The genetic manipulation of plants
At the time of writing (2006) a relatively small number of types of GM crop have been grown globally, in a limited number of countries. The take-up of these crops has been relatively high in countries like the USA and Canada, but very much lower in Europe. However, there is a very rapid increase in the growth of GM crops in developing countries.
The technique most commonly used to introduce new genetic material into dicots has involved the use of a modified soil bacterium, Agrobacterium tumefaciens. This naturally occurring bacterium contains a large Ti plasmid which consists of the genes required to facilitate the transfer of DNA to plant cells, alongside the T-DNA region, which is the region actually transferred and incorporated into the plant cell's chromosomes. Novel genes can be spliced into the T-DNA region, and the machinery of the Agrobacterium used to transfer them into plant cells. Difficulties in modifying the Agrobacterium itself mean that scientists usually create and clone a modified Ti plasmid in E. coli, and then transfer this into A. tumefaciens. Modified plant cells can be induced, under the right stimuli, to produce entire genetically modified plants.
Commercially grown GM crops currently all display either herbicide tolerance or insect resistance, or both traits combined. Bt crops are insect-resistant crops that have been genetically modified to produce the Bt protoxin, a protein toxic to certain insects. The protein is derived from another soil bacterium, Bacillus thuringiensis, and the modification involves the transfer of a single gene coding for the protoxin. Herbicide-tolerant plants have been modified to show greater tolerance for glyphosate. This herbicide acts by inhibiting a key plant enzyme, EPSP synthase, involved in the production of certain amino acids. The modification again involves the transfer of a single gene – in this case, one derived from bacteria – for a novel version of EPSP synthase which is active in the presence of glyphosate.
A GM crop currently under development is Golden Rice, which has been modified to produce β-carotene. It is suggested that this rice can play a role in alleviating vitamin A deficiency in developing countries. Golden Rice was produced by Agrobacterium-mediated transfer of several genes into immature rice embryos, which later developed into fertile plants. The process was more complex than the production of either Bt or glyphosate-tolerant crops, in that it involved the transfer of more genes. The potential of Golden Rice to alleviate vitamin A deficiency has been the subject of controversy. Those who oppose GM crops argue that it is not an appropriate or practical solution, and dispute whether the rice can provide enough vitamin A.
Genetic modification using Agrobacterium tumefaciens often involves the use of a binary vector system – using two different plasmids. (a) What are the roles of the two plasmids? (b) Which parts of the plasmids are incorporated into the plant's genome?
(a) The two plasmids are the artificial Ti plasmid and the disarmed Ti plasmid or helper vector:
- The artificial Ti plasmid carries the foreign DNA that is to be transferred, a selectable marker sequence (these are both inserted between the left and right border sequences, and form the artificial T-DNA sequence) and origins of replication (ORIs) which allow it to be replicated in both A. tumefaciens and E. coli. This plasmid incorporates the genes that are to be transferred into the plant cell.
- The disarmed Ti plasmid (helper vector) contains the virulence region and an ORI that allows replication in A. tumefaciens. The virulence region codes for the proteins that are necessary to effect transfer of the T-DNA sequence, i.e. this plasmid facilitates transfer of T-DNA into the plant cell and its integration into the nuclear genome.
(b) Only the T-DNA sequence is actually incorporated into the plant cell's genome, i.e. the foreign DNA, the selectable marker sequence, and anything else between the left and right border of the T-DNA sequence.
(a) In what way is the protein produced by Bacillus thuringiensis toxic to insects? (b) Why isn't this protein toxic to humans and farm animals?
(a) The protein produced by Bacillus thuringiensis is a protoxin – when converted into its active form it becomes incorporated into the membranes of the cells lining the insect's gut. A chain of processes is initiated that cause the cells to die. The insect can no longer absorb food or water, and quickly dies from dehydration.
(b) The protoxin is converted into the active toxin only in alkaline conditions and in the presence of specific proteases. Humans and other vertebrates lack these specific proteases. In humans, the protoxin is likely to be destroyed by other proteases and the acid conditions in the stomach.
(a) How does the herbicide glyphosate kill plants? (b) Describe two methods that have been used to attempt to genetically engineer plants to tolerate the effects of glyphosate.
(a) Glyphosate inhibits the enzyme EPSP synthase and prevents the synthesis of aromatic amino acids via the shikimic acid pathway. Without these amino acids, the plants die.
(b) The two methods attempted to genetically modify plants to tolerate glyphosate were to engineer the plants so that they produced:
- massive amounts of normal EPSP synthase.
- normal amounts of an EPSP synthase that remains active in the presence of glyphosate.
In what ways do the science and social issues surrounding Golden Rice differ from those surrounding glyphosate-tolerant and Bt crops?
Golden Rice differed scientifically in that its production was more complex – it involved the introduction of more than one new gene. The social issues surrounding it were different too. Glyphosate-tolerant and Bt crops obviously benefited large multinationals, but Golden Rice appeared to address an urgent humanitarian need -vitamin A deficiency in developing countries.
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