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.

Impact Evaluation of Insect-Resistant Transgenic Rice on the Feeding and Oviposition Behavior of Its Non-Target Insect, the Brown Planthopper, Nilaparvata lugens (Homptera: Delphacidae)

The feeding and oviposition behavior of the brown planthopper (BPH), Nilaparvata lugens on two transgenic indica rice homogenous genotypes (B1 and B6) with cry1Ab gene from Bacillus thuringiensis and transgenic restored line of hybrid rice (MSA) with SCK gene (a modified CpTI gene) were measured, compared with those on their corresponding non transgenic parental cultivars Jiazao935 and Minghui86 performed by BPH. Under the selection condition of host plants by BPH, loading percentage, oviposition preference and laying egg number of BPH both on transgenic cry1Ab rice and transgenic SCK rice were not significantly different from those on their controls, while their total number of probing wound caused by PBH expect for feeding on B1 plants was markedly more than that on the control. In contrast, under the non selection condition, total number of probing wound caused by BPH on either transgenic cry1Ab rice or transgenic SCK rice was pronouncedly more than those on their controls. Conversely, their honeydew amount excreted by BPH after feeding for 24 h was significantly less than those on the control. As a conclusion, three tested transgenic rice genotypes with insect resistance acted adverse effect on BHP feeding, and no marked effect on BPH oviposition.

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.

Impact of insect-resistant transgenic rice on target insect pests and non-target arthropods in China

Progress on the research and development of insect-resistant transgenic rice, especially expressing insecticidal proteins from Bacillus thuringiensis （Bt）, in China has been rapid in recent years. A number of insect-resistant transgenic rice lines/varieties have passed restricted and enlarged field testing, and several have been approved for productive testing since 2002 in China, although none was approved for commercial use until 2006. Extensive laboratory and field trials have been conducted for evaluation of the efficiency of transgenic rice on target lepidoteran pests and potential ecological risks on non-target arthropods. The efficacy of a number of transgenic rice lines currently tested in China was excellent for control of the major target insect pests, the rice stem borers （Chilo suppressalis, Scirpophaga incertulas, Sesamia inferens） and leaffolder （ Cnaphalocrocis medinalis）, and was better than most insecticides extensively used by millions of farmers at present in China. No significantly negative or unintended effects of transgenic rice on non-target arthropods were found compared with non-transgenic rice. In contrast, most of the current insecticides used for the control of rice stem borers and leaffolders proved harmful to natural enemies, and some insecticides may directly induce resurgence of rice planthoppers. Studies for developing a proactive insect resistance management of transgenic rice in the future are discussed to ensure the sustainable use of transgenic rice.

Transfer of Lysine-rich Protein Gene into Rice and Production of Fertile Transgenic Plants

Lysine-rich protein gene (lys) was cloned from Psophocarpus tetragonolobus (L.) DC. A plant expression plasmid was constructed and lys gene was under the control of maize ubiquitin promoter which is the highest efficient monocotyledon promoter. The plasmid was introduced into rice embryogenic calli by microprojectile bombardment. The regenerated fertile plants were obtained by effective selection for hygromycin B resistance. Genomic PCR and Southern blotting analyses showed that the lys gene has been integrated into rice genome. Simultaneously, the results of GUS histochemical assay demonstrated that gus report gene is also expressed in leaves, stems and roots of the transgenic rice plants. Data analysis showed that lysine content in most of the 11 transgenic plants is differently improved, and in one of them increased by 16.04%.

Photosynthetic Characteristics of Transgenic Rice Plants Overexpressing Maize Phosphoenopyruvate Carboxylase

用转PEPC、PPDK、NADP_ME、PEPC +PPDK 双基因水稻 (OryzasativaL .)及原种为材料 ,以光合酶活性、饱和光合速率及PSⅡ光化学效率 (Fv/Fm)为指标 ,研究了转PEPC基因水稻的光合生理特征。结果如下 :转PEPC基因水稻PEPC活性比原种提高 2 0倍 ;饱和光合速率比原种高 5 5 % ;用高光强或人工光氧化剂甲基紫精 (MV)处理后 ,与原种相比 ,转PEPC基因水稻光化学效率下降较少 ,证明其耐光抑制、耐光氧化能力增强 ,测定其光合日变化看出 :在 1d中不同时间 ,转PEPC基因水稻的光合速率均高于原种 ,且与PEPC活性的日变化有相似的趋势。上述结果为转PEPC基因水稻的生理机制和育种研究提供了依据和途径。

