Cowpea Trypsin Inhibitor Research Articles

Analysis of T-DNA integration events in transgenic rice

Agrobacterium-mediated plant transformation has been widely used for introducing transgene(s) into a plant genome and plant breeding. However, our understanding of T-DNA integration into rice genome remains limited relative to that in the model dicot Arabidopsis. To better elucidate the T-DNA integration into the rice genome, we investigated extensively the T-DNA ends and their flanking rice genomic sequences from two transgenic rice plants carrying Cowpea Trypsin Inhibitor (CpTI)-derived gene Signal-CpTI-KDEL (SCK) and Bacillus thuringiensis (BT) gene, respectively, by TAIL-PCR method. Analysis of the junction sequences between the T-DNA ends and rice genome DNA indicated that there were three joining patterns of microhomology, filler DNA sequences, and exact joining, and both the T-DNA ends tend to adopt identical manner to join the rice genome. After T-DNA integration, there were several variations of rice genomic sequences, including small deletions at the integration sites, superfluous DNA inserted between T-DNA and genome, and translocation of genomic DNA in the flanking regions. The translocation block could be from a noncontiguous region in the same chromosome or different chromosomes at the integration sites, and the originating position of the translocated block resulted in comparable deletion based on a cut/paste mechanism rather than a replication mechanism. Our study may lead to a better understand of T-DNA integration mechanism and facilitate functional genomic studies and further crop improvement.

Protein quality of autoclaved cowpea varieties as influenced by anti-nutritional factors

One hundred and ten (110) weanling male winstar strain rats were used to determine the protein efficiency ration (PER) and net protein ration (NPR) of five cowpea samples. PER ranged from 1.06 in IT 81D-1137 to 1.91 in 1784E-1-108 while values in autoclaved samples ranged from 1.55 in IT81D-1137 to 2.78 in 1784E-1-108. for NPR, raw samples gave values that ranged between 2.44 and 3.57 in 1781D-1137 and 1784E-1-108 respectively. Autoclaved samples gave NPR values that ranged from 2.72 to 4.47 in IT 8ID-1137 and IT84E-1-108 respectively. In the raw and autoclaved cowpea diets, trypsin inhibitor, lectin and tannic acid were negatively correlated with PER and NPR while HCN and phytic acid had positive relationship with these indicators of protein quality. In autoclaved samples cowpea variety IT84E-1-108 had the highest PER and NPR values showing that it would support growth to a reasonable extent.

Transgenic Cry1Ac/CpTI cotton assessment finds no detrimental effects on the insect predator Chrysoperla sinica

The widespread commercialization of genetically modified (GM) cotton makes it important to assess the potential impact of this recombinant crop on non-target organisms. As important natural enemies of cotton field predators, green lacewing Chrysoperla sinica larvae are exposed to Bt insecticidal proteins expressed by GM cotton by feeding on herbivorous pests, and adults are directly exposed to Bt proteins by cotton pollen consumption. However, potential impacts of transgenic Bt cotton on C. sinica remain unclear. In this study, we evaluated the effects of two transgenic cotton varieties, CCRI41 and CCRI45, which express Cry1Ac (Bt toxin) and CpTI (Cowpea Trypsin Inhibitor), on C. sinica larvae and adults. After being fed with cotton aphids Aphis gossypii reared on transgenic cotton, the survival rate, developmental duration, pupation rate, and emergence rate of larvae were not adversely affected. After being fed two types of transgenic cotton pollen, the 7-day weight of adults and the preoviposition period and the cumulative oviposition of females were not significantly different from control specimen. Taken together, these results indicate that the potential risks of the two tested GM cotton varieties for the predator C. sinica are negligible. CapsuleOur study indicated that GM cotton varieties CCRI41 and CCRI45 have no adverse effects on insect predator C. sinica.

