Insect-resistant Transgenic Rice Research Articles

MiRNA-mediated insect-resistant transgenic rice poses no risk to a non-target parasitoid, Cotesia chilonis, via direct feeding or through its target host.

MicroRNAs (miRNAs) have started to play an important role in pest control, and novel miRNA-based transgenic insect-resistant plants are now emerging. However, an environmental risk assessment of these novel transgenic plants expressing insect miRNAs must be undertaken before promoting their application. Here, transgenic miR-14 rice, which has high resistance to the rice stem borer Chilo suppressalis, was used as an example for evaluation in this study. Taking the tier1 risk assessment method in Bacillus thuringiensis (Bt) crops as a reference, the effects of the direct exposure of a non-target parasitoid, Cotesia chilonis, to a high concentration of miRNA were evaluated. The results showed that direct feeding with miR-14 at high concentration had no significant effects on the biological parameters of Co.chilonis, whereas when miR-14 was injected into Ch.suppressalis-parasitized larvae, the development duration of Co.chilonis was significantly affected. In combination with the real conditions of the rice paddy field, it could be inferred that transgenic miR-14 rice has no significant negative effects on the important non-target parasitoid, Co.chilonis. These results will provide a foundation for the establishment of a new safety evaluation system for novel RNAi-based transgenic plants.

Individual and combined effects of land use and weeds on Cry1Ab/c protein expression and yield of transgenic cry1Ab/c rice

ABSTRACT Considering the anticipated commercial exploitation of insect-resistant transgenic rice and that the planting area of cultivated rice overlaps with wild rice, simulating an escape of transgenic rice from farmlands and exploring its fitness after entering semi-natural or natural ecosystems through uncontrolled seed dispersal or gene flow are critical to understand the resulting potential long-term environmental risks. The expression of foreign Cry1Ab/c protein and vegetative and reproductive fitness of insect-resistant transgenic rice Huahui1 (HH1) and its parental-line Minghui63 (MH63) were studied under four treatments combining land use and weed competition: farmland and uncultivated land under weed control (F-NW and U-NW, respectively), and farmland and uncultivated land without weed control (F-W and U-W, respectively). The expression of Cry1Ab/c was significantly lower in U-NW, F-W, and U-W than that in the control treatment, F-NW. Except for plant height, key vegetative (tiller number and biomass) and reproductive (grain number and grain weight per plant) growth indices of transgenic HH1 were significantly lower than those of the parental-line MH63 in F-NW and U-NW, indicating a significant fitness cost. In F-W and U-W, vegetative growth indices (plant height, tiller number, and biomass) were similar in HH1 and MH63; however, key reproductive indices including seed-set rate were significantly higher in HH1 than in MH63, indicating significant fitness benefits. Although these results support large-scale cultivation of insect-resistant transgenic rice in China, the ecological risk involved is high in farmland or uncultivated land without weed control (F-W and U-W).

Cellular Localization of Exogenous Cry1Ab/c and its Interaction with Plasma Membrane Ca2+-ATPase in Transgenic Rice.

The cellular localization of exogenous proteins expressed in transgenic crops not only determines their stability, but also their effects on crop growth and development, including under stressful conditions; however, the underlying molecular mechanisms remain unknown. Here, we determined the cellular distribution of exogenously expressed Cry1Ab/c protein in insect-resistant transgenic rice Huahui-1 (HH1) cells through subcellular localization, immunohistochemistry, immunofluorescence, and western blot analyses. Interaction between the Cry1Ab/c protein and the preliminarily screened endogenous plasma membrane protein Ca2+-ATPase was investigated through yeast two-hybrid, bimolecular fluorescence complementation (BIFC), and co-immunoprecipitation analyses. The potential interaction mechanism was analyzed by comparing the cellular localization and interaction sites between Cry1Ab/c and Ca2+-ATPase. Phenotypic indices and Ca2+-ATPase activity, which may be regulated by the Cry1Ab/c–Ca2+-ATPase interaction, were determined in transgenic HH1 and the parental line Minghui-63 under stress-free and salt-stress conditions. The results showed that Cry1Ab/c was not only distributed in the cytoplasm and nucleus but was also distributed on the plasma membrane, where it interacted with plasma membrane Ca2+-ATPase. This interaction partially retain plasma membrane protein Ca2+-ATPase in the nucleus by a BIFC experiment and thus may affect Ca2+-ATPase activity on the membrane by altering the cellular location of the protein. Consistently, our results confirmed that the presence of Cry1Ab/c in the transgenic HH1 resulted in a reduction in Ca2+-ATPase activity as well as causing detrimental effects on plant phenotype, including significantly reduced plant height and biomass, compared to parental MH63; and that these detrimental effects were more pronounced under salt stress conditions, impacting the salt resistance of the transgenic plants. We suggest that the Cry1Ab/c–Ca2+-ATPase interaction may explain the plasma membrane localization of Cry1Ab/c, which lacks a signal peptide and a transmembrane domain, and the adverse effects of Cry1Ab/c expression on the growth and development of transgenic HH1 plants under salt stress. This information may clarify the molecular mechanisms of these unintended effects and demonstrate the feasibility of evaluating the success and performance of genetic modification of commercially vital crops.

