Efficacy of insect-resistance Bt/Cp TI transgenes in F5–F7generations of rice crop–weed hybrid progeny: implications for assessing ecological impact of transgene flow

Ecological impact of transgene flow into pop- ulations 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 experi- ment with Fs-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 the efficacy of transgenes Fs-F7 populations, suggesting for insect resistance. Fitness benefits of the transgenes were similar among the Fs-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-effec- tiveness, 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.

Research on Frequency of Exogenous Gene Flow from Marber-free Insect-resistant Transgenic Rice to Conventional Rice Varieties

[Objective] This study aimed to investigate the frequency of exogenous gene flow to non-transgenic conventional rice cultivars and assess the potential risks of marker-free of insect-resistant transgenic rice to agricultural ecological environment. [Method] Insect-resistant transgenic rice variety HUAHUI No.1 was planted as the experimental material and surrounded by several non-transgenic conventional rice cultivars. F1 non-transgenic rice seeds were collected according to different distances and identified by using PCR technology, the frequency of exogenous gene flow from insect-resistant transgenic rice to non-transgenic conventional rice cultivars was counted and analyzed. [Result] The average frequency of exogenous Bt gene flow to P13381 and CHUNJIANG063 was 0. Transgene flow occurred to varying degrees from insect-resistant transgenic rice HUAHUI No.1 to several non-transgenic rice lines including HEX122-2, TIANXlANG, MINGHUI63 and Pl157, with the maximum average gene flow frequency of 0.875%. The frequency of gene flow was gradually reduced with the increase of distance, and the average transgene flow frequency de- creased to 0 in all the sampling points 7 m away from transgenic rice material. [Conclusion] This study revealed that the exogenous gene flow frequency of insect-re- sistant transgenic rice variety HUAHUI No.1 was very low, leading to very small risk to the eco-environment. Rational distribution in the field for physical isolation, keeping the appropriate distance and scientific farming arrangement to avoid the synchronization of flowering can effectively control the exogenous gene flow from transgenic rice and reduce he ecological risks caused by transgene escape.

Effect of Different Sowing Dates and Densities on Individual Morphological Development of Super Short-season Insect-resistant Cotton

[Objective] The paper was to explore the effect of different sowing dates and densities on individual morphological development of super short-season insect-resistant cotton,confirm their effects on vegetative and reproductive growth of cotton,so as to provide theoretical and practical guidance for sowing date and density management of cotton planting in Jidong cotton growing region in Yellow River Basin.[Method] With super short-season insect-resistant cotton＂546＂as materials,the effects of different sowing dates（sowing dateⅠ：May 20;sowing dateⅡ：June 2;sowing date Ⅲ：June 14）and densities（low density：120 000 plants/hm2;middle density：150 000 plants/hm2;high density：180 000 plants/hm2）on individual morphological development of super short-season insect-resistant cotton were explored.[Result] Different sowing dates and density treatments significantly affected the individual morphological development of super short-season insect-resistant cotton＂546＂.The effectiveness of sowing date was higher than the effectiveness of density,and the effectiveness of sowing date on development of number of individual fruit branches was higher than that on plant height and stem diameter.[Conclusion] The regulation of sowing date and density during the cultivation process of super short-season insect-resistant cotton ＂546＂ in Jidong cotton growing region in Yellow River Basin could effectively promote vegetative and reproductive growth of cotton,strengthening its production base.

Study on Insect-resistant Transgenic Cotton Harbouring Double-gene and Its Resistance to Insect Pests

By using the method of pollen tube pathway,the synthesized GFM CryIA gene and modified CpTI gene were transfered into the elite cotton(Gossypium hirsutun L.)varieties(lines).Through the field and lab identifications,the insect-resistant transgenic plants were obtained.PCR analysis indicated that both the synthesized GFM CryIA gene and modified CpTI gene presented positive reaction.In R1 the boliworm resistance of each transformant was different,and the insect-resistance of R3 of ZGK9708 was stable.