Transgenic insect-resistant Bt cotton expressing Cry1Ac/CpTI does not affect the mirid bug Apolygus lucorum

Common varieties of genetically modified (GM) cotton increasingly display insect-resistant properties via expression of bacterial-derived toxins from Bacillus thuringiensis (Bt). This necessitates a deeper understanding of the possible effects of these crops on non-target insects. The mirid bug Apolygus lucorum is a major pest in cotton production in China, however, the effect of GM cotton on this non-target species is currently virtually unknown. This insect is exposed to these transgenic plants by consuming genetically modified (GM) leaves. In this study, laboratory experiments were conducted to assess the toxicity of CCRI41 and CCRI45, (genetically modified cotton varieties which express the toxins Cry1Ac and CpTI (Cowpea Trypsin Inhibitor)) on nymphs and adults of A. lucorum. There was no detectable increase in mortality after A. lucorum fed on GM cotton leaves for 20 days. While we detected trace amounts of Cry1Ac proteins in both A. lucorum nymphs and adults (<10 ng/g fresh weight), the expression of genes related to detoxification did not detectably differ from those feeding on non-GM cotton. Our binding assays did not show Cry1Ac binding to receptors on the midgut brush border membrane from either A. lucorum nymphs or adults. Our findings collectively indicate that feeding on leaves of the GM cotton varieties CCRI41 and CCRI45 have few toxic effects on A. lucorum.

Expressing a modified cowpea trypsin inhibitor gene to increase insect tolerance against Pieris rapae in Chinese cabbage

To increase the tolerance of Chinese cabbage (Brassica campestris L. ssp. pekinensis) to Pieris rapae, we introduced a modified cowpea trypsin inhibitor (CpTI) gene, SCK, into various cultivars. SCK was derived from CpTI, an insect-resistance gene. The protein accumulating capacity of CpTI can be improved by adding a signal peptide sequence at the 5′ end and an endoplasmic reticulum-detained signal sequence at the 3′ end. Using an optimized Agrobacterium tumefaciens-mediated transformation system in Chinese cabbage, we obtained a maximum transformation efficiency of ~6.83%. Insect resistance tests and CpTI enzymatic assays showed that most of the transgenic plants had significant resistance to cabbage worm (Pieris rapae) larvae and that the plants with the highest levels of insect resistance had the greatest CpTI-related capacity, indicating a high correlation between SCK expression and insect resistance. An evaluation of segregation patterns in the independent transgenic line with the highest insect resistance, ‘ZB-08-04’, showed that kanamycin resistant versus sensitive plants segregated in a 3:1 Mendelian fashion. This study provides a potential germplasm resource for Chinese cabbage breeding in the future.

Transgenic Tobacco Expressing the TAT-Helicokinin I-CpTI Fusion Protein Show Increased Resistance and Toxicity to Helicoverpa armigera (Lepidoptera: Noctuidae).

Insect kinins were shown to have diuretic activity, inhibit weight gain, and have antifeedant activity in insects. In order to study the potential of the TAT-fusion approach to deliver diuretic peptides per os to pest insects, the HezK I peptide from Helicoverpa zea, as a representative of the kinin family, was selected. The fusion gene TAT-HezK I was designed and was used to transform tobacco plants. As a means to further improve the stability of TAT-HezK I, a fusion protein incorporating HezK I, transactivator of transcription (TAT), and the cowpea trypsin inhibitor (CpTI) was also designed. Finally, the toxicity of the different tobacco transgenic strains toward Helicoverpa armigera was compared. The results demonstrated that TAT-HezK I had high toxicity against insects via transgenic expression of the peptide in planta and intake through larval feeding. The toxicity of the fusion TAT-HezK I and CpTI was higher than the CpTI single gene in transgenic tobacco, and the fusion TAT-HezK I and CpTI further enhanced the stability and bioavailability of agents in oral administration. Our research helps in targeting new genes for improving herbivore tolerance in transgenic plant breeding.

