Effects of Transgenic Bt+CpTI Cotton on Field Abundance of Non-Target Pests and Predators in Xinjiang, China

Transgenic insect-resistant cotton is being increasingly planted in Xinjiang cotton-planting regions, where geographical climate conditions and species composition of pests and natural enemies are greatly unique in China. Limited studies have been conducted on the ecological impacts of transgenic insect-resistant cotton, especially for transgenic double genes （Bt＋CpTI） cotton, in this region. In this study, the potential effects of transgenic Bt＋CpTI cotton on the seasonal abundance of non-target pests and predators were assessed from 2009 to 2011 in Korla, Xinjiang. The results showed that species composition and seasonal abundance of 5 groups of pests and 5 groups of predators were not significantly different between transgenic Bt＋CpTI cotton and non-transgenic cotton every year. It suggests that transgenic Bt＋CpTI cotton per se does not affect the population dynamics of non-target pests and predators on this crop in Xinjiang.

Modified accumulation of selected heavy metals in Bt transgenic rice

Safety assessment of genetically modified crops generally does not take into account the potential hazard of altered patterns of heavy metal accumulation in plants. A pot experiment was conducted under greenhouse conditions to evaluate the impact of heavy metal amendments on the accumulation of Cd, Cu, Pb and Zn in a Bt transgenic rice Ke-Ming-Dao （KMD） and its wild-type Xiushui 11 （Xs11）. In control soils, significant difference was only found in contents of Cu （p 〈 0.01） and Pb （p 〈 0.05） in straw between KMD and Xs11. At three levels of Cd amendments （5, 10, and 20 mg/kg）, the Cd contents in grain and straw of KMD were significantly higher than those of Xs11, and all grain Cd contents were significantly higher than the International Criteria （0.2-0.4 mg/kg） as specified by the Codex Alimentarius Commission （CAC）. These results implied that it may be unsafe for growing Bt transgenic rice in heavily Cd-polluted areas. No significant difference in Zn was found between the two varieties with the exception of roots at Zn amendment level of 600 mg/kg, while Pb contents in KMD were much higher in the straw at the lead amendment level of 1000 mg/kg and in the root at 250 mg Pb/kg. Data on the heavy metal accumulation patterns for the genetically modified rice may be used for the selection of growing areas as well as for plant residue management for Bt rice.

NaCl-Induced Changes of Ion Fluxes in Roots of Transgenic Bacillus thuringiensis (Bt) Cotton (Gossypium hirsutum L.)

Bacillus thuringiensis （Bt） cotton is grown worldwide, including in saline soils, but the effect of salinity on ion fluxes of Bt cotton remains unknown. Responses of two transgenic Bt cotton genotypes （SGK321 and 29317） and their corresponding receptors, Shiyuan 321 （SY321） and Jihe 321 （J321）, to 150 mmol L-1 NaCl stress were studied in a growth chamber. The root dry weight of SGK321 and 29317 under NaCl treatment was decreased by 30 and 31%, respectively. However, their corresponding receptor cultivars SY321 and J321 were less affected （19 and 24%, respectively）. The root length and surface area of the Bt cultivars were significantly decreased relative to their receptors under salt stress. NaCl treatment significantly increased CrylAc mRNA transcript levels in SGK321 and 29317 but did not affect Bt protein content in leaves or roots of either cultivar at 1 and 7 d after NaCl treatment. Fluxes of Na^＋, K^＋, and H^＋ in roots were investigated using the scanning ion-selective electrode technique. Both mean K^＋ efflux rate and transient K^＋ efflux of the Bt cultivars increased four-fold compared to their corresponding receptors when exposed to salinity stress. There were no significant differences in Na^＋ efflux between Bt and non-Bt cottons. Furthermore, the Na^＋ contents in roots and leaves of all genotypes dramatically increased under salt stress, whereas K^＋ contents decreased. Our results suggested that Bt cotton cultivars are more sensitive to salt stress than their receptor genotypes.

