Generation Of Transgenic Plants Research Articles

Marker-free transgenic cucumber expressing Arabidopsis CBF1 gene confers chilling stress tolerance

Marker-free transgenic cucumber (Cucumis sativus L.) cv. Poinsett 76 SR plants were produced by Agrobacterium mediated transformation. A transformation efficiency of 1.62 was observed on using Agrobacterium tumefaciens strain LBA4404 harbouring Arabidopsis cbf1 gene driven by the inducible promoter RD29A in a binary vector system pCAMBIA. Transgene integration and single copy insert in transgenic cucumber was confirmed by polymerase chain reaction (PCR) and Southern blot analysis in T0 lines and also confirmed marker-free status in T1 generation. Transgene expression was confirmed by reverse transcription (RT)-PCR in T1 generation transgenic cucumber and advanced to T2 generation. Upon exposure to chilling stress (4 °C), the T2 generation transgenic plants survived up to 36 h; however, wild-type plants could not survive and gradually died. A significant decrease in membrane injury index (MII), increase in activities of antioxidant enzymes (SOD and CAT), free proline content and relative water content (RWC) in the leaves were observed in transgenic cucumber as compared to wild-type under chilling stress. Thus, the transgenic cucumber plants expressing Arabidopsis cbf1 gene conferred protection against chilling stress.

Minimizing the unpredictability of transgene expression in plants: the role of genetic insulators

The genetic transformation of plants has become a necessary tool for fundamental plant biology research, as well as the generation of engineered plants exhibiting improved agronomic and industrial traits. However, this technology is significantly hindered by the fact that transgene expression is often highly variable amongst independent transgenic lines. Two of the major contributing factors to this type of inconsistency are inappropriate enhancer-promoter interactions and chromosomal position effects, which frequently result in mis-expression or silencing of the transgene, respectively. Since the precise, often tissue-specific, expression of the transgene(s) of interest is often a necessity for the successful generation of transgenic plants, these undesirable side effects have the potential to pose a major challenge for the genetic engineering of these organisms. In this review, we discuss strategies for improving foreign gene expression in plants via the inclusion of enhancer-blocking insulators, which function to impede enhancer-promoter communication, and barrier insulators, which block the spread of heterochromatin, in transgenic constructs. While a complete understanding of these elements remains elusive, recent studies regarding their use in genetically engineered plants indicate that they hold great promise for the improvement of transgene expression, and thus the future of plant biotechnology.

Molecular breeding of transgenic white clover (Trifolium repens L.) with field resistance to Alfalfa mosaic virus through the expression of its coat protein gene

Viral diseases, such as Alfalfa mosaic virus (AMV), cause significant reductions in the productivity and vegetative persistence of white clover plants in the field. Transgenic white clover plants ectopically expressing the viral coat protein gene encoded by the sub-genomic RNA4 of AMV were generated. Lines carrying a single copy of the transgene were analysed at the molecular, biochemical and phenotypic level under glasshouse and field conditions. Field resistance to AMV infection, as well as mitotic and meiotic stability of the transgene, were confirmed by phenotypic evaluation of the transgenic plants at two sites within Australia. The T(0) and T(1) generations of transgenic plants showed immunity to infection by AMV under glasshouse and field conditions, while the T(4) generation in an agronomically elite 'Grasslands Sustain' genetic background, showed a very high level of resistance to AMV in the field. An extensive biochemical study of the T(4) generation of transgenic plants, aiming to evaluate the level and composition of natural toxicants and key nutritional parameters, showed that the composition of the transgenic plants was within the range of variation seen in non-transgenic populations.

Development of an Agrobacterium-mediated stable transformation method for industrial oilseed crop Crambe abyssinica ‘BelAnn’

