Modeling gene flow distribution within conventional fields and development of a simplified sampling method to quantify adventitious GM contents in maize.

Genetically modified (GM) crops have been commercially grown for two decades. GM maize is one of 3 species with the highest acreage and specific events. Many countries established a mandatory labeling of products containing GM material, with thresholds for adventitious presence, to support consumers' freedom of choice. In consequence, coexistence systems need to be introduced to facilitate commercial culture of GM and non-GM crops in the same agricultural area. On modeling adventitious GM cross-pollination distribution within maize fields, we deduced a simple equation to estimate overall GM contents (%GM) of conventional fields, irrespective of its shape and size, and with no previous information on possible GM pollen donor fields. A sampling strategy was designed and experimentally validated in 19 agricultural fields. With 9 samples, %GM quantification requires just one analytical GM determination while identification of the pollen source needs 9 additional analyses. A decision support tool is provided.

Overexpression of a Calcium-Dependent Protein Kinase Confers Salt and Drought Tolerance in Rice by Preventing Membrane Lipid Peroxidation.

The OsCPK4 gene is a member of the complex gene family of calcium-dependent protein kinases in rice (Oryza sativa). Here, we report that OsCPK4 expression is induced by high salinity, drought, and the phytohormone abscisic acid. Moreover, a plasma membrane localization of OsCPK4 was observed by transient expression assays of green fluorescent protein-tagged OsCPK4 in onion (Allium cepa) epidermal cells. Overexpression of OsCPK4 in rice plants significantly enhances tolerance to salt and drought stress. Knockdown rice plants, however, are severely impaired in growth and development. Compared with control plants, OsCPK4 overexpressor plants exhibit stronger water-holding capability and reduced levels of membrane lipid peroxidation and electrolyte leakage under drought or salt stress conditions. Also, salt-treated OsCPK4 seedlings accumulate less Na+ in their roots. We carried out microarray analysis of transgenic rice overexpressing OsCPK4 and found that overexpression of OsCPK4 has a low impact on the rice transcriptome. Moreover, no genes were found to be commonly regulated by OsCPK4 in roots and leaves of rice plants. A significant number of genes involved in lipid metabolism and protection against oxidative stress appear to be up-regulated by OsCPK4 in roots of overexpressor plants. Meanwhile, OsCPK4 overexpression has no effect on the expression of well-characterized abiotic stress-associated transcriptional regulatory networks (i.e. ORYZA SATIVA DEHYDRATION-RESPONSIVE ELEMENT BINDING PROTEIN1 and ORYZA SATIVA No Apical Meristem, Arabidopsis Transcription Activation Factor1-2, Cup-Shaped Cotyledon6 genes) and LATE EMBRYOGENESIS ABUNDANT genes in their roots. Taken together, our data show that OsCPK4 functions as a positive regulator of the salt and drought stress responses in rice via the protection of cellular membranes from stress-induced oxidative damage.

Constitutive expression of transgenes encoding derivatives of the synthetic antimicrobial peptide BP100: impact on rice host plant fitness.

BACKGROUND: The Biopeptide BP100 is a synthetic and strongly cationic α-helical undecapeptide with high, specific antibacterial activity against economically important plant-pathogenic bacteria, and very low toxicity. It was selected from a library of synthetic peptides, along with other peptides with activities against relevant bacterial and fungal species. Expression of the BP100 series of peptides in plants is of major interest to establish disease-resistant plants and facilitate molecular farming. Specific challenges were the small length, peptide degradation by plant proteases and toxicity to the host plant. Here we approached the expression of the BP100 peptide series in plants using BP100 as a proof-of-concept. RESULTS: Our design considered up to three tandemly arranged BP100 units and peptide accumulation in the endoplasmic reticulum (ER), analyzing five BP100 derivatives. The ER retention sequence did not reduce the antimicrobial activity of chemically synthesized BP100 derivatives, making this strategy possible. Transformation with sequences encoding BP100 derivatives (bp100der) was over ten-fold less efficient than that of the hygromycin phosphotransferase (hptII) transgene. The BP100 direct tandems did not show higher antimicrobial activity than BP100, and genetically modified (GM) plants constitutively expressing them were not viable. In contrast, inverted repeats of BP100, whether or not elongated with a portion of a natural antimicrobial peptide (AMP), had higher antimicrobial activity, and fertile GM rice lines constitutively expressing bp100der were produced. These GM lines had increased resistance to the pathogens Dickeya chrysanthemi and Fusarium verticillioides, and tolerance to oxidative stress, with agronomic performance comparable to untransformed lines. CONCLUSIONS: Constitutive expression of transgenes encoding short cationic α-helical synthetic peptides can have a strong negative impact on rice fitness. However, GM plants expressing, for example, BP100 based on inverted repeats, have adequate agronomic performance and resistant phenotypes as a result of a complex equilibrium between bp100der toxicity to plant cells, antimicrobial activity and transgene-derived plant stress response. It is likely that these results can be extended to other peptides with similar characteristics.

