The use of enzymes to cut and splice DNA, producing new gene sequences and the use of plasmids as vectors of these sequences to produce transgenic bacteria, is part of the core syllabus requirement. This higher tier material begins by developing the concept of transgenic organisms further.

It is very important throughout that students appreciate the clear distinction between the use, and efficacy' of selective breeding (which still has a very important role - see core syllabus, page 20) and of the recently developed gene manipulation technology, in producing new strains of organism and improving managed stock.
Definition
*   A transgenic organism is one which contains genetic material, usually from another species, placed there using DNA manipulation techniques.

It is recommended that teachers use one or more of the applications of the Agrobacterium system (see next Background box) to illustrate a technique of producing transgenic plants. This is one way in which the requirements of this syllabus section, to describe the process of gene transfer via a vector in the context of food production (syllabus page 29) could be met. The system is well documented in a number of GCSE texts, though there are differences in the procedures described (see below). It is immaterial which procedural variant is studied,

(The study of gene transfer in the context of insulin production using transgenic bacteria, suggested in the core syllabus (page 20) is extended by consideration of the Agrobacterium system, but would not in itself satisfy the requirement of this part of the syllabus extension.)

Information for students

The bacterium Agrobacterium (tumifaciens) is found naturally in soil. It is able to infect over 1 000 species of broad-leaved plants, entering by wounds (for example, those made by insects). It enters the cells and reproduces. The bacteria carry a T_i (tumour-inducing) plasmid. The plasmid DNA enters one of the plant's chromosomes and stimulates the production of substances which are required by the bacterium for its own growth, but which cause rapid and disorganised division of the plant cells, producing a disfiguring mass of hyperplastic tumour tissue, often known as crown gall.

The plasmid can be used to introduce new genes artificially into plants in the following way:

• Remove the plasmid from Agrobacterium and use restriction enzymes to open it and to remove the T_i genes.
  
• Cut out the desired gene from the donor organism, again using restriction enzymes.
  
• Insert the desired gene into the plasmid, using ligase enzymes to close the ring.
  
• Place the modified plasmid into a dish containing leaf discs cut from the plant to be modified. Leaf disc cells take up the modified plasmid.
  
• Place the leaf disc on sterile jelly containing nutrients and other substances. Each group of cells produces plantlets, whose cells contain the desired gene.
  
• Remove the transgenic plantlets and grow them normally.
  

(In one variant of this procedure, the tumour-inducing genes are not cut from the plasmid which is used directly to 'infect' healthy plants, to form crown galls. Groups of cells from these galls, each carrying the added gene, are then grown on into mature transgenic plants via tissue culture. )

Background

The Agrobacterium system has been used to transfer a number of single, prokaryote genes into a range of crop species, including a system which confers insecticidal properties on potato plants. Another confers upon crop plants, such as sugar beet, soya bean, tobacco and oilseed rape, resistance to a herbicide, enabling the chemical to act selectively.

Agrobacterium does not infect monocotyledonous plants and the T_i plasmid does not integrate into their genome, thus many important crop species cannot be manipulated using this system. (See next Background box).

Possible homework and/or discussion assignment

Projects are under way to enable non-leguminous crop plants to fix nitrogen by incorporation of the appropriate functional gene cluster. Students might be asked to suggest the benefits which might follow this achievement, to consider limitations and to think of possible problems which might arise.

Background

The nitrogen fixation (nif) gene cluster includes some 20-30 genes. It is unlikely that Agrobacterium can be used to transfer the entire cluster, as the amount of DNA involved exceeds the capacity of the T_i plasmid. There are particular obstacles, too, in bringing about the expression of multiple prokaryote genes in a eukaryote environment, although individual genes of the nif cluster have been transferred successfully to plants.

Alternative, even more ambitious means of transferring nitrogen fixation ability to non-legumes are currently being investigated.

Interested teachers may wish to explore the updated information in this field available at the Gene Web address on the Internet (see Other Resource Materials, page 40).