Biosafety management and commercial use of genetically modified crops in China

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

Enhanced yield performance of <Emphasis Type="Italic">Bt</Emphasis> rice under target-insect attacks: implications for field insect management

The rapid development of transgenic biotechnology has greatly promoted the breeding of genetically engineered (GE) rice in China, and many GE rice lines are in the pipeline for commercialization. To understand field performances of GE rice, key agronomic traits of two insect-resistant Bt rice lines that have been granted biosafety certificates for commercial production in China were evaluated together with their nontransgenic counterparts under environmental conditions with significant differences in insect pressure. Results from the experiments showed enhanced field performances of the Bt GE rice lines compared with the non-GE counterparts for yield-related traits such as number of panicles and filled seeds per plant, under environmental conditions with no insecticide application. No detectable underlying cost of the Bt transgene was observed in the two insect-resistant GE rice lines, particularly in the GE hybrid rice line. Results further indicated significantly greater yield performances of the two insect-resistant GE rice lines under environmental conditions with non-target insect control compared with no insect control. It is concluded from this study that insect-resistant Bt GE rice, particularly the hybrid line, has great potential to maintain its high yield when ambient insect pressure is high. In addition, proper application of insecticides to control non-target insects will guarantee optimal performance of insect-resistant Bt GE rice.     

Effects of seed mixture sowing with transgenic Bt rice and its parental line on the population dynamics of target stemborers and leafrollers,and non‐target planthoppers

The widespread planting of insect‐resistant crops has caused a dramatic shift in agricultural landscapes, thus raising concerns about the potential impacts on both target and non‐target pests. In this study, we examined the potential effects of intra‐specific seed mixture sowing with transgenic Bt rice (Bt) and its parental non‐transgenic line (Nt) (100% Bt rice [Bt₁₀₀], 5% Nt+95% Bt [Nt₀₅Bt₉₅], 10% Nt+90% Bt [Nt₁₀Bt₉₀], 20% Nt+80% Bt [Nt₂₀Bt₈₀], 40% Nt+60% Bt [Nt₄₀Bt₆₀] and 100% Nt rice [Nt₁₀₀]) on target and non‐target pests in a 2‐year field trial in southern China. The occurrence of target pests, Sesamia inferens, Chilo suppressalis and Cnaphalocrocis medinalis, decreased with the increased ratio of Bt rice, and the mixture ratios with more than 90% Bt rice (Bt₁₀₀ and Nt₀₅Bt₉₅) significantly increased the pest suppression efficiency, with the lowest occurrences of non‐target planthoppers, Nilaparvata lugens and Sogatella furcifera in Nt₁₀₀ and Nt₀₅Bt₉₅. Furthermore, there were no significant differences in 1000‐grain dry weight and grain dry weight per 100 plants between Bt₁₀₀ and Nt₀₅Bt₉₅. Seed mixture sowing of Bt rice with ≤10% (especially 5%) of its parent line was sufficient to overcome potential compliance issues that exist with the use of block or structured refuge to provide most effective control of both target and non‐target pests without compromising the grain yield. It is also expected that the strategy of seed mixture sowing with transgenic Bt rice and the non‐transgenic parental line would provide rice yield stability while decreasing the insecticide use frequency in rice production.     

Does Bt rice pose risks to non‐target arthropods? Results of a meta‐analysis in China

Transgenic Bt rice expressing the insecticidal proteins derived from Bacillus thuringiensis Berliner (Bt) has been developed since 1989. Their ecological risks towards non‐target organisms have been investigated; however, these studies were conducted individually, yielding uncertainty regarding potential agroecological risks associated with large‐scale deployment of Bt rice lines. Here, we developed a meta‐analysis of the existing literature to synthesize current knowledge of the impacts of Bt rice on functional arthropod guilds, including herbivores, predators, parasitoids and detritivores in laboratory and field studies. Laboratory results indicate Bt rice did not influence survival rate and developmental duration of herbivores, although exposure to Bt rice led to reduced egg laying, which correctly predicted their reduced abundance in Bt rice agroecosystems. Similarly, consuming prey exposed to Bt protein did not influence survival, development or fecundity of predators, indicating constant abundances of predators in Bt rice fields. Compared to control agroecosystems, parasitoid populations decreased slightly in Bt rice cropping systems, while detritivores increased. We draw two inferences. One, laboratory studies of Bt rice showing effects on ecological functional groups are mainly either consistent with or more conservative than results of field studies, and two, Bt rice will pose negligible risks to the non‐target functional guilds in future large‐scale Bt rice agroecosystems in China.     