Crambe abyssinica is a fast growing non-food high biomass crop with significant potential for biofuel production and for phytoremediation of heavy metal contaminated soils and sediments. We have developed an efficient Agrobacterium-mediated transformation system for C. abyssinica ‘BelAnn’ using β-glucuronidase (gus), bacterial arsenate reductase (ArsC), and γ-glutamylcysteine synthetase (γ-ECS) genes. Pre-cultured hypocotyl explants were infected with Agrobacterium tumefaciens strain LBA4404 harboring binary vector pCAMBIA1300 containing gus, ArsC, and γ-ECS genes under the control of CaMV35S, leaf-specific SRS1p, and constitutive Act2p promoters, respectively. Following co-cultivation and selection, regenerated shoot buds were sub-cultured on MS medium containing GA3 for shoot elongation. Elongated shoots were transferred to root induction medium for 1 week. Semi-quantitative RT-PCR analysis confirmed the expression of mRNA transcripts for gus, ArsC and γ-ECS genes in T0 generation transgenic plants. Histochemical assays showed the gus expression in both vegetative and reproductive tissues of stably transformed T1 generation plants. Germinating seeds from T1 transgenic plants grown on MS medium containing hygromycin revealed a 3:1 Mendelian inheritance pattern for each transgene. Immunoblotting assays further confirmed the stable transformation and expression of the ArsC and γ-ECS proteins in T2 generation plants using anti-ArsC and γ-ECS antibodies. This method achieved an overall frequency of 50–70% regeneration and 6.7–8.3% transformation with three different gene constructs. The method showed higher transformation efficiency than the previously reported methods and will be useful for gene manipulation in crambe to enable biomaterial and biofuel production, as well as for potential oil content modification and heavy metal phytoremediation.

An improved particle bombardment for the generation of transgenic plants by direct immobilization of relleasable Tn5 transposases onto gold particles

We have developed a modified particle bombardment method for plant transgenesis. An intein-tag and a 6×Cys-tag were successively fused to the N-terminus of a hyperactive Tn5 transposase. The modified transposase was immobilized on bare gold microscopic particles via covalent binding of a 6×Cys-tag sulfydryl groups to the gold surface. The tethered transposase can bind the transposon DNA in vitro to form the transposome in the absence of Mg²⁺ ions. After bombardment of the gold particles carrying the transposomes into the plant cells, the transposomes will be released from the carrier due to the activated self-cleavage function of intein-tag. Our data showed this procedure integrated foreign DNA into the plant genome with an increased transformation frequency as compared to the conventional particle bombardment method. A single copy insertion can also be obtained by decreasing of the assembled transposon DNA amount in relation to plant cell biomass.

Ectopic expression of an <em>AGAMOUS</em> homolog <em>NTAG1</em> from Chinese narcissus accelerated earlier flowering and senescence in Arabidopsis

There are two cultivating varieties of Chinese narcissus, named as Yulinglong ( Narcissus tazetta var. chinensis Roem. Florepleno) and Jinzhanyutai ( Narcissus tazetta var. chinensis M. Roem.) well known in China. Yulinglong plants exhibit double flower resulted from petaloid stamens. However, the molecular basis of double flower formation is known little and unclear. Based on the flowering ABCDE model, double flower formation is commonly used to link to C functional genes. In this present paper, the isolation and characterization of NTAG1 gene, an AGAMOUS homolog from Chinese narcissus varieties mentioned above are reported. Sequence and expression pattern of NTAG1 gene exhibited the same in both tested varieties. It expresses only in the reproductive organs. Furthermore, functional analysis by using ectopic tests in Arabidopsis showed that NATG1 might be involved in the carpel identity and floral transition The effects of ectopic expression of NTAG1 mainly include dwarfing , early flowering, losing inflorescence indeterminacy,branch number increasing, and advancing senescence, whereas some of homeotic phenotypes gradually sisappeared in higher generation of transgenic plants. The utilizations of NATG1 gene in the future gene engineering were also discussed in the paper.

Heat Shock Factor 8 Introducing into Soybean (<i>Glycine max</i>) by <i>Agrobacterium</i>-Mediated Transformation

Environmental stress seriously affects growth and development of crops. Heat shock transcription factor (heat shock factor 8, HSF8 ) is a class of proteins that play important roles in the reactions of heat shock, the main functions of heat shock gene are binding the corresponding heat shock element in the process of expression, initiating the gene transcription process and, ultimately, facilitating the expression of heat shock protein (HSP) gene. In this paper, we inserted hsf8 gene into dicotyledon expression vector pCAMBIA3300 that contains the selection marker, bar gene. The construct, named pCAMBIA3300- HSF8 , has been transfered into new soybean lines, Hajiao5337 and Hajiao5489, mediated by Agrobacterium tumefaciens . In the practice of soybean genetic transformation, we explored the impact factors on the transformation of soybean cotyledon mediated by Agrobacterium , and optimized the transformation conditions. It is the way to increase the selection efficiency by delaying the screening after co-culture with optimal concentration of glufosinate-ammonium 3.5 mg/L in selection culture medium. We obtained the T1 generation transgenic plants with pCAMBIA3300- HSF8 by Agrobacterium -mediated aprroch derived from Hajiao5337 and Hajiao5489 , of which 17 plants were proved by PCR to be positive transgenic plants. Furthermore, transcription level of hsf8 gene in T1 transgenic plants with glufosinate-ammonium resistance was detected by Real-time PCR approach, the results showed that 9 of 17 plants had much higher expression level than that of the reference of Hajiao5337, whereas one transgenic plant had lower level than that of another reference Hajiao5489.