Consumption of miRNA-Mediated Insect-Resistant Transgenic Rice Pollen Does Not Harm Apis mellifera Adults.

RNAi-based insect-resistant genetically engineered (IRGE) crops represent a promising approach for pest management by suppressing gene expressions or translation. A developed microRNA-mediated IRGE rice line expressing endogenous Chilo suppressalis Csu-novel-260 shows significant resistance to target pests. The nontarget insect Apis mellifera is an important pollinator used as a surrogate species for the ecological risk assessment of IRGE plants. To simulate a worst-case scenario, the full-length C. suppressalis and A. mellifera disembodied (dib) cDNAs were cloned. The dib 3'-untranslated regions shared 58.06% nucleotide sequence similarity between C. suppressalis and A. mellifera. No potential Csu-novel-260 binding site in Amdib was detected through the bioinformatics analysis. A dietary RNAi toxicity assay of the impacts of ingested Csu-novel-260 on A. mellifera adults showed that the survival rates of RNAi-treated A. mellifera did not significantly differ from those in the blank control (CK) and negative control (NC) treatments. The Csu-novel-260 uptake by A. mellifera peaked at 8 days postfeeding and then gradually decreased. The Amdib expression was not affected by the RNAi assay days or treatments. These results suggest that A. mellifera adults are not susceptible to high doses of Csu-novel-260 in the dietary RNAi assay and that the impact of miRNA-mediated IRGE plants on A. mellifera is negligible.

Foreign Cry1Ab/c Delays Flowering in Insect-Resistant Transgenic Rice via Interaction With Hd3a Florigen.

Genetic modifications in rice, which resulted in insect resistance, have been highly efficacious. However, they have also induced undesirable secondary phenotypes, such as delayed flowering. The molecular mechanisms associated with these unwanted effects remain unclear. Here, we showed that the flowering time for insect-resistant transgenic cry1Ab/c rice Huahui-1 (HH1) was delayed, compared with that for the parental rice Minghui-63 (MH63), cultivated on farmland and saline–alkaline soils. In contrast, the insect-resistant transgenic cry1C^* rice cultivars T1C-19 and MH63 had similar flowering times under the same conditions. We quantified the following: the expression of five major flowering genes in HH1, T1C-19, and MH63; florigen Hd3a protein expression levels in HH1 and MH63; interactions between Cry1Ab/c and the five main flowering proteins; and the effects of E3s ubiquitin ligase-mediated Cry1Ab/c expression on florigen Hd3a. Hd3a transcription was significantly lower in HH1 but not in T1C-19, compared with that in MH63. The results of yeast two-hybrid, complementary bimolecular fluorescence, and co-immunoprecipitation assays revealed that florigen Hd3a interacted with the exogenous Cry1Ab/c expressed in HH1 and not the exogenous Cry1C^* expressed in T1C-19. When Cry1Ab/c, Hd3a, and E3s fusion proteins were transiently co-expressed in tobacco cells, the Hd3a expression level was significantly lower than the level of Cry1Ab/c and Hd3a co-expression. Thus, the downregulation of Hd3a expression and the interaction between Cry1Ab/c and Hd3a interfere with Hd3a protein expression and might cooperatively delay HH1 flowering time. To the best of our knowledge, this study is the first to explain the delay in flowering time in insect-resistant transgenic rice, mediated by interactions between exogenous and endogenous proteins. This information might help elucidate the molecular mechanisms associated with these unwanted phenotypes effects and improve the process of biosafety assessment of transgenic rice.