Impacts of Environmental Factors on Degradation of CrylAb Insecticidal Protein in Leaf-Blade Powders of Transgenic Bt Rice

The determination of the environmental fate of Bt insecticidal protein released by Bt rice plants in paddy soils is a key issue in its ecological risk assessment. In this study, the impacts of soil water content, pH, and temperature on the degradation of CrylAb protein expressed in the leaves of Bt rice KMD2 were studied in the laboratory. Three types of paddy soils were used, i.e., blue clayey paddy soil, pale paddy soil on quaternary red soil, and marine-fluvigenic yellow loamy paddy soil. Ground powders of KMD2 leaf blades were mixed with each type of soil, and degradation dynamics of Cry lAb were measured using enzyme-linked immunosorbent assay （ELISA）. The degradation rate of CrylAb was high at the early experimental stage, but slowed down steadily at middle and later stages, which could be described by exponential equations, with the half-life period of degradation determined as 1.8-4.0 d. The soil water content, pH, and temperature could affect the degradation of CrylAb, but the effects of soil pH and temperature were relatively greater. In general, CrylAb degradations were slower under lower soil pH and temperature conditions, especially for marine-fluvigenic yellow loamy paddy soil.

Combining Ability and Heterosis Between High Strength Lines and Transgenic Bt (Bacillus thuringiensis) Bollworm-Resistant Lines in Upland Cotton (Gossypium hirsutum L.)

To analyse the combining ability and heterosis between high-strength lines and transgenic Bt bollworm-resistant lines in upland cotton, 5 high-strength lines were crossed as female lines with 12 transgenic Bt bollworm-resistant lines according to NCII design. It was demonstrated that the compositions of variance in various traits were quite different. For seed cotton yield, lint yield, boll numbers per plant and boll weight, the dominant (special combining ability) effects were the major effects, accounting for 87.38, 84. 40, 80. 04 and 64. 46% of the total phenotypic variances, respectively, while for fibre strength and micronaire value, the additive (general combining ability) effects had the major effects, with a ratio of additive variance to phenotypic variance of 78.85 and 43.80%. As for lint percent and 2.5% span length, the dominant and additive variances had similar effects, in phenotypic variances (54.94 and 40.11% for lint percent, 45.76 and 42.49% for 2. 5% span length, respectively). The mid-parent heterosis (Hpm), surpassing parent heterosis (Hpb) and competitive heterosis (Hck) for seed cotton yield and lint yield were both extremely significant. For fibre properties, the Hck and Hpm of 2.5 % fibre span length were extremely significant, the Hck of fibre strength was significant, and the favorable negative Hck of micronaire was also extremely significant. The increments of hybrid over common variety were 17% for lint yield and fibre strength, 7% for fibre span length, and 4 % for fineness.

Effects of Transgenic Bt+CpTI Cotton Cultivation on Functional Diversity of Microbial Communities in Rhizosphere Soils

[Objective] This study aimed to investigation the effects of tranagenic Bt ＋ CpTI cotton cultivation on functional diversity of microbial communities in rhizospbere soils. E Method] By using the Biolog method, a comparative study was conducted on the utilization level of single carbon source by microbes in the rhi- zosphere soils of transgenic Bt ＋ CpTI cotton sGK321 and its parental conventional cotton ＇ Shiyuan 321＇ at different growth stages. [ Result ] The results showed that, compared with the parental conventional cotton, the average well-color development （AWCD） value of micmhial communities in rhizospbere soils of transgenie Bt ＋ CpTI cotton were significantly higher （P 〈 O. 05） at seedling stage and budding stage while significantly lower at flower and boll stage and bell opening stage. Shannon-Wiener diversity index （H） and Simpson dominance index （D） of microbial communities in rhlzesphere soils of transgenic cotton and conventional cotton varied with the different growth stages, whereas the Shannon-Wiener evenness index （E） showed no significant difference between transgenie cotton and convention- al cotton at four growth stages. Principal component analysis indicated that the patterns of carbon source utilization by microbial communities in rhizospbere soils were similar among transgenic cotton at seeding stage and flower and boll stage and parental conventional cotton at seeding stage and budding stage, which were also similar between tranagenic cotton at budding stage and parental conventional cotton at flower and boll stage. [ Conclusion] Analysis of different carbon sources indi- cated that the main carbon sources utilized by soil microbes were carbohydrates, amino acids, carboxylie acids and polymers.