SOIL ECOLOGICAL SAFETY EVALUATION FOR BIVALENT TRANSGENIC COTTON PLANTS: ROOT EXUDATES VERSUS SOIL ENZYME ACTIVITIES AND SOIL MICROBIAL DIVERSITY

Pot experiments were conducted to assess the possibly adverse effect of transgenic Bt- and CpTI-cotton (Bacillus thuringiensis and cowpea trypsin inhibitor cotton) on soil ecosystem. Soil enzymes activities and microbial molecular community and diversity based on 16s- and 18s-PCR-DGGE (polymerase chain reaction-denaturing gradient gel electrophoresis) profiles were determined by adding root exudates of Bt- and CpTI-cotton into soil. Results showed that the root exudates of transgenic cotton seedlings increased the soil catalase activity by 21.8% to 32.7%. The soil urease activity was decreased by 11.0% while the activity in the treatment of 1ml/350g soil was increased by 18.1% compared to control. The invertase activity in the treatment of 1 ml/350g soil was increased by 54.2%, however, the activity in the treatments of 2 and 4 ml/350 g soil was decreased by from 25% to 29.2%. The total 16sPCR-DGGE lane bands of bacterial community and diversity were almost not different. However, the effect pattern of root exudates of transgenic cotton on soil fungal community and diversity was differed from bacteria. The fungal community number and composition were changed. Though some dominant community disappeared and other newly dominant community appeared, the fungal stability index was increased. Results suggested there was little significantly adverse effect of Bt- and CpTI-cotton on soil ecosystem.

Effects of water management practices on residue decomposition and degradation of Cry1Ac protein from crop-wild Bt rice hybrids and parental lines during winter fallow season

Rice is the staple diet of over half of the world's population and Bacillus thuringiensis (Bt) rice expressing insecticidal Cry proteins is ready for deployment. An assessment of the potential impact of Bt rice on the soil ecosystem under varied field management practices is urgently required. We used litter bags to assess the residue (leaves, stems and roots) decomposition dynamics of two transgenic rice lines (Kefeng6 and Kefeng8) containing stacked genes from Bt and sck (a modified CpTI gene encoding a cowpea trypsin inhibitor) (Bt/CpTI), a non-transgenic rice near-isoline (Minghui86), wild rice (Oryza rufipogon) and crop-wild Bt rice hybrid under contrasting conditions (drainage or continuous flooding) in the field. No significant difference was detected in the remaining mass, total C and total N among cultivars under aerobic conditions, whereas significant differences in the remaining mass and total C were detected between Kefeng6 and Kefeng8 and Minghui86 under the flooded condition. A higher decomposition rate constant (km) was measured under the flooded condition compared with the aerobic condition for leaf residues, whereas the reverse was observed for root residues. The enzyme-linked immunosorbent assay (ELISA), which was used to monitor the changes in the Cry1Ac protein in Bt rice residues, indicated that (1) the degradation of the Cry1Ac protein under both conditions best fit first-order kinetics, and the predicted DT50 (50% degradation time) of the Cry1Ac protein ranged from 3.6 to 32.5 days; (2) the Cry1Ac protein in the residue degraded relatively faster under aerobic conditions; and (3) by the end of the study (~154 days), the protein was present at a low concentration in the remaining residues under both conditions. The degradation rate constant was negatively correlated with the initial carbon content and positively correlated with the initial Cry1Ac protein concentration, but it was only correlated with the mass decomposition rate constants under the flooded condition. No Cry1Ac protein was detected in the soils surrounding the buried residue. Our results did not reveal any evidence that the stacked genes (Bt/CpTI) or the presence of the Cry1Ac protein influenced the decomposition dynamics of the rice residues. Furthermore, our results suggested that field drainage after residue incorporation would promote Cry1Ac protein degradation.

A real-time PCR method for detection of CpTI (Cowpea Trypsin Inhibitor) gene in the genetically modified rice originating from China

Labelling and traceability of genetically modified organisms (GMOs) are necessary for trade and regulation in the world and Vietnam. Cowpea Trypsin Inhibitor (CpTI) gene encodes a trypsin inhibitor which is considered as a suitable candidate for developing insect-resistant transgenic plants, especially transgenic rice lines originating from China. In this study, we established a real-time PCR protocol to detect the CpTI gene in transgenic rice. The protocol with CpTI-F and CpTI-R primers, 300 nM primers, 0.5 X SYBR Green I, annealing temperature at 62 0C showed the best results. Amplification efficiency is 94.64 % and the limit of detection is 50 copies. Moreover, PCR product of CpTI gene was cloned into pBluescript plasmid using as a positive control

Identification of top-down forces regulating cotton aphid population growth in transgenic Bt cotton in central China.