Fitness of Insect-resistant transgenic rice T1C-19 under four growing conditions combining land use and weed competition

ABSTRACT Transgene escape into natural ecosystems through seed spraying or transgene introgression may potentially cause environmental biosafety problems. In this study, we assessed the environmental risk of insect-resistant transgenic rice entering farmland margins or natural ecosystems adjacent to farmland. Transgenic Cry1C* rice (T1C-19) was used to study the effects of exogenous Cry1C* expression on vegetative and reproductive growth indices under different growing conditions using the following four combined treatments of land use and weeds: farmland and uncultivated land without weeds (F–NW and U–NW, respectively), and farmland and uncultivated land with weeds (F–W and U–W, respectively). The expression of Cry1C* protein under the U–NW, F–W, and U–W conditions was significantly lower than under the control condition, F–NW. Tiller number, biomass, filled grain number, filled grain weight, and other vegetative and reproductive indices were significantly lower in the rice line TIC-19 than in MH63 under F–NW and U–NW conditions, indicating a significant fitness cost. However, under F–W and U–W conditions, vegetative growth indices such as plant height, tiller number, and biomass, as well as reproductive growth indices such as filled grain number per plant, filled grain weight per plant, and seed setting rate in TIC-19 were similar to those in MH63, indicating a long-term coexistence. These results indicate a lower ecological risk of T1C-19 compared to MH63 under F–NW and U–NW, although their long-term coexistence may lead to potential ecological risks under F–W and U–W.

Differences in midgut transcriptomes between resistant and susceptible strains of Chilo suppressalis to Cry1C toxin

BackgroundChilo suppressalis is a widespread rice pest that poses a major threat to food security in China. This pest can develop resistance to Cry toxins from Bacillus thuringiensis (Bt), threatening the sustainable use of insect-resistant transgenic Bt rice. However, the molecular basis for the resistance mechanisms of C. suppressalis to Cry1C toxin remains unknown. This study aimed to identify genes associated with the mechanism of Cry1C resistance in C. suppressalis by comparing the midgut transcriptomic responses of resistant and susceptible C. suppressalis strains to Cry1C toxin and to provide information for insect resistance management.ResultsA C. suppressalis midgut transcriptome of 139,206 unigenes was de novo assembled from 373 million Illumina HiSeq and Roche 454 clean reads. Comparative analysis identified 5328 significantly differentially expressed unigenes (DEGs) between C. suppressalis Cry1C-resistant and -susceptible strains. DEGs encoding Bt Cry toxin receptors, aminopeptidase-P like protein, the ABC subfamily and alkaline phosphatase were downregulated, suggesting an association with C. suppressalis Cry1C resistance. Additionally, Cry1C resistance in C. suppressalis may be related to changes in the transcription levels of enzymes involved in hydrolysis, digestive, catalytic and detoxification processes.ConclusionOur study identified genes potentially involved in Cry1C resistance in C. suppressalis by comparative transcriptome analysis. The assembled and annotated transcriptome data provide valuable genomic resources for further study of the molecular mechanisms of C. suppressalis resistance to Cry toxins.

Exogenous Cry1Ab/c Protein Recruits Different Endogenous Proteins for Its Function in Plant Growth and Development.