Expression of the maize ZmGF14-6 gene in rice confers tolerance to drought stress while enhancing susceptibility to pathogen infection.

14-3-3 proteins are found in all eukaryotes where they act as regulators of diverse signalling pathways associated with a wide range of biological processes. In this study the functional characterization of the ZmGF14-6 gene encoding a maize 14-3-3 protein is reported. Gene expression analyses indicated that ZmGF14-6 is up-regulated by fungal infection and salt treatment in maize plants, whereas its expression is down-regulated by drought stress. It is reported that rice plants constitutively expressing ZmGF14-6 displayed enhanced tolerance to drought stress which was accompanied by a stronger induction of drought-associated rice genes. However, rice plants expressing ZmGF14-6 either in a constitutive or under a pathogen-inducible regime showed a higher susceptibility to infection by the fungal pathogens Fusarium verticillioides and Magnaporthe oryzae. Under infection conditions, a lower intensity in the expression of defence-related genes occurred in ZmGF14-6 rice plants. These findings support that ZmGF14-6 positively regulates drought tolerance in transgenic rice while negatively modulating the plant defence response to pathogen infection. Transient expression assays of fluorescently labelled ZmGF14-6 protein in onion epidermal cells revealed a widespread distribution of ZmGF14-6 in the cytoplasm and nucleus. Additionally, colocalization experiments of fluorescently labelled ZmGF14-6 with organelle markers, in combination with cell labelling with the endocytic tracer FM4-64, revealed a subcellular localization of ZmGF14-6 in the early endosomes. Taken together, these results improve our understanding of the role of ZmGF14-6 in stress signalling pathways, while indicating that ZmGF14-6 inversely regulates the plant response to biotic and abiotic stresses.

Only half the transcriptomic differences between resistant genetically modified and conventional rice are associated with the transgene.

Besides the intended effects that give a genetically modified (GM) plant the desired trait, unintended differences between GM and non-GM comparable plants may also occur. Profiling technologies allow their identification, and a number of examples demonstrating that unintended effects are limited and diverse have recently been reported. Both from the food safety aspect and for research purposes, it is important to discern unintended changes produced by the transgene and its expression from those that may be attributed to other factors. Here, we show differential expression of around 0.40% transcriptome between conventional rice var. Senia and Senia-afp constitutively expressing the AFP antifungal protein. Analysis of one-fifth of the regulated sequences showed that around 35% of the unintended effects could be attributed to the process used to produce GM plants, based on in vitro tissue culture techniques. A further ∼15% were event specific, and their regulation was attributed to host gene disruption and genome rearrangements at the insertion site, and effects on proximal sequences. Thus, only around half the transcriptional unintended effects could be associated to the transgene itself. A significant number of changes in Senia-afp and Senia are part of the plant response to stress conditions, and around half the sequences for which up-regulation was attributed to the transgene were induced in conventional (but not transgenic) plants after wounding. Unintended effects might, as such, putatively result in widening the self-resistance characteristics because of the transgene in GM plants.

Natural variation explains most transcriptomic changes among maize plants of MON810 and comparable non-GM varieties subjected to two N-fertilization farming practices.