Relevance of Bt toxin interaction studies for environmental risk assessment of genetically modified crops

In recent years, different Bacillus thuringiensis (Bt) toxin‐encoding genes have been combined or ‘stacked’ in genetically modified (GM) crops. Synergism between Bt proteins may occur and thereby increase the impact of the stacked GM event on nontarget invertebrates compared to plants expressing a single Bt gene. On the basis of bioassay data available for Bt toxins alone or in combination, we argue that the current knowledge of Bt protein interactions is of limited relevance in environmental risk assessment (ERA).     

An approach for post‐market monitoring of potential environmental effects of Bt‐maize expressing Cry1Ab on natural enemies

Post‐market monitoring (PMM) consistent with Swiss and European Union legislation should ensure the detection and prevention of adverse effects on the environment possibly deriving from commercial cultivation of genetically modified (GM) crops. Insect‐resistant GM crops (such as Bt‐maize) raise particular questions regarding disturbances of biological control functions provided by beneficial insects such as predators and parasitoids (so‐called natural enemies). Consensus among regulators, scientists and the agricultural biotech industry on appropriate PMM plans allowing the detection and possibly prevention of such adverse effects is still lacking. The aims of this study were to identify the necessity for PMM of Bt‐maize expressing Cry1Ab on natural enemies and to develop an appropriate PMM plan. The approach chosen consisted in determining what type of monitoring is most appropriate to address potential effects of Bt‐maize on natural enemies during commercial cultivation. This included identifying whether there remain substantial scientific uncertainties that would support case‐specific monitoring. Existing pre‐market risk assessment data indicate that Bt‐maize (Cry1Ab) comprises a negligible risk for disturbances in biological control functions of natural enemies. As a consequence, a faunistic monitoring of specific groups of natural enemies is not considered an appropriate approach to detect failures in biological control functions. Alternatively, an approach is proposed that consists in indirectly analysing biological control functions by surveying outbreaks of maize herbivores. Unusual herbivore outbreaks could indicate failures in biological control functions of natural enemies. Data could be collected via questionnaires addressed to farmers growing Bt‐maize. Significant correlations between unusual occurrences of specific maize herbivores and the cultivation of Bt‐maize would subsequently need specific studies to determine possible causalities in more detail. The here proposed approach has the advantage of covering different natural enemy groups. It represents a cost‐effective strategy to obtain scientifically sound data as a basis for regulatory decision‐making.     

The impact of secondary pests on Bacillus thuringiensis (Bt) crops

The intensification of agriculture and the development of synthetic insecticides enabled worldwide grain production to more than double in the last third of the 20th century. However, the heavy dependence and, in some cases, overuse of insecticides has been responsible for negative environmental and ecological impacts across the globe, such as a reduction in biodiversity, insect resistance to insecticides, negative effects on nontarget species (e.g. natural enemies) and the development of secondary pests. The use of recombinant DNA technology to develop genetically engineered insect‐resistant crops could mitigate many of the negative side effects of insecticides. One such genetic alteration enables crops to express toxic crystalline (Cry) proteins from the soil bacteria Bacillus thuringiensis (Bt). Despite the widespread adoption of Bt crops, there are still a range of unanswered questions concerning longer term agro‐ecosystem interactions. For instance, insect species that are not susceptible to the expressed toxin can develop into secondary pests and cause significant damage to the crop. Here, we review the main causes surrounding secondary pest dynamics in Bt crops and the impact of such outbreaks. Regardless of the causes, if nonsusceptible secondary pest populations exceed economic thresholds, insecticide spraying could become the immediate solution at farmers’ disposal, and the sustainable use of this genetic modification technology may be in jeopardy. Based on the literature, recommendations for future research are outlined that will help to improve the knowledge of the possible long‐term ecological trophic interactions of employing this technology.     

Comparison of three transgenic Bt rice lines for insecticidal protein expression and resistance against a target pest,Chilo suppressalis (Lepidoptera: Crambidae)

