The Alternatives
Contents
Methods of TransformationProtoplasts
Particle Bombardment
Tissue Electroporation
Silicon Carbide Whiskers
Direct Injection
Other Factors Affecting Transformation
There are many different approaches to plant genetic manipulation, some of which are more successful than others, and some have been superseded by more up to date methods. Two classes of plant transformation technology currently exist. These are non natural or in vitro methods, or natural methods. Non natural methods include microinjection and direct DNA uptake, whilst natural methods include technologies such as the use of viral vectors and A. tumefaciens. Some of the main methods of in vitroplant transformation are listed below.
Method of Transformation |
Species Used |
Reference |
| Agrobacterium Transformation | ||
| Wounding with Glass Beads | Sunflower | Grayburn and Vick (1995) |
| Wounding by Bombardment | Carnation | Zuker et al. (1999) |
| Floral Dip | Arabidopsis thaliana | Clough and Bent (1998) |
| Germinating seed imbibition | Arabidopsis thaliana | Feldman and Marks (1987) |
| Direct DNA Delivery | ||
| PEG Fusion | Tobacco | Krens et al. (1982) |
| Electroporation | Carrot, Tobacco, Maize | Fromm et al. (1985) |
| Microinjection | Tobacco | Crossway et al. (1986) |
| Gunpowder Charge | Onion | Klein et al. (1987) |
| Electric Discharge | Soybean, Cotton, Rice | McCabe and Christou (1993) |
| Compressed Air Gun | Maize, Rice | Oard (1993) |
| Other Methods | ||
| Tissue Electroporation | Pea | Chowrira et al. (1995) |
| Laser | Rice | Guo et al. (1995) |
| Silicon Carbide Whiskers | Chlamydomonas, Maize | Wang et al. (1995) |
| Adapted from Newell (2000). | ||
Protoplasts
Plant cell protoplasts are simply a plant cell in which the outer cellulose cell wall has been removed, to leave a cell membrane layer that is easier to work with and is more amenable to cell fusion. Several methods have been successfully used to introduce DNA into plant protoplasts, such as fusion using PEG (polyethylene glycol), electroporation and microinjection. Although this method can and does work, regeneration of plants from protoplasts is a time consuming exercise and isn't always successful, as the process is very much a 'hit and miss' affair.
Particle Bombardment
One method that avoids the host range restriction of agrobacterium and the regeneration problems when using protoplast techniques is the use of particle bombardment. The DNA is coated onto many small micro projectiles, which are then accelerated into the plant cells, usually either in culture or whole plant parts. This process is much simpler than agrobacterium mediated transformation as it avoids the complex T-DNA transfer process thus eliminating the need for DNA sequences necessary for T-strand replication and transfer. Follow the link below to the Purdue Agricultural Biotechnology Website, to see an animated demonstration of how particle bombardment can be used to genetically modify plants.
There are many different methods of particle bombardment available, with many scientific labs adapting the same basic theory to suit their own particular needs and interests. Traditionally, DNA is coated onto inert particles such as gold and tungsten, but biological projectiles such as E.coli, yeast and phage have been complexed with tungsten and used as particles with some success (Kikkert et al., 1999). The method by which particles are accelerated into the plant also differs. Some devices use compressed air, others use a magnetic field to accelerate the particles, whilst some of the earlier methods actually used a controlled explosion for propulsion. Although this method has been shown to work successfully (Luthra et al. 1995) care must to be taken to ensure that the process does not irreparably damage the plant tissue. Therefore the technology is regarded as being relatively inefficient as relatively few numbers of cells are stably transformed (Gelvin, 1998).
Tissue Electroporation
This technique uses an electrical pulse of high voltage but low amperage to create pores in the cell membrane through which DNA can pass. The cells must be in solution for this to take place, therefore no-one has actually seen the pores form so the knowledge is theoretical, but it does appear to work. This method was previously only used for protoplast transformation, but also works on whole cells, even tissues. This method has been successfully used to produce transgenic maize (D'Halluin et al., 1992) and transgenic legumes (Chowrira et al., 1995). However, the main drawback of this technology is that it depends on protoplasts being regenerated to form whole plants, a process which is difficult to carry out, and has a low success rate for many species (Gelvin, 1998).
Silicon Carbide Whiskers
These are very small hollow silicon tubes, which are agitated in a solution of the target cells and a supply of DNA. The theory is that the whiskers will pierce the plant cell, thus allowing the DNA to enter. However, care must be taken when handling the whiskers as they are very small and are very similar to asbestos fibres. Whilst not widely used, they have been proven to work by Wang & Colleagues (1995). The risk of causing tissue damage however is great, so transformation efficiency is relatively low.
Direct Injection
Direct injection of DNA has the potential to be species independent (Newell, 2000) and has been successful at producing transgenic plants. However, it is a very difficult process to carry out as it has to be done under a microscope, and the person carrying out the procedure needs to be very highly skilled with lots of experience. Therefore it is not a routinely used procedure as it involves large amounts of expenditure and effort for very limited returns (Jones-Villeneuve et al., 1995).
Other Factors Affecting the Efficiency of Plant Transformation
Selectable markers allow plants that are expressing the new foreign DNA to be selected for quickly and efficiently. One of the most common marker system used is antibiotic resistance, whereby a gene for antibiotic resistance is also inserted into the plant genome, and the resultant plant cells are cultured in the presence of the antibiotic. The most widely used system for dicotyledonous plants is NPTII (Neomycin Phosphotransferase II) and kanamycin, although there are currently doubts as to the effects these proteins may have further down the line, especially where the consumer is concerned (Fuchs et al., 1993).
Scorable markers have evolved from the early days of plant transformation to confirm whether or not transformation has really taken place. Recently, utillisation of the green fluorescent protein (GFP) from the jellyfish Aequorea Victoria has allowed visualisation of those plants expressing the new genes by excitation with light, without the need to supply any additional substrates to the plant (Sheen et al., 1995).
Many improvements have also been made to the vectors which aid the integration of DNA into the plant genome during transformation. Some examples include pBECKS, an updated version of pBIN19, development of smaller, simpler vectors and the use of yeast (YAC) and bacterial (BAC) artificial chromosomes to increase the size of DNA fragments that can be integrated. Another suggested improvement is to couple the required transgene to a gene that would render the hybrid less able to compete and cross with wild species, thus reducing the spread of the transgenes into the gene pool (Gressel, 1999).