The introduction of genetically modified organisms (GMO) in many countries follows strict regulations to ensure that only safety-tested products are marketed. Over the last few years, targeted approaches have been complemented by profiling methods to assess possible unintended effects of transformation. Here we used a commercial (Affymertix) microarray platform (i.e. allowing assessing the expression of approximately 1/3 of the genes of maize) to evaluate transcriptional differences between commercial MON810 GM maize and non-transgenic crops in real agricultural conditions, in a region where about 70% of the maize grown was MON810. To consider natural variation in gene expression in relation to biotech plants we took two common MON810/non-GM variety pairs as examples, and two farming practices (conventional and low-nitrogen fertilization). MON810 and comparable non-GM varieties grown in the field have very low numbers of sequences with differential expression, and their identity differs among varieties. Furthermore, we show that the differences between a given MON810 variety and the non-GM counterpart do not appear to depend to any major extent on the assayed cultural conditions, even though these differences may slightly vary between the conditions. In our study, natural variation explained most of the variability in gene expression among the samples. Up to 37.4% was dependent upon the variety (obtained by conventional breeding) and 31.9% a result of the fertilization treatment. In contrast, the MON810 GM character had a very minor effect (9.7%) on gene expression in the analyzed varieties and conditions, even though similar cryIA(b) expression levels were detected in the two MON810 varieties and nitrogen treatments. This indicates that transcriptional differences of conventionally-bred varieties and under different environmental conditions should be taken into account in safety assessment studies of GM plants.

Gene expression profiles of MON810 and comparable non-GM maize varieties cultured in the field are more similar than are those of conventional lines.

Maize is a major food crop and genetically modified (GM) varieties represented 24% of the global production in 2007. Authorized GM organisms have been tested for human and environmental safety. We previously used microarrays to compare the transcriptome profiles of widely used commercial MON810 versus near-isogenic varieties and reported differential expression of a small set of sequences in leaves of in vitro cultured plants of AristisBt/Aristis and PR33P67/PR33P66 (Coll et al. 2008). Here we further assessed the significance of these differential expression patterns in plants grown in a real context, i.e. in the field. Most sequences that were differentially expressed in plants cultured in vitro had the same expression values in MON810 and comparable varieties when grown in the field; and no sequence was found to be differentially regulated in the two variety pairs grown in the field. The differential expression patterns observed between in vitro and field culture were similar between MON810 and comparable varieties, with higher divergence between the two conventional varieties. This further indicates that MON810 and comparable non-GM varieties are equivalent except for the introduced character.

Effect of volunteers on maize gene flow.

Regulatory approvals for deliberate release of GM maize events into the environment have lead to real situations of coexistence between GM and non-GM, with some fields being cultivated with GM and conventional varieties in successive seasons. Given the common presence of volunteer plants in maize fields in temperate areas, we investigated the real impact of GM volunteers on the yield of 12 non-GM agricultural fields. Volunteer density varied from residual to around 10% of plants in the field and was largely reduced using certain cultural practices. Plant vigour was low, they rarely had cobs and produced pollen that cross-fertilized neighbour plants only at low--but variable--levels. In the worst-case scenario, the estimated content of GMO was 0.16%. The influence of GM volunteers was not enough to reach the 0.9% adventitious GM threshold but it could potentially contribute to adventitious GM levels, especially at high initial densities (i.e. above 1,000 volunteers/ha).

The Arabidopsis AtNPR1 inversely modulates defense responses against fungal, bacterial, or viral pathogens while conferring hypersensitivity to abiotic stresses in transgenic rice.

The nonexpressor of pathogenesis-related (PR) genes (NPR1) protein plays an important role in mediating defense responses activated by pathogens in Arabidopsis. In rice, a disease-resistance pathway similar to the Arabidopsis NPR1-mediated signaling pathway one has been described. Here, we show that constitutive expression of the Arabidopsis NPR1 (AtNPR1) gene in rice confers resistance against fungal and bacterial pathogens. AtNPR1 exerts its protective effects against fungal pathogens by priming the expression of salicylic acid (SA)-responsive endogenous genes, such as the PR1b, TLP (PR5), PR10, and PBZ1. However, expression of AtNPR1 in rice has negative effects on viral infections. The AtNPR1-expressing rice plants showed a higher susceptibility to infection by the Rice yellow mottle virus (RYMV) which correlated well with a misregulation of RYMV-responsive genes, including expression of the SA-regulated RNA-dependent RNA polymerase 1 gene (OsRDR1). Moreover, AtNPR1 negatively regulates the expression of genes playing a role in the plant response to salt and drought stress (rab21, salT, and dip1), which results in a higher sensitivity of AtNPR1 rice to the two types of abiotic stress. These observations suggest that AtNPR1 has both positive and negative regulatory roles in mediating defense responses against biotic and abiotic stresses.

Lack of repeatable differential expression patterns between MON810 and comparable commercial varieties of maize.