Current risk assessments of transgenic crops do not take into consideration whether exogenous proteins interact with endogenous proteins and thereby induce unintended effects in the crops. Therefore, the unintended effects through protein interactions in insect-resistant transgenic rice merit investigation. Here, a yeast two-hybrid assay was used to evaluate interactions between Bacillus thuringiensis (Bt) protein-derived Cry1Ab/c insect resistance rice Huahui-1 and the endogenous proteins of its parental rice Minghui-63. The authenticity of the strongest interactions of Cry1Ab/c and 14 endogenous proteins involved in photosynthesis and stress resistance, which may be primarily responsible for the significant phenotypic differences between transgenic Huahui-1 and parental Minghui-63, were then analyzed and validated by subcellular co-localization, bimolecular fluorescence complementation and co-immunoprecipitation. As the exogenous full-length Cry1Ab/c protein was found to have self-activating activity, we cleaved it - into three segments based on its three domains, and these were screened for interaction with host proteins using the yeast two-hybrid assay. Sixty endogenous proteins related to the regulation of photosynthesis, stress tolerance, and substance metabolism were found to interact with the Cry1Ab/c protein. The results of bimolecular fluorescence complementation and co-immunoprecipitation verified the interactions between the full-length Cry1Ab/c protein and 12 endogenous proteins involved in photosynthesis 23KD, G, PSBP, Rubisco, Trx, THF1 and stress resistance CAMTAs, DAHP, E3s, HKMTs, KIN13A, FREE1. We used a combination of yeast two-hybrid, bimolecular fluorescence complementation, and co-immunoprecipitation to identify Cry1Ab/c interacting with rice proteins that seem to be associated with the observed unintended effects on photosynthesis and stress resistance between Huahui-1 and Minghui-63 rice plants, and analyze the possible interaction mechanisms by comparing differences in cell localization and interaction sites between these interactions. The results herein provide a molecular analytical system to qualify and quantify the interactions between exogenous proteins and the endogenous proteins of the recipient crop. It could help elucidate both the positive and negative effects of creating transgenic plants and predict their potential risks as well as net crop quality and yield.

Fitness Cost of Transgenic cry1Ab/c Rice Under Saline-Alkaline Soil Condition.

The environmental release and biosafety of transgenic Bt crops have attracted global attention. China has a large area of saline-alkali land, which is ideal for large-scale production of Bt transgenic rice. Therefore an understanding of the fitness of Bt transgenic rice in saline-alkaline soils and the ability to predict its long-term environmental effects are important for the future sustainable use of these crops. In the present study, we aimed to evaluate the fitness of cry1Ab/c transgenic rice in both farmland and natural ecosystems. Transgenic cry1Ab/c rice Huahui1, for which a national biosafety certificate was obtained, was grown on normal farmland and saline-alkaline soils in a glass greenhouse. The expression pattern of exogenous Cry1Ab/c protein, and vegetative and reproductive fitness of rice were assessed. The expression of the exogenous Cry1Ab/c protein in the transgenic rice grown on saline-alkaline soil was lower than that in the strain grown on farmland soil. Under both the soil conditions, vegetative growth abilities, as evaluated by tiller number and biomass, and reproductive growth abilities, as measured by filled grain number and filled grain weight per plant, showed a significantly higher fitness cost for Huahui1 than that for the parental rice Minghui63 grown under the same soil conditions. In saline-alkaline soil, the fitness cost of Huahui1was moderately higher than that of Minghui63. Therefore, the ecological risk of cry1Ab/c transgenic rice is not expected to be higher than that of parental rice Minghui63 if the former escapes into natural saline-alkaline soil. The results of the present study provide a scientific basis to improve environmental safety assessment of the insect-resistant transgenic rice strain Huahui1 before commercialization.

Coexistence of Bacillus thuringiensis (Bt)-transgenic and conventional rice affects insect abundance and plant fitness in fields.

As genetically modified (GM) crops are cultivated worldwide, concerns are emerging about the ecological consequences of the coexistence of transgenic and non-transgenic crops in fields. We first conducted field experiments using insect-resistant transgenic rice expressing Bacillus thuringiensis (Bt-transgenic rice) and its counterpart conventional rice (Oryza sativa L.) with or without insecticide spraying in 2013 and 2014. In 2015 and 2016, Bt-transgenic and conventional rice plants were employed in pure and mixed cages, with an infestation of the target insect (Chilo suppressalis) and with insecticide spraying as the control treatment to prevent target insect infestation. The presence of Bt-transgenic rice decreased the abundance of target insects but did not affect non-target insects and predators in fields. Compared with conventional rice, Bt-transgenic rice showed more empty seeds but comparable seed production in cages. The infestation of target insects significantly decreased the plant fitness of conventional rice in pure cages, but did not affect its fitness when conventional rice coexisted with Bt-transgenic rice. In mixed cages, the presence of Bt-transgenic rice decreased the abundance of target insects and the percentage of dead sheaths in conventional rice. The presence of Bt-transgenic rice benefits the growth and reproduction of non-transgenic rice in fields because of a decreased abundance of target insects. © 2018 Society of Chemical Industry.

