Release of transgenic bacterial inoculants - rhizobia as a case study

The current debate on the release of genetically modified organisms to the environment must be informed by scientific data obtained from field studies. Many of the microorganisms that have potential applications outside the laboratory, especially in agriculture and horticulture, could be improved by genetic modification. Rhizobia, the bacteria that form N₂-fixing symbioses with leguminous plants, have a long history of safe use as seed inoculants, their biology is relatively well known, and they represent a relevant model system. There have been several field releases of genetically modified (GM) rhizobia in the USA and Europe, which provide information on various aspects of their ecology and efficacy. This review summarises the rationale for each release, details of the methods used for monitoring, and the results. Novel properties of rhizobia did not always have the predicted effects. Most studies revealed that rhizobial numbers dropped rapidly after application to soil or seeds but then numbers stabilised for months or years. The monitoring of survival and spread of rhizobia was greatly improved by the presence of novel marker genes. Tagging of rhizobia with marker genes provided more accurate information compared to the use of conventional strains, illustrating an important application of genetic modification, for tracking bacteria in the environment.     

Monitoring genetically modified rhizobia in field soils using the polymerase chain reaction

Monitoring genetically modified (GM) bacterial inoculants after field release using conventional culture methods can be difficult. An alternative is the detection of marker genes in DNA extracted directly from soil, using specific oligonucleotide primers with the polymerase chain reaction (PCR). The PCR was used to monitor survival of two GM Rhizobium leguminosarum bv. viciae inoculants after release in the field at Rothamsted. One strain, RSM2004, is marked by insertion of transposon Tn 5 ; the second strain, CT0370, released at the same site, is modified by chromosomal integration of a single copy of the gene from E. coli conferring GUS activity. Both GM strains provide a realistic case study for the development of PCR-based detection techniques. Specific primers were developed to amplify regions of the Tn 5 and GUS genetic markers using PCR and conditions optimized for each primer set to routinely detect a signal from 10 fg of purified template DNA, the equivalent of one cell per reaction. Procedures to improve the sensitivity of detection are described, to detect fewer than 50 cells g⁻¹ soil in soil-extracted DNA.     

Effect of a Sinorhizobium meliloti Strain with a Modified putA Gene on the Rhizosphere Microbial Community of Alfalfa

The success of a rhizobial inoculant in the soil depends to a large extent on its capacity to compete against indigenous strains. M403, a Sinorhizobium meliloti strain with enhanced competitiveness for nodule occupancy, was recently constructed by introducing a plasmid containing an extra copy of a modified putA (proline dehydrogenase) gene. This strain and M401, a control strain carrying the same plasmid without the modified gene, were used as soil inoculants for alfalfa in a contained field release experiment at León, Spain. In this study, we determined the effects of these two strains on the indigenous microbial community. 16S rRNA genes were obtained from the rhizosphere of alfalfa inoculated with strain M403 or strain M401 or from noninoculated plants by amplification of DNA from soil with bacterial group-specific primers. These genes were analyzed and compared by restriction fragment length polymorphism and temperature gradient gel electrophoresis. The results allowed us to differentiate between alterations in the microbial community apparently caused by inoculation and by the rhizosphere effect and seasonal fluctuations induced by the alfalfa plants and by the environment. Only moderate inoculation-dependent effects could be detected, while the alfalfa plants appeared to have a much stronger influence on the microbial community.     

Molecular evidence of genetic modification of Sinorhizobium meliloti: enhanced PCB bioremediation

The genome of the nitrogen-fixing soil bacterium Sinorhizobium meliloti does not possess genes for bioremediation of aromatic pollutants. It has the well-known ability to interact specifically with the leguminous alfalfa plant, Medicago sativa. Our previous work has shown enhanced degradation of the nitroaromatic compound 2,4-dinitrotoluene (DNT) when a plasmid containing degradative genes was introduced in it. In this study we report molecular evidence of the transfer of a polychlorinated biphenyl (PCB)-biodegradative plasmid pE43 to S. meliloti strain USDA 1936. Several standard analytical tests and plant growth chamber studies were conducted to test the ability of S. meliloti to degrade 2′,3,4-PCB congener. Alfalfa plant alone was able to degrade 30% of PCBs compared with control. No enhanced dechlorination was noted when alfalfa plant was grown with wild-type S. meliloti, and when alfalfa plant was grown with the S. meliloti electrotransformants (genetically modified) dechlorination of PCBs was more than twice that when alfalfa plant was grown with wild-type S. meliloti. When alfalfa plant was grown with uncharacterized mixed culture (containing nodule formers), almost equally significant PCB degradation was observed. The significance of this work is that the naturally occurring nitrogen-fixing soil bacterium S. meliloti (genetically modified) has the ability to enhance fertility of soil in association with the leguminous alfalfa plant while simultaneously enhancing bioremediation of PCB-contaminated soils. Enhanced bioremediation of PCB and robust alfalfa plant growth was also noted when uncharacterized mixed cultures containing alfalfa plant nodule formers were used.

