BIOSAFETY IN A GLOBALIZED WORLD
Hans Hoenicka

Human beings and their activities have increased the changes occurring in the environment. Until recently the arrival of new organisms into geographically isolated environments was a rare event. Modern means of transportation have broken old geographic boundaries. Non-native organisms, when introduced to a new area, can out-compete native species, cause disease to, or destabilize and occasionally ultimately wipe out native species. Invasive species constitute a threat for biodiversity at the local and regional levels, since the spread of newcomers can alter the richness and abundance of the flora and fauna of the original ecosystem. As a result, native species are abruptly faced with new species and new environments with which they have had no evolutionary history.

Some species have developed the capacity to adapt to new sites and to displace original populations. This phenomenon has been widely recorded and it is known as "biological invasion." Many non-native plants have allowed increasing productivity in agriculture, becoming a fundamental part of human economy, whereas others have developed into serious ecological problems. However, besides (a) non-native plants, potential biosafety risk factors comprise two additional groups: (b) taxa resulting from traditional breeding, and (c) genetically modified plants (GMPs).

The term "biosafety," defined as the prevention of large-scale loss of biological diversity and integrity due to human activities¹, has been often reduced to potential risks due to GMPs. However, non-native plants and pathogens, taxa resulting from traditional breeding and also GMPs are important issues for the biosafety sector.

Non-native plants and plant pathogens
The ecological impact of introducing non-native plants, insects, and microorganisms has been dramatic for some plant species. However, only a low proportion of plants introduced into a new environment becomes invasive. It has been found that about 62 percent of the crops that had come under APHIS (Animal and Plant Health Inspection Service, US Department of Agriculture) oversight were not persistent in native environments and thus could be considered non-invasive. Another 21 percent persist for a few generations in native environments but eventually disappear. These have slightly higher native fitness than the non-persistent crops, but they can still be considered non-invasive, because they do not spread outside the agro-ecosystem. About 17 percent fall into the persistent category; these can be ranked as invasive, because they readily reproduce outside the agro-ecosystem and spread.

The investigation program SCOPE (Scientific Committee on Problems of the Environment, 1982) studied the ecology of biological invasions worldwide for ten years. SCOPE results indicate that invasiveness of a plant species into a new ecological system is a very complex and often barely predictable phenomenon.

Taxa resulting from traditional breeding
Natural and induced mutations play an important role in traditional plant breeding. Many different abiotic factors such as ionizing and UV radiation, various chemicals, or even changes in sodium chloride concentration and light intensity are known to destabilize the plant genome. Environmental conditions and many stress factors may promote polyploidy and the activation of retrotransposons and other mobile elements². Hybridization is also a frequent and important component of plant evolution and speciation. More than 70% of plant species may be descended from hybrids. However, hybridization and mutations can catalyze the evolution of invasiveness. Concerns regarding uncontrolled "Vertical Gene Transfer" (VGT), i.e., gene flow between organisms that are sexually compatible (like non-native and bred species), have prompted the development of genetic conservation programs in many countries.

Genetically modified plants
Introduced non-native plant species or new taxa resulting from traditional breeding have never been as important to the public opinion as GMPs. Despite promising aspects of genetic transformation, this technology has been the subject of considerable controversy, with concerns raised mainly from ecological and ethical arguments. Amongst the potential biosafety threats argued against GMPs are:

Fitness of a plant species might be changed in an unpredictable way by genetic modification. The potential impact of individual transgenes might be estimated by evaluating their phenotypic effect³. Genes improving stress tolerance to detrimental biotic or abiotic factors fall into a group whose incorporation into natural populations could increase fitness. Transgenes already deployed that fall into this category are, for example, Bt toxin genes for insect resistance or those conferring tolerance against drought, salinity, or high temperature.