Generation of insect resistant marker-free transgenic rice with a novel cry2AX1 gene

A synthetic cry2AX1 gene, consisting sequence of cry2Aa and cry2Ac genes, driven by green tissue specific rbcS promoter was used to develop insect resistant marker-free transgenic rice events. The gene of interest cry2AX1 and the selectable marker gene hpt were present in two independent T-DNAs of the Agrobacterium strain, C58C1. Seventy seven hygromycin and GUS positive T0 transgenic plants were recovered. Among them, 48 were found to be co-transformed with cry2AX1. The Cry2AX1 protein expression level in T0 transgenic events ranged from 0.027 to 0.110 μg/g of fresh leaf tissue. The toxicity level of Cry2AX1 protein was assessed through in vitro bioassay against target insects. The larval mortality ranged from 46.66 to 66.67 per cent against rice leaffolder (C. medinalis) while it ranged from 60 to 100 per cent against rice yellow stem borer (S. incertulas) in T0 transgenic plants. A marker free transgenic event, which expressed 0.043 μg/g of Cry2AX1 in fresh leaf tissue exhibited 45 and 55 per cent larval mortality against rice leaffolder and rice yellow stem borer, respectively in T2 generation

Production of insect-resistant transgenic rice plants for use in practical agriculture

Plant biotechnology provides a powerful solution to boost agricultural productivity and nutritional quality. The development process of a transgenic crop includes multiple steps that consist of gene isolation for a target trait, generation of T0 transgenic crops, characterization of the transgene, evaluation of agronomic performance of transgenic crops, selection of elite transgenic lines and assessment of target trait efficacy. Here, we developed elite insect-resistant transgenic rice plants that may satisfy the standards of biosafety assessments. We made a construct with the insecticide cry1Ac gene for a target trait. A total of 310 T0 transgenic lines were generated and underwent extensive analysis. We selected four T3 lines that contain a single-copy transgene inserted into intergenic regions of the rice genome. During this process, we critically analyzed the transgenic lines with five checkpoints that include single copy of transgene, its integration into intergenic region, clean T-DNA arrangement, stability of transgene through generations and substantial equivalence of transgenic plants in agronomic traits other than insect resistance. Consequently, we obtained insect-resistant transgenic rice plants that can be used in practical agriculture.

Transgenic restorer rice line T1c-19 with stacked cry1C*/bar genes has low weediness potential without selection pressure

Stacked (insect and herbicide resistant) transgenic rice T1c-19 with cry1C*/bar genes, its receptor rice Minghui 63 (herein MH63) and a local two-line hybrid indica rice Fengliangyou Xiang 1 (used as a control) were compared for agronomic performance under field conditions without the relevant selection pressures. Agronomic traits (plant height, tiller number, and aboveground dry biomass), reproductive ability (pollen viability, panicle length, and filled grain number of main panicles, seed set, and grain yield), and weediness characteristics (seed shattering, seed overwintering ability, and volunteer seedling recruitment) were used to assess the potential weediness without selection pressure of stacked transgene rice T1c-19. In wet direct-seeded and transplanted rice fields, T1c-19 and its receptor MH63 performed similarly regarding vegetative growth and reproductive ability, but both of them were significantly inferior to the control. T1c-19 did not display weed characteristics; it had weak overwintering ability, low seed shattering and failed to establish volunteers. Exogenous insect and herbicide resistance genes did not confer competitive advantage to transgenic rice T1c-19 grown in the field without the relevant selection pressures.

Ambient insect pressure and recipient genotypes determine fecundity of transgenic crop-weed rice hybrid progeny: Implications for environmental biosafety assessment.