Genetic Transformation of NGc Insect-resistant Gene in Hi-Ⅱ Maize

In order to promote the research of transgenic insect-resistant maize,the target gene were transferred into maize material Hi-Ⅱ by Agrobacterium-mediated genetic transformation of maize embryos,and maize plants with CryNGc insect-resistant genes were cultured by explant infection,co-culture and differentiation screening to study the genetic expression and resistance of exogenous genes in the offspring.The results showed that the infection effect was the best when the size of young maize embryo was 1.2-1.8 mm.Ten positive transformed plants with CryNGc insect-resistant genes were successfully obtained,and the transformation efficiency was 1.428‰.

Indication of Genetic Linkage Map for Sunflower(Helianthus annuus L.) by SSR Markers

In order to investigate the possibility and efficiency of exogenous gene spread in nature and potential ecological risk of transgenic rice, as well as analyze the effect of exogenous Bt gene insertion on ecological fitness of transgenic rice plants, a experiment was carried out with three insect-resistant Bt transgenic rice cultivars Bt63, R1 and R2 and one conventional rice line 11-838 as experimental materials, the insect-resistant transgenic and non-transgenic rice plants were inter- cropped pair-wisely under high and low insect-infestation pressures, and the vegeta- tive growth, seed-setting and the resistance to rice stem borers were compared be- tween transgenic and non-transgenic lines. According to the experimental results, both the tiller number and fresh weight of Bt transgenic rice plants under low insect- infestation pressure showed no significant differences compared with the control, but the plant height, spike length and spike weight were all lower than those of non- transgenic rice plant, and Bt63 and R2 were significantly different compared with the control. On the contrary, under high insect-infestation pressure, the tiller number, spike length and spike weight of three Bt transgenic rice cultivars were significantly higher than those of the control, while the plant height showed different fitness ef- fects among various transgenic rice cultivars, which might be related to the charac- teristics of the receptive cultivars. The individual filled grain number and 1 000-grain weight of three transgenic rice cultivars showed no significant difference compared with the control under two different insect-infestation pressures, suggesting that the effect of exogenous Bt gene on seed setting was not significant. Under insect-infes- tation pressure, the resistance of three Bt transgenic rice cultivars against rice stem borer was significantly superior to non-transgenic rice, indicating that the effect of exogenous Bt gene on insect resistance of receptive plants was distinctly. Further- more, experimental results showed that the fitness cost of Bt transgenic rice was rel- atively low, which implied that exogenous Bt gene in insect-resistant transgenic rice might escape under certain environmental conditions, but this risk was very low.

Cotton Plants Transformed with the Activated Chimeric Cry1Ac and API-B Genes

A chimeric gene, Bt29K, composed of coding sequences of activated Cry1Ac insecticidal protein and an endoplasm reticulum-retarding signal peptide, was synthesized. A plant expression vector containing two expression cassettes for the Bt29K and API-B genes was constructed. These two insect-resistant genes were transferred into two cotton ( Gossypium hirsutum L.) varieties ( or lines) via Agrobacterium-mediated transformation and nine homozygous transgenic cotton lines showing a mortality of 90.0% - 99.7% to cotton ballworm (Heliothis armigera) larvae and good agronomic traits were selected through six generations. Molecular biology analysis revealed that one or two copies of the insecticidal protein genes were integrated into the transgenic cotton genome and activated Cry1Ac and API-B protein expression was at a level of 0.17% and 0.09% of the total soluble protein in the transgenic cotton leaves, respectively. Comparison of the insect-resistance of the homozygous lines expressing the activated chimeric Cry1Ac and API-B with that expressing Cry1Ac only revealed that the insect-resistance of the former is apparently higher than the latter. These results also indicate that the strategy to construct a plant expression vector expressing two different insect-resistant genes reported here is reasonable.

