Synthetic microbe-to-plant communication channels.

Plants and microbes communicate to collaborate to stop pests, scavenge nutrients, and react to environmental change. Microbiota consisting of thousands of species interact with each other and plants using a large chemical language that is interpreted by complex regulatory networks. In this work, we develop modular interkingdom communication channels, enabling bacteria to convey environmental stimuli to plants. We introduce a "sender device" in Pseudomonas putida and Klebsiella pneumoniae, that produces the small molecule p-coumaroyl-homoserine lactone (pC-HSL) when the output of a sensor or circuit turns on. This molecule triggers a "receiver device" in the plant to activate gene expression. We validate this system in Arabidopsis thaliana and Solanum tuberosum (potato) grown hydroponically and in soil, demonstrating its modularity by swapping bacteria that process different stimuli, including IPTG, aTc and arsenic. Programmable communication channels between bacteria and plants will enable microbial sentinels to transmit information to crops and provide the building blocks for designing artificial consortia.

Generation, analysis, and transformation of macro-chloroplast Potato (Solanum tuberosum) lines for chloroplast biotechnology.

Chloroplast biotechnology is a route for novel crop metabolic engineering. The potential bio-confinement of transgenes, the high protein expression and the possibility to organize genes into operons represent considerable advantages that make chloroplasts valuable targets in agricultural biotechnology. In the last 3 decades, chloroplast genomes from a few economically important crops have been successfully transformed. The main bottlenecks that prevent efficient transformation in a greater number of crops include the dearth of proven selectable marker gene-selection combinations and tissue culture methods for efficient regeneration of transplastomic plants. The prospects of increasing organelle size are attractive from several perspectives, including an increase in the surface area of potential targets. As a proof-of-concept, we generated Solanum tuberosum (potato) macro-chloroplast lines overexpressing the tubulin-like GTPase protein gene FtsZ1 from Arabidopsis thaliana. Macro-chloroplast lines exhibited delayed growth at anthesis; however, at the time of harvest there was no significant difference in height between macro-chloroplast and wild-type lines. Macro-chloroplasts were successfully transformed by biolistic DNA-delivery and efficiently regenerated into homoplasmic transplastomic lines. We also demonstrated that macro-chloroplasts accumulate the same amount of heterologous protein than wild-type organelles, confirming efficient usage in plastid engineering. Advantages and limitations of using enlarge compartments in chloroplast biotechnology are discussed.

MoChlo: A Versatile, Modular Cloning Toolbox for Chloroplast Biotechnology.

Plant synthetic biology is a rapidly evolving field with new tools constantly emerging to drive innovation. Of particular interest is the application of synthetic biology to chloroplast biotechnology to generate plants capable of producing new metabolites, vaccines, biofuels, and high-value chemicals. Progress made in the assembly of large DNA molecules, composing multiple transcriptional units, has significantly aided in the ability to rapidly construct novel vectors for genetic engineering. In particular, Golden Gate assembly has provided a facile molecular tool for standardized assembly of synthetic genetic elements into larger DNA constructs. In this work, a complete modular chloroplast cloning system, MoChlo, was developed and validated for fast and flexible chloroplast engineering in plants. A library of 128 standardized chloroplast-specific parts (47 promoters, 38 5' untranslated regions [5'UTRs], nine promoter:5'UTR fusions, 10 3'UTRs, 14 genes of interest, and 10 chloroplast-specific destination vectors) were mined from the literature and modified for use in MoChlo assembly, along with chloroplast-specific destination vectors. The strategy was validated by assembling synthetic operons of various sizes and determining the efficiency of assembly. This method was successfully used to generate chloroplast transformation vectors containing up to seven transcriptional units in a single vector (∼10.6-kb synthetic operon). To enable researchers with limited resources to engage in chloroplast biotechnology, and to accelerate progress in the field, the entire kit, as described, is available through Addgene at minimal cost. Thus, the MoChlo kit represents a valuable tool for fast and flexible design of heterologous metabolic pathways for plastid metabolic engineering.

Pollen-mediated gene flow from transgenic to non-transgenic switchgrass (Panicum virgatum L.) in the field.