Transgene introgression into crop weedy/wild relatives can provide natural selective advantages, probably causing undesirable environmental impact. The advantages are likely associated with factors such as transgenes, selective pressure, and genetic background of transgene recipients. To explore the role of the environment and background of transgene recipients in affecting the advantages, we estimated the fitness of crop‐weed hybrid lineages derived from crosses between marker‐free insect‐resistant transgenic (Bt/CpTI) rice with five weedy rice populations under varied insect pressure. Multiway anova indicated the significant effect of both transgenes and weedy rice genotypes on the performance of crop‐weed hybrid lineages in the high‐insect environment. Increased fecundity was detected in most transgene‐present F1 and F2 hybrid lineages under high‐insect pressure, but varied among crop‐weed hybrid lineages with different weedy rice parents. Increased fecundity of transgenic crop‐weed hybrid lineages was associated with the environmental insect pressure and genotypes of their weedy rice parents. The findings suggest that the fitness effects of an insect‐resistant transgene introgressed into weedy populations are not uniform across different environments and genotypes of the recipient plants that have acquired the transgene. Therefore, these factors should be considered when assessing the environmental impact of transgene flow to weedy or wild rice relatives.

Development of leaffolder resistant transgenic rice expressing cry2AX1 gene driven by green tissue-specific rbcS promoter.

The insecticidal cry genes of Bacillus thuringiensis (Bt) have been successfully used for development of insect resistant transgenic rice plants. In this study, a novel cry2AX1 gene consisting a sequence of cry2Aa and cry2Ac gene driven by rice rbcS promoter was introduced into a rice cultivar, ASD16. Among 27 putative rice transformants, 20 plants were found to be positive for cry2AX1 gene. The expression of Cry2AX1 protein in transgenic rice plants ranged from 5.95 to 122.40 ng/g of fresh leaf tissue. Stable integration of the transgene was confirmed in putative transformants of rice by Southern blot hybridization analysis. Insect bioassay on T0 transgenic rice plants against rice leaffolder (Cnaphalocrosis medinalis) recorded larval mortality up to 83.33%. Stable inheritance and expression of cry2AX1 gene in T1 progenies was demonstrated using Southern and ELISA. The detached leaf bit bioassay with selected T1 plants showed 83.33-90.00% mortality against C. medinalis. The whole plant bioassay for T1 plants with rice leaffolder showed significant level of resistance even at a lower level of Cry2AX1 expression varying from 131 to 158 ng/g fresh leaf tissue during tillering stage.

Efficacy of insect-resistance Bt/CpTI transgenes in F5–F7 generations of rice crop–weed hybrid progeny: implications for assessing ecological impact of transgene flow

Ecological impact of transgene flow into populations of wild/weedy relatives is associated with fitness effects in hybrid progeny. Most studies assessing fitness effects focus essentially on early-generation hybrid progeny. However, whether the transgenes remain effective and durable in advanced generations of hybrid progeny remains unclear. We conducted a common garden experiment with F5–F7 hybrid progeny derived from crosses between insect-resistant transgenic (Bt/CpTI) rice and weedy rice, to examine their insect resistance and fitness effects of transgenes on progeny. Hybrid progeny were grown under different insect pressures and cultivation modes where insect damage and fitness-related traits were measured in the same growth season. Plants with transgenes showed significantly lower insect damage (10% vs. 32%) and higher fecundity (551 vs. 392 seeds/plant) than those without transgenes in F5–F7 populations, suggesting the efficacy of transgenes for insect resistance. Fitness benefits of the transgenes were similar among the F5–F7 populations, indicating the stability of transgenic effects. A positive correlation between insect index and fecundity change was detected, stressing the important role of ambient insect pressures in assessing fitness effects caused by insect-resistance transgenes. Our results have important implications for assessing ecological impacts caused by transgene flow to wild/weedy relatives. For cost-effectiveness, the experimental estimation of fitness effects is probably sufficient based on data from hybrids in early generations. Given that fitness effects of insect-resistance transgenes are associated with ambient insect pressure, ecological risk assessment on transgene flow should consider this variable in experimental design, reasonably reflecting actual situations in wild/weedy populations.

Characterization of competitive interactions in the coexistence of Bt-transgenic and conventional rice.