Transgene flow from GMPs to their wild relatives is one of the major concerns in relation to the commercial release of transgenic plants. Most transgenic crops commercialized thus far, such as corn and soybean in the United States, have no wild relatives growing nearby so hybridization does not occur. However, there are still concerns about transgenic crops being hybridized with crops that are grown in organic farming, or in regions where transgenic plants have wild relatives. The development of transgene containment strategies, using sterility or apomixis genes, might reduce or even avoid VGT of transgenes into non-transgenic relatives.

Gene transfer between organisms that are sexually non-compatible is called "Horizontal Gene Transfer" (HGT). The list of potentially naturally occurring HGT vectors include: transposable elements, plasmids, viruses, bacteria, protozoans, nematodes, fungi, and insects. To gain a perspective when assessing the potential environmental impact of DNA from GMPs, it is important to consider the amount of DNA in the environment from non-GM origins. The impact of free DNA of transgenic origin is negligible when compared with the total amount of free DNA available in the environment. Pollen, leaves, and fruit alone result in thousands of tons of DNA per year being released into the environment, in addition to the contribution from decaying plant and animal matter and release from microorganisms. DNA sequences from plants, animals, bacteria, and viruses have been present in human and animal feed throughout history. Therefore, most sequences found in GMPs will have entered the mammalian gut before present time.

Many papers on annual crops have shown that expression of transgenes is less stable than had originally been thought. Most of these events fall into the class of homology-dependent gene silencing, which involves mechanisms that function at the level of transgene transcription or post-transcriptionally. The stability of transgene expression has a decisive influence on the efficiency of strategies for biological confinement of transgenic plants. However, gene instability is not an exclusive phenomenon of transgenic plants. Adverse abiotic and biotic stresses have been shown to induce genome instability in non-transgenic plants.

– Promoters
The 35S promoter of the Cauliflower Mosaic Virus (CaMV) is currently widely used in plant transformation. A study supports that there is a ‘recombination hotspot’ in this promoter⁵. The CaMV 35S promoter seems to be prone to recombination during the biolistic transformation process. Some NGOs (Non-Governmental Organizations) that are opposed to biotechnology argue that the CaMV 35S might be also prone to recombine with other DNA sequences in the host genome, including dormant viral DNA, as well as with other viruses in the host cell. This opinion has been criticized because pararetroviral sequences, such as the CaMV 35S promoter, are not exotic to plant genomes. Many plants contain already integrated pararetroviral sequences and related retrotransposons⁶. The 35S promoter has been proposed to represent a lower biosafety risk factor than viruses, endogene retrovirus/pararetrovirus, and retrotransposons present in all plants. Indeed, in some plants such elements constitute up to 90% of the genome.

– Antibiotic resistance
Due to concerns from some environmental groups, scientists, and NGOs, antibiotic resistance genes in transgenic plants came to the attention of politicians and the public because antibiotic resistance genes might be transmitted to bacteria in nature. The FDA (Food and Drug Administration, USA) suggested ranking the antibiotic resistance genes in transgenic plants with the kanamycin resistance gene on one end as the most acceptable, and the vancomycin resistance gene on the other. Genes conferring resistance to antibiotics important in human medicine, like amikacin and tetracyclines, should not be present in GMPs at all.

Concluding remarks
The complicated process of invasion ecology and the high number of invasive non-native organisms integrated into our ecosystems are certainly the most important reasons why these processes have been ignored in most countries, when compared with GMPs. The effective collapse of world ecological barriers is a phenomenon, as far as we know, without precedent in the entire history of life. The introduction of non-native and traditional bred plants is changing the "gene pool" of many important plant species. Geographical isolation had avoided hybridization in many of these species for thousands of years.