     

Influence of arbuscular-mycorrhizal fungi, Rhizobium meliloti strains and PGPR inoculation on the growth of Medicago arborea used as model legume for re-vegetation and biological reactivation in a semi-arid mediterranean area

Medicago arborea can be used for re-vegetationpurposes under semiarid conditions. These woody legumes have the ability toforman association with arbuscular mycorrhizal (AM) fungi and rhizobial bacteria,which can be maximised by microorganisms producing certain stimulatingmetabolites acting as plant growth promoting rhizobacteria (PGPR). The effectsof single and combined inoculations using microorganisms with different andinteractive metabolic capacities, namely three Glomusspecies, two Rhizobium meliloti strains (a wild type, WTand its genetically modified derivative GM) and a plant growth promotingrhizobacterium, (PGPR), were evaluated. All three inoculated AM fungi affectedMedicago growth in different ways. Differences weremaintained when soil was co-inoculated with each of the rhizobial strains (WTorGM) and the PGPR. Mycorrhizal fungi were effective in all cases, but the PGPRonly affected plant growth specific microbial situations. PGPR increased growthof G. mosseae-colonised plants associated withRhizobium WT strain by 36% and those infected byG. deserticola when associated with the rhizobial GMstrainby 40%. The most efficient microbial treatments involved mycorrhizalinoculation, which was an indication of the AM dependency of this plantspecies.Moreover, PGPR inoculation was only effective when associated with specificmycorrhizal endophytes (G. mosseae plus WT andG.deserticola plus GM rhizobial strain). The reduced root/shoot (R/S)ratio resulting from PGPR inoculation, was an indication of more effective rootfunction in treated plants. AM colonisation and nodule formation wereunaffectedby the type of AM fungus or bacteria (rhizobial strain and/or PGPR). AM fromnatural soil were less infective and effective than those from the collection.The results supported the existence of selective microbial interactionsaffecting plant performance. The indigenous AM fungi appeared to be ineffectiveand M. arborea behaved as though it was highly dependentonAM colonisation, which implied that it must have a mycorrhizal association toreach maximum growth in the stressed conditions tested. Optimum growth ofmycorrhizal M. arborea plants was associated with specificmicrobial groups, accounting for a 355% increase in growth overnodulatedcontrol plants. The beneficial effect of PGPR in increasing the growth of awoody legume, such as M. arborea under stress, was onlyobserved with co-inoculation of specific AM endophytes. As a result of theinteraction, only shoot biomass was enhanced, but not as a consequence ofenhancing of the colonising abilities of the endophytes. The growthstimulation,occurring as a consequence of selected microbial groups, may be critical anddecisive for the successful establishment of plants under Mediterraneanclimaticand soil conditions.     

Plant–microbe interactions promoting plant growth and health: perspectives for controlled use of microorganisms in agriculture

Plant-associated microorganisms fulfill important functions for plant growth and health. Direct plant growth promotion by microbes is based on improved nutrient acquisition and hormonal stimulation. Diverse mechanisms are involved in the suppression of plant pathogens, which is often indirectly connected with plant growth. Whereas members of the bacterial genera Azospirillum and Rhizobium are well-studied examples for plant growth promotion, Bacillus, Pseudomonas, Serratia, Stenotrophomonas, and Streptomyces and the fungal genera Ampelomyces, Coniothyrium, and Trichoderma are model organisms to demonstrate influence on plant health. Based on these beneficial plant–microbe interactions, it is possible to develop microbial inoculants for use in agricultural biotechnology. Dependent on their mode of action and effects, these products can be used as biofertilizers, plant strengtheners, phytostimulators, and biopesticides. There is a strong growing market for microbial inoculants worldwide with an annual growth rate of approximately 10%. The use of genomic technologies leads to products with more predictable and consistent effects. The future success of the biological control industry will benefit from interdisciplinary research, e.g., on mass production, formulation, interactions, and signaling with the environment, as well as on innovative business management, product marketing, and education. Altogether, the use of microorganisms and the exploitation of beneficial plant–microbe interactions offer promising and environmentally friendly strategies for conventional and organic agriculture worldwide.     