Main Agronomic Characters and Grain Quality of Rice Blast Resistance Gene Pi-d2 Transgenic Rice

[Objective] The aim of this study was to provide metabolic evidence for the analysis of the ecological and safety assessment of Pi-d2-transgenic rice.[Method] The main agronomic characters of Pi-d2-transgenic rice were observed in field experiment and the grain chemical characters and amino acid content were measured.[Results] Introduction of foreign gene Pi-d2 resulted in stably hereditable variation in agronomic characteristics in the descents.Most of the transgenic lines grew normally and orderly.Compared with the control（wild type plants）,about half of transgenic plants showed an increased or reduced plant height.There was no observable difference between transgenic plants and controls in tiller number,length of panicle,panicles per plant,seed-setting rate and 1 000-grain weight.Total amino acid content in transgenic rice was reduced,while the starch content,GC and GT were not altered in comparison with the control.[Conclusion] Introduction of foreign gene Pi-d2 has remarkable influence on plant height,while little on grain chemical characters.

Characteristics of CO_2 Exchange and Chlorophyll Fluorescence of Transgenic Rice with C_4 Genes

The responses of photosynthesis of phosphoenopyruvate carboxylase (PEPC), pyrurate dikinase (PPDK), NADP-malic enzyme (NADP-ME) and PPDK+PEPC transgenic rice (Oryza saltiva L.) plant to light, temperature, CO 2 and the characteristics of chlorophyll fluorescence under photoinhibition conditions were studied. The results were as follows: 1. The light-saturated photosynthetic rates of transgenic rice plants were higher than that of wild type, in which the light-saturated point of PEPC and PPDK+PEPC transgenic rice plants was 200 μmol·m -2·s -1 higher than that of untransformed rice and the light-saturated photosynthetic rates were 51.6% and 58.5% respectively. The carboxylation efficiency of PEPC transgenic rice plant increased by 49.3% and the CO 2 compensation point decreased by 26.2% than that of untransformed rice. Under high temperature (35 ℃), the photosynthetic rate of PEPC transgenic rice plant was higher over 17.5% than that of untransformed rice. 2. On the 8th day after photoinhibition treatment, the PSⅡ photochemical efficiency (F v/F m) and photochemical quenching (qP) of PEPC and PPDK+PEPC transgenic rice plants decreased by about 20%-30% while the non-photochemical quenching (qN) increased by approximately 30%. But F v/F m and qP of untransformed rice decreased by over 50% while qN increased by less than 10%. The result suggested that transgenic rice plants were more tolerant to photoinhibition.

Expression of Bt Protein in Transgenic Pest-resistant Rice

[Objective] The aim of this study was to study on expression of Bt protein in transgenic pest-resistant rice. [Method] Enzyme-linked immunosorbent assay （ELISA） was used to measure Bt protein expression in different tissues of transgenic pest-resistant rice at same growth stage. [Result] Absolute content of Bt protein from high to low was as follows： leaves 〉 immature seeds and glumes 〉 roots 〉 stems in different tissues of transgenic rice in grain-filling stage; Bt protein content of trans- genic rice changed a little in different growth stages （including tillering stage, booting stage, and grain-filling stage）; in general, its level declined a little in later growth stage, but the resistibility would not be influenced significantly. [Conclusion] The ex- periment is significant for pest prevention and transgenic rice breeding.

Development of Transgenic Glyphosate-Resistant Rice with G6 Gene Encoding 5-Enolpyruvylshikimate-3-Phosphate Synthase

Glyphosate-resistant crops have been a huge economic success for genetic engineering. The creating of new glypbosateresistant plants would increase the available choices for planting and lower the price of genetically modified crop seeds. A novel G6 gene from Pseudomonas putida that encoded 5-enolpyruvylshikimate-3-phosphate synthase （EPSPS） was previously isolated. The G6 gene was transfected into rice via Agrobacterium-mediated transformation. The transgenic rice obtained was confirmed by PCR, Southern, and Western blots. The lab experiment and field trials further confirmed that the transgenic rice can survive glyphosate spraying at a dose of 8 g L^-1. In contrast, conventional rice was killed at a weed control glyphosate spray dose of 1 g L^-1. Altogether, the present study showed that the G6 gene works well in rice in vivo for glyphosate-resistance.