Temporal and spatial changes in Bt toxin expression in Bt-transgenic poplar and insect resistance in field tests

Extensive planting of Bacillus thuringiensis （Bt）-transgenic plants economically benefits society; how-ever, the potential risk they pose is receiving increasing attention. This study used enzyme-linked immunosorbent assay and fluorescence quantitative PCR （RT-PCR） to monitor the temporal and spatial dynamics of the expression of Bt toxic protein in a forest of 6- to 8-year-old trees of transgenic insect-resistant poplar 741 for three consec- utive years. The enrichment, distribution, and degradation of Bt toxic protein and the influence of transgenic poplars on the targeted insect population, Hyphantria cunea, were investigated. The content of CrylAc toxic protein dynamically changed in transgenic poplar. During the annual growth cycle, the content initially increased, then decreased in the long and the short branches of the crown and in the root system, peaking in August. During the study, the protein did not accumulate overtime. The mRNA transcription of gene CrylAc was almost consistent with the level of the protein, but transcription peaked in July. In the transgenic and control forestland, microscale levels of the CrylAc toxic protein were detected from the soil, but increased accumulation was not observed with the planting year of transgenic poplar. Meanwhile, Bt was isolated and detected molecularly from the soil in the experimental forestland. A systematic investigation of the density of H. cunea in the experimental transgenic poplar forest indi- cated that transgenic Pb29 poplar could resist insects to a certain degree. At peak occurrence of the targeted insects, the density of H. cunea in the experimental forest was significantly lower than in the nontransgenic poplar forest.

Screening of Brown Planthopper Resistant miRNAs in Rice and Their Roles in Regulation of Brown Planthopper Fecundity

MicroRNAs(miRNAs)can participate in plant-insect interactions,which regulate plant defense networks.In this study,we analyzed the miRNA expression profiles of six rice varieties before and after brown planthopper(BPH)-feeding.We identified 45 differentially expressed miRNAs between BPH-susceptible and BPH-resistant rice varieties and 144 miRNAs that responded to BPH-feeding.Thus,miRNAs may be involved in multiple pathways regulating rice defense response against BPH.In addition,we found that the genetic history of rice varieties determined the regulation mode of the miRNA and affected the amounts,types,changing trends and response periods of miRNAs in response to BPH-feeding.To conclude,we scanned seven potential cross-kingdom miRNAs,of which miR5795 may target the vitellogenin gene in BPH,causing a 16.07%reduction in BPH oviposition.The results provide new miRNA information of rice-BPH interactions and BPH-resistant rice variety breeding.

Molecular Cloning of a Thiol Proteinase Inhibitor Gene and Its Expression in E.coli

A cDNA library was constructed with 1.5×10~6 pfu from rice immature seeds,fromwhich a cDNA clone for rice thiol proteinase inhibitor,oryzacystatin(OC),was isolated byscreening with synthesized oligodeoxynucleotide probe,which contained a 309bp open read-ing frame,84bp 5′-end noncoding region and a poly(A)signal AATAAA at the 3′-end fol-lowed by 31Nt poly(A).Then the coding region of OC was amplified and inserted into thedownstream of λP_RP_L promoter for thermal-inducible expression in E.coli.Shifting the cul-ture temperature from 30℃ to 42℃ led to a high level expression of OC,which exhibited adistinct band of 12.0 kDa and accounted for at least 10% of the total soluble proteins fromSDS-PAGE.The papain-inhibitory activity of the expressed OC was further confirmed.

Performance of Hybrids between Weedy Rice and Insect-resistant Transgenic Rice under Field Experiments: Implication for Environmental Biosafety Assessment

Transgene escape from genetically modified （GM） rice into weedy rice via gene flow may cause undesired environmental consequences. Estimating the field performance of crop-weed hybrids will facilitate our understanding of potential introgression of crop genes （including transgenes） into weedy rice populations, allowing for effective biosafety assessment. Comparative studies of three weedy rice strains and their hybrids with two GM rice lines containing different insect- resistance transgenes （CpTI or Bt/CpTI） indicated an enhanced relative performance of the crop-weed hybrids, with taller plants, more tillers, panicles, and spikelets per plant, as well as higher 1000-seed weight, compared with the weedy rice parents, although the hybrids produced less filled seeds per plant than their weedy parents. Seeds from the F1 hybrids had higher germination rates and produced more seedlings than the weedy parents, which correlated positively with 1000-seed weight. The crop-weed hybrids demonstrated a generally enhanced relative performance than their weedy rice parents in our field experiments. These findings indicate that transgenes from GM rice can persist to and introgress into weedy rice populations through recurrent crop-to-weed gene flow with the aid of slightly increased relative fitness in F1 hybrids.