Characterization of two bioluminescentRhizobium meliloti strains constructed for field releases

The deliberate release of genetically engineered microorganisms requires a thorough characterization of the microbes in question. For the two bioluminescentRhizobium meliloti strains, L1 and L33 [Selbitschka et al. (1992) Mol Ecol 1: 9–19; Selbitschka et al. (1995) FEMS Microbiol Ecol 16:223–232], designated for field release, the sites of genetic modifications in the chromosomes were sequenced from amplified genomic DNA. This indicated no unexpected alterations in the nucleotide sequence. The bioluminescent phenotype was stably inherited over more than 100 generations in liquid cultures. The presence of the luciferase gene in both strains did not have secondary effects on a variety of metabolic pathways as assessed by the Biolog GN system. A specific polymerase chain reaction amplification, based on the chromosomal insertion site of theluc cassette, allowed the discrimination between the two strains and thus simplifies monitoring. The RecA-deficient strain L1 showed a strongly (more than 90%) reduced ability to nodulate alfalfa in competition with its parent strainR. meliloti 2011 and its RecA⁺ counterpart L33.     

Plant Growth-Promoting Rhizobacteria Allow Reduced Application Rates of Chemical Fertilizers

The search for microorganisms that improve soil fertility and enhance plant nutrition has continued to attract attention due to the increasing cost of fertilizers and some of their negative environmental impacts. The objectives of this greenhouse study with tomato were to determine (1) if reduced rates of inorganic fertilizer coupled with microbial inoculants will produce plant growth, yield, and nutrient uptake levels equivalent to those with full rates of the fertilizer and (2) the minimum level to which fertilizer could be reduced when inoculants were used. The microbial inoculants used in the study were a mixture of plant growth-promoting rhizobacteria (PGPR) strains Bacillus amyloliquefaciens IN937a and Bacillus pumilus T4, a formulated PGPR product, and the arbuscular mycorrhiza fungus (AMF), Glomus intraradices. Results showed that supplementing 75% of the recommended fertilizer rate with inoculants produced plant growth, yield, and nutrient (nitrogen and phosphorus) uptake that were statistically equivalent to the full fertilizer rate without inoculants. When inoculants were used with rates of fertilizer below 75% of the recommended rate, the beneficial effects were usually not consistent; however, inoculation with the mixture of PGPR and AMF at 70% fertility consistently produced the same yield as the full fertility rate without inoculants. Without inoculants, use of fertilizer rates lower than the recommended resulted in significantly less plant growth, yield, and nutrient uptake or inconsistent impacts. The results suggest that PGPR-based inoculants can be used and should be further evaluated as components of integrated nutrient management strategies.     

Bacterial diversity in roots of conventional and genetically modified hybrid maize

Cultivated surfaces of genetically modified (GM) crops increased year by year, becoming in 2012 more extensive in developed than in industrialized countries. Furthermore, it has been postulated that the plant is which leads to the selection of the microorganisms on its root exudates, creating specific conditions which in turn regulate the specific microbial structure of each plant. In this study, our main objective was to examine whether the introduction of transgenic maize herbicide-tolerant plants will impact the microbial structures that inhabit at the rhizosphere and rhizoplane with respect to conventional hybrid maize plants. Bacterial populations were determined (CFU/g) using four different semi-selective media. The bacterial genera isolated from the rhizoplane and rhizosphere were identified by sequencing its 16S ribosomal DNA. Although minor differences were found in bacterial populations, our results indicated that there was not a strong change of the microorganisms populations that interact at the rhizosphere of an either conventional hybrid or genetically modified maize. However, we found some bacteria that were only isolated in the either genetically modified [Chryseobacterium sp. (4.39%) and Micrococcus sp. (3.72%)] or conventional maize [Sphingobium sp. (13.17%) and Microbacterium sp. (14.81%)].     