The cotton aphid Aphis gossypii Glover is the main aphid pest in cotton fields in the Yangtze River Valley Cotton-planting Zone (YRZ) in central China. Various natural enemies may attack the cotton aphid in Bt cotton fields but no studies have identified potential specific top-down forces that could help manage this pest in the YRZ in China. In order to identify possibilities for managing the cotton aphid, we monitored cotton aphid population dynamics and identified the effect of natural enemies on cotton aphid population growth using various exclusion cages in transgenic Cry1Ac (Bt)+CpTI (Cowpea trypsin inhibitor) cotton field in 2011. The aphid population growth in the open field (control) was significantly lower than those protected or restricted from exposure to natural enemies in the various exclusion cage types tested. The ladybird predator Propylaea japonica Thunberg represented 65% of Coccinellidae predators, and other predators consisted mainly of syrphids (2.1%) and spiders (1.5%). The aphid parasitoids Aphidiines represented 76.7% of the total count of the natural enemy guild (mainly Lysiphlebia japonica Ashmead and Binodoxys indicus Subba Rao & Sharma). Our results showed that P. japonica can effectively delay the establishment and subsequent population growth of aphids during the cotton growing season. Aphidiines could also reduce aphid density although their impact may be shadowed by the presence of coccinellids in the open field (likely both owing to resource competition and intraguild predation). The implications of these results are discussed in a framework of the compatibility of transgenic crops and top-down forces exerted by natural enemy guild.

Effects of Transgenic Cry1Ac + CpTI Cotton on Non-Target Mealybug Pest Ferrisia virgata and Its Predator Cryptolaemus montrouzieri

Recently, several invasive mealybugs (Hemiptera: Pseudococcidae) have rapidly spread to Asia and have become a serious threat to the production of cotton including transgenic cotton. Thus far, studies have mainly focused on the effects of mealybugs on non-transgenic cotton, without fully considering their effects on transgenic cotton and trophic interactions. Therefore, investigating the potential effects of mealybugs on transgenic cotton and their key natural enemies is vitally important. A first study on the effects of transgenic cotton on a non-target mealybug, Ferrisia virgata (Cockerell) (Hemiptera: Pseudococcidae) was performed by comparing its development, survival and body weight on transgenic cotton leaves expressing Cry1Ac (Bt toxin) + CpTI (Cowpea Trypsin Inhibitor) with those on its near-isogenic non-transgenic line. Furthermore, the development, survival, body weight, fecundity, adult longevity and feeding preference of the mealybug predator Cryptolaemus montrouzieri Mulsant (Coleoptera: Coccinellidae) was assessed when fed F. virgata maintained on transgenic cotton. In order to investigate potential transfer of Cry1Ac and CpTI proteins via the food chain, protein levels in cotton leaves, mealybugs and ladybirds were quantified. Experimental results showed that F. virgata could infest this bivalent transgenic cotton. No significant differences were observed in the physiological parameters of the predator C. montrouzieri offered F. virgata reared on transgenic cotton or its near-isogenic line. Cry1Ac and CpTI proteins were detected in transgenic cotton leaves, but no detectable levels of both proteins were present in the mealybug or its predator when reared on transgenic cotton leaves. Our bioassays indicated that transgenic cotton poses a negligible risk to the predatory coccinellid C. montrouzieri via its prey, the mealybug F. virgata.