BACKGROUND: Switchgrass is C4 perennial grass species that is being developed as a cellulosic bioenergy feedstock. It is wind-pollinated and considered to be an obligate outcrosser. Genetic engineering has been used to alter cell walls for more facile bioprocessing and biofuel yield. Gene flow from transgenic cultivars would likely be of regulatory concern. In this study we investigated pollen-mediated gene flow from transgenic to nontransgenic switchgrass in a 3-year field experiment performed in Oliver Springs, Tennessee, U.S.A. using a modified Nelder wheel design. The planted area (0.6 ha) contained sexually compatible pollen source and pollen receptor switchgrass plants. One hundred clonal switchgrass 'Alamo' plants transgenic for an orange-fluorescent protein (OFP) and hygromycin resistance were used as the pollen source; whole plants, including pollen, were orange-fluorescent. To assess pollen movement, pollen traps were placed at 10 m intervals from the pollen-source plot in the four cardinal directions extending to 20 m, 30 m, 30 m, and 100 m to the north, south, west, and east, respectively. To assess pollination rates, nontransgenic 'Alamo 2' switchgrass clones were planted in pairs adjacent to pollen traps. RESULTS: In the eastward direction there was a 98% decrease in OFP pollen grains from 10 to 100 m from the pollen-source plot (Poisson regression, F1,8 = 288.38, P < 0.0001). At the end of the second and third year, 1,820 F1 seeds were collected from pollen recipient-plots of which 962 (52.9%) germinated and analyzed for their transgenic status. Transgenic progeny production detected in each pollen-recipient plot decreased with increased distance from the edge of the transgenic plot (Poisson regression, F1,15 = 12.98, P < 0.003). The frequency of transgenic progeny detected in the eastward plots (the direction of the prevailing wind) ranged from 79.2% at 10 m to 9.3% at 100 m. CONCLUSIONS: In these experiments we found transgenic pollen movement and hybridization rates to be inversely associated with distance. However, these data suggest pollen-mediated gene flow is likely to occur up to, at least, 100 m. This study gives baseline data useful to determine isolation distances and other management practices should transgenic switchgrass be grown commercially in relevant environments.

Antimicrobial activity of Hibiscus sabdariffa aqueous extracts against Escherichia coli O157:H7 and Staphylococcus aureus in a microbiological medium and milk of various fat concentrations.

Hibiscus sabdariffa L. calyces are widely used in the preparation of beverages. The calyces contain compounds that exhibit antimicrobial activity, yet little research has been conducted on their possible use in food systems as antimicrobials. Aqueous extracts prepared from the brand "Mi Costenita" were sterilized by membrane filtration (0.22-µm pore size) or autoclaving (121 °C, 30 min) and tested for antimicrobial activity against the foodborne pathogens Escherichia coli O157:H7 strains ATCC 43894 and Cider and Staphylococcus aureus strains SA113 and ATCC 27708 in a microbiological medium and ultrahigh-temperature-processed milk with various fat percentages. Extracts heated by autoclaving exhibited greater activity than did filtered extracts in a microbiological medium. Against E. coli, results of 20 mg/ml filtered extract were not different from those of the control, whereas autoclaved extracts reduced viable cells ca. 3 to 4 log CFU/ml. At 60 mg/ml, both extracts inactivated cells after 24 h. There were reduced populations of both strains of S. aureus (ca. 2.7 and 3 log CFU/ml, respectively) after 24 h of incubation in 40 mg/ml filtered extracts. When grown in autoclaved extracts at 40 mg/ml, both strains of S. aureus were inactivated after 9 h. Autoclaved extracts had decreased anthocyanin content (2.63 mg/liter) compared with filtered extracts (14.27 mg/liter), whereas the phenolic content (48.7 and 53.8 mg/g) remained similar for both treatments. Autoclaved extracts were then tested for activity in milk at various fat concentrations (skim [<0.5%], 1%, 2%, and whole [>3.25%]) against a 1:1 mixture of the two strains of E. coli O157:H7 and a 1:1 mixture of the two strains of S. aureus. Extracts at 40 mg/ml inactivated S. aureus after 168 h in skim and whole milk, and E. coli was inactivated after 96 h in 60 mg/ml extract in all fat levels. These findings show the potential use of Hibiscus extracts to prevent the growth of pathogens in foods and beverages.

An orange fluorescent protein tagging system for real-time pollen tracking.

