In the last lecture I described transformation of bacteria and yeast using plasmid vectors. These plasmids are not integrated into the chromosome(s) of the host and remain as separate, independent DNA molecules. Even in these microbes, the plasmids will be lost gradually from the population if no selection is applied (i.e. antibiotic in the medium) because in most cases there is a cost to the organism to maintain this extra piece of DNA. For most eukaryotes, extra-chromosomal plasmid DNA molecules have either not been identified or not been developed as vectors for transformation. Therefore, most of the transformation methods used for higher eukaryotes rely on integration of the DNA that is transferred into a chromosome. When the DNA is integrated into the chromosome it will be reliably transferred through mitosis and meiosis, and stably inherited.
Transformation methods can be divided into 2 groups. Some use purely physical methods to get DNA into an organism. The methods I described for E. coli and yeast, using calcium chloride or other chemical treatments to make cells competent, fall into this category. Other methods use a biological agent, such as a bacterium or virus, as the vector to transfer the DNA.
Whichever method is used, the DNA must cross a number of physical barriers for transformation to be successful.
Overhead of barriers to transformation.
For transformation of a plant, these barriers include the cell wall, plasmamembrane, contents of the cytoplasm, nuclear membrane, ultimately leading to integration into a chromosome.
All of the transformation methods for plants follow the general approach described below.
Overhead of general approach to plant transformation.
Notice that this scheme is very similar to that used with bacteria through the first three steps, until you have to regenerate a multicellular organism. Unfortunately, the methods for transforming plants are not as simple as for E. coli. I will first describe one of the biological methods used to transform plants, and the limitations of this method. In the next lecture, I will discuss the physical methods of transformation which have been developed in part to overcome these limitations of the Agrobacterium transformation methods.
As an aside, plant transformation has a murky history. In the 1970s several researchers claimed success at transforming plant cells using viruses that infect bacteria. These viruses carried genes that allowed bacteria to grow on lactose. It was claimed that the transformed plant cells were now able to grow on medium containing lactose as the carbon source. From what you now know about the regulation of gene expression in bacteria and plants, would you expect genes from a bacterial virus to function in plant cells? Genes in bacteria and their viruses use different methods to regulate transcription, so genes from bacteria will not be correctly expressed in plants. Methods to detect the physical presence of a specific gene were not available at that time. These results have now been generally discredited. In the early 1980s reliable methods of plant transformation were developed and proven to work.
The first reliable method for plant transformation was based on a pathogen that attacks plants, especially grape and olive, and causes crown gall disease - formation of galls at the crown of a plant, the junction between root and shoot at the soil surface. The organism that causes this disease is Agrobacterium tumefaciens, loosely translated from the Latin as "soil-bacterium tumor-maker". The galls are produced at the site of infection and consist of a mass of undifferentiated cells, also known as tumors. Agrobacterium produces these tumors by transferring a piece of DNA from the bacterium to the plant. This is called the T-DNA for "transferred DNA". This natural transfer of DNA from a prokaryote into a eukaryote is, as far as I am aware, unique in the world of biology.
The T-DNA carries a number of genes with promoters and other control sequences that are designed to function in plants rather than in bacteria. The genes on the T-DNA are then expressed in the plant. The T-DNA genes encode enzymes to make the plant hormones auxin and cytokinin. What happens if you alter the concentration and balance between auxin and cytokinin? Higher levels of auxin promote root growth, while cytokinin promotes shoot development. But the T-DNA genes result in very high levels of both hormones, and this leads to the proliferation of undifferentiated cells - a tumor.
The T-DNA in the bacterium is part of a large plasmid known as the tumor-inducing or Ti plasmid. Bacteria which do not have the Ti plasmid cannot produce galls and are not pathogenic.
As another aside, what does the bacterium gain from causing this disease on a plant? Is the production of tumors on plants just an act of malicious thuggery on the part of the bacterium, or is there some good biological reason for why the bacterium causes these galls? The T-DNA also carries genes encoding enzymes for the production of opines, unusual amino acid derivatives. The tumor produces opines and bacteria living in the tumor, outside the plant cells, use these compounds as their source of carbon and nitrogen. Some of the other genes carried by the Ti plasmid encode enzymes to utilise these opines in metabolism. By producing opines, the tumor provides an ecological niche for the Agrobacterium to grow.
Agrobacterium has developed this unique method to transfer DNA from the bacterium into plants. This has now been manipulated to develop systems for transformation of plants.
The T-DNA region of the Ti plasmid, the portion that is transferred into plant cells, is physically defined by specific sequences of approximately 20 base pairs on ither end of the T-DNA. Any DNA that lies between these border sequences will be transferred into the plant. The T-DNA normally contains genes for hormone synthesis and production of opines, but these genes are not required for the transfer of DNA from the bacterium. The bacterium is not aware of the sequence of DNA that is being transferred. This is important for two reasons. First, any piece of DNA can be placed between the border sequences and will be transferred form bacterium to plant. Second the genes that result in altered synthesis of hormones, and thereby production of tumors, can be removed. Therefore, it is possible to obtain transformed cells which have normal levels of hormones and appear to be quite normal.
While plant cells can be readily transformed by Agrobacterium, there must be some method to distinguish between transformed cells and those that are not transformed. The fraction of cells that are transformed is small. Without any selection, most of the regenerated plants would not be transformed and some other method would have to be used to identify transformed cells and plants. In transformation of bacteria, plasmid vectors carry genes for resistance to antibiotics. This allows transformed bacteria to be identified based, for example, on their ability to grow in the presence of ampicillin. Similarly, the T-DNA that is tranferred to plants must also carry a gene giving a selective advantage to transformed plant cells under specific conditions.
A selectable marker must be included in the T-DNA. For plants, the first selectable marker gene was for kanamycin resistance. The gene for resistance was found in bacteria. This gene encodes a protein that adds a phosphate group to kanamycin, thereby inactivating the antibiotic. Growth of plant cells is also inhibited by kanamycin. If the bacterial protein that inactivated kanamycin could be expressed in plants, then plants should be able to grow in the presence of kanamycin. To express the protein encoded by this bacterial gene in plants, the gene first had to be modified. These modifications included removing the promoter and terminator sequences, which direct the transcription of this gene in bacteria, and replacing them with a promoter from a plant gene which directs a high level of expression of this gene in all plant cells and a transcription terminator which also works in plant cells.
Overhead of making kanamycin resistance gene for plant transformation.
This chimeric resistance gene is inserted into the T-DNA of the Ti plasmid so that it will be transferred from bacterium to plant. The Ti plasmid can be further manipulated so that:
With this modified Ti plasmid in Agrobacterium tumefaciens, how do we go about producing transgenic plants? The procedure is quite straightforward:
Here is an animation that describes how Agrobacterium is used to produce transgenic plants.
This method is very widely used for plant transformation, especially many of the model systems used to study plants - tobacco, petunia, and others.
It used to be thought that Agrobacterium tumefaciens was not very efficient at infecting monocots, including wheat, rice, maize etc., and many legumes such as soybean. These are the crop plants for which there is the greatest economic incentive to develop transgenic varieties. Therefore, other methods were developed to transform these plants. All of these are based on physical methods to get DNA inside in a cell.
However, in the last two years there have been a number of reports showing that Agrobacterium can be used to transform rice and maize at high efficiency.