Two transgenic rice lines (T2A‐1 and T1C‐19b) expressing cry2A and cry1C genes, respectively, were developed in China, targeting lepidopteran pests including Chilo suppressalis (Walker) (Lepidoptera: Crambidae). The seasonal expression of Cry proteins in different tissues of the rice lines and their resistance to C. suppressalis were assessed in comparison to a Bt rice line expressing a cry1Ab/Ac fusion gene, Huahui 1, which has been granted a biosafety certificate. In general, levels of Cry proteins were T2A‐1 > Huahui 1 > T1C‐19b among rice lines, and leaf > stem > root among rice tissues. The expression patterns of Cry protein in the rice line plants were similar: higher level at early stages than at later stages with an exception that high Cry1C level in T1C‐19b stems at the maturing stage. The bioassay results revealed that the three transgenic rice lines exhibited significantly high resistance against C. suppressalis larvae throughout the rice growing season. According to Cry protein levels in rice tissues, the raw and corrected mortalities of C. suppressalis caused by each Bt rice line were the highest in the seedling and declined through the jointing stage with an exception for T1C‐19b providing an excellent performance at the maturing stage. By comparison, T1C‐19b exhibited more stable and greater resistance to C. suppressalis larvae than T2A‐1, being close to Huahui 1. The results suggest cry1C is an ideal Bt gene for plant transformation for lepidopteran pest control, and T1C‐19b is a promising Bt rice line for commercial use for tolerating lepidopteran rice pests.     

Bt rice could provide ecological resistance against nontarget planthoppers

Genetically engineered (GE) rice lines expressing Lepidoptera‐active insecticidal cry genes from the bacterium Bacillus thuringiensis (Bt) have been developed in China. Field surveys indicated that Bt rice harbours fewer rice planthoppers than non‐Bt rice although planthoppers are not sensitive to the produced Bt Cry proteins. The mechanisms underlying this phenomenon remain unknown. Here, we show that the low numbers of planthoppers on Bt rice are associated with reduced caterpillar damage. In laboratory and field‐cage experiments, the rice planthopper Nilapavata lugens had no feeding preference for undamaged Bt or non‐Bt plants but exhibited a strong preference for caterpillar‐damaged plants whether Bt or non‐Bt. Under open‐field conditions, rice planthoppers were more abundant on caterpillar‐damaged non‐Bt rice than on neighbouring healthy Bt rice. GC–MS analyses showed that caterpillar damage induced the release of rice plant volatiles known to be attractive to planthoppers, and metabolome analyses revealed increased amino acid contents and reduced sterol contents known to benefit planthopper development. That Lepidoptera‐resistant Bt rice is less attractive to this important nontarget pest in the field is therefore a first example of ecological resistance of Bt plants to nontarget pests. Our findings suggest that non‐Bt rice refuges established for delaying the development of Bt resistance may also act as a trap crop for N. lugens and possibly other planthoppers.     

Normal expression of insect-resistant transgene in progeny of common wild rice crossed with genetically modified rice: its implication in ecological biosafety assessment

Transgene outflow from genetically modified (GM) rice to its wild relatives may cause undesirable ecological consequences. Understanding the level of transgene expression in wild rice following gene flow is important for assessing such consequences, providing that transgene escape from GM rice cannot be prevented. To determine the expression of a transgene in common wild rice (Oryza rufipogon), we analyzed the content of Cry1Ac protein in three GM rice lines containing a Bt transgene, their F₁ hybrids with common wild rice and F₂ progeny at different growth stages, using the sandwich enzyme-linked immunosorbent assay. The average content of Cry1Ac protein in leaf samples of the wild rice lines ranged between 0.016 and 0.069% during the entire growth period, whereas that in stems varied between 0.12 and 0.39%. A great variation in Cry1Ac protein content was detected among individuals of F₁ hybrids and F₂ progeny, with some wild individuals showing higher level of Bt toxin than the cultivated GM rice. The results suggest that the Bt transgene can express normally in the interspecific hybrids between insect-resistant GM rice and common wild rice, and may have similar effects on the target insects as in GM rice.     

Molecular characterization of lepidopteran pest-resistant transgenic rice events expressing synthetic Cry1Ac

The insecticidal toxin gene of Bacillus thuringiensis (Bt) is one of the most commonly used in the development of genetically modified (GM) crops. In this research, we analyzed Bt rice showing lepidopteran pest-resistance. The Bt gene is a synthetic Cry1Ac composed of optimal codons for plants, and the Bt protein is targeted to the chloroplast by a transit peptide. Three Cry1Ac rice events (C103-3, C127-1, and C7-1) were analyzed for molecular characterization. C103-3 contains two copies of T-DNA where the left border (LB) region is truncated. Both C7-1 and C127-1 have a single copy of T-DNA, but a part of the vector backbone DNA is inserted into the genome of C127-1; thus, only C7-1 had intact T-DNA. Progenies of C7-1 crossed with the original cultivar, Nakdong, and double-haploid lines from anther culture of lines crossed with the elite cultivar, Dongjin, were analyzed for T-DNA flanking genomic DNA and genotyping. Results showed that an intact T-DNA region without the vector backbone was inserted into the genome and was stably inherited through generations. The C7-1 homozygous event could be used as breeding material to develop GM rice with pest resistance.     