EXTRACTION, PURIFICATION, AND CHARACTERIZATION OF A TRYPSIN INHIBITOR FROM COWPEA SEEDS (Vigna unguiculata)

Protease inhibitors against trypsin were extracted from cowpea seeds, purified, and characterized. After the seed powder was defatted with hexane, the cowpea trypsin inhibitor (CpTI) was extracted with 0.15 M NaCl for 30 min. The crude extracts were then heated at 90°C for 10 min, followed by precipitation with 40–65% saturation ammonium sulfate, by which the protein purity increased approximately 15-fold. The CpTI had approximate 88-fold and 186-fold purification after anion-exchange chromatography (Super-Q) and gel filtration (Sephadex G-200), respectively. A broad band of the purified CpTI on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) indicates a degree of heterogeneity and partial denaturation of CpTI, having a molecular mass of ∼8000 kD. Multiple peaks between 7451 and 8898 by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectroscopy also suggest heterogeneity. The purified CpTI was stable at 90°C for 60 min, pH 5–10, and 0–3.0% of NaCl. The purification method described here can be used to obtain highly purified CpTI for its studies such as risk assessment of CpTI genetically modified foods.

Evaluation of real-time PCR detection methods for detecting rice products contaminated by rice genetically modified with a CpTI–KDEL–T-nos transgenic construct

Genetically modified (GM) rice (Oryza sativa) lines, such as insecticidal Kefeng and Kemingdao, have been developed and found unauthorised in processed rice products in many countries. Therefore, qualitative detection methods for the GM rice are required for the GM food regulation. A transgenic construct for expressing cowpea (Vigna unguiculata) trypsin inhibitor (CpTI) was detected in some imported processed rice products contaminated with Kemingdao. The 3′ terminal sequence of the identified transgenic construct for expression of CpTI included an endoplasmic reticulum retention signal coding sequence (KDEL) and nopaline synthase terminator (T-nos). The sequence was identical to that in a report on Kefeng. A novel construct-specific real-time polymerase chain reaction (PCR) detection method for detecting the junction region sequence between the CpTI–KDEL and T-nos was developed. The imported processed rice products were evaluated for the contamination of the GM rice using the developed construct-specific real-time PCR methods, and detection frequency was compared with five event-specific detection methods. The construct-specific detection methods detected the GM rice at higher frequency than the event-specific detection methods. Therefore, we propose that the construct-specific detection method is a beneficial tool for screening the contamination of GM rice lines, such as Kefeng, in processed rice products for the GM food regulation.

Does transgenic Cry1Ac + CpTI cotton pollen affect hypopharyngeal gland development and midgut proteolytic enzyme activity in the honey bee Apis mellifera L. (Hymenoptera, Apidae)?

The transgenic Cry1Ac (Bt toxin) + CpTI (Cowpea Trypsin Inhibitor) cotton cultivar CCRI41 is increasingly used in China and potential side effects on the honey bee Apis mellifera L. have been documented recently. Two studies have assessed potential lethal and sublethal effects in young bees fed with CCRI41 cotton pollen but no effect was observed on learning capacities, although lower feeding activity in exposed honey bees was noted (antifeedant effect). The present study aimed at providing further insights into potential side effects of CCRI41 cotton on honey bees. Emerging honey bees were exposed to different pollen diets using no-choice feeding protocols (chronic exposure) in controlled laboratory conditions and we aimed at documenting potential mechanisms underneath the CCRI41 antifeedant effect previously reported. Activity of midgut proteolytic enzyme of young adult honey bees fed on CCRI41 cotton pollen were not significantly affected, i.e. previously observed antifeedant effect was not linked to disturbed activity of the proteolytic enzymes in bees' midgut. Hypopharyngeal gland development was assessed by quantifying total extractable proteins from the glands. Results suggested that CCRI41 cotton pollen carries no risk to hypopharyngeal gland development of young adult honey bees. In the two bioassays, honey bees exposed to 1 % soybean trypsin inhibitor were used as positive controls for both midgut proteolytic enzymes and hypopharyngeal gland proteins quantification, and bees exposed to 48 ppb (part per billion) (i.e. 48 ng g(-1)) imidacloprid were used as controls for exposure to a sublethal concentration of toxic product. The results show that the previously reported antifeedant effect of CCRI41 cotton pollen on honey bees is not linked to effects on their midgut proteolytic enzymes or on the development of their hypopharyngeal glands. The results of the study are discussed in the framework of risk assessment of transgenic crops on honey bees.