BACKGROUND: Monitoring gene flow could be important for future transgenic crops, such as those producing plant-made-pharmaceuticals (PMPs) in open field production. A Nicotiana hybrid (Nicotiana. tabacum × Nicotiana glauca) shows limited male fertility and could be used as a bioconfined PMP platform. Effective assessment of gene flow from these plants is augmented with methods that utilize fluorescent proteins for transgenic pollen identification. RESULTS: We report the generation of a pollen tagging system utilizing an orange fluorescent protein to monitor pollen flow and as a visual assessment of transgene zygosity of the parent plant. This system was created to generate a tagged Nicotiana hybrid that could be used for the incidence of gene flow. Nicotiana tabacum 'TN 90' and Nicotiana glauca were successfully transformed via Agrobacterium tumefaciens to express the orange fluorescent protein gene, tdTomato-ER, in pollen and a green fluorescent protein gene, mgfp5-er, was expressed in vegetative structures of the plant. Hybrids were created that utilized the fluorescent proteins as a research tool for monitoring pollen movement and gene flow. Manual greenhouse crosses were used to assess hybrid sexual compatibility with N. tabacum, resulting in seed formation from hybrid pollination in 2% of crosses, which yielded non-viable seed. Pollen transfer to the hybrid formed seed in 19% of crosses and 10 out of 12 viable progeny showed GFP expression. CONCLUSION: The orange fluorescent protein is visible when expressed in the pollen of N. glauca, N. tabacum, and the Nicotiana hybrid, although hybrid pollen did not appear as bright as the parent lines. The hybrid plants, which show limited ability to outcross, could provide bioconfinement with the benefit of detectable pollen using this system. Fluorescent protein-tagging could be a valuable tool for breeding and in vivo ecological monitoring.

Assessing the bioconfinement potential of a Nicotiana hybrid platform for use in plant molecular farming applications.

BACKGROUND: The introduction of pharmaceutical traits in tobacco for commercial production could benefit from the utilization of a transgene bioconfinement system. It has been observed that interspecific F1Nicotiana hybrids (Nicotiana tabacum × Nicotiana glauca) are sterile and thus proposed that hybrids could be suitable bioconfined hosts for biomanufacturing. We genetically tagged hybrids with green fluorescent protein (GFP), which was used as a visual marker to enable gene flow tracking and quantification for field and greenhouse studies. GFP was used as a useful proxy for pharmaceutical transgenes. RESULTS: Analysis of DNA content revealed significant genomic downsizing of the hybrid relative to that of N. tabacum. Hybrid pollen was capable of germination in vitro, albeit with a very low frequency and with significant differences between plants. In two field experiments, one each in Tennessee and Kentucky, we detected outcrossing at only one location (Tennessee) at 1.4%. Additionally, from 50 hybrid plants at each field site, formation of 84 and 16 seed was observed, respectively. Similar conclusions about hybrid fertility were drawn from greenhouse crosses. In terms of above-ground biomass, the hybrid yield was not significantly different than that of N. tabacum in the field. CONCLUSION: N. tabacum × N. glauca hybrids show potential to contribute to a bioconfinement- and biomanufacturing host system. Hybrids exhibit extremely low fertility with no difference of green biomass yields relative to N. tabacum. In addition, hybrids are morphologically distinguishable from tobacco allowing for identity preservation. This hybrid system for biomanufacturing would optimally be used where N. glauca is not present and in physical isolation of N. tabacum production to provide total bioconfinement.

Isolation and chemical analysis of nanoparticles from English ivy (Hedera helix L.).

Bio-inspiration for novel adhesive development has drawn increasing interest in recent years with the discovery of the nanoscale morphology of the gecko footpad and mussel adhesive proteins. Similar to these animal systems, it was discovered that English ivy (Hedera helix L.) secretes a high strength adhesive containing uniform nanoparticles. Recent studies have demonstrated that the ivy nanoparticles not only contribute to the high strength of this adhesive, but also have ultraviolet (UV) protective abilities, making them ideal for sunscreen and cosmetic fillers, and may be used as nanocarriers for drug delivery. To make these applications a reality, the chemical nature of the ivy nanoparticles must be elucidated. In the current work, a method was developed to harvest bulk ivy nanoparticles from an adventitious root culture system, and the chemical composition of the nanoparticles was analysed. UV/visible spectroscopy, inductively coupled plasma mass spectrometry, Fourier transform infrared spectroscopy and electrophoresis were used in this study to identify the chemical nature of the ivy nanoparticles. Based on this analysis, we conclude that the ivy nanoparticles are proteinaceous.

Bio-synthesis of gold nanoparticles using English ivy (Hedera helix).