Aspects of Marker/Reporter Stability and Selectivity in Soil Microbiology

Based on several experiences of microbial release using genetically modified Rhizobium leguminosarum, we have highlighted a number of aspects related to the suitability of introduced markers such as resistance to mercury and β-galactosidase activity, the latter serving the function of high-expression level reporter gene obtained by the introduction of a synthetic promoter conferring strong inducible expression in Gram-negative bacteria. In vitro expression and in vivo performances of the chosen examples have been followed in model strains comparing gene dosage and expression levels. The technical possibility of unambiguously monitoring the marked GMM has been evaluated in medium- and long-term experiments carried out both in microcosms and soil, also including the presence of the plant symbiotic host. Marker stability, regardless the nature of the gene, was shown to be dependent on the location of the genetic modification and on its degree of gene expression regulation. Reporter strength was found to be an advantage allowing the distinction of marker-bearing bacterial while negatively affecting their genetic stability. Plasmid-borne regulated reporters were found to be stable up to the stages of rhizosphere colonization, but were more critically selected against upon symbiotic host invasion.     

The host niches of soybean rather than genetic modification or glyphosate application drive the assembly of root-associated microbial communities

Plant roots significantly influence soil microbial diversity, and soil microorganisms play significant roles in both natural and agricultural ecosystems. Although the genetically modified (GM) crops with enhanced insect and herbicide resistance are thought to have unmatched yield and stress resistance advantages, thorough and in-depth case studies still need to be carried out in a real-world setting due to the potential effects of GM plants on soil microbial communities. In this study, three treatments were used: a recipient soybean variety Jack, a triple transgenic soybean line JD321, and the glyphosate-treated JD321 (JD321G). Three sampling stages (flowering, seed filling and maturing), as well as three host niches of soybean rhizosphere [intact roots (RT), rhizospheric soil (RS) and surrounding soil (SS)] were established. In comparison to Jack, the rhizospheric soil of JD321G had higher urease activity and lower nitrite reductase at the flowering stage. Different treatments and different sampling stages existed no significant effects on the compositions of microbial communities at different taxonomic levels. However, at the genus level, the relative abundance of three plant growth-promoting fungal genera (i.e. Mortierella, Chaetomium and Pseudombrophila) increased while endophytic bacteria Chryseobacterium and pathogenic bacteria Streptomyces decreased from the inside to the outside of the roots (i.e. RT → RS → SS). Moreover, two bacterial genera, Bradyrhizobium and Ensifer were more abundant in RT than in RS and SS, as well as three species, Agrobacterium radiobacter, Ensifer fredii and Ensifer meliloti, which are closely related to nitrogen-fixation. Furthermore, five clusters of orthologous groups (COGs) associated to nitrogen-fixation genes were higher in RT than in RS, whereas only one COG annotated as dinitrogenase iron-molybdenum cofactor biosynthesis protein was lower. Overall, the results imply that the rhizosphere host niches throughout the soil–plant continuum largely control the composition and function of the root-associated microbiome of triple transgenic soybean.     

Microbial inoculants with higher capacity to colonize soils improved wheat drought tolerance

Microbial inoculants have gained increasing attention worldwide as an eco-friendly solution for improving agriculture productivity. Several studies have demonstrated their potential benefits, such as enhanced resistance to drought, salinity, and pathogens. However, the beneficial impacts of inoculants remain inconsistent. This variability is attributed to limited knowledge of the mechanisms by which microbial inoculants affect crop growth and a lack of ecological characteristics of these inoculants that limit our ability to predict their beneficial effects. The first important step is believed to be the evaluation of the inoculant's ability to colonize new habitats (soils and plant roots), which could provide crops with beneficial functions and improve the consistency and efficiency of the inoculants. In this study, we aimed to investigate the impact of three microbial inoculants (two bacterial: P1 and P2, and one fungal: P3) on the growth and stress responses of three wheat varieties in two different soil types under drought conditions. Furthermore, we investigated the impact of microbial inoculants on soil microbial communities. Plant biomass and traits were measured, and high-throughput sequencing was used to characterize bulk and rhizosphere soil microbiomes after exposure to drought stress. Under drought conditions, plant shoot weight significantly increased (11.37%) under P1 treatments compared to uninoculated controls. In addition, total nitrogen enzyme activity increased significantly under P1 in sandy soil but not in clay soil. Importantly, network analyses revealed that P1, consisting of Bacillus paralicheniformis and Bacillus subtilis, emerged as the keystone taxa in sandy soil. Conversely, P2 and P3 failed to establish as keystone taxa, which may explain their insignificant impact on wheat performance under drought conditions. In conclusion, our study emphasizes the importance of effective colonization by microbial inoculants in promoting crop growth under drought conditions. Our findings support the development of microbial inoculants that robustly colonize plant roots for improved agricultural productivity.     