The introduction of genetically modified organisms (GMO) in many countries follows strict regulations to assure that only products that have been safety tested in relation to human health and the environment are marketed. Thus, GMOs must be authorized before use. By complementing more targeted approaches, profiling methods can assess possible unintended effects of transformation. We used microarrays to compare the transcriptome profiles of widely commercialized maize MON810 varieties and their non-GM near-isogenic counterparts. The expression profiles of MON810 seedlings are more similar to those of their corresponding near-isogenic varieties than are the profiles of other lines produced by conventional breeding. However, differential expression of approximately 1.7 and approximately 0.1% of transcripts was identified in two variety pairs (AristisBt/Aristis and PR33P67/PR33P66) that had similar cryIA(b) mRNA levels, demonstrating that commercial varieties of the same event have different similarity levels to their near-isogenic counterparts without the transgene (note that these two pairs also show phenotypic differences). In the tissues, developmental stage and varieties analyzed, we could not identify any gene differentially expressed in all variety-pairs. However, a small set of sequences were differentially expressed in various pairs. Their relation to the transgenesis was not proven, although this is likely to be modulated by the genetic background of each variety.

Sucrose-mediated priming of plant defense responses and broad-spectrum disease resistance by overexpression of the maize pathogenesis-related PRms protein in rice plants.

Expression of pathogenesis-related (PR) genes is part of the plant's natural defense response against pathogen attack. The PRms gene encodes a fungal-inducible PR protein from maize. Here, we demonstrate that expression of PRms in transgenic rice confers broad-spectrum protection against pathogens, including fungal (Magnaporthe oryzae, Fusarium verticillioides, and Helminthosporium oryzae) and bacterial (Erwinia chrysanthemi) pathogens. The PRms-mediated disease resistance in rice plants is associated with an enhanced capacity to express and activate the natural plant defense mechanisms. Thus, PRms rice plants display a basal level of expression of endogenous defense genes in the absence of the pathogen. PRms plants also exhibit stronger and quicker defense responses during pathogen infection. We also have found that sucrose accumulates at higher levels in leaves of PRms plants. Sucrose responsiveness of rice defense genes correlates with the pathogen-responsive priming of their expression in PRms rice plants. Moreover, pretreatment of rice plants with sucrose enhances resistance to M. oryzae infection. Together, these results support a sucrose-mediated priming of defense responses in PRms rice plants which results in broad-spectrum disease resistance.

Assessment of real-time PCR based methods for quantification of pollen-mediated gene flow from GM to conventional maize in a field study.

Maize is one of the main crops worldwide and an increasing number of genetically modified (GM) maize varieties are cultivated and commercialized in many countries in parallel to conventional crops. Given the labeling rules established e.g. in the European Union and the necessary coexistence between GM and non-GM crops, it is important to determine the extent of pollen dissemination from transgenic maize to other cultivars under field conditions. The most widely used methods for quantitative detection of GMO are based on real-time PCR, which implies the results are expressed in genome percentages (in contrast to seed or grain percentages). Our objective was to assess the accuracy of real-time PCR based assays to accurately quantify the contents of transgenic grains in non-GM fields in comparison with the real cross-fertilization rate as determined by phenotypical analysis. We performed this study in a region where both GM and conventional maize are normally cultivated and used the predominant transgenic maize Mon810 in combination with a conventional maize variety which displays the characteristic of white grains (therefore allowing cross-pollination quantification as percentage of yellow grains). Our results indicated an excellent correlation between real-time PCR results and number of cross-fertilized grains at Mon810 levels of 0.1-10%. In contrast, Mon810 percentage estimated by weight of grains produced less accurate results. Finally, we present and discuss the pattern of pollen-mediated gene flow from GM to conventional maize in an example case under field conditions.

Enhanced resistance to the rice blast fungus Magnaporthe grisea conferred by expression of a cecropin A gene in transgenic rice.

Cecropins are a family of antimicrobial peptides, which constitute an important key component of the immune response in insects. Here, we demonstrate that transgenic rice (Oryza sativa L.) plants expressing the cecropin A gene from the giant silk moth Hyalophora cecropia show enhanced resistance to Magnaporthe grisea, the causal agent of the rice blast disease. Two plant codon-optimized synthetic cecropin A genes, which were designed either to retain the cecropin A peptide in the endoplasmic reticulum, the ER-CecA gene, or to secrete cecropin A to the extracellular space, the Ap-CecA gene, were prepared. Both cecropin A genes were efficiently expressed in transgenic rice. The inhibitory activity of protein extracts prepared from leaves of cecropin A-expressing plants on the in vitro growth of M. grisea indicated that the cecropin A protein produced by the transgenic rice plants was biologically active. Whereas no effect on plant phenotype was observed in ER-CecA plants, most of the rice lines expressing the Ap-CecA gene were non-fertile. Cecropin A rice plants exhibited resistance to rice blast at various levels. Transgene expression of cecropin A genes was not accompanied by an induction of pathogenesis-related (PR) gene expression supporting that the transgene product itself is directly active against the pathogen. Taken together, the results presented in this study suggest that the cecropin A gene, when designed for retention of cecropin A into the endoplasmic reticulum, could be a useful candidate for protection of rice plants against the rice blast fungus M. grisea.