Gold nanoparticles (AuNPs) have drawn significant interest in recent years due to unique properties that make them advantageous in biomedical applications, including drug delivery and tissue engineering. In this paper, we have developed multiple methods for the synthesis of AuNPs using English ivy as the substrate. In the first method, we have used actively growing English ivy shoots to develop a sustainable system for the production of ivy nanoparticles. The second method was developed using the extract from the adventitious roots of English ivy. The nanoparticles formed using both methods were compared to determine the size distribution, morphology, and chemical structure of the nanoparticles. Characterization of the AuNPs was conducted using ultraviolet-visible (UV-Vis) spectroscopy, dynamic light scattering (DLS), scanning electron microscopy (SEM), and energy-dispersive X-ray spectroscopy (EDS). In addition to the structural differences between the AuNPs formed from the different methods, details of the methods in terms of yield, duration, and speed of AuNP formation are also discussed. Further, this paper will show that AuNPs formed using both methods demonstrated efficient uptake in mammalian cells, which provides the potential for biomedical applications. The two methods developed through this research for eco-friendly synthesis of AuNPs present an alternative to traditional chemical synthesis methods.

Characterization of physicochemical properties of ivy nanoparticles for cosmetic application.

BACKGROUND: Naturally occurring nanoparticles isolated from English ivy (Hedera helix) have previously been proposed as an alternative to metallic nanoparticles as sunscreen fillers due to their effective UV extinction property, low toxicity and potential biodegradability. METHODS: This study focused on analyzing the physicochemical properties of the ivy nanoparticles, specifically, those parameters which are crucial for use as sunscreen fillers, such as pH, temperature, and UV irradiation. The visual transparency and cytotoxicity of ivy nanoparticles were also investigated comparing them with other metal oxide nanoparticles. RESULTS: Results from this study demonstrated that, after treatment at 100°C, there was a clear increase in the UV extinction spectra of the ivy nanoparticles caused by the partial decomposition. In addition, the UVA extinction spectra of the ivy nanoparticles gradually reduced slightly with the decrease of pH values in solvents. Prolonged UV irradiation indicated that the influence of UV light on the stability of the ivy nanoparticle was limited and time-independent. Compared to TiO2 and ZnO nanoparticles, ivy nanoparticles showed better visual transparency. Methylthiazol tetrazolium assay demonstrated that ivy nanoparticles exhibited lower cytotoxicity than the other two types of nanoparticles. Results also suggested that protein played an important role in modulating the three-dimensional structure of the ivy nanoparticles. CONCLUSIONS: Based on the results from this study it can be concluded that the ivy nanoparticles are able to maintain their UV protective capability at wide range of temperature and pH values, further demonstrating their potential as an alternative to replace currently available metal oxide nanoparticles in sunscreen applications.

Nanoparticle biofabrication using English ivy (Hedera helix).

BACKGROUND: English ivy (Hedera helix) is well known for its adhesive properties and climbing ability. Essential to its ability to adhere to vertical surfaces is the secretion of a nanocomposite adhesive containing spherical nanoparticles, 60-85 nm in diameter, produced exclusively by root hairs present on adventitious roots. These organic nanoparticles have shown promise in biomedical and cosmetic applications, and represent a safer alternative to metal oxide nanoparticles currently available. RESULTS: It was discovered that the maximum adventitious root production was achieved by a 4 h application of 1 mg/ml indole-3 butyric acid (IBA) to juvenile English ivy shoot segments cultured in custom vessels. After incubation of the shoots under continuous light at 83 µmol/m2 s at 20°C for 2 weeks, the adventitious roots were harvested from the culture system and it was possible to isolate 90 mg of dry weight nanoparticles per 12 g of roots. The nanoparticle morphology was characterized by atomic force microscopy, and found to be similar to previous studies. CONCLUSIONS: An enhanced system for the production of English ivy adventitious roots and their nanoparticles by modifying GA7 Magenta boxes and identifying the optimal concentration of IBA for adventitious root growth was developed. This system is the first such platform for growing and harvesting organic nanoparticles from plants, and represents an important step in the development of plant-based nanomanufacturing. It is a significant improvement on the exploitation of plant systems for the formation of metallic nanoparticles, and represents a pathway for the generation of bulk ivy nanoparticles for translation into biomedical applications.

Aqueous extracts of yerba mate (Ilex paraguariensis) as a natural antimicrobial against Escherichia coli O157:H7 in a microbiological medium and pH 6.0 apple juice.

