. One is with a day after the inoculation, you put the plant in high heat, 45 degrees. A second plant you put in high heat only after three or four days, same 45 degrees. The plant that was exposed to high heat after one day of inoculation, does not form any galls. Whilst the plant that was exposed to high heat after four or five days, does start growing the galls even though the bacteria have been killed. In other words, the biobacteria are no longer necessary several days past inoculation. The scientists at this time speculated that somehow or another the bacteria transferred what they called a factor into the host plant, which cause the gall to be formed. We need to know one other property of these galls. These galls, which are a sort of type of tumerous cancerous tissue, can grow without the addition of any chemicals. If you take a normal plant leaf or part of a plant stem, and put it on a petri dish, you can only maintain their growth by the addition of plant hormones such as auxin or cytokynin. But if you take part of the gall tissue and put it in a petri dish, it continuously grows without any addition of any plant hormones. So how does the agrobacterium transform the plant tissue, such that it can maintain growth, even in the absence of the hormones? And even in the eventual absence of the bacteria? The agrobacterium manages to do this because what it actually does, is it injects part of its own DNA into the host plant. If we look inside the agrobacteria, it has two types of DNA. It has its chromosome, which contains most of its genetic information which it needs for living. And it has another small, circular chromosome, which is called a plasmid. This plasmid, which we call the Ti plasmid, is transferred from the bacteria into the host plant cells. The bacteria injects this TI plasmid into the host plant cell. And when it goes into the host plant cell, it becomes incorporated into the chromosome of the host plant. It becomes incorporated, part of the plant, so that it an continue growing, even without the bacteria. So what is this Ti plasmid, what does it contain? The Ti plasmid contains a very small number of genes. First, it contains genes, which are called virulence genes. Which encode the factors, which enable the bacteria to literally inject the plasmid into the plant. But then it contains another set of genes, which we'll call the T-DNA and this is the part of the DNA, a part of the plasmid, which actually becomes incorporated, physically is transferred into the genome of the plant. In the agrobacterium, the T-DNA contains three types of genes. It contains genes for auxinsynthesis. That's why when you put the gall tumor on a plate you no longer need to add auxin, because these cells make their own auxin. It contains genes for cytokinin synthesis. Again, this is why cytokinin is not needed to be added to the medium if you isolate these cells. And it also contains genes which lead to the production of a chemical called opines, or nopanines, which are the food of the agrobacterium. So the agrobacterium is actually very smart. What has it done? It's hijacked the plant cells so that they will always grow and always produce the food that it needs to survive. But what scientists realize, is that we can utilize this Ti plasmid to insert any gene that we want into plants. What scientists can do, is that we can remove the genes for the auxin synthesis, remove the genes from the cytokinin synthesis, reduce the genes for the opine synthesis. And insert whatever gene we want and the same T-DNA will be transferred into the host plant. So we disarmed the bacteria, it no longer makes a gall. But on the other hand, it will transform the plant with the gene that we want. So what we do in the laboratory is we can a isolate the Ti plasmid. Literally cut out the genes that are normally in the Ti plasmid. Put in the genes that we want through recombinant DNA technology, and then transform a plant and get a plant with a new trait. This T-DNA then must contain both the gene we want and what is called a selectable marker gene.
