转Bt基因水稻CrylAb杀虫蛋白在水稻土中的降解

室内研究了转Bt基因水稻克螟稻2号和华池B6叶片中的CrylAb杀虫蛋白在稻田水稻土中的降解动态。分别将克螟稻2号的粉碎叶片和华池B6的整张叶片埋入3种土壤(即老黄筋泥田、青紫泥田和黄松田)中，此后每隔6～10d测定土壤或叶片中的CrylAb含量。克螟稻2号粉碎叶片中的CrylAb均以处理后的前36d，尤其是前6d的降解较快，其中在青紫泥田中最快，黄松田中最慢。在处理后6～36d内，不同土壤中的CrylAb残留量有显著差异，黄松田中的明显最高，老黄筋泥田中的次之，青紫泥田中的为最低；此后土壤间的残留量差异逐渐缩小，至处理后78d差异已不明显。土壤淹水可显著加快华池B6叶片中CrylAb的降解，且淹水后其降解动态在不同土壤之间十分相似；淹水对CrylAb降解的促进作用仅发生在前12d之内，此后多数时间内残留量在淹水与非淹水处理间无显著差异。建立了各处理中CrylAb降解的动力学指数方程，相应地得出了其降解的半衰期。还讨论了土壤有机质和微生物等因子对土壤中CrylAb降解的影响。     

转Bt基因水稻克螟稻对黑尾叶蝉种群增长的影响

以克螟稻1和克螟稻2及其亲本秀水11为材料,研究了转Bt基因水稻上黑尾叶蝉种群的表现。田间种群动态调查表明,转 Bt 基因水稻克螟稻2上的黑尾叶蝉（Nephotettix cincticeps）的种群数量比其母本秀水11高0.5~1.0倍以上。室内试验结果表明克螟稻1上取食的黑尾叶蝉和克螟稻2上的相比,两者在卵、若虫发育历期、雌成虫寿命、产卵量、产卵持续时间及种群内禀增长力方面均存在明显的差异。其中,取食克螟稻1的黑尾叶蝉的雌成虫寿命、产卵量、产卵持续时间均显著长于或高于以秀水11为食的黑尾叶蝉。以克螟稻为食的黑尾叶蝉的净生殖率为以秀水11为食的黑尾叶蝉的4~8倍。     

稻蓟马危害转Bt基因水稻克螟稻2号研究初报

 用卷叶率、为害级别和严重度3个指标评价了克螟稻2号及其亲本秀水11对稻蓟马的抗感性。结果表明,克螟稻2号较秀水11在3种指标上存在着极显著的差异,克螟稻2号较感虫;两品种上单位叶面积成虫数量差异均不显著,而卵量和若虫数量的差异显著;说明高密度的稻蓟马若虫将对克螟稻2号为害更严重。     

水稻胡麻斑类似病危害转Bt稻“克螟稻”初报

 于田间转Bt稻“克螟稻”稻叶上观测到一种病斑，病斑中心和周围均呈褐色，长椭圆形与稻胡麻斑病类似，平板上分离到的病原菌菌丝呈灰褐色，菌丝有多个隔膜，分生孢子梗成丛，但在PDA培养基上，产生的分生孢子特征与稻胡麻斑病不吻合。从为害级别和严重度上分析克螟稻对胡麻斑类似病的感病程度远远超过其亲本秀水11。克螟稻叶片间发病速率增长迅速，而秀水11叶片间发病速率增长缓慢。     

5—氮胞苷介导的转基因cry1Ab的阶段复活及其对水稻的生物学效应

用不同浓度的和5－氮胞苷溶液配合不同时间处理了发生的在转录水平的转基因cry1Ab沉默水稻。结果表明，5－氮胞苷处理能使8％－30％转基因水稻植株的转基因cry1Ab恢复表达活性，复活性基因表达的Cry1Ab蛋白含量可达0.147％，45mg/L5-氮胞苷处理1d可使转基因cry1Ab的复活率及Cry1Ab蛋白含量达最高。同时，5－氮胞苷处理使转基因水稻植株出现矮化，分蘖数、单株粒重下降，穗长减小及开花期延迟等发育异常现象。5－氮胞苷作为一种弱诱变剂在克服转基因沉默及创造具优良农艺性状的转基因植物中具有一定的应用价值。     