Pollen-mediated gene flow in maize in real situations of coexistence.

We present the first study on cross-fertilization between Bt and conventional maize in real situations of coexistence in two regions in which Bt and conventional maize were cultivated. A map was designed and the different crops were identified, as were the sowing and flowering dates, in Bt and conventional maize fields. These data were used to choose the non-transgenic fields for sampling and analysis by the real-time quantification system-polymerase chain reaction (RTQ-PCR) technique. In general, the rate of cross-fertilization was higher in the borders and, in most of the fields, decreased towards the centre of the field. Nine fields had values of genetically modified organism DNA to total DNA of much lower than 0.9%, whereas in three the rate was higher. Some differences were found when comparing our results with those of common field trials. In real conditions of coexistence and in cropping areas with smaller fields, the main factors that determined cross-pollination were the synchronicity of flowering and the distances between the donor and receptor fields. By establishing an index based on these two variables, the rate of the adventitious presence of genetically modified maize could be predicted, as well as the influence of other factors. By applying this index, and in the case of a fully synchronous flowering time, a security distance between transgenic and conventional fields of about 20 m should be sufficient to maintain the adventitious presence of genetically modified organisms as a result of pollen flow below the 0.9% threshold in the total yield of the field.

Pathogen-induced production of the antifungal AFP protein from Aspergillus giganteus confers resistance to the blast fungus Magnaporthe grisea in transgenic rice.

Rice blast, caused by Magnaporthe grisea, is the most important fungal disease of cultivated rice worldwide. We have developed a strategy for creating disease resistance to M. grisea whereby pathogen-induced expression of the afp (antifungal protein) gene from Aspergillus giganteus occurs in transgenic rice plants. Here, we evaluated the activity of the promoters from three maize pathogenesis-related (PR) genes, ZmPR4, mpi, and PRms, in transgenic rice. Chimeric gene fusions were prepared between the maize promoters and the beta-glucuronidase reporter gene (gus A). Histochemical assays of GUS activity in transgenic rice revealed that the ZmPR4 promoter is strongly induced in response to fungal infection, treatment with fungal elicitors, and mechanical wounding. The ZmPR4 promoter is not active in the seed endosperm. The mpi promoter also proved responsiveness to fungal infection and wounding but not to treatment with elicitors. In contrast, no activity of the PRms promoter in leaves of transgenic rice was observed. Transgenic plants expressing the afp gene under the control of the ZmPR4 promoter were generated. Transformants showed resistance to M. grisea at various levels. Our results suggest that pathogen-inducible expression of the afp gene in rice plants may be a practical way for protection against the blast fungus. Most agricultural crop species suffer from a vast array of fungal diseases that cause severe yield losses all over the world. Rice blast, caused by the fungus Magnaporthe grisea (Herbert) Barr (anamorph Pyricularia grisea), is the most devastating disease of cultivated rice (Oryza sativa L.), due to its

Expression of the maize proteinase inhibitor (mpi) gene in rice plants enhances resistance against the striped stem borer (Chilo suppressalis): effects on larval growth and insect gut proteinases.