Ilex paraguariensis is popularly used in the preparation of a tea infusion (yerba mate), most commonly produced and consumed in the South American countries of Uruguay, Paraguay, Argentina, and Brazil. In this study, aqueous extracts of commercial tea, derived from the holly plant species I. paraguariensis were evaluated for their ability to inhibit or inactivate Escherichia coli O157:H7 in a microbiological medium and modified apple juice. Dialyzed, lyophilized aqueous extracts were screened for antimicrobial activity against E. coli O157:H7 strains ATCC 43894 and 'Cider' in tryptic soy broth (TSB) and apple juice (adjusted to pH 6.0 to allow for growth of the bacterium). A mixture of the two strains was used as the inoculum when apple juice was used as the medium. MBCs were determined to be ca. 5 and 10 mg/ml for ATCC 43894 and 'Cider', respectively, in TSB. Higher concentrations of the extract were required to inactivate E. coli O157:H7 in pH-adjusted apple juice. An approximate 4.5-log reduction was observed for E. coli O157:H7 treated with 40 mg/ml extract. It was concluded that aqueous extracts from commercial yerba mate have potential to be used as antimicrobials in foods and beverages against pathogenic E. coli O157:H7.

Gene flow matters in switchgrass (Panicum virgatum L.), a potential widespread biofuel feedstock.

There currently exists a large push for the use, improvement, and expansion via landscape modification of dedicated biofuel crops (feedstocks) in the United States and in many parts of the world. Ecological concerns have been voiced because many biofuel feedstocks exhibit characteristics associated with invasiveness, and due to potential negative consequences of agronomic genes in native wild populations. Seed purity concerns for biofuel feedstock cultivars whose seeds would be harvested in agronomic fields also exist from the agribusiness sector. The common thread underlying these concerns, which have regulatory implications, is gene flow; thus detailed knowledge of gene flow in biofuel crop plants is important in the formulation of environmental risk management plans. Here, we synthesize the current state of knowledge of gene flow in an exemplary biofuel crop, switchgrass (Panicum virgatum L.), which is native to eastern North America and is currently experiencing conventional and technological advances in biomass yields and ethanol production. Surprisingly little is known regarding aspects of switchgrass pollen flow and seed dispersal, and whether native populations of conspecific or congeneric relatives will readily cross with current agronomic switchgrass cultivars. We pose that filling these important gaps will be required to confront the sustainability challenges of widespread planting of biofuel feedstocks.

An efficient and rapid transgenic pollen screening and detection method using flow cytometry.

Assaying for transgenic pollen, a major vector of transgene flow, provides valuable information and essential data for the study of gene flow and assessing the effectiveness of transgene containment. Most studies have employed microscopic screening methods or progeny analyses to estimate the frequency of transgenic pollen. However, these methods are time-consuming and laborious when large numbers of pollen grains must be analyzed to look for rare transgenic pollen grains. Thus, there is an urgent need for the development of a simple, rapid, and high throughput analysis method for transgenic pollen analysis. In this study, our objective was to determine the accuracy of using flow cytometry technology for transgenic pollen quantification in practical application where transgenic pollen is not frequent. A suspension of non-transgenic tobacco pollen was spiked with a known amount of verified transgenic tobacco pollen synthesizing low or high amounts of green fluorescent protein (GFP). The flow cytometric method detected approximately 75% and 100% of pollen grains synthesizing low and high amounts of GFP, respectively. The method is rapid, as it is able to count 5000 pollen grains per minute-long run. Our data indicate that this flow cytometric method is useful to study gene flow and assessment of transgene containment.

Keeping the genie in the bottle: transgene biocontainment by excision in pollen.

Gene flow from transgenic plants is an environmental and regulatory concern. While biocontainment might be achieved using male sterility or transgenic mitigation tools, we believe that perhaps the optimal solution might be simply to remove transgenes from pollen. Male sterility might not be ideal for many pollinators, and might not be implementable using standardized genes. Transgenic mitigation might not be useful to control conspecific gene flow (e.g. crop to crop), and relies on competition and not biocontainment per se. Site-specific recombination systems could allow highly efficient excision of transgenes in pollen to eliminate, or at least minimize, unwanted transgene movement via pollen dispersal. There are other potential biotechnologies, such as zinc finger nucleases, that could be also used for transgene excision.

'GM-gene-deletor': fused loxP-FRT recognition sequences dramatically improve the efficiency of FLP or CRE recombinase on transgene excision from pollen and seed of tobacco plants.