Expression of two insect-resistant genes cryIA (b&c)/GNA in transgenic tobacco plants results in added protection against both cotton bollworm and aphids

The synthesized Bacillus thuringiensis insecticidal protein gene crylA(b&c) and the synthesized gene GNA, (the mannose specific lectin from snowdrop ( Galanthus nivalis)), tumefaciens have been inserted into plant expression vector pGW4BAI. Leave stripes of Nico-tiana tabacum var. K326 have been transformed with Agrobacterium tumefaciens strain LBA4404 harboring the plant expression vector. 28 kanamycin resistant tobacco plants

Impact of insect-resistant GM rice on pesticide use and farmers' health in China

The economic benefits of insect-resistant genetically modified(GM) crops have been well documented, but the impact of such crops and the consequent reduction in pesticide use on farmers' health remains largely unknown. Through the analysis of the data collected from the physical examination from farmers in China, we show that GM rice significantly reduces pesticide use and the resultant not only visible but also invisible adverse effects on farmers' neurological, hematological, and electrolyte system. Hence, the commercialization of GM rice is expected to improve the health of farmers in developing countries, where pesticide application is necessary to mitigate crop loss.

Commercial production of transgenic Bt insect-resistant cotton varieties and the resistance management for bollworm (Helicoverpa armigera Hubner)

There are currently three kinds of transgenic Bt insect-resistant cotton germplasm lines, Shanxi 94-24, Zhongxin 94 and R19, in China. They showed high resistance to the neonate larvae of bollworm (Helicoverpa armigera). Transgenic Bt insect-resistant cotton varieties or hybrids have been bred using the three kinds of germplasm lines as parents. Our researches reveal that there exist different expressions in resistant level at different developmental stages in the three categories of germplasm lines. When neonate larvae are fed with leaves of cotton plant at the seeding stage with less than 10 leaves on the main stem, the mortality of the neonate larvae is 100%, but the resistance level will decline at later season. When Bt gene has been transferred to the cotton genome, it can be steadily transferred to the progeny, the level of resistance to bollworm keeps fundamentally uniform. Such insects as tobacco budworm (Heliothis virencens) in laboratory directive selection are very apt to produce resistance

Integration and inheritance stability of foreign Bt toxin gene in the bivalent insect-resistant transgenic cotton plants

Genetic and expressional stability of Bt toxin gene is crucial for the breeding of insect-resistant transgenic cotton varieties and their commercialization. Genomic Southern blot analysis of R3, R4 and R5 generations of bivalent transgenic insect-resistant cotton plants was done in order to determine the integration, the copy number and the inheritance stability of Bt toxin gene in the transgenic cotton plants. The results indicated that there was a 4.7 kb positive band in the Southern blot when the genomic DNA of the bivalent transgenic insect-resistant cotton plants and the positive control (the plasmid) were digested with HindⅢ respectively. This result proved that the Bt toxin gene had been integrated into the genome of the cotton in full length. There is only one XhoⅠ restriction site in the Bt toxin gene. Southern blot analysis indicated that many copies of Bt toxin gene had been integrated into the genome of the cotton when the genomic DNA of transgenic plants was digested with XhoⅠ. Among them, there were four copies (about 17.7, 8, 5.5 and 4.7 kb in size) existing in all the tested plants of R3, R4 and R5 generations. The preliminary conclusion was that there were more than four copies of Bt toxin gene integrated into the genome of the cotton, among them, more than one copy can express and inherit steadily. This result provides a scientific basis for the breeding of the bivalent insect-resis- tant transgenic cotton plants and its commercialization.

Ray Wu,Cornell's acclaimed pioneer of genetic engineering and developer of insect-resistant rice

ITHACA, N.Y. -- Ray J. Wu, Cornell University professor of molecular biology and genetics, who was widely recog-nized as one of the fathers of genetic engineering and who developed and sought to feed the world with a higher yield-ing rice that resists insects and drought, died of cardiac arrest in Ithaca, Feb. 10.