Risk assessment of Bacillus thuringiensis in wild Brassica rapa: A field simulation of introgressed transgenes

Abstract

Brassica crops are able to hybridize with closely related wild and weedy species such as Brassica rapa and therefore these species could become recipients of transgenes from GM brassica crops, such as oilseed rape. Transgenes which protect against herbivory, such as a gene conferring the production of a Bt toxin, could increase the recipient's fitness and therefore enhance its competitiveness. We used microbial Bt and several other pesticides to exclude several guilds of herbivores and thus simulate the effect of transgene introgression on the performance of wild B. rapa. There were only minor negative effects of excluding insect herbivores on the performance of B. rapa and it appears that vertebrate herbivory has a more significant effect. The advantages and disadvantages of simulating the risks associated with GM plants are discussed. If we apply this simulation to GM risk assessment and make inferences from these findings, the implications of this for gene flow from insect-resistant transgenic plants are that it will be of little significance should it occur.     

Assessment of transgene flow in tomato and potential effects of genetically modified tomato expressing Cry3Bb1 toxins on bumblebee feeding behaviour

One of the concerns surrounding the commercial release of genetically modified (GM) crops is the escape of transgenes into agricultural or semi‐natural habitats through vertical gene flow, as this may cause environmental or economic problems. There is also the concern that GM crops may affect pollinators and the pollination services they provide. Despite the growing commercial interest of GM tomato (Solanum lycopersicum), gene flow has been assessed only sparsely in tomato. To evaluate the likelihood of gene flow from GM tomato plants to sexually compatible plants, and to assess whether bumblebee activity is affected by GM tomato, three experiments were conducted under greenhouse conditions, using a Bt‐tomato expressing the insecticidal Cry3Bb1 protein as model system: (a) artificial crosses between a GM tomato line, two wild tomato relatives (Solanum hirsutum and Solanum nigrum) and a non‐GM tomato variety; (b) bumblebee‐mediated crosses between GM and non‐GM tomato plants and (c) visual observations of bumblebees' feeding behaviour. No hybrids were obtained between the GM tomato line and S. hirsutum and S. nigrum. In an experimental design where non‐GM receptor plants outnumbered GM plants by approximately 3:1, the bumblebee‐mediated cross‐fertilisation rate between GM and non‐GM tomato plants was measured at 4.3 ± 5.47%. No significant differences in feeding behaviour of bumblebees foraging on GM and non‐GM tomato plants were observed. Therefore, we conclude that: (a) the probability of transgene introgression between the GM tomato line used in this study and its wild relatives S. hirsutum and S. nigrum is negligible; (b) bumblebee activity can mediate cross‐fertilisation between GM and non‐GM tomato and (3) the Cry3Bb1‐expressing tomato line tested does not adversely affect the feeding behaviour of bumblebees.     

A multidisciplinary approach directed towards the commercial release of transgenic herbicide-tolerant rice in Costa Rica

This review discusses a multidisciplinary and multicomponent approach leading to the development and commercial release of transgenic Costa Rican rice varieties tolerant to the herbicide gluphosinate ammonium. We describe the field evaluations of the transgenic lines and their potential environmental impact, focusing on gene flow, particularly in relation to native wild Oryza species and weedy rice, based on trials performed in compliance with the national regulatory requirements of the country. We also present a socio-economic analysis of rice production in Costa Rica and the economic benefits of genetically modified (GM) rice as well as an environmental risk-benefit analysis for the deployment of GM rice. Additionally, food safety evaluation, intellectual property management, requirements for deregulation, and options for the commercialization of the new varieties are discussed. We also present results from a national survey aimed at assessing the level of support for GM crops in Costa Rica as this forms an integral component of our approach. Taken together, our results demonstrate that the adoption of these genetically improved rice varieties will provide clear benefits to Costa Rican rice growers and consumers.     

CRISPR/Cas9‐mediated knockout of Ms1 enables the rapid generation of male‐sterile hexaploid wheat lines for use in hybrid seed production

The development and adoption of hybrid seed technology have led to dramatic increases in agricultural productivity. However, it has been a challenge to develop a commercially viable platform for the production of hybrid wheat (Triticum aestivum) seed due to wheat's strong inbreeding habit. Recently, a novel platform for commercial hybrid seed production was described. This hybridization platform utilizes nuclear male sterility to force outcrossing and has been applied to maize and rice. With the recent molecular identification of the wheat male fertility gene Ms1, it is now possible to extend the use of this novel hybridization platform to wheat. In this report, we used the CRISPR/Cas9 system to generate heritable, targeted mutations in Ms1. The introduction of biallelic frameshift mutations into Ms1 resulted in complete male sterility in wheat cultivars Fielder and Gladius, and several of the selected male‐sterile lines were potentially non‐transgenic. Our study demonstrates the utility of the CRISPR/Cas9 system for the rapid generation of male sterility in commercial wheat cultivars. This represents an important step towards capturing heterosis to improve wheat yields, through the production and use of hybrid seed on an industrial scale.     

