I described how Agrobacterium tumefaciens is able to transfer DNA into plants, and how this mechanism of DNA transfer has been manipulated to develop methods to introduce specific genes into plants. The following modifications were made to the Ti plasmid:
Agrobacterium carrying this modified Ti plasmid is then used to infect pieces of plant tissue. The T-DNA is excised from the Ti-plasmid, moved across the various barriers that I described, and then inserts into the chromosome of the host. Many details of the molecular events involved in this process are now understood. The T-DNA has now transformed a small number of cells. Transformed cells grow in the presence of the selection agent (kanamycin, or others) and fertile plants are then regenerated from these cells.
When bacteria or yeast are transformed with a plasmid, every cell that takes up the plasmid is identical. This is not true when plant cells are transformed with Agrobacterium. Each plant that is obtained from a separate transformation event should be regarded as unique with a distinct genotype.
One major difference is that the T-DNA must integrate into a chromosome, rather than replicate extra-chromosomally. The site of integration appears to be random. Because the T-DNA is integrated at different sites in different cells, each transformed cell is now unique. There are two reasons why this random integration is important.
In addition to the variation produced by transformation, the process of tissue culture - growing plant material in culture, exposing it to hormones, antibiotics, and other exogenous agents, and regenerating differentiated plants - also produces variation. This is known as somaclonal variation. Starting with a single plant, you place this in culture and produce callus - a mass of undifferentiated cells. From this you then regenerate many plants which should be genetically identical to the starting material. But the plants show variation - unusual phenotypes or forms that are unlike the original starting plant. This phenomenon is well documented, but not well understood.
So we have two major contributors to variation among transgenic plants, even though they were transformed with the same DNA and are obtained from the same original plant host:
Why is this variation between transgenic plants important to consider? Because you need to look at many transgenics before you understand the typical, average effect that the introduced gene has on the plant. If you are trying to obtain a transgenic plant variety with a novel property, you will probably need to examine and test 100 or more independent transformants to identify suitable transgenic plants for further development.
Agrobacterium has not been used successfully to transform all plant species that have been tested. Many important agronomic crops cannot be transformed using this method. There are two major reasons for the failure of Agrobacterium to be used with all species:
Since Agrobacterium transformation is not successful with some plant species, a variety of other methods have been developed. These all use physical methods to deliver DNA into plant cells.
The first method is virtually identical in principle to the methods used to transform microbes. Plant tissues can be reduced to a collectiom of individual cells that lack cell walls. This is done by treating plant tissue with a collection of enzymes that break down the cell wall polymers. These cells without any wall are called protoplasts. One of the major barriers to getting DNA into plant cells, the cell wall, has been removed in the preparation of protoplasts.
Protoplasts can then be transformed in a number of ways. In electroporation, the protoplasts are mixed with the DNA - the genes to be introduced - and are then given an electric shock. In some way this high voltage makes temporary holes or pores in the plasmamembrane which allows DNA to enter the cell. The DNA then somehow gets to the nucleus and is integrated into a chromosome. Electroporation is generally the most efficient treatment to get DNA inside plant protoplasts, but other chemical treatments, such as addition of polyethylene glycol (PEG) or calcium chloride, can also be used.
After transformation, plants must be regenerated from transformed cells. The procedures used are quite similar to those used in Agrobacterium-mediated transformation:
The goal of these various methods is to produce fertile, transgenic plants from transformed cells.
Another physical method to get DNA into protoplasts is by microinjection, using very fine needles and syringes to directly inject a solution of DNA into the nucleus of a cell. One at a time, individual protoplasts are held in place by suction from a pipette, while a needle is inserted through the cytoplasm into the nucleus. A few nanoliters (1 millionth of a milliliter) of DNA solution are injected - using a larger volume would make the cells burst. The needle is removed, hopefully the cell does not burst, and you then attempt to regenerate plants from these injected, transformed cells.
One advantage of this method is that the frequency of getting DNA into the protoplasts is very high - essentially all of the cells that are injected will get the DNA. However, the number of cells that can be transformed is limited. Other problems with this method include the level of technical sophistication that is required to carry out these experiments.
No matter which method is used to get the DNA into the plant cells, one central problem remains with all of these transformation methods that require the preparation of protoplasts: regeneration of plants from single cells. While you have all learned about the totipotency of plant cells in an early biology class, it is very difficult to achieve this with many plant species
How can plants be transformed without having to go through all of these tissue culture procedures, which are only successful with some species and also inadvertently introduce undesirable variation? These concerns were one of the reasons behind development of particle bombardment for transformation of plants.
In this method the DNA is delivered into cells on microscopic particles of gold or tungsten.
Click here to view an animation showing transformation of plants using particle bombardment.
The original method used an explosive charge (a blank cartridge) to accelerate the plastic bullet (the macroprojectile) loaded with DNA-coated microprojectiles. Because the gunpowder was found to damage the plant tissue at the end of the barrel, it has been replaced with a number of other methods. The most widely used is gas pressure, normally provided by compressed helium, but other methods have been used successfully.
The primary advantage of particle bombardment is that it has an unlimited host range. Any cell can be physically penetrated by these particles, and thereby transformed. In fact, this method is not limited to plants and has been applied to animals.
What "target tissue" can be used? Living tissue of any species is suitable, so long as plants can be obtained or regenerated from it. Plant cells growing in culture, leaf tissue such as leaf disks, and immature embryos, removed from developing seeds, have all been used. The ability to regenerate from single cells in culture is not limiting. For example, immature embryos which have been removed from a developing seed are in the process of developing into a seedling, and can continue on this path after they have been bombarded with these particles. Particle bombardment can be used to transform cells in organized morphogenic tissues, such as meristems or embryos. These have the advantage that they are already in the process of developing into differentiated tissues, and so are not dependent on tissue culture procedures to induce differentiation, organogenesis or embryogenesis.
In the last two years, it has been shown that two important crop plants, rice and maize, could be transformed with Agrobacterium after all. Successful use of Agrobacterium for transformation of maize and rice depended on improving several facets of the procedure including the choice of tissue for infection (immature embryos at a specific stage of development), the vector and strain of Agrobacterium (with a super-active T-DNA transfer mechanism), and the conditions used for culture of embryos after transformation. It is clear is that this important breakthrough will have a dramatic effect on the development of transformation systems in the future. Agrobacterium-mediated transformation would be the method of choice for most crops if this can be developed.
In summary, a number of methods have been developed for transformation of plants. These utilise either biological or physical methods to deliver DNA into plant cells. Selectable marker genes, e.g. for resistance to kanamycin, have been constructed to allow identification of transformed cells. Regeneration of plants from transformed cells is not always straightforward. Improvements in this aspect of plant biotechnology continue to be made, but some important crops can still not be transformed reliably.