5-氮胞苷介导的转基因crylAb的阶段复活及其对水稻的生物学效应

用不同浓度的5-氮胞苷溶液配合不同时间处理了发生在转录水平的转基因cry1Ab沉默水稻。结果表明，5-氮胞苷处理能使8%～30%转基因水稻植株的转基因cry1Ab恢复表达活性，复活转基因表达的Cry1Ab蛋白含量最高可达0.147%，45mg/L5-氮胞苷处理1d可使转基因cry1Ab的复活率及Cry1Ab蛋白含量达最高。同时，5-氮胞苷处理使转基因水稻植株出现矮化，分蘖数、单株粒重下降，穗长减小及开花期延迟等发育异常现象。5-氮胞苷作为一种弱诱变剂在克服转基因沉默及创造具优良农艺性状的转基因植物中具有一定的应用价值。     

抗虫抗除草剂转基因水稻B1C893的获得与鉴定

利用农杆菌介导法将Cry1Ca#和Bar基因转化水稻恢复系R893,获得6株再生植株。经过分子鉴定,发现Bar基因和Cry1Ca#基因成功整合到水稻基因组并稳定表达。测定其中单拷贝且可育的转基因株系苗期不同器官中Cry1Ca蛋白的表达量,发现根系中最低而叶片中最高,其中最高的第6号株系叶片中含量达到35.07μg/g。获得的转基因株系对除草剂草铵膦和稻纵卷叶螟均具有良好的抗性。     

转sck/cry1Ac 双基因抗虫水稻对二化螟和稻纵卷叶螟的抗虫效果

通过室内测定和田间观察 ,就转sck/cry1Ac双基因抗虫水稻MSB和MSA对我国主要水稻害虫二化螟和稻纵卷叶螟的抗虫效果进行了研究。室内离体植株测定结果表明 ,MSB、MSA高抗两种害虫 ,抗性水平受水稻苗龄影响。 6 5～71d苗龄水稻上害虫校正死亡率达 98.9%～ 10 0 % ,10 5～ 10 9d时抗性水平有所降低 ,但除MSB对二化螟的校正死亡率 (仍达 79.3% )降低明显 (P <0 .0 5 )外 ,MSA对二化螟及MSB、MSA对稻纵卷叶螟的校正死亡率 (92 .8%～ 98.1% )均无明显下降(P>0 .0 5 )。选取MSB进行的田间实验进一步明确 ,MSB对两种害虫有显著控虫效果 ,抗性水平与抗虫对照克螟稻 1号(含cry1Ab)无明显差异(P >0 .0 5 ) ,且略优于施药亲本明恢 86。相对于未施药明恢 86 ,MSB对两种害虫的防治效果分别为 92 .6 %、97.3% ,施药明恢 86则分别为 87.7%、89.8%。     

水稻免耕抛秧除草剂残留监测分析

通过对免耕稻田使用的除草剂农民乐747、10%草甘膦、克无踪在土壤和稻米、米糠中的残留量进行监测分析,结果表明,1年2造、2年4造、3年6造、4年5造5、年10造、6年12造使用农民乐747、10%草甘膦、克无踪在土壤、稻米、米糠中的残留量均未检出。同时对农民乐747、10%草甘膦、克无踪除草剂本身的镉(Cd)、汞(Hg)、砷(As)、铅(Pb)、铬(Cr)、铜(Cu)6个农作物无公害生产主要重金属残留量进行测定分析,结果3种除草剂的6个重金属含量均未超标,说明农民乐747、草甘膦、克无踪对稻田环境和稻米生产是安全的。     