GMPs, the new biosafety risk group, are convulsing public opinion more than any other biosafety risk group before. Important concerns regarding GMPs, like horizontal and vertical gene transfer, invasive potential, gene instability, and impact on other organisms, are not an exclusive problem of these plants. Novel risks introduced by new technologies are presumed greater than established ones, even if the latter are less well characterized. Perhaps the most difficult hurdle facing the advance of genetic modification is not a technical but a psychological one. The public has witnessed the consequences of deficient risk assessments in the past, e.g., waste disposals, air pollution, mad cow disease, and many others. The experience has resulted in a lack of confidence in, and mistrust of political institutions, corporations, and scientists as sources of reliable information.

The application of any new technology, not only genetic modification, should occur after a meticulous safety assessment. Biosafety problems detected in the past show clearly the importance for avoidance of risks to the environment and human health of a prior case-by-case evaluation, before their release and according to the precautionary principle, of non-native species, new taxa, and also genetically modified plants.

The importance of independent and objective biosafety research has been often pointed out. Biosafety research from private companies and the increasing cooperation between transnational corporations and public research institutions have raised ethical questions related to the integrity of research and the objectivity of scientists. Restrictions applied to public sector biosafety research by some governments are additional burden for an independent biosafety research.

Scientific uncertainties and the relative lack of information on genome and ecology dynamics will pose challenges to the existing methods of analyzing risks and benefits. Biosafety research will probably never be able to detect all possible genome instabilities in GMPs, just as it will not be possible for wild or traditionally bred plants. The development of efficient biosafety standards may reduce impacts in the environment caused by human beings in the future, but it will not completely eliminate potential risks. Biosafety represents a profound and global potential challenge to our economic system, to our technical conservation skills, and to our ethics.

Owing to the predominant role of cereal crops in the human diet, food security in the future cannot be achieved without major increases in cereal production. Therefore, in recent years, genetic transformation of cereals has become an important tool for cultivar improvement with desirable traits. Despite tremendous successes in genetic transformation of cereal crops, one of the major technical challenges facing cereal transformation is the development of methods to produce a high proportion of plants routinely showing stable and precise transgene expression without collateral genetic damage.

Agrobacterium-based systems and direct gene transfer via microprojectile bombardment have both been successfully used in genetic transformation of cereals. Although the method of introducing DNA into cells by microprojectile bombardment has revolutionized the field of genetic transformation of crop plants, a major drawback of this system is the considerable variation seen in stability, integration, and expression of the introduced transgene. The Agrobacterium-mediated transformation system, on the other hand, facilitates the precise integration of a small number of gene copies into the plant genome and shows a greater degree of stability for the transgene. Although the delivery of foreign genes to rice plants via Agrobacterium tumefaciens has now become a routine technique, there are still serious handicaps with Agrobacterium-mediated transformation of other major cereals. Several factors influencing Agrobacterium-mediated transformation of cereals have been investigated and elucidated (reviewed by Cheng et al. 2004, Shrawat and Lörz, 2006). Recently, the identification and molecular characterization of the plant genes involved for successful Agrobacterium-mediated transformation have opened up new avenues for better understanding of the plant response to Agrobacterium infection (Veena et al. 2003). Such information may help to develop methods to enhance the transformation frequency of economically important plant species, including cereals.

Agrobacterium-mediated transformation of cereals
In 1994 in a landmark report, Hiei et al. (1994) provided unequivocal evidence for stable transformation of Japonica rice with Agrobacterium after molecular and genetic analysis of large numbers of R₀, R₁ and R₂ progeny. This report opened the possibility of using Agrobacterium for genetic transformation of recalcitrant cereals plants. A superbinary vector in the Agrobacterium strain LBA4404 was demonstrated to be the most effective for transformation of all three japonica cultivars tested. Agrobacterium-mediated transformation of rice has now emerged as a reliable and highly reproducible method for transferring genes of interest into the rice genome.

