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Chapter: Environmental Biotechnology: Genetic Manipulation

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Examples of developments in plant GE(Genetic Engineering)

The purpose of these examples is to illustrate the potential plant genetic engi-neering could bring to future practical applications in the field of environmental biotechnology.

Examples of developments in plant GE

The purpose of these examples is to illustrate the potential plant genetic engi-neering could bring to future practical applications in the field of environmental biotechnology. In some cases the intention is to reduce the amount of herbicide and pesticides, or other agricultural chemicals required to produce a given crop yield, in others it is to improve tolerance of harsh conditions or to protect the plants from attack thus reducing wastage. The intention is to note the technical details here, while the effects such developments may have on the environment as a whole, feature elsewhere throughout this book.

Broad range protection

A general strategy to protect plants from various viruses, fungi and oxidative damage by a range of agents, has been proposed using tobacco plants as a model. The transgenics express the iron-binding protein, ferritin, in their cells which appears to afford them far-ranging protection (Deak´ et al. 1999).

Resistance to herbicides

‘Glyphosate’, one of the most widely used herbicides, is an analogue of phos-phoenol pyruvate and shows herbicidal activity because it inhibits the enzyme 5-enolpyruvylshikimate-3-phosphate synthase. The gene coding for this enzymehas been identified, isolated and inserted into a number of plants includingpetunias. In this case, the gene was expressed behind a CaMV promoter and introduced using A. tumefaciens, leading to very high levels of enzyme expres-sion. As a consequence, the recombinant plants showed significant resistance to the effects of glyphosate (Shah et al. 1986). Developments in this strategy include the formation of a chimaericsynthase enzyme, the analysis of which should lead to improved herbicide resistance in transgenic crops using this strategy (He 2001).

 An alternative approach but still using A. tumefaciens has been to transfer the genes for mammalian cytochrome P450 monooxygenases, known to be involved in the detoxification (and activation) of many xenobiotics including pesticides, into tobacco plants. These transgenics displayed resistance to two herbicides, chlortoluron andchlorsulphuron (Yordanova, Gorinova and Atanassov 2001).

Improved resistance to pests

Plants have an inbuilt defence mechanism protecting them from attack by insects but the damage caused by the pests may still be sufficient to reduce the com-mercial potential of the crop. The usual procedure is to spray the crop with insecticides but in an effort to reduce the amount of chemical insecticides being used, plants are being engineered to have an increased self-defence against pests. Attack by insects not only causes damage to the plant but also provides a route for bacterial or fungal infection in addition to the role played in the spread of plant viruses. With a view to increasing resistance to sustained attack, the genes coding for the δ-endotoxin of the bacterium,Bacillus thuringiensis (Bt) have been transferred into plants. Examples are of synthetic B. thuringiensisδ-endotoxin genes transferred, in the first case, by A. tumefaciensinto Chinese cabbage (Cho et al. 2001) and in the second, bybiolistic bombardment into maize (Koziel et al. 1993). In both cases, the trans-genic plants showed greatly improved resistance to pest infestation. There are, however, some problems with crop performance of some genetically engineered plants highlighted in Magg et al. (2001). Insects are able to develop resistance to Bt products which is a problem addressed by insertion of δ-endotoxin genes into the chloroplast genome rather than into that of the plant’s nucleus, with promising early results (Kota et al. 1999).

 It may be recalled, that for each amino acid incorporated into a protein there is usually a choice of three or four codons all of which code for that same amino acid. Different organisms have distinct preferences for a particular codon, thus Bacillus thuringiensis tends to use codons richer in thymidine and adenine thanthe plant cells into which the gene is placed. There are also signals controlling the expression of these genes relevant to bacteria, rather than eukaryotes, which will not function very well, if at all, in the plant cell. For these reasons, expression may benefit from modification of the DNA sequence to compensate for these differences while maintaining the information and instructions. This may account in part for the very high levels of expression and stability of the Bt proteins whose genes have been introduced, by (biolistic) microbombardment, into chloroplasts(De Cosa et al. 2001) which, because of their prokaryotic ancestry, have ‘protein synthesising machinery’ more in keeping with prokaryotes than the eukaryotic cell in which they cohabit.

 Attempts to improve virus resistance have led to the introduction, by A. tumefaciens, of the genes expressing antibodies to the coat protein of TobaccoMosaic Virus (TMV). Expression of these in the plant led to complete immunity against TMV (Bajrovic et al. 2001).