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.

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

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

Recent Advances in Development of Herbicide Resistant Transgenic Hybrid Rice in China

In addition to weed control in direct seeding field of hybrid rice, herbicide resistance genes were used by Chinese scientists to increase and identify the purity of hybrid seeds, and to realize the mechanization of hybrid seed production. The elite restorer lines, such as Minghui 63, R752, T461, R402, D68 and E32 were transformed directly with herbicide resistance genes, in which D68 and E32 are restorer lines of two-line system and the others are of three-line system. Because almost all of important restorer lines are indica varieties and are recalcitrant in transformation, many herbicide resistant near-isogenic restorer lines were developed by sexual hybridization of indica and japonica varieties and backcross with indica restorer lines later, such as Ce 64, Minghui 63, Teqing, Milyang 46, R402 and 9311, in which 9311 is a restorer line of two-line system. The elite photoperiod-sensitive/thermo-sensitive genic male sterile lines, such as Pei＇ai 64S, P88S, 4008S and 7001S, were transformed with herbicide resistance genes. A few herbicide resistant male sterile lines were developed through sexual hybridization and subsequently systemic selection, such as Bar1259S, Bar2172S, 05Z221A and 05Z227A. With the employment of herbicide resistant male sterile lines or herbicide resistant restorer lines, a few herbicide resistant hybrid rice combinations were developed, such as Xiang 125S/Bar 68-1 and Pei＇ai 64S/Bar 9311. Based on herbicide resistance, the research was marching on to investigate the parental lines of hybrid rice with insect resistance, drought tolerance, etc.

Resistance of Antimicrobial Peptide Gene Transgenic Rice to Bacterial Blight

Antimicrobial peptide is a polypeptide with antimicrobial activity. Antimicrobial peptide genes Np3 and Np5 from Chinese shrimp （Fenneropenaeus Chinensis） were integrated into Oryza sativa L. subsp, japonica cv. Aichi ashahi by Agrobacterium mediated transformation system. PCR analysis showed that the positive ratios of Np3 and Np5 were 36% and 45% in To generation, respectively. RT-PCR analysis showed that the antimicrobial peptide genes were expressed in T1 generation, and there was no obvious difference in agronomic traits between transgenic plants and non-transgenic plants. Four Np3 and Np5 transgenic lines in T1 generation were inoculated with Xanthomonas oryzae pv. oryzae strain CR4, and all the four transgenic lines had significantly enhanced resistance to bacterial blight caused by the strain CR4. The Np5 transgenic lines also showed higher resistance to bacterial blight caused by strains JS97-2, Zhe 173 and OS-225. It is suggested that transgenic lines with Np5 gene might possess broad spectrum resistance to rice bacterial blight.

Enhanced tolerance to drought in transgenic rice plants overexpressing C_4 photosynthesis enzymes

Maize-specific pyruvate orthophosphate dikinase(PPDK) was overexpressed in rice independently or in combination with the maize C4-specific phosphoenolpyruvate carboxylase(PCK). The wild-type(WT) cultivar Kitaake and transgenic plants were evaluated in independent field and tank experiments. Three soil moisture treatments,well-watered(WW), moderate drought(MD) and severe drought(SD), were imposed from 9d post-anthesis till maturity. Leaf physiological and biochemical traits, root activities,biomass, grain yield, and yield components in the untransformed WT and two transgenic rice lines(PPDK and PCK) were systematically studied. Compared with the WT, both transgenic rice lines showed increased leaf photosynthetic rate: by 20%–40% under WW, by45%–60% under MD, and by 80%–120% under SD. The transgenic plants produced 16.1%,20.2% and 20.0% higher grain yields than WT under the WW, MD and SD treatments,respectively. Under the same soil moisture treatments, activities of phosphoenolpyruvate carboxylase(PEPC) and carbonic anhydrase(CA) in transgenic plants were 3–5-fold higher than those in WT plants. Compared with ribulose-1,5-bisphosphate carboxylase, activities of PEPC and CA were less reduced under both MD and SD treatments. The transgenic plants also showed higher leaf water content, stomatal conductance, transpiration efficiency, and root oxidation activity and a stronger active oxygen scavenging system than the WT under all soil moisture treatments, especially MD and SD. The results suggest that drought tolerance is greatly enhanced in transgenic rice plants overexpressing C4photosynthesis enzymes. This study was performed under natural conditions and normal planting density to evaluate yield advantages on a field basis. It may open a new avenue to droughttolerance breeding via overexpression of C4enzymes in rice.