Evaluation of Impact of Pollen Grains from Bt,Bt/CpTITransgenic Cotton and Bt Corn Plants on the Growth andDevelopment of the Mulberry Silkworm,Bombyx moriLinnaeus (Lepidoptera:Bombycidae)

The S-endotoxin genes of Bacillus thuringiensis (Bt) and proteinase inhibitor (PI) genes are two kinds of genes popularly used for developing transgenic plants resistant to insect pests. To clarify whether there is any risk concerning the effects of pollens from these transgenic crops on non-target insects with economic importance, such as the effects on the growth and development as well as cocoon quality of the silkworm, Bombyx mori Linnaeus, a series of feeding experiments were conducted, using pollens from transgenic cotton or corn containing cry 1Ac, cry1A+CpTI or crylAb genes, compared with pollens from non-transgenic normal cotton and corn as well as the non-pollen treatment. In contrast to the latter ones, pollens from transgenic plants showed no significant adverse effects on larval mortality, cocoon weight, pupa weight, cocoon shell weight, pupation rate, emergence rate and fecundity of the silkworm after neonates were fed with the pollens for 72 h. In addition, no dosage effects of pollens were found. Though the duration of 1st instar larvae was prolonged in the case of feeding with transgenic pollens as compared with those of the non-pollen treatment , but they were not significantly different from those fed with pollens from non-transgenic cotton or corn. Meanwhile, the body weight of the 3rd instar molters fed with transgenic pollens was obviously different from those for non-pollen treatment, and was all significantly heavier than that of the controls. Consequently, it is considered that the adverse effect of pollens from transgenic insect-resistant cotton and corn on the growth and development of the silkworm is negligible.

AFLP Analysis and Trait Evaluation of Bt Transgenic Sugarcane Lines

[ Objective] A large number of high-quality Bt transgenic sugarcane lines were obtained using transgenic technologies. This study aimed to explore the genetic background, genetic diversity, important traits and relationship between the heredity and phenotype of these transgenic sugarcane lines, thereby providing basis for further effective utilization and preservation of insect-resistant germplasm resources and improving breeding efficiency of insect-resistant sugarcane in China. [Method] By using six AFLP primers with high polymorphism, the genetic diversity of 13 Bt transgenic sugarcane lines and their donor varieties was analyzed. Growth assay and insect-resistance identification were carried out in a quarantine greenhouse with conventional methods. Evaluation of important economic traits was conducted in the field with conventional methods. [ Result] The polymorphism rate was relatively low. Specifically, the polymorphism rate of the first group was 21.00%, the polymorphism rate of the second group was 31.80%, and the total polymorphism rate was 42.50%. Cluster analysis showed that 13 Bt transgenic sugarcane lines and their donor varieties were divided into two categories, the genetic similarity coefficient between two categories was 0.54. All the six materials in the first group were clustered into category Ⅰ, with genetic similarity of 0. 921 ; nine materials in the second group were clustered into category Ⅱ, with genetic similarity of 0. 898. Bt transgenic sugarcane lines showed abundant phenotypic types and significant trait variations, and the economic traits were generally worse than donor varieties. The insect resistance of 13 Bt transgenic sugarcane lines was divided into five grades. [ Conclusion] The first-generation Bt transgenic sugarcane lines have low genetic diversity, abundant trait variations, high insect resistance and generally poor economic traits, which can only be used as germplasm resources for hybridization. Introducing Bt gene into multiple donors will to some extent improve the genetic diversity of sugarcane germplasm resources, but the increasing rate is relatively low. It is preliminarily considered that the genetic diversity of insect-resistant sugarcane germplasm resources should be improved by continuous hybridization and backcrossing of Bt transgenic lines with high-quality parents, thus laying foundation for breeding high-quality insect-resistant sugarcane varieties.

