The transfer of genes from plants to other types of organisms cannot occur through cross pollination. The most likely means by which this could occur is via horizontal gene transfer — the transfer of genetic material from one organism (the donor) to another organism (the recipient) which is not sexually compatible with the donor (Conner et al. 2003). Horizontal gene transfer is not an abstract theoretical process (Jain et al. 1999). There is growing evidence that horizontal gene transfer has been a principal force in the evolution of genomes, particularly in bacterial genome evolution (Ochman et al. 2000; Jain et al. 1999; Smalla et al. 2000; Stanhope et al. 2001).
Section 2.1 Nature of the gene transfer hazard
Potential hazards, with respect to the specific gene sequences, are as follows:
ACC synthase genes and related constructs:
This is unlikely to present a risk to human health or the environment, in the extremely unlikely event that it occurred, because it is highly unlikely that the genes would function or produce functioning proteins.
Antibiotic resistance genes:
Microorganisms could become resistant to the antibiotics. The consequences of this for human health and safety and the environment would depend on:
the pathogenicity of the microorganism;
the use and significance of the antibiotic in clinical and/or veterinary practice;
whether resistance to the antibiotic is already widespread in the microbial population.
The anitbiotic resistance genes occur within naturally occurring genetic elements (transposons and plasmids) that are readily transferable between bacterial species (US FDA 1998; Flavell et al. 1992; Langridge 1997; Pittard 1997). Gene transfer between bacteria via these elements, through well documented mechanisms for horizontal transfer (Doblhoff-Dier et al. 2000; Nielsen et al. 1998; Nielsen 1998), is far more likely than transfer of the same gene from GM papaya.
GUS reporter gene (uidA):
This would not present a risk to human health or the environment, in the extremely unlikely event that it occurred. The GUS gene occurs naturally in some soil bacteria and transfer from these bacteria to other bacteria is much more likely than from the GM papayas proposed for release.
CaMV 35S promoter and other regulatory sequences:
The expression of endogenous genes in the recipient microorganism could be altered. If a change in normal gene expression did occur, the hazard to the recipient microorganism and to the environment would depend on the specifics of the resultant phenotypic change.
Some of these sequences are derived from plant pathogens (cauliflower mosaic virus, figwort mosaic virus, Agrobacterium tumefaciens). The possibility was considered that the sequences might have pathogenic properties.
The possibility that the regulatory sequences could recombine with the genome of another virus infecting the plants to create a novel recombinant virus has also been considered.
The CaMV 35S promoter is already ubiquitous in the environment and in the human diet (Hodgson, 2000). This promoter and the other bacterial regulatory sequences could be transferred to other microorganisms by their native bacterial hosts.
Section 2.2 Likelihood of gene transfer from the GM papayas to microorganisms
Horizontal Gene transfer can occur between sexually incompatible organisms. Most gene transfers have been identified through analyses of gene sequences (Ochman et al. 2000; Worobey & Holmes 1999). In general, gene transfers are detected over evolutionary time scales of millions of years (Lawrence & Ochman 1998). Most gene transfers have been from virus to virus (Lai 1992), or between bacteria (Ochman et al. 2000).
In contrast, transfers of plant genes to other organisms such as bacteria, fungi or viruses is exceedingly rare (Mayo & Jolly 1991; Nielsen et al. 1998; Nielsen et al. 2000; Harper et al. 1999; Schoelz & Wintermantel 1993; Greene & Allison 1994; Pittard 1997; Aoki & Syono 1999; Worobey & Holmes 1999). The transfer of plant genes to bacteria and viruses has been observed in laboratory and glasshouse experiments (Nielsen et al. 1998; Nielsen et al. 2000; Schoelz & Wintermantel 1993; Greene & Allison 1994; Pittard 1997; Worobey & Holmes 1999). However, in all cases this was achieved only under controlled conditions with the presence of related gene sequences (homologous recombination), and using highly sensitive or powerful selection methods to detect rare gene transfer events.
Natural transformation is a mechanism by which transfer of DNA from plants to microorganisms could have occurred during evolution (Bertolla & Simonet 1999) and is the mechanism that is most likely to contribute to a horizontal gene transfer from transgenic plants to bacteria (Smalla et al. 2000). Natural transformation enables competent bacteria to generate genetic variability by taking up and integrating free DNA that is present in their surroundings. This uptake of DNA does not necessarily depend on DNA sequence, thus indicating the potential of gene transfer from divergent donor organisms (Nielsen, 1998).
Bertolla and Simonet (1999) identified several steps that would be required for natural transformation to occur:
Release of the DNA molecules from plant cells into the environment;
Protection of the free DNA from enzymatic activities;
Presence of bacterial genotypes capable of developing competence for natural transformation;
Appropriate biotic and abiotic conditions for the development of the competent stage.
Efficient adsorption of the DNA to the bacterial cell surface.
Efficient DNA uptake.
Chromosomal integration via recombination or autonomous replication of the transforming DNA.
Expression of the genes by the recipient bacterium.