BackgroundTransgene flow through pollen and seeds leads to transgenic volunteers and feral populations in the nature, and consumer choice and economic incentives determine whether transgenic crops will be cultivated in the field. Transgenic and non-transgenic plants are likely to coexist in the field and natural habitats, but their competitive interactions are not well understood.MethodsField experiments were conducted in an agricultural ecosystem with insecticide spraying and a natural ecosystem, using Bt-transgenic rice (Oryza sativa) and its non-transgenic counterpart in pure and mixed stands with a replacement series.ResultsInsect damage and competition significantly decreased plant growth and reproduction under the coexistence of transgenic and conventional rice. Insect-resistant transgenic rice was not competitively superior to its counterpart under different densities in both agricultural and natural ecosystems, irrespective of insect infection. Fitness cost due to Bt-transgene expression occurred only in an agroecosystem, where the population yield decreased with increasing percentage of transgenic rice. The population yield fluctuated in a natural ecosystem, with slight differences among pure and mixed stands under plant competition or insect pressure. The presence of Chilo suppressalis infection increased the number of non-target insects.ConclusionsPlant growth and reproduction patterns, relative competition ability and population yield indicate that Bt-transgenic and non-transgenic rice can coexist in agroecosystems, whereas in more natural habitats, transgenic rice is likely to outcompete non-transgenic rice.

Crossbreeding of cry1Ac/bar Transgenic Progeny with a Non-transgenic Elite Rice Cultivar for Minimizing the Underlying Grain Cost of Bt Transgenic Rice

Most Bt transgenic rice events have an underlying grain cost depending on the insect pressure in the field. This study was conducted (1) to determine if the cry1Ac/bar genetically modified (GM) rice (designated Agb0101) based on a Korean japonica rice cultivar has any underlying grain cost and (2) to develop cry1Ac/bar GM rice lines with yield performance similar to those of Korean elite cultivars via crossbreeding. A three-year field experiment showed that Agb0101 had a high underlying grain cost equivalent to 18% grain yield loss, and this cost was mainly due to the grain number per panicle. Moreover, it was found that the cry1Ac/bar transgene was inherited as a single dominant gene. BC1F4―F5 crossbred progeny derived from an anther culture of Agb0101―showed phenotypic resemblance to non-transgenic Korean elite cultivars, resulting in a decrease in the grain yield cost of Agb0101 from 27% to 10%. These results suggest that Bt-GM rice-oriented cross-fertilization with a non-GM elite cultivar followed by two- or threefold selection is a feasible method to minimize or remove the underlying grain cost of the Bt transgenic insect-resistant rice line.

Analysis of key nutrients and anti-nutrients in insect-resistant transgenic rice

Content of key nutrients and anti-nutrients of the insect-resistant transgenic rice (Btt12R) developed in Korea that contains a cryIIIA insecticidal gene was compared with those of its non-transgenic counterpart (Oryza sative L. cv. Nakdongbyeo). Grains of Btt12R, its parent cultivar, and two commercial rice plants (cv. Yeonganbyeo and Hwaseongbyeo) grown in the adjoining fields under the same environmental conditions and field management were used for this study. Among the analyzed 47 nutrients (8 proximates, 17 amino acids, 8 fatty acids, 9 minerals, and 5 vitamins) and two anti-nutrients (trypsin inhibitors and phytic acid), although the levels of 17 components differed between Nakdongbyeo and Btt12R, all of the measured values from Btt12R were within the ranges of values observed in the two typical Korean varieties and commercial rice provided by the OECD. These results confirm that the nutritional quality of rice grains was not affected by the insertion of the cryIIIA gene.

Creation of Bt rice expressing a fusion protein of Cry1Ac and Cry1I-like using a green tissue-specific promoter.

The insecticidal genes from Bacillus thuringiensis Berliner (Bt) have long been successfully used for development of insect-resistant rice. However, commercial planting of Bt rice has been delayed by the concern over food safety, although no scientific evidence is ever found to justify the concern. To address this safety concern, we developed a transgenic insect-resistant rice line using a green tissue promoter to minimize the Bt protein expression in the rice seeds. The Bt protein expressed in the rice was a fusion protein of two different Bt toxins, Cry1Ac and Cry1I-like protein. The fusion of the two toxins may be helpful to delay the development of insect resistance to Bt rice. Laboratory and field bioassays demonstrated that the transgenic rice plants created by this study were highly active against the rice leaf folder Cnaphalocrocis medinalis (Guenée) and the striped stem borer Chilo suppressalis (Walker). Western analysis indicated that the fusion protein was specifically expressed in green tissues but not in seeds. Therefore, the transgenic rice created in this study should be useful to mitigate the food safety concern and to delay the development of insect resistance.