Characterization of two novel Rhizobium leguminosarum bacteriophages from a field release site of genetically-modified rhizobia

Two Rhizobium leguminosarum biovar viceae bacteriophages with contrasting properties were isolated from a field site in which the survival of genetically modified R. leguminosarum inoculants had been monitored for several years. Inoculant strain RSM2004 was used as the indicator for phage isolation and propagation. One phage, RL1RES, was temperate and could not replicate in any of the 42 indigenous R. leguminosarum field isolates tested although nested PCR indicated that phage sequences were present in six of the isolates. The second phage, RL2RES, was virulent, capable of generalised transduction, contained DNA with modified cytosine residues, and was capable of infecting all field isolates tested although the GM inoculant strain CT0370 was resistant. Sequence with homology to RL2RES was detected by nested PCR in six of the 42 field-isolates. These were not the same isolates that showed homology to RL1RES. The implication of these findings for the survival of rhizobial inoculants, and the ecology of phages and their host bacteria, are discussed.     

Relative genetic structure of a population of Rhizobium meliloti isolated directly from soil and from nodules of alfalfa (Medicago sativa) and sweet clover (Melilotus alba)

Insertion sequence (IS) hybridization was used to define the structure of a population of Rhizobium meliloti isolated directly from soil and from nodules of Medicago sativa (alfalfa) and Melilotus alba (sweet clover) grown under controlled conditions and inoculated with a suspension of the same soil. The detection of R. meliloti isolated from soil on agar plates was facilitated by use of a highly species specific DNA probe derived from ISRm5. All R. meliloti obtained directly from soil proved to be symbiotic (i.e. nodulated and fixed nitrogen with alfalfa). Analysis of 293 R. meliloti isolates revealed a total of 17 distinct IS genotypes of which 9, 9 and 15 were from soil, M. alba and M. sativa, respectively; 8 genotypes were common to soil and both plant species. The frequency of R. meliloti genotypes from soil differed markedly from that sampled from nodules of both legume species: 5 genotypes represented about 90% of the isolates from soil whereas a single genotype predominated among isolates from nodules accounting for more than 55% of the total. The distribution of genotypes differed between M. sativa and M. alba indicating species variation in nodulation preferences for indigenous R. meliloti. The data are discussed in the context of competition for nodulation of the host plant and the selection of Rhizobium strains for use in legume inoculants. This study has ecological implications and suggests that the composition of R. meliloti populations sampled by the traditionally used host legume may not be representative of that actually present in soil.     

Not all GMOs are crop plants: non-plant GMO applications in agriculture

Since tools of modern biotechnology have become available, the most commonly applied and often discussed genetically modified organisms are genetically modified crop plants, although genetic engineering is also being used successfully in organisms other than plants, including bacteria, fungi, insects, and viruses. Many of these organisms, as with crop plants, are being engineered for applications in agriculture, to control plant insect pests or diseases. This paper reviews the genetically modified non-plant organisms that have been the subject of permit approvals for environmental release by the United States Department of Agriculture/Animal and Plant Health Inspection Service since the US began regulating genetically modified organisms. This is an indication of the breadth and progress of research in the area of non-plant genetically modified organisms. This review includes three examples of promising research on non-plant genetically modified organisms for application in agriculture: (1) insects for insect pest control using improved vector systems; (2) fungal pathogens of insects to control insect pests; and (3) virus for use as transient-expression vectors for disease control in plants.     

Biosafety management and commercial use of genetically modified crops in China

As a developing country with relatively limited arable land, China is making great efforts for development and use of genetically modified (GM) crops to boost agricultural productivity. Many GM crop varieties have been developed in China in recent years; in particular, China is playing a leading role in development of insect-resistant GM rice lines. To ensure the safe use of GM crops, biosafety risk assessments are required as an important part of the regulatory oversight of such products. With over 20 years of nationwide promotion of agricultural biotechnology, a relatively well-developed regulatory system for risk assessment and management of GM plants has been developed that establishes a firm basis for safe use of GM crops. So far, a total of seven GM crops involving ten events have been approved for commercial planting, and 5 GM crops with a total of 37 events have been approved for import as processing material in China. However, currently only insect-resistant Bt cotton and disease-resistant papaya have been commercially planted on a large scale. The planting of Bt cotton and disease-resistant papaya have provided efficient protection against cotton bollworms and Papaya ringspot virus (PRSV), respectively. As a consequence, chemical application to these crops has been significantly reduced, enhancing farm income while reducing human and non-target organism exposure to toxic chemicals. This article provides useful information for the colleagues, in particular for them whose mother tongue is not Chinese, to clearly understand the biosafety regulation and commercial use of genetically modified crops in China.     