Competence in bacteria is not usually constitutively expressed and bacterial cells that are transformable need to enter a physiologically regulated state of competence for the uptake of exogenous DNA (Lorenz & Wackernagel 1994). The major limiting factor for natural transformation remains the presence of competent bacteria and the development of competence (Smalla et al, 2000). Few bacteria induced to express competence in the laboratory have subsequently been shown able to express competence under natural conditions (Nielsen, 1998).
All of the steps identified by Bertolla & Simonet (1999) would need to occur simultaneously in the same place to enable gene transfer to occur via this mechanism. Barriers such as the development of competence make this scenario highly unlikely. It is yet to be demonstrated that plant-bacterium transfer occurs under natural conditions.
Several studies have demonstrated the persistence of plant DNA in the soil (Gebhard & Smalla 1999; Paget & Simonet 1994; Widmer et al. 1996; Paget & Simonet 1997; Widmer et al. 1997). Bacteria residing on the plant surface can access nutrients leaking from the leaf or exuded from the root and they often aggregate in biofilms that can facilitate cell-to-cell contact and may, thereby, possibly transfer DNA. Several studies have also demonstrated the persistence of plant DNA in the gastrointestinal tract of animals (see Section 2.3.1), in contact with the microorganisms that colonise the whole length of the gastrointestinal tract and aid in the digestive process. However, the proportion of DNA which may derive from the introduced genes of GM plants in the animal diet is extremely low (see Section 2.3.1).
Horizontal gene transfer from plants to bacteria has not been demonstrated under natural conditions (Syvanen 1999) and deliberate attempts to induce such transfers have so far failed (eg Schlüter et al. 1995; Coghlan 2000). Transfer of plant DNA to bacteria has been demonstrated only under highly artificial laboratory conditions, between homologous sequences and under conditions of selective pressure (Mercer et al. 1999; Gebhard & Smalla 1998; De Vries & Wackernagel 1998; De Vries et al. 2001) and even then only, at a very low frequency.
Using antibiotic selection to detect extremely rare events, Acinobacter sp. cells containing a defective copy of the neomycin resistance (nptII) gene (with 10 bp or 317 bp of DNA deleted) were observed to incorporate DNA from GM plants (sugarbeet, tomato, potato or oilseed rape) carrying the intact nptII gene, leading to restoration of neomycin resistance. Without the artificially introduced homology in the recipient strain, no uptake of DNA could be detected in Acinobacter sp. (Nielsen et al. 2000; De Vries et al. 2001) or in Pseudomonas stutzeri (De Vries et al. 2001).
Electrical fields and current are also known to be capable of permeabilising bacterial cell membranes under laboratory conditions, facilitating experimental transformation. Given that the environment is subjected to regular thunderstorms and lightning discharges that induce enormous electrical perturbations, the possibility of natural electro-transformation of bacteria has been investigated. Bacteria added to soil have been transformed via simulated lightning in the laboratory (Demaneche et al. 2001).
Integration of genes into the genome of recipient bacteria is known to be dependent on sequence homology between the captured DNA and that of the recipient bacteria. It seems that heterology between these sequences is the main barrier to the stable introduction of diverged DNA in bacteria (Baron et al. 1968; Rayssiguier et al. 1989; Matic et al. 1995; Vulic et al. 1997). There is a decreasing exponential relationship between recombination frequencies in enterobacteria and increasing sequence divergence of the introduced DNA (Vulic et al. 1997). Although there is a higher probability of recombination when the sequences become more similar, the risks of adverse effects resulting from such recombination is reduced because the likelihood of novel and hazardous recombinants being generated is less.
Even if transfer and establishment barriers were overcome, there are also barriers to expression of the exogenous genes. Gene promoters have to be compatible with expression in prokaryotes. Probably the single most important factor in determining whether the exogenous DNA would be integrated into bacteria is the strength of selection pressure. Prokaryotes have efficient genomes and generally do not contain extraneous sequences. If the genes are not useful to the organism then there will be no selective advantage in either integrating the genes or maintaining them in the genome.
There is a theoretical possibility of recombination between sequences that have been introduced into the genome of GM plants and the genome of viruses that infect the plants (Hodgson 2000a; Ho et al. 2000; Hodgson 2000b). Recombination between viral genomes and plant DNA has only been observed at very low levels, and only between homologous sequences under conditions of selective pressure, eg regeneration of infectious virus by complementation of a defective virus by viral sequences introduced into a GM plant genome (Greene & Allison 1994; Teycheney & Tepfer 1999).
Fungi are known to be transformable, and horizontal gene transfer from plants to plant associated fungi has been claimed. Uptake of DNA from the host plant by Plasmodiophora brassicae (Bryngelsson et al. 1988; Buhariwalla & Mithen 1995) and uptake of the hygromycin gene from a GM plant by Aspergillus niger (Hoffman et al. 1994) have been reported. However, stable integration and inheritance of the plant DNA in the genome of these fungi has not been substantiated by experimental evidence (Nielsen 1998).