The success of Hiei and colleagues ignited a significant interest in transforming other agronomically important crop species, such as maize, barley, and wheat. Using an approach similar to the one developed by Hiei and colleagues, maize transformation was accomplished with freshly isolated immature embryos (Ishida et al. 1996). In maize, transformation frequency was further improved with the addition of silver nitrate in the medium, modification of medium components, and optimization of co-culture and resting timing periods. For the first time in 2002, it was demonstrated that maize can be transformed by using a combination of standard binary vector and the antioxidant cysteine in the co-culture medium. Relatively high transformation efficiency was obtained. In the same year, other researchers successfully showed that elite maize cultivars could also be transformed fairly efficiently using Agrobacterium. This report demonstrated possibility of extending the maize transformation beyond cultivar A188.

The successful Agrobacterium-mediated transformation of wheat and barley soon followed maize. Compared to rice and maize, progress with wheat and barley has been slower. Since the first report of Agrobacterium-mediated transformation of wheat in 1997, various factors that influence T-DNA delivery have been further investigated and modified (reviewed by Jones et al. 2005). The use of surfactant such as Silwet L-77 and desiccation treatment during co-culture significantly increased T-DNA delivery.

Following the success of Tingay and colleagues (Tingay et al. 1997) in Agrobacterium-mediated transformation of barley, a number of laboratories around the world reported the successful production of transgenic barley plants (reviewed by Shrawat and Lörz, 2006). Because the majority of the successful reports on the Agrobacterium-mediated transformation of barley are restricted with model genotypes ‘golden promise’ and ‘Igri’, further efforts are required to extend the Agrobacterium-mediated transformation systems for elite barley cultivars.

Despite successful reports on Agrobacterium-mediated transformation of sorghum (reviewed by Shrawat and Lörz, 2006), sorghum is the least successfully manipulated for tissue culture and Agrobacterium-mediated transformation. Therefore, optimization of parameters that are considered crucial for cereal transformation and screening of highly competent explants and genotypes should broaden the scope for the genetic transformation with genes of interest.

Transgene integration, expression and characteristics of transgenic plants
The stable inheritance and expression of foreign genes are of critical importance in the application of genetically engineered cereals to agriculture. Many factors can contribute to variations in transgene expression, including tissue culture induced variation, transgene copy number, transgene mutation, and epigenetic gene silencing. Gene silencing can occur at the transcriptional or post-transcriptional level and the phenomenon is often associated with a high transgene copy number. High promoter activity is also correlated with hypermethylation and the abolishment of gene transcription in both monocot and dicot plant species. Without any doubt, the problem of transgene silencing raises serious concerns regarding selection of transgenic lines for crop improvement with specific trait(s). Therefore, it now appears imperative that transgenic lines carrying gene(s) of economic importance need to be carefully tested for gene expression levels over many generations.

The patterns of integration, inheritance, and expression of transgenes in plants after Agrobacterium-mediated and direct delivery-mediated transformation have been reported by many researchers. Both of these methods have advantages and disadvantages and can be used to produce transgenic plants with agronomically important traits. Although both of these methods result in integration of multiple copies of transgene, the direct DNA delivery systems tend to result in integration of multiple copies of transgenes at single loci and rearrangement of transgenes more frequently (Dai et al. 2001). Agrobacterium-mediated transformation also results in small or large-scale deletions, duplications or rearrangement of nearby plant DNA sequences, and in the integration of non-contiguous plant DNA at the site of T-DNA insertion. Studies on cereal transformation indicate that particle bombardment-mediated transformation facilitates a wide range of transformation strategies with a wide range of gene expression, has no biological constraints or host limitations, and diverse cell types can be targeted efficiently for foreign DNA delivery (reviewed by Altpeter et al. 2005). Despite the fact that the Agrobacterium-mediated gene transfer system also results integration of multiple elements in different patterns of inverted or tandem repeat, Agrobacterium-mediated transformation will remain a method of choice for obtaining transgenic plants with lower copy number, intact foreign genes, and stable gene expression.