水稻萌发耐淹性鉴定评价方法研究及种质资源筛选

低氧胁迫下出苗率下降是限制直播稻安全生产的关键问题之一。建立准确、高效的水稻萌发耐淹性鉴定方法，并筛选强萌发耐淹性的水稻种质对直播稻新品种选育具有重要意义。本研究以20份水稻品种为供试材料，设置不同淹水深度（3 cm、6 cm、9 cm、12 cm、15 cm和18 cm）、不同处理温度（20℃、25℃和30℃）和不同淹水天数（3 d、5 d、7 d、9 d、11 d、13 d和15 d）进行萌发耐淹性鉴定，以胚芽鞘长度为指标进行萌发耐淹性评价，旨在明确水稻萌发耐淹性的评价方法。结果表明，水稻种质在淹水临界深度上存在籼粳间差异，粳稻的淹水临界深度为6 cm，而籼稻的淹水临界深度为9 cm；在温度梯度处理中，30℃时种质间的胚芽鞘长四分位差最大，因此认为30℃是水稻种质萌发耐淹性鉴定的最适温度；调查时期梯度试验表明，淹水11 d时参试种质的胚芽鞘长四分位差最大，因此认为淹水11 d是水稻萌发耐淹性调查的最适时期。本研究建立了一种以纯水隔绝空气，在温度30℃、淹水深度9 cm以上、第11 d调查胚芽鞘长度进行水稻萌发耐淹性鉴定的简便方法，为水稻种质资源的大规模鉴定提供了方法基础。依据上述确...     

Susceptibility of Brazilian populations of Diatraea saccharalis to Cry1Ab and response to selection for resistance

The sugarcane borer, Diatraea saccharalis (F.), is a key pest of sugarcane and other grasses, which is a current focus of research aiming to develop transgenically resistant genotypes. However, the current susceptibility of Brazilian populations of the sugarcane borer to Bt toxins is unknown. Laboratory assays were carried out to characterize the susceptibility of sugarcane borer to the Bt toxin Cry1Ab and to select a resistant strain for additional studies on Cry1Ab resistance. Susceptibility was characterized by exposing neonates of different Brazilian colonies of sugarcane borer to different concentrations in meridic diet using surface application of Cry1Ab toxin. Selection for Cry1Ab resistance was carried out by exposing neonates to Cry1Ab-expressing maize (MON 810). The resistance of the borer populations to Cry1Ab was variable with LC₅₀ and EC₅₀ values reaching about 30-fold. The larvae responded positively to Cry1Ab selection exhibiting a 55-fold increase in resistance after four generations. This suggests the suitability of using leaves containing Bt-expressing genes for selection and the existence of variability of Bt-resistance in populations of the sugarcane borer.     

Performance of Cry1A.105-selected fall armyworm (Lepidoptera: Noctuidae) on transgenic maize plants containing single or pyramided Bt genes

Cry1A.105 is a Cry protein expressed in some transgenic Bacillus thuringiensis (Bt) maize products. In this study, performance of five populations of fall armyworm, Spodoptera frugiperda (J.E. Smith), were evaluated on four non-Bt and eight commercial and experimental Bt maize hybrids/lines (hereafter referred as maize products). The five insect populations included one Cry1A.105-susceptible strain, two Cry1A.105-resistant strains, and two F₁ heterozygous genotypes. The eight Bt maize hybrids/lines consisted of five single-gene Bt maize products containing Cry1A.105, Cry2Ab2, Cry1F, or Cry1Ab protein, and three pyramided Bt maize products expressing Cry1A.105/Cry2Ab2, Cry1A.105/Cry2Ab2/Cry1F, or Cry1Ab/Vip3A for targeting aboveground lepidopteran maize pests. In the study, neonates of each population were tested on leaf tissues in the laboratory and whole plants in the greenhouse. Cry1A.105 and Cry1F maize killed 92.2–100% susceptible larvae in both test methods, while resistant larvae survived well on these two maize products. Performance of the two F₁ populations on Cry1A.105 and Cry1F maize varied between the two test methods. In leaf tissue bioassay, Cry1Ab maize was marginally effective against the susceptible population. In contrast, few live larvae and little leaf injury from any of the five populations were observed on Cry2Ab2 and the three pyramided Bt maize products. The results of this study showed evidence of cross resistance of the Cry1A.105-resistant S. frugiperda to Cry1F and Cry1Ab maize, but not to the Bt maize products containing Cry2Ab2 or Vip3A. Data generated from this study will be useful in developing resistance management strategies for the sustainable use of Bt maize technology.     