Inheritance of resistance to Helicoverpa armigera of 3 kinds of transgenic Bt strains available in upland cotton in China

There are 3 kinds of transgenic Bt strains, Shanxi 94-24, Zhongxin 94, and R19, in upland cotton in China. Their transgenic Bt insect-resistance cultivars or hybrids have been developed and grown by farmers. Genetic studies indicate that the resistance of the 3 transgenic Bt cotton strains to Helicoverpa armigera is controlled by one pair of non-allelic dominant genes. Linkage relationship between the resistant genes of R19 and Shanxi 94-24 transgenic Bt strains shows that they may be inserted in the same chromosome. FT hybrids crossed among the 3 strains show that high levels of protection from feeding damage are the same as that of their parents. Therefore, there is no co-suppression phenomenon in many transgenic plants. The results presented here afford a fundamental reliance in developing transgenic Bt insect-resistant cultivars and exploiting the heterosis of hybrids in upland cotton.

Transgenic Tobacco Plants With Efficient Insect Resistance

Insecticidal protein gene CryIA(c)from Bacillus thuringiensis HD-1(B.t.toxin gene)with 5’-end modified and 3’-end deleted to 4 different lengths were inserted downstream of 35S promoterwith double enhancer and"Ω’"fragment of TMV-RNA cDNA in the binary vector pBin438 to constructthe chimeric expression vector of B.t.toxin gene.Leave stripes of tobacco plant var.NC89 widelygrown in China were transformed with A.tumefaciens LBA4404 harbouring the above expression vectorsrespectively,and kanamycin resistant tobacco plants were regenerated.Insect test with tobacco budwormH.assulta showed that insect-resistant transform.ants could be obtained from the regenerated plantstransformed with B.t.genes of different lengths though highest percentage(～50%)of plants with ahigh morality(90%-100%)to the testing insects is among those transformed with 1.8-kb toxin gene.Genetic,molecular and biological analyses of T1 and T2 progenies of plants with high efficient insect re-sistance showed that B.t.toxin gene and the character of insect resistance have been inherited in the pro-genies.Insect-resistant homozygotes D8-14 and D19-8 have been selected for small-scale field tests.

Variation induced by DNA rearrangement in a transgenic Bt+CpTI cotton strain

In the development, of transgenic Bt + CpTI cotton cultivars, one male and female sterile mutant has been found in a homozygous T4 strain in our laboratory. The mutant plant, as well as its leaves, buds and flowers, is only 1/2-1/3 as large as that of the wild transgenic Bt + CpTI bivalant cotton plants. Cytological observation found that the chromosome number of the mutant is 2n=52; however, there are 4-8 univalents observed in meiosis I of pollen mother cells. Laboratory bioassay indicated that the mutant was highly resistant to bollworm as the wild plants. PCRamplification revealed that Bt and CpTl genes in the mutant were still intactly inserted. However, small deletion of flanked area had been observed in the mutant by Southern blotting analysis. So it is proposed that the mutant pheno-type might result from either the DNA deletion or T-DNA transferring in plant genome. No such report has been presented that the rearrangement of chromosome structure in a homozygous transgenic line occurred.

Construction of a rice immature seeds cDNA library and molecular cloning of oryzacystatin cDNA

Total RNA was extracted from rice immature seeds harvested 2 weeks after flowering; then mRNA was purified. cDNA with NotI and SaiI cohesive ends was synthesized and inserted into λgt22A. After packaged in vitno, the cDNA library was constructed with 1.5×106pfu. A 21-mer oligodeoxynucleotide was synthesized according to the 5’-end conserved coding sequence of oryzacystatin (a thiol proteinase inhibitor) and labeled as a probe. From 2.1 × 104 pfu, 9 positive dones have been isolated, 8 of which contain the entire coding region of oryzacystatin. λOC1 has the longest cDNA insert, which contains an open reading frame of 309 bp coding sequence, 84 bp 5’-end non-coding region and a poly(A) signal AATAAA at the 3’-end followed by 31 Nt of poly(A). The coding sequence is the same compared with oryzacystatin genomic DNA sequence, while there are some obvious differences such as insertion and variation in the non-coding region, especially lots of nonsucoessive insertion in the 3’ region after poly(A) signal.