Existing problems and future prospects
In comparison to rice, Agrobacterium-mediated transformation of other major cereal crops lags significantly behind. Tissue browning and necrosis following Agrobacterium infection is still a major obstacle in genetic transformation of cereals. Subsequent to Agrobacterium infection, wheat embryos and root cells may produce hydrogen peroxide (H₂O₂), display altered cell wall composition, and result in higher levels of cellular necrosis and subsequent cell death. A correlation between the reduction in cell death and the improved transformation frequency has been demonstrated in cereals. Anti-necrotic treatment of the target tissues might provide an adequate environment for the interaction of Agrobacterium with plant cells by inhibiting necrosis and result in increased efficiency of transformation.

In Agrobacterium-mediated transformation of cereals, apart from necrosis, a number of factors such as genotype, explant, Agrobacterium strain, binary vector, selectable marker gene and promoter, inoculation and co-culture conditions, inoculation and co-culture medium, osmotic treatment, desiccation, Agrobacterium density and surfactants, tissue culture, and regeneration medium may influence the recovery of stable plant cells after Agrobacterium infection (reviewed by Shrawat and Lörz, 2006, Cheng et al. 2004). Of these factors, the difference in the competence of Agrobacterium to infect a specific tissue, genotype, or species is still a major drawback not only in extending the Agrobacterium-mediated transformation system to elite cultivars of economically important cereals but also in utilizing Agrobacterium routinely for introducing gene(s) of interest in major cereal crops.

Explant type, quality, and source have also been correlated with reports of successful Agrobacterium-mediated genetic transformation of cereals. For example, work with freshly isolated immature embryos with and without pre-treatment have comprised the majority of successful reports on genetic transformation of cereals and are considered the best explant type. Embryogenic callus derived from mature seed was reported to be the best explant for Agrobacterium-mediated transformation of rice due to its active cell division (Hiei et al. 1994). The difference in the competence of Agrobacterium to infect a particular tissue, genotype, or species has also been a major drawback in genetic transformation of elite cultivars of cereals. For example, an efficient transformation system in maize and sorghum was established only with super-binary vectors in LBA4404, while a standard binary vector in a super-virulent strain showed low transformation frequency even with improved co-culture conditions in maize.

Recently, a number of plant genes that were differentially expressed during early stages of Agrobacterium-mediated transformation have also been identified. The majority of these genes showed induced expression during early stages of infection with various strains of Agrobacterium (Veena et al. 2003). Interestingly, Agrobacterium infection triggered changes in the host cell’s gene expression pattern, inducing or repressing specific sets of plant genes. Veena and colleagues also demonstrated the involvement of T-DNA and/or Vir proteins as factors that resulted in differential expression of these genes during Agrobacterium infection. Screening of T-DNA insertion mutant lines of Arabidopsis for recalcitrance to transformation following bacterial infection showed a large degree of variation in transformation among ecotypes (Nam et al. 1999). This study suggested that many plant genes might be involved in this process, and therefore, screening of such mutagenized lines would be an important tool for understanding the role of host genes during interaction with Agrobacterium. Therefore, it is necessary to identify more plant factors participating in T-DNA transformation in order to better understand the underlying processes accounting for the host range and the susceptibility of plant cells to Agrobacterium infection.

Recently, several non-Agrobacterium species of bacteria have also been used successfully for genetic transformation of three plant species, including rice (Broothaerts et al. 2005). Analysis of seed progeny of all three plant species transformed with Sinorhizobium meliloti showed stable inheritance of the transgenic GUS and hygromycin resistance phenotypes. This study suggests that a number of diverse plant-associated bacteria can be successfully used for gene transfer to crop plants.

In conclusion, the in-depth studies and evaluation of genes responsible for stimulating plant cell division and for stimulating the competency of plant cells to Agrobacterium might increase not only the extension of transformation protocols to elite cultivars but also the transformation efficiency in cereals. Understanding mechanisms by which treatments such as desiccation and antioxidants impact T-DNA delivery and stable transformation will undoubtedly facilitate development of efficient transformation systems in cereals.

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