Bt rice in China — focusing the nontarget risk assessment

Bt rice can control yield losses caused by lepidopteran pests but may also harm nontarget species and reduce important ecosystem services. A comprehensive data set on herbivores, natural enemies, and their interactions in Chinese rice fields was compiled. This together with an analysis of the Cry protein content in arthropods collected from Bt rice in China indicated which nontarget species are most exposed to the insecticidal protein and should be the focus of regulatory risk assessment.     

Transgene flow to hybrid rice and its male-sterile lines

Gene flow from genetically modified (GM) crops to the same species or wild relatives is a major concern in risk assessment. Transgenic rice with insect and/or disease resistance, herbicide, salt and/or drought tolerance and improved quality has been successfully developed. However, data on rice gene flow from environmental risk assessment studies are currently insufficient for the large-scale commercialization of GM rice. We have provided data on the gene flow frequency at 17 distances between a GM japonica line containing the bar gene as a pollen donor and two indica hybrid rice varieties and four male-sterile (ms) lines. The GM line was planted in a 640m² in an isolated experimental plot (2.4 ha), which simulates actual conditions of rice production with pollen competition. Results showed that: (1) under parallel plantation at the 0-m zone, the transgene flow frequency to the ms lines ranged from 3.145 to 36.116% and was significantly higher than that to hybrid rice cultivars (0.037–0.045%). (2) Gene flow frequency decreased as the distance increased, with a sharp cutoff point at about 1–2 m; (3) The maximum distance of transgene flow was 30–40 m to rice cultivars and 40–150 m to ms lines. We believe that these data will be useful for the risk assessment and management of transgenic rice lines, especially in Asia where 90% of world's rice is produced and hybrid rice varieties are extensively used. Shirong Jia, Feng Wang and Lei Shi contributed equally to this investigation.     

Insecticidal potential of Bacillus thuringiensis for the biological control of neotropical brown stink bug

Euschistus heros (Hemiptera: Pentatomidae) is considered a major insect pest in soybean production in Brazil. The indiscriminate application of pesticides to fields leads to reduction in biodiversity, selection of resistant populations, emergence of new pest outbreaks, and damage to non‐target organisms. The objective of this research was to evaluate the effects of exposure to Cry proteins present in new isolates of Bacillus thuringiensis Berliner, commercial formulations, and Bt soybean on E. heros development. The feeding preference of E. heros for Bt‐ vs. non‐Bt soybean was also evaluated. All treatments caused significant mortality to E. heros except the Bt soybean. Tests with combinations of isolated Bt‐toxins indicated that combinations had greater efficacy than other treatments (>98% mortality). The results demonstrate that E. heros is susceptible to B. thuringiensis toxins, which may contribute to the management of this insect in soybean agro‐ecosystems.     

Effectiveness in the field of Bt rice lines against target pests under various cultural regimes

China has a long history of rice cultivation, incorporating several cultural practices known to influence damage by insect pests. Transgenic Bt rice expresses lepidopteran‐specific insecticidal proteins that primarily target lepidopteran insect pests. However, the effectiveness of Bt rice against target insect pests under different cultural regimes has not been evaluated. In this study, the effectiveness of Bt rice lines against rice leaffolder, Cnaphalocrocis medinalis (Guenée) (Lepidoptera: Pyralidae), and striped stem borer, Chilo suppressalis Walker (Lepidoptera: Crambidae), was evaluated under various transplanting densities, crop establishment methods, and planting times. The results showed that Bt rice lines (T2A‐1 and T1C‐19, containing Cry2A and Cry1C, respectively) could prevent damage by these target pests under a range of cultural practices. Injury by C. medinalis or C. suppressalis on rice did not differ with the rice lines under various transplanting densities. Direct‐seeded non‐Bt rice MH63 suffered heavier injury by C. medinalis and C. suppressalis than it did with transplanting, whereas injury to the two Bt rice lines did not differ with planting methods. Planting time significantly affected injury by C. medinalis or C. suppressalis on non‐Bt rice, whereas injury to Bt rice lines did not differ with planting time. These results suggest that transplanting density, planting method, and planting time did not significantly affect the resistance of two Bt rice lines, due to their high insecticidal activity against target insects.