Improved resistance to disease

Bacteria communicate with each other by way of small diffusible molecules such as the N-acylhomoserine lactones (AHLs) of Gram negative organisms. In this way, described as ‘quorum sensing’, they are able to detect when a critical minimum number of organisms is present, before reacting. These responses are diverse and include the exchange of plasmids and production of antibiotics and other biologically active molecules. Plants are susceptible to bacterial pathogens such as Erwinia carotovora, which produces enzymes capable of degrading its cell walls. The synthesis of these enzymes is under the control of AHLs and so they are made only once the appropriate threshold level of this chemical has been reached. The rationale behind using AHLs for plant protection is to make transgenic plants, tobacco in this case, which express this signal themselves. The consequent high level of AHL presented to the pathogenic bacteria, wrongly indicates a very high number of similar organisms in the vicinity, and triggers the bacteria into responding. As a consequence, they produce enzymes able to degrade the plant cell walls and continue infection. The plant will mount its normal response to invasion but on a far greater scale than necessary to destroy the few bacteria actually causing the infection, thus improving the plant’s resis-tance to the disease. It seems complicated, but research into the validity of the hypothesis is under way (Fray et al. 1999).

Improved tolerance

Plant – microbe interactions are addressed. Among the examples given are that of Pseudomonas syringae which colonises the surface of leaves. This example is of bacterial rather than plant modification but impinges on interaction between the two. Pseudomonas syringae produces a protein which promotes the formation of ice crystals just below 0 ◦C thus increasing the risk of frost damage. Lindow et al. (1989) have identified and isolated the gene for this protein. They transferred it to the bacterium Eschericia coli to simplify the genetic manipulations. This required the deletion of sufficient regions so that a truncated, and therefore nonfunctional, ice mediating protein was expressed. They reintroduced this mutated gene into Pseudomonas syringae and selected for ice− mutants which were no longer able to produce the ice nucleating protein. Many such regimes fail in practice because it is difficult to maintain a population ofmutant bacteria in a community dominated by the wild type as, frequently, the latter will soon outnumber the mutant by competition for nutrients, since it is usually better adapted to the particular environment than the mutant. However, in this case, due to massive application of Pseudomonas syringae ice− to straw-berry plants, the mutants were able to compete with the wild type and protect this particularly susceptible crop against frost damage.

 Salt tolerance in tomatoes has been established by introducing genes involved in Na+/H+ antiport, the transport of sodium and hydrogen ions in opposite direc-tions across a membrane. The quality of the fruit was maintained by virtue of the fact that the sodium accumulation caused by the antiport occurred in leaves only and not in the fruit (Zhang and Blumwald 2001).

 Improved tolerance to drought, salt and freezing in Arabidopsis has been achieved by overexpressing a protein which induces the stress response genes. However, if too much of this factor is produced, which was the case when the 35S CaMV was employed, severe growth retardation was observed. No such problem existed when instead, the overexpression was under the control of a promoter which was only switched on when stressful conditions existed (Kasuga et al. 1999).

Improved plants for phytoremediation

Genetic modification of a poplar to enable mercury to be removed from the soil and converted to a form able to be released to the atmosphere. This process is termed ‘phytovolatilisation’ (Rugh et al. 1998). The modification required a gene to be constructed, styled on the bacterial mer A gene, by making a copy reflecting the codon bias found in plants using PCR technique. The mer A gene is one of a cluster of genes involved in bacterial detoxification of mercury, and is the one coding for the enzyme,mercuric ion reductase, which converts mercury from an ionic to a volatile form. Initially the constructed merAgene was expressed in Arabidopsis thalia (rape) where resistance to mercurywas observed, and in this study, the gene was transferred by microprojectile bombardment (‘gene guns’) to poplar tree (Liriodendrn tulipifera)embryogenic material. When the resulting yellow poplar plantlets were allowed to develop, they were found to exhibit tolerance to mercury and to volatalise it at 10 times the rate observed in untransformed yellow poplar plantlets. This study demonstrated the possibility that trees can be modified to become useful tools in the detoxification of soil contaminated with mercury. These studies were pursued in Arabidopsisthaliawhere it was observed that successful remediation also required the mer B genes coding for a lyase (Bizily, Rugh and Meagher 2000).

 A bacterial gene encoding pentaerythritol tetranitrate reductase, an enzyme involved in the degradation of explosives, has been transferred into tobacco plants. The transgenics have been shown to express the correct enzyme and trials are under way to determine their ability to degrade TNT (French et al. 1999).

 Developments in the use of transgenic plants for bioremediation have been reviewed (Francova et al. 2001).

New products from plants

The rape plant, Arabidopsis thalia has become a popular choice for the production of recombinant species. One such recombinant is a rape plant, the fatty acid com-position in the seed of which has been modified. It now produces triacylglycerols containing elevated levels of trierucinic acid suitable for use in the polymer indus-try (Brough et al. 1996) and, in a separate project, polyhydroxybutyrate suitable for the production of biodegradable plastics (Hanley, Slabas and Elborough 2000). Synthesis of the copolymer poly(3-hydroxybutyrate – co – 3-hydroxyvalerate) by Arabidopsis, is another example of the application of Agrobacterium tume-faciens technology and the use of the 35S promoter from Cauliflower MosaicVirus (Slater et al. 1999). This copolymer can be produced by bacterial fer-mentation, but due to cost considerations, it is normally synthesised chemically.

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