Differential Expression of Bt Protein in Transgenic Bt Cotton

[Objective] The paper was to study the temporal and spatial dynamics of Bt protein expression in transgenic Bt cotton and to determine the inner relationship of Bt protein expression and transgenic Bt cotton. [Method] With transgenic cotton cultivar（ GK19） as the test material,Bt protein contents in different organs,main stem functional leaves at different growth stages and different positions of main stem leaves at different growth stages were studied by enzyme-linked immunosorbent assay. [Result] There were differences in Bt protein content among different organs of transgenic Bt cotton; the Bt protein content of leaves at seedling stage was the highest,followed by flowers,bubs and bolls,and those of roots and stems were relatively low. The Bt protein content of main stem function leaves gradually decreased with the progressing development. There were great differences in Bt protein content among different positions of main stem leaves at different growth stages; the Bt protein content of the 1^（st）-7^（th） top leaves at seedling stage and full budding stage gradually decreased,while those at full flowering stage and full bolling stage first slowly increased then gradually stabilized. [Conclusion] Bt protein expression was found in all organs of transgenic cotton at all growth stages,and the expression level presented temporal and spatial dynamics.

The Influence of Transgenic cry1Ab/cry1Ac,cry1C and cry2A Rice on Non-Target Planthoppers and Their Main Predators Under Field Conditions

Transgenic Bt rice has been shown to be an effective means of controlling Lepidoptera pests of rice. However, the potential roles of transgenic rice on planthoppers and their predators need to be investigated before its commercialization. Population density, species dominance and population dynamics are important parameters of arthropods populations in field. So the impacts of three transgenic Bt rice strains expressing crylAb/crylAc, crylC and cry2A on population density, species dominance and population dynamics of three species of planthoppers （Nilaparvata lugens, Sogatella furcifera and Laodelphax striatellus） and their three main predators （ Cyrtorhinus lividipennis, Pirata subpiraticus and Theridium octomaculatum） were evaluated at three sites in Hubei Province, China, in the current study. The results showed that among three species of planthoppers, both in transgenic and non-transgenic rice field, the predominant species ofplanthoppers within phytophagous guild was S. furcifera at any site either growing season （46-50%）. Significantly higher population density ofN. lugens was observed in T2A-1 field relative to Minghui 63 field at Wuxue in 2010. The species dominance of predator, P. subpiraticus, in TT51 field was significantly higher than that in T 1 C-19 and T2A-1 fields in 2009 at Xiaogan site. Sampling date significantly influenced six arthropods except for P. subpiraticus in 2010. The interaction between rice strain^sampling date had no significant adverse effects on the population dynamics of three species of planthoppers and their predators, except for several individual species in 2009. The interaction among rice strain^sampling date^sampling site also had no significant effect on six arthropods except for S. furcifera in 2009. The results indicated that transgenic Bt rice expressing crylAb/crylAc, cry2A and crylC had no significant adverse effects on the population dynamics of three planthoppers and their predators in most investigated data and sampling site.

Effects of Pesticide Applications on the Biochemical Properties of Transgenic cry 2A Rice and the Life History Parameters of Nilaparvata lugens Stl (Homptera: Delphacidae)

Effects of transgenic Bt rice on non-target pests following pesticide applications plays an important role in evaluating the ecological safety of transgenic rice. Changes of the life history parameters of laboratory population of Nilaparvata lugens feeding on transgenic Bt rice T2A-1 and its parental line cv. MH63 as the control, and the contents of oxalic acid and soluble sugar following three pesticides （triazophos, jinggangmycin and chlorantraniliprole） treatments were investigated in this paper. Results showed that the population parameters of N. lugens and the physiological and biochemical parameters of rice did not differ significantly between T2A-1 and MH63 without pesticide application. But, the emergence rate （ER）, the nymphal number of next generation （NN）, and the index of population trend （IP） on T2A-1 treated with 10 mg L^-1 of triazophos were significantly higher than those on MH63, respectively, while the hatchability （HB） on T2A-1 treated with the three pesticides were obviously lower than those on MH 63. Furthermore, the content of oxalic acid in MH63 treated with 20 mg L-1 triazophos and, 40 and 80 mg L-1 chlorantraniliprole was significantly higher than that in T2A-1, while the content of soluble sugar in T2A-1 treated with 400 mg L-1 jinggangmycin was significantly higher than that in MH63. The results above indicated that triazophos in a low dose could cause a more favor of T2A-1 to population growth of N. lugens compared with MH 63.

