P1: SBT 9780521850063c13 CUUS128/Nicholl 978 0 521 85006 3 March 11, 2008 13:31 Chapter 13 summary Aims r To outline the range and scope of transgenic technology r To describe the methods used to produce transgenic plants and animals r To discuss the current and potential uses of transgenic organisms r To present the scientific, commercial, and ethical issues surrounding transgenic organism technology Chapter summary/learning outcomes When you have completed this chapter you will have knowledge of: r The definition of the term ‘transgenic’ r The range of methods used to generate tranegenic organisms r The uses and potential applications of transgenic plants r The uses and potential applications of transgenic animals r Issues affecting the development of transgenics to commercial success r Ethical issues surrounding the development and use of transgenic organisms Key words Transgenic, genetically modified foods, selective breeding, genetically modified organisms (GMOs), polygenic trait, calumoviruses, geminiviruses, Agrobacterium tumefaciens,crowngall disease, Ti plasmid, opines, octopine, nopaline, T-DNA, octopine synthase, tripartite (triparental) cross, cointegration, disarmed vector, binary vector, mini-Ti, ‘Frankenfoods’, ice-forming bacteria, Pseudomonas syringae, ice-minus bacteria, deliberate release, Bacillis thuringiensis, Bt plants, glyphosate, RoundupTM, TumbleweedTM, EPSP synthase, Roundup- ready, Flavr Savr, antisense technology, polygalacturonase, iron deficiency, vitamin A deficiency, micronutrient, β-carotene, Golden rice, gene protection technology, technology protection system, terminator technology, genetic use restriction technology (GURT), genetic trait control technology, traitor technology, plant-made pharmaceuticals (PMPs), pharm animal, pharming, nuclear transfer, pronuclei, ‘supermouse’, mosaic, chimaera, oncomouse, c-myc oncogene, mouse mammary tuour (MMT) virus, prostate mouse, severe combined immunodeficiency syndrome (SCIDS), Alzheimer disease, knockout mouse, knockin mouse, Mouse Knockout and Mutation Database (MKMD), tissue plasminogen activator (TPA), blood coagulation factor IX, whey acid protein (WAP), β-lactoglobulin (BLG), α-1-antitrypsin, fibrinogen, xenotransplantation, green fluorescent protein (GFP). 256 P1: SBT 9780521850063c13 CUUS128/Nicholl 978 0 521 85006 3 March 11, 2008 13:31 Chapter 13 Transgenic plants and animals The production of a transgenic organism involves altering the genome so that a permanent change is effected. This is different from somatic cell gene therapy, in which the effects of the transgene are restricted to the individual who receives the treatment. In fact, the whole point of generating a transgenic organism is to alter the germ line so that the genetic change is inherited in a stable pattern fol- lowing reproduction. This is one area of genetic engineering that has caused great public concern, and there are many complex issues sur- rounding the development and use of transgenic organisms. In addi- tion, the scientific and technical problems associated with genetic engineering in higher organisms are often difficult to overcome. This is partly due to the size and complexity of the genome, and partly due to the fact that the development of plants and animals is an extremely complex process that is still not yet fully understood at the molecular level. Despite these difficulties, methods for the gener- ation of transgenic plants and animals are now well established, and the technology has already had a major impact in a range of differ- ent disciplines. In this chapter we will consider some aspects of the development and use of transgenic organisms. 13.1 Transgenic plants All life on earth is dependent on the photosynthetic fixation of car- bon dioxide by plants. We sometimes lose sight of this fact, as most It is often easy to forget that we people are removed from the actual process of generating our food, are dependent on the and the supermarket shelves have all sorts of exotic processed foods photosynthetic reaction for our and pre-prepared meals that seem to swamp the vegetable section. foods, and plants are therefore Despite this, the generation of transgenic plants, particularly in the the most important part of our context of genetically modified foods, has produced an enormous food supply chain. public reaction to an extent that no one could have predicted. We will return to this aspect of the debate in Chapter 15. In this section, we will look at the science of transgenic plant production. 