Pollen- and seed-mediated transgene flow is a concern in plant biotechnology. We report here a highly efficient 'genetically modified (GM)-gene-deletor' system to remove all functional transgenes from pollen, seed or both. With the three pollen- and/or seed-specific gene promoters tested, the phage CRE/loxP or yeast FLP/FRT system alone was inefficient in excising transgenes from tobacco pollen and/or seed, with no transgenic event having 100% efficiency. When loxP-FRT fusion sequences were used as recognition sites, simultaneous expression of both FLP and CRE reduced the average excision efficiency, but the expression of FLP or CRE alone increased the average excision efficiency, with many transgenic events being 100% efficient based on more than 25,000 T(1) progeny examined per event. The 'GM-gene-deletor' reported here may be used to produce 'non-transgenic' pollen and/or seed from transgenic plants and to provide a bioconfinement tool for transgenic crops and perennials, with special applicability towards vegetatively propagated plants and trees.

Expression of green fluorescent protein in pollen of oilseed rape (Brassica napus L.) and its utility for assessing pollen movement in the field.

Transgene movement via pollen is an important component of gene flow from transgenic plants. Here, we present proof-of-concept studies that demonstrate the monitoring of short distant movement of pollen expressing a genetically encoded fluorescent tag in oilseed rape (Brassica napus L. cv. Westar). Transgenic oilseed rape plants were produced using Agrobacterium-mediated transformation method with the pBINDC1 construct containing a green fluorescent protein (GFP) variant, mGFP5-ER, under the control of the pollen-specific LAT59 promoter from tomato. Transgenic pollen was differentiated from non-transgenic pollen in vivo by a unique spectral signature, and was shown to be an effective tool to monitor pollen movement in the greenhouse and field. GFP-tagged pollen also served as a practical marker to determine the zygosity of plants. In a greenhouse pollen flow study, more pollen was captured at closer distances from the source plant plot with consistent wind generated by a fan. Under field conditions, GFP transgenic pollen grains were detected up to a distance of 15 m, the farthest distance from source plants assayed. GFP-tagged pollen was easily distinguishable from non-transgenic pollen using an epifluorescence microscope.

Transgene dispersal through pollen.

Techniques used for the transfer of novel genes into host plant genomes have created new possibilities for crop improvement. The implementation of transgenic crop species into agriculture has introduced the possibility of transgene escape into the environment via pollen dispersal. Although the movement of pollen is a critical step in transgene escape, there is currently no system to monitor transgenic pollen movement under field conditions. The development of an effective in vivo monitoring system suitable for use under field conditions is needed for research and commercial purposes so potential risks can be quantified and evaluated. This chapter describes the development of a model system using green fluorescent protein (GFP) expression in pollen as a marker to monitor pollen distribution patterns. A pollen specific promoter was used to express the GFP gene in tobacco (Nicotiana tabacum L.). GFP was visualized in pollen and growing pollen tubes using fluorescent microscopy. Furthermore, the goal of this research was to compare the dynamics of pollen movement with that of gene flow by using another method of whole plant expression of GFP to estimate out-crossing frequencies by progeny analysis. Pollen movement and gene flow were quantified under field conditions. Pollen traps were collected and screened for presence of GFP-tagged pollen using fluorescence microscopy. Progeny from wild type plants were screened with a hand held ultraviolet light for detection of the GFP phenotype.

Larvicidal Cry proteins from Bacillus thuringiensis are released in root exudates of transgenic B. thuringiensis corn, potato, and rice but not of B. thuringiensis canola, cotton, and tobacco.

Larvicidal proteins encoded by cry genes from Bacillus thuringiensis were released in root exudates from transgenic B. thuringiensis corn, rice, and potato but not from B. thuringiensis canola, cotton, and tobacco. Nonsterile soil and sterile hydroponic solution in which B. thuringiensis corn, rice, or potato had been grown were immunologically positive for the presence of the Cry proteins; from B. thuringiensis corn and rice, the soil and solution were toxic to the larva of the tobacco hornworm (Manduca sexta), and from potato, to the larva of the Colorado potato beetle (Leptinotarsa decemlineata), representative lepidoptera and coleoptera, respectively. No toxin was detected immunologically or by larvicidal assay in soil or hydroponic solution in which B. thuringiensis canola, cotton, or tobacco, as well as all near-isogenic non-B. thuringiensis plant counterparts or no plants, had been grown. All plant species had the cauliflower mosaic virus (CaMV) 35S promoter, except rice, which had the ubiquitin promoter from maize. The reasons for the differences between species in the exudation from roots of the toxins are not known. The released toxins persisted in soil as the result of their binding on surface-active particles (e.g. clay minerals, humic substances), which reduced their biodegradation. The release of the toxins in root exudates could enhance the control of target insect pests, constitute a hazard to nontarget organisms, and/or increase the selection of toxin-resistant target insects.