Development of lepidopteran pest-resistant transgenic japonica rice harboring a synthetic cry2A* gene

A synthetic cry2A^＊ gene enco ding Bacillus thuringiensis（Bt） δ-endotoxi n that resi st ance to lepidopteran pest was transformed into japonica rice variety Jijing 88, which is the most widely cultivated variety in Jilin Province, Northeast China, by Agrobacterium-mediated transformation. A total of 106 independent transformants overexpressing cry2A^＊ gene driven by ubiquitin（Ubi） promoter was produced. Three single-copy homozygous transgenic lines were finally selected based on the results of PCR analysis, se gregation ratio of Bast a resistance, and Southern hybridiza tion analyse s. RT-PCR and enzyme linke dimmune sorbent assay（ELISA） revealed that cry2A^＊ transcripts and protein were highly expressed in these lines. The high level of Cry2A^＊ protein expression resulted in high resistance to rice striped stem borer as evidence d by insect feeding bioassays. Our results demonst rate that cry2A^＊ transgenic japonica rice confers resistance to the rice striped stem borer in the laboratory conditions.

Effect of vegetation of transgenic Bt rice lines and their straw amendment on soil enzymes, respiration, functional diversity and community structure of soil microorganisms under field conditions

With the development of transgenic crops, there is an increasing concern about the possible adverse effects of their vegetation and residues on soil environmental quality. This study was carried out to evaluate the possible effects of the vegetation of transgenic Bt rice lines Huachi B6 （HC） and TT51 （TT） followed by the return of their straw to the soil on soil enzymes （catalase, urease, neutral phosphatase and invertase）, anaerobic respiration activity, microbial utilization of carbon substrates and community structure, under field conditions. The results indicated that the vegetation of the two transgenic rice lines （HC and TT） and return of their straw had few adverse effects on soil enzymes and anaerobic respiration activity compared to their parent and distant parent, although some transient differences were observed. The vegetation and subsequent straw amendment of Bt rice HC and TT did not appear to have a harmful effect on the richness, evenness and community structure of soil microorganisms. No different pattern of impact due to plant species was found between HC and TT. It could be concluded that the vegetation of transgenic Bt rice lines and the return of their straw as organic fertilizer may not alter soil microbe-mediated functions.

Effects of Elevated Carbon Dioxide on the Growth and Foliar Chemistry of Transgenic Bt Cotton

A field study was carried out to quantify plant growth and the foliar chemistry of transgenic Bacillus thuringiensis （Bt） cotton （cv. GK-12） exposed to ambient CO2 and elevated （double-ambient） CO2 for different lengths of time （1, 2 and 3 months） in 2004 and 2005. The results indicated that CO2 levels significantly affected plant height, leaf area per plant and leaf chemistry of transgenic Bt cotton. Significantly, higher plant height and leaf area per plant were observed after cotton plants that were grown in elevated CO2 were compared with plants grown in ambient CO2 for 1, 2 and 3 months in the investigation. Simultaneously, significant interaction between CO2 level x investigating year was observed in leaf area per plant. Moreover, foliar total amino acids were increased by 14%, 13%, 11% and 12%, 14%, 10% in transgenic Bt cotton after exposed to elevated CO2 for 1, 2 or 3 months compared with ambient CO2 in 2004 and 2005, respectively. Condensed tannin occurrence increased by 17%, 11%, 9% in 2004 and 12%, 11%, 9% in 2005 in transgenic Bt cotton after being exposed to elevated CO2 for 1, 2 or 3 months compared with ambient CO2 for the same time. However, Bt toxin decreased by 3.0%, 2.9%, 3.1% and 2.4%, 2.5%, 2.9% in transgenic Bt cotton after exposed to elevated CO2 for 1, 2 or 3months compared with ambient CO2 for same time in 2004 and 2005, respectively. Furthermore, there was prominent interaction on the foliar total amino acids between the CO2 level and the time of cotton plant being exposed to elevated CO2. It is presumed that elevated CO2 can alter the plant growth and hence ultimately the phenotype allocation to foliar chemistical components of transgenic Bt cotton, which may in turn, affect the plant-herbivore interactions.