257 P1: SBT 9780521850063c13 CUUS128/Nicholl 978 0 521 85006 3 March 11, 2008 13:31 258 GENETIC ENGINEERING IN ACTION 13.1.1 Why transgenic plants? For thousands of years humans have manipulated the genetic char- acteristics of plants by selective breeding. This approach has been extremely successful and will continue to play a major part in agricul- ture. However, classical plant breeding programmes rely on being able to carry out genetic crosses between individual plants. Such plants must be sexually compatible (which usually means that they have to be closely related); thus, it has not been possible to combine genetic traits from widely differing species. The advent of genetic engineering has removed this constraint and has given the agricultural scientist a very powerful way of incorporating defined genetic changes into plants. Such changes are often aimed at improving the productivity and ‘efficiency’ of crop plants, both of which are important to help feed and clothe the increasing world population. There are many diverse areas of plant genetics, biochemistry, In agriculture, several aspects of physiology, and pathology involved in the genetic manipulation of plant growth are potential plants. Some of the prime targets for the improvement of crop plants targets for improvement, either are listed in Table 13.1. In many of these, success has already been by traditional plant breeding achieved to some extent. However, many people are concerned about methods or by gene the possible ecological effects of the release of genetically modified manipulation. organisms (GMOs) into the environment, and there is much debate about this aspect. The truth of the matter is that we simply do not know what the long-term consequences might be -- a very small alter- ation to the balance of an ecosystem, caused by a more vigorous or disease-resistant plant, might have a considerable knock-on effect over an extended time scale. There are two main requirements for the successful genetic manip- ulation of plants: (1) a method for introducing the manipulated gene into the target plant and (2) a detailed knowledge of the molecu- lar genetics of the system that is being manipulated. In many cases the latter is the limiting factor, particularly where the characteristic under study involves many genes (a polygenic trait). However, despite the problems, plant genetic manipulation is already having a consid- erable impact on agriculture. 13.1.2 Ti plasmids as vectors for plant cells Introducing cloned DNA into plant cells is now routine practice in The Agrobacterium Ti many laboratories worldwide. A number of methods can be used to plasmid-based vector is the most achieve this, including physical methods such as microinjection or commonly used system for the biolistic DNA delivery. Alternatively a biological method can be used introduction of recombinant in which the cloned DNA is incorporated into the plant by a vector. DNA into plant cells. Although plant viruses such as calumoviruses or geminiviruses may be attractive candidates for use as vectors, there are several problems with these systems. Currently the most widely used plant cell vectors are based on the Ti plasmid of Agrobacterium tumefaciens, which is a soil bacterium that is responsible for crown gall disease. The bacterium infects the plant through a wound in the stem, and a tumour of cancerous tissue develops at the crown of the plant. P1: SBT 9780521850063c13 CUUS128/Nicholl 978 0 521 85006 3 March 11, 2008 13:31 TRANSGENIC PLANTS AND ANIMALS 259 Table 13.1. Possible targets for crop plant improvement Target Benefits Resistance to: Improve productivity of crops and reduce Diseases losses due to biological agents Herbicides Insects Viruses Tolerance of: Permit growth of crops in areas that are Cold physically unsuitable at present Drought Salt Reduction of Increase efficiency of energy conversion photorespiration Nitrogen fixation Capacity to fix atmospheric nitrogen extended to a wider range of species Nutritional value Improve nutritional value of storage proteins by protein engineering Storage properties Extend shelf-life of fruits and vegetables Consumer appeal Make fruits and vegetables more appealing with respect to colour, size, shape, etc. The agent responsible for the formation of the crown gall tumour is not the bacterium itself, but a plasmid known as the Ti plasmid (Ti stands for tumour-inducing). Ti plasmids are large, with a size range in the region of 140 to 235 kb. In addition to the genes responsi- ble for tumour formation, the Ti plasmids carry genes for virulence functions and for the synthesis and utilisation of unusual amino acid derivatives known as opines. Two main types of opine are commonly found, these being octopine and nopaline, and Ti plasmids can be characterised on this basis. A map of a nopaline Ti plasmid is shown in Fig. 13.1. The region of the Ti plasmids responsible for tumour formation is known as the T-DNA. This is some 15--30 kb in size and also car- ries the gene for octopine or nopaline synthesis. On infection, the T-DNA becomes integrated into the plant cell genome and is there- fore a possible avenue for the introduction of foreign DNA into the plant genome. Integration can occur at many different sites in the plant genome, apparently at random. Nopaline T-DNA is a single seg- ment, wheras octopine DNA is arranged as two regions known as the left and right segments. The left segment is similar in structure to nopaline T-DNA, and the right is not necessary for tumour forma- tion. The structure of nopaline T-DNA is shown in Fig. 13.2. Genes for tumour morphology are designated tms (‘shooty’ tumours), tmr (‘rooty’ tumours), and tml (‘large’ tumours).