Comparison of chemical composition of different transgenic insect-resistant cotton seeds using Fourier transform infrared spectroscopy (FTIR)

The comparison of chemical composition of genetic modification (GM) crops to their conventional counterparts forms the basis of safety assessment process for GM crops. In this study, we used Fourier transform infrared spectroscopy (FTIR) to detect the chemical and conformational changes between transgenic cotton seeds and their non-transgenic counterparts. The assignment of absorption bands and comparison in band areas of four regions from original FTIR spectra indicated that the contents of the compounds did not differ significantly between transgenic cotton seeds and their non-transgenic counterparts (P > 0.05). The comparison of Fourier self-deconvolution (FSD) and after peak-smoothing spectra in the region between 2000 and 1000 cm-1showed that the differences in band pattern can be observed obviously between transgenic cotton seeds and its counterpart, depending on the varieties. The changes in the protein profile of transgenic Bacillus thuringiensis (Bt) cotton seeds Z30 were significant, both in multi-peaks-fitted protein structures and its ratios, compared with non-transgenic cotton seeds Z16 (P < 0.05). However, transgenic Bt + cowpea trypsin inhibitor (CpTI) cotton seeds SGK321, did not show significant changes, in comparison with non-transgenic cotton seeds SY321. These results indicated that both the indigenous and exogenous proteins structural changes in genetically modified organism (GMO) are worth being detected in detail for research related to its’ safety assessment. Key words: Transgenic, cotton seeds, safety assessment, Fourier transform infrared spectroscopy (FTIR).

Relationships among persistence of Bacillus thuringiensis and Cowpea trypsin inhibitor proteins, microbial properties and enzymatic activities in rhizosphere soil after repeated cultivation with transgenic cotton

We evaluated the effect of transgenic cottons (Bacillus thuringiensis (Bt) and Bt with cowpea trypsin inhibitor (Bt–CpTI)) on soil ecosystems after three and four years of growth. The persistence and the relationships among insecticidal crystal proteins 1Ac (Cry1Ac) and cowpea trypsin inhibitor (CpTI) proteins derived from transgenic cottons, microbial properties, and enzymatic activities in the rhizosphere soil were investigated in the repeated cultivation experiment. The Cry1Ac and CpTI proteins persisted in the rhizosphere soil of transgenic cotton, whereas no proteins were detected in the rhizosphere soil of non-transgenic cotton. The persistence of CpTI inhibited the degradation of Cry1Ac protein in the rhizosphere soil of Bt–CpTI cotton. Repeated measures analysis of variance indicated that the year of cultivation, the variety of cotton, the transgenic trait, and their interactions significantly influenced the microbial properties and the enzymatic activities in rhizosphere soil. Overall, the repeated cultivation of transgenic cotton had significantly negative effects on the microbial properties and the enzymatic activities in rhizosphere soil compared to those in the rhizosphere soil of non-transgenic cotton.

Construction and Application of a Reference Plasmid Suitable for Determination of CpTI and cry1A Gene Dosages in Genetically Modified Cottons

A reference plasmid, pMD-CCS, was constructed to detect multiple target genes in the insect resistant cotton (Gossypium hirsutum L.) varieties in China. This plasmid contained cowpea trypsin inhibitor (CpTI), Bacillus thuringiensis insecticidal crystal protein (cry1A), and cotton endogenous gene Stearoyl-acyl carrier protein desaturase (Sad1) sequences targeting the key exogenous gene types. The real-time quantitative PCR methods for CpTI and cry1A were established using pMD-CCS as the reference material (RM). The dosagess of CpTI and cry1A from 9 insect resistant cotton varieties were determined. The average CpTI dosages were 0.018–0.020 copies per genome and the average cry1A dosages were 1.377–2.136 copies per genome in the 3 insect resistant cotton varieties including Kemian 3. The average cry1A dosages were 0.887–2.564 copies per genome in 6 varieties including Ezamian 1-F1. The standard deviations of the quantitative measurement ranged from 0.001 to 0.049. These results demonstrated that pMD-CCS could be used as the RM for the quantitative measurement of CpTI and cry1A genes in insect resistant cotton varieties.