Influences of elevated CO2 and pest damage on the allocation of plant defense compounds in Bt-transgenic cotton and enzymatic activity of cotton aphid

Plant allocation to defensive compounds by elevated CO2-grown nontransgenic and transgenic Bt cotton in response to infestation by cotton aphid, Aphis gossypii （Glover） in open-top chambers under elevated CO2 were studied. The results showed that significantly lower foliar nitrogen concentration and Bt toxin protein occurred in transgenic Bt cotton with and without cotton aphid infestation under elevated CO2. However, significantly higher carbon/nitrogen ratio, condensed tannin and gossypol were observed in transgenic Bt cotton ＂GK-12＂ and non-transgenic Bt cotton ＇Simian-3＇ under elevated CO2. The CO2 level and cotton variety significantly influenced the foliar nitrogen, condensed tannin and gossypol concentrations in the plant leaves after feeding by A. gossypii. The interaction between CO2 level x infestation time （24 h, 48 h and 72 h） showed a significant increase in cotton condensed tannin concentrations, while the interaction between CO2 level x cotton variety significantly decreased the true choline esterase （TChE） concentration in the body ofA. gossypi. This study exemplified the complexities of predicting how transgenic and non-transgenic plants will allocate defensive compounds in response to herbivorous insects under differing climatic conditions. Plant defensive compound allocation patterns and aphid enzyme changes observed in this study appear to be broadly applicable across a range of plant and herbivorous insect interactions as CO2 atmosphere rises.

Azotobacter inoculation can enhance the resistance of Bt cotton to cotton bollworm, Helicoverpa armigera

The rising trend in the cultivation of Bacillus thuringiensis (Bt) transgenic crops may cause a destabilization of agroecosystems, thus increasing concerns about the sustainability of Bt crops as a valid pest management method. Azotobacter can be used as a biological regulator to increase environmental suitability and improve the soil nitrogen utilization efficiency of crops, especially Bt cotton. A laboratory test investigated effects on the development and food utilization of Helicoverpa armigera fed with different Cry1Ab/Cry1Ac proteins and nitrogen metabolism-related compounds from cotton (transgenic variety SCRC 37 vs non-Bt cotton cv. Yu 2067) inoculated with Azospirillum brasilense (Ab) and Azotobacter chroococcum (Ac). The findings indicate that inoculation with Azotobacter significantly decreased the partial development and food utilization indexes (pupal weight;pupation rate;adult longevity;fecundity;relative growth rate, RGR;efficiency of conversion of digested food, ECD;and efficiency of conversion of ingested food, ECI) of H. armigera fed on Bt cotton, but contrasting trends were found among these indexes in H. armigera fed on non-Bt cotton inoculated with Azotobacter, as a result of differences in Bt toxin production. Overall, the results showed that inoculation with Azotobacter had negative effects on the development and food utilization of H. armigera fed on Bt cotton, leading to enhanced target insect resistance. Presumably, Azotobacter inoculation can be used to stimulate plant soil nitrogen uptake to increase nitrogen metabolism-related compounds and promote plant growth for Bt and non-Bt cotton, simultaneously raising Bt protein expression and enhancing resistance efficacy against cotton bollworm in Bt cotton.