Development of a transgenic hairy root system in jute (Corchorus capsularis L.) with gusA reporter gene through Agrobacterium rhizogenes mediated co-transformation

Transgenic hairy root system is important in several recalcitrant plants, where Agrobacterium tumefaciens-mediated plant transformation and generation of transgenic plants are problematic. Jute (Corchorus spp.), the major fibre crop in Indian subcontinent, is one of those recalcitrant plants where in vitro tissue culture has provided a little success, and hence, Agrobacterium-mediated genetic transformation remains to be a challenging proposition in this crop. In the present work, a system of transgenic hairy roots in Corchorus capsularis L. has been developed through genetic transformation by Agrobacterium rhizogenes harbouring two plasmids, i.e. the natural Ri plasmid and a recombinant binary vector derived from the disarmed Ti plasmid of A. tumefaciens. Our findings indicate that the system is relatively easy to establish and reproducible. Molecular analysis of the independent lines of transgenic hairy roots revealed the transfer of relevant transgenes from both the T-DNA parts into the plant genome, indicating the co-transformation nature of the event. High level expression and activity of the gusA reporter gene advocate that the transgenic hairy root system, thus developed, could be applicable as gene expression system in general and for root functional genomics in particular. Furthermore, these transgenic hairy roots can be used in future as explants for plantlet regeneration to obtain stable transgenic jute plants.

Cloning the bacterial bphC gene into Nicotiana tabacum to improve the efficiency of phytoremediation of polychlorinated biphenyls

The aim of this work was to construct transgenic plants with increased capabilities to degrade organic pollutants, such as polychlorinated biphenyls. The environmentally important gene of bacterial dioxygenase, the bphC gene, was chosen to clone into a plant of Nicotiana tabacum. The chosen bphC gene encodes 2,3-dihydroxybiphenyl-1,2-dioxygenase, which cleaves the aromatic ring of dihydroxybiphenyl, and we cloned it in fusion with the gene for b-glucuronidase (GUS), luciferase (LUC) or with a histidine tail. Several genetic constructs were designed and prepared and the possible expression of desired proteins in tobacco plants was studied by transient expression. We used genetic constructs successfully expressing dioxygenase's genes we used for preparation of transgenic tobacco plants by agrobacterial infection. The presence of transgenic DNA, mRNA and protein was determined in parental and the first filial generation of transgenic plants with the bphC gene. Properties of prepared transgenic plants will be further studied.

Generation of transgenic plants expressing plasma membrane-bound antibodies to the environmental pollutant microcystin-LR

In this paper we describe the engineering and regeneration of transgenic tobacco plants expressing a recombinant plasma membrane-retained antibody specific to microcystin-LR (MC-LR), the environmental toxin pollutant produced by cyanobacteria. The antibody was created by a genetic fusion of the antigen binding regions of the microcystin-specific single chain antibody, 3A8, with the constant regions from the murine IgG1κ, Guy's 13, including a membrane retention sequence at the C-terminal end of the antibody heavy chain. The antibody produced in the leaves was shown to be functional by binding to MC-LR in an ELISA with antibody yields in transgenic plant leaves reaching a maximum of 1.2 μg g(-1) leaf f.wt (0.005% total soluble protein). Antibody-MC-LR complexes formed in leaves after addition of MC-LR to hydroponic medium around the roots of transgenic plant cultures.