The maize proteinase inhibitor (mpi) gene was introduced into two elite japonica rice varieties. Both constitutive expression of the mpi gene driven by the maize ubiquitin 1 promoter and wound-inducible expression of the mpi gene driven by its own promoter resulted in the accumulation of MPI protein in the transgenic plants. No effect on plant phenotype was observed in mpi-expressing lines. The stability of transgene expression through successive generations of mpi rice lines (up to the T(4) generation) and the production of functional MPI protein were confirmed. Expression of the mpi gene in rice enhanced resistance to the striped stem borer (Chilo suppressalis), one of the most important pests of rice. In addition, transgenic mpi plants were evaluated in terms of their effects on the growth of C. suppressalis larvae and the insect digestive proteolytic system. An important dose-dependent reduction of larval weight of C. suppressalis larvae fed on mpi rice, compared with larvae fed on untransformed rice plants, was observed. Analysis of the digestive proteolytic activity from the gut of C. suppressalis demonstrated that larvae adapted to mpi transgene expression by increasing the complement of digestive proteolytic activity: the serine and cysteine endoproteinases as well as the exopeptidases leucine aminopeptidase and carboxypeptidases A and B. However, the induction of such proteolytic activity did not prevent the deleterious effects of MPI on larval growth. The introduction of the mpi gene into rice plants can thus be considered as a promising strategy to protect rice plants against striped stem borer.

Transgenic rice plants expressing the antifungal AFP protein from Aspergillus giganteus show enhanced resistance to the rice blast fungus Magnaporthe grisea.

The Aspergillus giganteus antifungal protein (AFP), encoded by the afp gene, has been reported to possess in vitro antifungal activity against various economically important fungal pathogens, including the rice blast fungus Magnaporthe grisea. In this study, transgenic rice ( Oryza sativa ) constitutively expressing the afp gene was generated by Agrobacterium -mediated transformation. Two different DNA constructs containing either the afp cDNA sequence from Aspergillus or a chemically synthesized codon-optimized afp gene were introduced into rice plants. In both cases, the DNA region encoding the signal sequence from the tobacco AP24 gene was N-terminally fused to the coding sequence of the mature AFP protein. Transgenic rice plants showed stable integration and inheritance of the transgene. No effect on plant morphology was observed in the afp -expressing rice lines. The inhibitory activity of protein extracts prepared from leaves of afp plants on the in vitro growth of M. grisea indicated that the AFP protein produced by the trangenic rice plants was biologically active. Several of the T(2) homozygous afp lines were challenged with M. grisea in a detached leaf infection assay. Transformants exhibited resistance to rice blast at various levels. Altogether, the results presented here indicate that AFP can be functionally expressed in rice plants for protection against the rice blast fungus M. grisea.

Bt rice harbouring cry genes controlled by a constitutive or wound-inducible promoter: protection and transgene expression under Mediterranean field conditions.

Seven homozygous transgenic lines of two European commercial cultivars of rice (Ariete (A) and Senia (S)), harbouring the cry1B or cry1Aa Bacillus thuringiensis (Bt) delta-endotoxin genes, were field evaluated for protection from striped stem borer (SSB) (Chilo suppressalis) damage during the 2001 and 2002 summer crop seasons in the Delta de l'Ebre region, Spain. The plant codon-optimized toxin gene was placed under the control of the promoter of either the constitutive ubi1 gene or the wound-inducible mpi gene from maize. Stable, high-level, insecticidal protein accumulation was observed throughout root, leaf and seed tissues of field-grown plants harbouring the cry1B (lines A64.1, A33.1, A3.4 and S98.9) or cry1Aa (lines S05.1 and A19.14) genes under the control of the ubi1 promoter. Conversely, no toxin was detected in unwounded vegetative tissues of the A9.1 line harbouring the cry1B gene controlled by the mpi promoter, indicating that natural environmental stresses did not trigger the activity of the wound-inducible promoter. However, the toxin accumulated at 0.2% total soluble proteins in A9.1 sheath tissue exhibiting brown lesions resulting from SSB damage. The agronomical traits and performance of the transgenic lines were generally comparable with parental controls, except in the two lines accumulating Cry1Aa, which exhibited a high frequency of plants non-true to type. Natural infestation was assisted with manual infestations of L2/L3 SSB larvae in border control plants surrounding the experimental plots, which served as a reservoir for the second-cycle SSB population. The observation of damage (brown lesions and dead hearts) during the crop season and dissection of plants at harvest stage revealed a range of protection amongst the transgenic lines, which was highly consistent with the level of toxin accumulation and with previous experience in greenhouse assays. Lines A3.4 and S05.1 were found to exhibit stable and full protection against SSB attacks, mediated by the accumulation of Cry1B and Cry1Aa toxin, respectively, which was comparable with that afforded by the spraying of chemical insecticides on control plants. The wound-induced A9.1 line exhibited a satisfactory level of protection, with a notably low level of penetration of SSB larvae in the stems, but higher external symptoms than constitutive lines, probably due to the time lag to benefit from the protective effect of Cry1B.