Regulatory and associated political issues with respect to Bt transgenic maize in the European union

Legislation at the national level in Europe as well as that developed by the European Union (EU) generally permits release and commercialization of genetically modified organisms (GMOs). However, only 10 plant/event combinations were registered as of 2002: three maize events (Bt176, Mon810, and Bt11), with the other seven divided among carnation (3), oil-seed rape (2), tobacco (1), and raddiccio (1). Of these, only one maize event (Bt176) has been registered as a legal variety, and this was in Spain, where 22,000ha have been planted annually since 1998. In this paper, we first provide an overview on the complexity of EU GMO legislation. Then we discuss the minor role that results of EU-funded biosafety research have had on governmental policy. Finally, we provide information about initiatives for post-commercialization monitoring plans of Bt maize in Europe. As a result of the slow progress to date, we conclude that commercialization of GMOs will be seriously delayed in the EU for the next several years.     

Relevance of genetically modified crops in light of future environmental and legislative challenges to the agri-environment

A key challenge for countries like Ireland up to 2030 is to produce sufficient supplies of food, feed and fuel, without compromising on public health or negatively impacting the environment. As we progress through the technology era, certain agricultural technologies [e.g. genetically modified (GM) crops] have been championed to maximise production while minimising environmental impact. Yet, multiple arguments have been made to counter such a claim, which has led to a polarisation of opinions and a plethora of generic commentaries being made in regard to the impact of this technology. Yet, few studies within the European Union (EU) have conducted a critical needs analysis to assess the potential of specific GM traits in light of issues, such as climate change, increased environmental legislation (e.g. EU Water Framework, Nitrates Directive, proposed reform to the Pesticide Directive and Common Agricultural Policy reform), mitigating biodiversity loss and sustainable biofuel production. The goal of this study is to collate a register of GM traits such that a list of potential GM crops could be prioritised against the backdrop of the challenges facing the tillage sector. Clearly, the crops with the most significant potential for genetic modification are those that are grown widely and/or receive high applications of pesticides and fertilisers (e.g. potato, wheat, barley and maize). GM traits with significant agronomic potential include late blight resistant potato, Fusarium head blight resistant wheat and Septoria resistant wheat and herbicide‐tolerant winter oilseed rape and maize. Following on from these, crops with enhanced nitrogen‐use efficiency could provide significant input to the tillage sector in light of EU‐based restrictions on nitrogen usage, crops with elevated protein content could offset the costs of imported animal feed and crops with modified oil content/lignocellulose composition could assist in biodiesel/bioenergy production at a regional level. This study is relevant to other European countries that cultivate similar crops and like Ireland, are facing multiple challenges to their tillage sector in the near future.     

Biosynthesis of compatible solutes in rhizobial strains isolated from <Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> nodules in Tunisian fields

Background  

Associated with appropriate crop and soil management, inoculation of legumes with microbial biofertilizers can improve food legume yield and soil fertility and reduce pollution by inorganic fertilizers. Rhizospheric bacteria are subjected to osmotic stress imposed by drought and/or NaCl, two abiotic constraints frequently found in semi-arid lands. Osmostress response in bacteria involves the accumulation of small organic compounds called compatible solutes. Whereas most studies on rhizobial osmoadaptation have focussed on the model species Sinorhizobium meliloti, little is known on the osmoadaptive mechanisms used by native rhizobia, which are good sources of inoculants. In this work, we investigated the synthesis and accumulations of compatible solutes by four rhizobial strains isolated from root nodules of Phaseolus vulgaris in Tunisia, as well as by the reference strain Rhizobium tropici CIAT 899^T.     

Gene stacking in transgenic plants--the challenge for 21st century plant biotechnology

One of the major technical hurdles impeding the advance of plant genetic engineering and biotechnology is the fact that the expression or manipulation of multiple genes in plants is still difficult to achieve. Although a small proportion of commercial genetically modified (GM) crops present 'stacked' or 'pyramided' traits, only a handful of products have been developed by introducing three or more novel genes. On the research front, a variety of conventional and more novel methods have been employed to introduce multiple genes into plants, but all techniques suffer from certain drawbacks. In this review, the potential and problems of these various techniques and strategies are discussed, and the prospects for improving these technologies in the future are presented.