Selectable marker-free transgenic orange plants recovered under non-selective conditions and through PCR analysis of all regenerants

Selectable marker (SM) genes have been considered necessary to achieve acceptable rates in the generation of transgenic plants. Genes encoding antibiotic or herbicide resistance are widely used for this purpose. In most cases, once transgenic plants have been regenerated, permanence of SM genes in the plant genome is no longer necessary, and it becomes a matter of public concern. Moreover, the removal of SM genes from transgenic plants could facilitate gene stacking through successive transformations, particularly when the availability of these markers is rather limited for most crop plants. In the genus Citrus, with highly heterozygotic species of long generation cycles, methods implying the segregation and removal of marker transgenes in the progeny are not feasible. Here, we have evaluated the direct production of SM-free citrus plants under non-selective conditions, using a “clean” binary vector carrying only the transgene of interest, and through the recovery of transformants by polymerase chain reaction (PCR) analysis of all regenerated shoots. The response of two different citrus genotypes, Carrizo citrange (intergeneric hybrid of C. sinensis L. Osb. X Poncirus trifoliata L. Raf.) and Pineapple sweet orange (C. sinensis L. Osb.), was evaluated. Our results indicate that, in this system, the competence between transgenic and non-transgenic cells is the main factor determining final transgenic regeneration frequencies. For Carrizo citrange, no transgenic plant could be recovered. For Pineapple sweet orange, marker-free transformation efficiency was 1.7%, paving the way for the viable production of orange transformants carrying only the transgene(s) of interest.

Transient gene expression in rose petals via Agrobacterium infiltration

The study of gene function in roses is hampered by the low efficiency of transformation systems and the long time span needed for the generation of transgenic plants. For some functional analyses, the transient expression of genes would be an efficient alternative. Based on current protocols for the transient expression of genes via the infiltration of Agrobacterium into plant tissues, we developed a transient expression system for rose petals. We used β-glucuronidase (GUS) as a marker gene to optimize several parameters with effects on GUS expression. The efficiency of expression was found to be dependent on the rose genotype, flower age, position of petals within a flower, Agrobacterium strain and temperature of co-cultivation. The highest GUS expression was recorded in petals of the middle whirls of half-bloomed flowers from cultivars of ‘Pariser Charme’ and ‘Marvel’.

Application of somatic embryos to rapid and reliable analysis of soybean seed components by RNA interference-mediated gene silencing

Soybean somatic embryos have attracted attention both as a model of zygotic embryos and as explants for the generation of transgenic plants. β-Conglycinin, which is composed of three subunits (α, α′, and β) that are encoded by a multigene family, is a major seed component of soybean, and accumulates in somatic embryos with their maturation. We subjected the somatic embryos to transformation with vectors encoding double-stranded RNA fragments of various sizes that correspond to the gene for the α′ subunit of β-conglycinin. These DNA fragments were put under the control of the promoter region of the gene for another major seed protein, the A2B1a subunit of glycinin. Transgenic somatic embryos were obtained within 2 months of transformation, and the resulting mature embryos manifested down-regulation of the α′ subunit of β-conglycinin in a manner dependent on the size of the RNAi vector insert. Accumulation of small interfering RNA and depletion of mRNA corresponding to the α′ subunit were indicative of RNAi. The amounts of α and β subunits of β-conglycinin, which share high sequence similarity with the α′ subunit, were also reduced in the mature somatic embryos. Moreover, the abundance of all β-conglycinin subunits was greatly reduced in the seeds of regenerated transgenic plants. Our results indicate that the application of RNAi to somatic embryos is a feasible and rapid option for functional studies of soybean seed components.

Generation of transgenic plants expressing antibodies to the environmental pollutant microcystin‐LR

We describe the engineering, regeneration, and characterization of transgenic tobacco plants expressing a recombinant antibody specific to microcystin-LR (MC-LR), the environmental toxin pollutant produced by species of cyanobacteria. The antibody was created by a genetic fusion of the antigen-binding regions of the microcystin-specific single-chain antibody, 3A8, with constant regions from the murine IgG1kappa, Guy's 13. IgG transgenes were controlled by a leader peptide that targets the transgene products to the secretory pathway and also allows for rhizosecretion. The antibody, extracted from the leaves or rhizosecreted into hydroponic medium by transgenic plants, was shown to have functional binding to MC-LR. Antibody yields in transgenic plant leaves reached a maximum of 64 microg/g leaf fresh weight (0.6% total soluble protein), and the rate of antibody rhizosecretion reached a maximum of 5 microg/g root dry weight/24 h. Rhizosecreted antibody bound to MC-LR in hydroponic medium, and transgenic plants grew more efficiently on medium containing MC-LR compared to wild-type controls. This proof of concept paves the way for applications to produce diagnostic antibodies to microcystin-LR, remove it from the environment by phytoremediation, or enhance yields in crops exposed to MC-LR.-Drake, P. M. W., Barbi, T., Drever, M. R., van Dolleweerd, C. J., Porter, A. J. R., Ma, J. K.-C. Generation of transgenic plants expressing antibodies to the environmental pollutant microcystin-LR.