Prospects for Using Proteinase Inhibitors to Protect Transgenic Plants Against Attack by Herbivorous Insects

Proteinase inhibitors which act on the digestive enzymes of insect herbivores are a basic mechanism of plant defence. Attempts to exploit this defence mechanism in plant genetic engineering have used over-expression of both endogenous and exogenous inhibitors. While significant protection against insect pests has been routinely achieved, the engineered plants do not show levels of resistance considered commercially viable. As a result of selective pressures, insect herbivores have developed multiple mechanisms of adaptation to overcome the defensive effects of plant proteinase inhibitors. Common polyphagous crop pests are well adapted to deal with a range of different inhibitors, which have only limited effects on fitness as a result. A range of strategies have been attempted to improve effectiveness of proteinase inhibitors as antimetabolites towards insects, including selection for inhibitory activity against insect digestive enzymes, mutagenesis for novel inhibitory activity, and engineering inhibitors with multiple functions. However, proteinase inhibitor genes have only been used in transgenic crops in combination with other insecticidal genes. In Chinese genetically engineered cotton varieties which express Bt toxins as an insecticidal protein against lepidopteran larvae, the CpTI (cowpea trypsin inhibitor) gene has been employed as a second transgene to improve protection. This gene combination represents the only commercial deployment of a proteinase inhibitor transgene to date, with Bt/CpTI cotton grown on over 0.5 million hectares in 2005. Future prospects for using proteinase inhibitor genes to enhance insect resistance in transgenic crops will require reassessment of their mechanisms of action, particularly in affecting processes other than digestion, as exemplified by effects on sap-feeding hemipteran pests.

Metabolic profiling of transgenic rice progeny using gas chromatography–mass spectrometry: the effects of gene insertion, tissue culture and breeding

The Bacillus thuringiensis δ-endotoxin and cowpea trypsin inhibitor genes have been introduced into the rice genome to improve its pest resistance via Agrobacterium-mediated transformation. A gas chromatography-mass spectrometry (GC–MS) based metabolic profiling method was employed to determine the unpredictable metabolic changes resulting from the gene insertion and tissue culture separately. Descendants of the same transformant were obtained from different breeding programs, including both the transgenic and null-segregant progeny. The comparison of the transgenic and respective null-segregant plants enabled the evaluation of variations caused by transgenes; also the null-segregant plants were compared with the wild-type control to identify the influence of tissue culture. Based on the GC–MS metabolic profiles, the principal component analysis and significant differences determined by Student’s t-test suggested that there were more metabolic changes from the tissue culture than those from the insertion of the transgenes. By comparing different breeding programs, it was clear that the progeny which was developed after several generations of backcross with the non-transformed rice as the recurrent parent, displayed fewer metabolic differences from the non-transformed parent. A GC–MS based metabolic profiling study confirmed that backcrossing can help to reduce unwanted variations that occur during transformation processes.

Transgenes for insect resistance reduce herbivory and enhance fecundity in advanced generations of crop-weed hybrids of rice.

Gene flow from transgenic crops allows novel traits to spread to sexually compatible weeds. Traits such as resistance to insects may enhance the fitness of weeds, but few studies have tested for these effects under natural field conditions. We created F2 and F3 crop–weed hybrid lineages of genetically engineered rice (Oryza sativa) using lines with two transgene constructs, cowpea trypsin inhibitor (CpTI) and a Bt transgene linked to CpTI (Bt/CpTI). Experiments conducted in Fuzhou, China, demonstrated that CpTI alone did not significantly affect fecundity, although it reduced herbivory. In contrast, under certain conditions, Bt/CpTI conferred up to 79% less insect damage and 47% greater fecundity relative to nontransgenic controls, and a 44% increase in fecundity relative to the weedy parent. A small fitness cost was detected in F3 progeny with Bt/CpTI when grown under low insect pressure and direct competition with transgene-negative controls. We conclude that Bt/CpTI transgenes may introgress into co-occurring weedy rice populations and contribute to greater seed production when target insects are abundant. However, the net fitness benefits that are associated with Bt/CpTI could be ephemeral if insect pressure is lacking, for example, because of widespread planting of Bt cultivars that suppress target insect populations.