Fitness of F1 hybrids between stacked transgenic rice T1c-19 with cry1C*/bar genes and weedy rice

Compared to single-trait transgenic crops, stacked transgenic plants may be more prone to become weedy, and transgene flow from stacked transgenic plants to weedy relatives may pose a potential environmental risk because these hybrids could be more advantageous under specific environmental conditions. Evaluation of the potential environmental risk caused by stacked transgenes is essential for assessing the environmental consequences caused by crop-weed transgene flow. The agronomic performance of fitness-related traits was assessed in F1+(transgene positive) hybrids(using the transgenic line T1 c-19 as the paternal parent) in monoculture and mixed planting under presence or absence glufosinate pressure in the presence or absence of natural insect pressure and then compared with the performance of F1–(transgene negative) hybrids(using the non-transgenic line Minghui 63(MH63) as the paternal parent) and their weedy rice counterparts. The results demonstrated that compared with the F1– hybrids and weedy rice counterparts, the F1+ hybrid presented higher performance(P<0.05) or non-significant changes(P>0.05) under natural insect pressure, respectively, lower performance(P<0.05) or non-significant changes(P>0.05) in the absence of insect pressure in monoculture planting, respectively. And compared to weedy rice counterparts, the F1+ hybrid presented higher performance(P<0.05) or non-significant changes(P>0.05) in the presence or absence of insect pressure in mixed planting, respectively. The F1+ hybrids presented nonsignificant changes(P>0.05) under the presence or absence glufosinate pressure under insect or non-insect pressure in monoculture planting. The all F1+ hybrids and two of three F1– hybrids had significantly lower(P<0.05) seed shattering than the weedy rice counterparts. The potential risk of gene flow from T1 c-19 to weedy rice should be prevented due to the greater fitness advantage of F1 hybrids in the majority of cases.

Western blot detection of PMI protein in transgenic rice

Phosphomannose isomerase （PMI） encoding gene manA is a desirable selective marker in transgenic research. Under- standing of its expression patterns in transgenic plant and establishing highly sensitive detection method based on immunoassay have great impacts on the application of PMI. In this study, PMI-specific monoclonal antibodies were generated using recombinant protein as immunogen, and could be used in Western blot to detect as little as 0.5 ng His-tagged PMI protein or rice expressed PMI protein in sample accounted for 0.4% of single rice grain （about 0.08 mg）. PMI protein driven by CaMV-35S promoter was detected in dozens of tested tissues, including root, stem, leaf, panicle, and seed at all developmental stages during rice growing, and PMI protein accounted for about 0.036% of total protein in the leaves at seedling stage. The established method potentially can be used to monitor PMI protein in rice grains.

Rapid Generation of Selectable Marker-Free Transgenic Rice with Three Target Genes by Co-Transformation and Anther Culture

The ＇double T-DNA＇ binary vector p13HSR which harbored two independent T-DNAs, containing hygromycin phosphotransferase gene （hpf） in one T-DNA region and three target genes （hLF, SB401, RZ10） in another T-DNA region, was used to generate selectable marker-free transgenic rice by Agrobacterium-mediated transformation. The regenerated plants with both the three target genes and the selectable marker gene hpt were selected for anther culture. RT-PCR analysis indicated that target genes were inserted in rice genomic DNA and successfully transcribed. It took only one year to obtain double haploid selectable marker-free transgenic plants containing the three target genes with co-transformation followed by anther culture technique, and the efficiency was 12.2%. It was also noted that one or two target genes derived from the binary vector were lost in some transgenic rice plants.

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.

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.