Enhanced ascorbic acid accumulation in transgenic potato confers tolerance to various abiotic stresses

L-ascorbic acid (Vitamin C, AsA) is an important component of human nutrition. Plants and several animals can synthesize their own ascorbic acid, whereas humans lack the gene essential for ascorbic acid biosynthesis and must acquire from their diet. In the present study, we developed transgenic potato (Solanum tuberosum L. cv. Taedong Valley) over-expressing L-gulono-gamma-lactone oxidase (GLOase gene; NCBI Acc. No. NM022220), isolated from rat cells driven by CaMV35S constitutive promoter that showed enhanced AsA accumulation. Molecular analyses of four independent transgenic lines performed by PCR, Southern and RT-PCR revealed the stable integration of the transgene in the progeny. The transformation frequency was ca. 7.5% and the time required for the generation of transgenic plants was 6-7 weeks. Transgenic tubers showed significantly enhanced AsA content (141%) and GLOase activity as compared to untransformed tubers. These transgenics were also found to withstand various abiotic stresses caused by Methyl Viologen (MV), NaCl or mannitol, respectively. The T(1) transgenic plants exposed to salt stress (100 mM NaCl) survived better with increased shoot and root length when compared to untransformed plants. The elevated level of AsA accumulation in transgenics was directly correlated with their ability to withstand abiotic stresses. These results further demonstrated that the overexpression of GLOase gene enhanced basal levels of AsA in potato tubers and also the transgenics showed better survival under various abiotic stresses.

Developing phosphinothricin-resistant transgenic sunflower (Helianthus annuus L.) plants

Most investigations on genetic transformations of sunflower have used the neomycin transferase (nptII) gene as the selectable marker. We previously reported a PPT-based selection system for sunflower transformation that uses the bialaphos resistance (bar) gene as the selectable marker and 20 mg/l of phosphinothricin (PPT) as the selective agent. Sunflower (Helianthus annuus L.) variety Skorospeliy 87 was genetically transformed via Agrobacterium tumefaciens strain EHA 105 harbouring the binary plasmid vector pBAR. Two-day-old explants from mature embryos competent for direct shooting were used. Southern blot and ELISA experiments confirmed the stability of expression in two generations of transgenic plants. Transformed plants transferred to soil in the greenhouse exhibited resistance to the herbicide Basta® at 3 l/ha.

종자 특이 프로모터와 대두 Ferritin 유전자에 의한 벼 종실의 철분강화

The recombinant DNAs, pGBF, pGTF, and pZ4F, using soybean ferritin gene have constructed with the promoters derived from seed proteins, glutelin, globulin, and zein. The recombinant ferritin genes were transformed into rice plant by Agrobacterium-mediated transformation. Iron contents and agronomic traits have been evaluated in the transgenic progenies. The embryogenic calli survived from second selection medium were regenerated at the rates of 19.2% with pGBF, 15.0% with pGTF, and 18.4% with pZ4F in Donganbyeo and 6.7% with pGBF, 11.7% with pGTF, and 3.4% with pZ4F in Hwashinbyeo. The introduction of ferritin gene in putative transgenic rice plants was confirmed by PCR and Southern blot analysis and also the expression of ferritin gene was identified by Northern blot and Western blot analysis. The iron accumulation in transgenic rice grains of the transgenic rice plant, T1-2, with zein promoter and ferritin gene contained 171.4 ppm showing 6.4 times higher than 26.7 ppm of Hwashinbyeo seed as wild type rice, but the transgenic plants with globulin and glutelin showed a bit higher iron contents with a range from 2.1 to 3.0 times compare to wild type grain. The growth responses of transgenic plants showed the large variances in plant height and number of tillers. However, there were some transgenic plants having similar phenotype to wild type plants. In the T1 generation of transgenic plants, plant height, culm length, panicle length, and number of tillers were similar to those of wild type plants, but ripened grain ratio ranged from 53.3% to 82.2% with relatively high variation. The transgenic rice plants would be useful for developing rice varieties with high iron content in rice grains.

Generation of vector backbone-free and selectable marker-free transgenic maize (<I>Zea mays</I> L.)<I>via</I> ovary-drip method

The presence of vector backbone sequences and selectable marker genes in transgenic plants has been the key concern for biosafety. A direct solution is to totally avoid the use of vector backbone sequences and selectable marker genes from the beginning of transgenic plant generation. In this study, the ovary-drip method was established and optimized. The key features of this method focused on the complete removal of the whole styles, and the subsequent application of a DNA solution directly to the ovaries. A vector backbone-free and selectable marker-free linear GFP cassette (Ubi-GFP -nos) was transformed into maize via the ovary-drip method. PCR analysis showed that suitable maize variety was 9818 and optimal transformation time was 18-20 h after pollination, which produced the highest PCR positive frequency (3.01%). Southern blotting analysis showed that the transgenic plants had simple integration patterns (1-2 bands). GFP transcription was de-tected by RT-PCR analysis. Green fluorescence was observed in roots and immature embryos of transgenic plants by a fluorescence microscopy.

Enhanced Resistance against the Rice Leaffolder (Cnaphalocrocis medinalis) in Transgenic Rice Plants containing the Potato Proteinase Inhibitor II Gene

Third generation transgenic plants of ten independently raised single-copy lines of Japonica (TNG67) and aromatic indica rice varieties (HBC19 and Pusa Basmati 1) containing potato proteinase inhibitor II gene (pinII) were evaluated for resistance against the rice leaffolder, Cnaphalocrocis medinalis (Guenee 1854) with emphasis on insect growth and development. Five of these transgenic lines (T-TNG-64, T-TNG-61, T-TNG-46b, T-TB-95 and T-TB-82) exhibited high level resistance against rice leaffolder. The highest larval mortality was noticed on T-TNG-46b (95.0%) followed by T-TB-82 (92.5%), T-TNG-64 (87.5%) and T-TNG-61 (87.5%) in comparison to only 4.0% in the control plants. The lowest larval feeding was observed on T-TNG-46b and T-TNG-61 (4.12 mm 2 and 4.50 mm 2 ) as against 19.1(TNG67), 18.6 (Pusa Basmati 1) and 23.0 (HBC19) mm 2 in control plants. In whole plant bioassay, the transgenic rice lines had 3 and 5 damage grades compared to 9 damage grade in control plants. Larval/ pupal growth and development (larval survival, larval period, larval weight, percent pupation, pupal period, pupal weight and moth emergence) were adversely affected when fed on transgenic leaf tissues. These results demonstrate that PinII in rice can provide effective control against rice leaffolder.

Transgenic plants and associated bacteria for phytoremediation of chlorinated compounds

Phytoremediation is the use of plants for the treatment of environmental pollution, including chlorinated organics. although conceptually very attractive, removal and biodegradation of chlorinated pollutants by plants is a rather slow and inefficient process resulting in incomplete treatment and potential release of toxic metabolites into the environment. In order to overcome inherent limitations of plant metabolic capabilities, plants have been genetically modified, following a strategy similar to the development of transgenic crops: genes from bacteria, fungi, and mammals involved in the metabolism of organic contaminants, such as cytochrome p-450 and glutathione substrate catabolic genes, natural or engineered, for the simultaneous remediation of a range of pollutants, such as usually found in contaminated sites, e.g., chlorinated solvent, metals, and nitroaromatics. In addition, biodegradation of many xenobiotics are catalyzed by similar, broad-substrate enzymes, such as cytochrome P-450 monoxygenases, glutathione S-transferases, and fungal peroxidases, that can potentially be used for the treatment of multiple pollutants. Moreover, the introduction of multiple transgenes involved in different phases of the metabolism of xenobiotics in plants, i.e., uptake by roots and the different phases of the green liver model, would allow enhancing both the removal and metabolism of several toxic compounds and could therefore help overcome a major limitation inherent to phytoremediation, i.e., the threat that accumulated toxic compounds would volatilize or otherwise contaminate the food chain. An important barrier to the application of transgenic plants for bioremediation in the field is associated with the true or perceived risk of horizontal gene transfer to related wild or cultivated plants. Therefore, it is likely that the next generation of transgenic plants will involve systems preventing such a transfer, for instance by the introduction of transgenes into chloroplastic DNA or the use of conditional lethality genes (Davison, 2005). Since bacteria naturally exchange plasmids via conjugation, endophytes that gain genes involved in pollutant degradation might not be considered ‘genetically modified’ and may be subject to fewer restrictions in usage.