Feeding disruption tests for monitoring the frequency of larval lepidopteran resistance to Cry1Ac, Cry1F and Cry1Ab

An alternative to traditional larval lepidopteran resistance-monitoring bioassays was developed. Feeding disruption tests were developed for detecting insects resistant to three Bacillus thuringiensis (Bt) proteins: Cry1Ac, Cry1F and Cry1Ab. The assays rely on a diagnostic dose of Bt toxin in 100-μl hydratable meal pads of artificial diet containing blue indicator dye. The assay was formatted as a portable (palm-sized) plastic plate containing an array of 16 test wells, each containing a single hydratable meal pad with one insect added per well. The diagnostic dose was the concentration of Bt in meal pad rehydration solution that reduced 24 h dyed fecal production of Bt-susceptible neonates to ≤2 fecal pellets per larva. Bt-resistant neonates were able to consume the diagnostic dose of the insecticidal protein and produce >2 blue fecal pellets. The feces were distinctly visible on the white background of the feeding disruption test plate. Diagnostic doses were determined with lab-strain Bt-susceptible Heliothis virescens and Helicoverpa zea. For H. virescens, the diagnostic doses were 10, 20 and 15 μg/ml for Cry1Ac, Cry1F and Cry1Ab, respectively. For H. zea, the diagnostic doses were 40, 200 and 500 μg/ml, respectively. The assays were validated against a lab-strain of Bt-resistant H. virescens and with susceptible larval H. virescens collected as eggs from field-grown tobacco in North Carolina.     

Damage and survivorship of fall armyworm (Lepidoptera: Noctuidae) on transgenic field corn expressing Bacillus thuringiensis Cry proteins

Field corn, Zea mays L., plants expressing Cry1Ab and Cry1F insecticidal crystal (Cry) proteins of Bacillus thuringiensis (Bt) Berliner are planted on considerable acreage across the Southern region of the United States. The fall armyworm, Spodoptera frugiperda (J.E. Smith), is an economically important pest during the mid-to-late season on non-Bt and some commercial Bt corn hybrids. The objective of this study was to quantify foliar injury and survivorship of fall armyworm on transgenic corn lines expressing Cry1Ab or Cry1F Bt proteins. Corn lines/hybrids expressing Cry1Ab, Cry1F, and a conventional non-Bt cultivar were evaluated against artificial infestations of fall armyworm in field trials. Larvae (second instars) of fall armyworm were placed on corn plants (V8-V10 stages). Leaf injury ratings were recorded 14 d after infestation. Hybrids expressing Cry1F had significantly lower feeding injury ratings than non-Bt corn plants. Development and survivorship of fall armyworm on Bt corn lines/hybrids were also evaluated in no-choice laboratory assays by offering freshly harvested corn leaf tissue to third instars. Transgenic corn hybrids expressing Cry1Ab or Cry1F significantly reduced growth, development, and survivorship of fall armyworm compared to those offered non-Bt corn tissue. However, 25-76% of third instars offered Bt corn leaf tissues successfully pupated and emerged as adults. These results suggest Cry1Ab has limited effects on fall armyworm; whereas Cry1F demonstrated significant reductions in foliar injury and lower survivorship compared to that on non-Bt corn tissues. Although fall armyworm is not considered a primary target for insect resistance management by the U.S. Environmental Protection Agency, these levels of survivorship could impact selection pressures across the farmscape, especially when considering that transgenic Bt cotton cultivars express similar Cry (Cry1Ac or Cry1F) proteins.     

Pest resistance to Cry1Ab Bt maize: Field resistance,contributing factors and lessons from South Africa

This paper documents the historical development of resistance of the African maize stem borer, Busseola fusca (Fuller) (Lepidoptera: Noctuidae) to Bt maize (Zea mays L.). This pest was one of the first to evolve resistance to Bt maize expressing Cry1Ab protein. A time-line of events and contributing factors are presented, from the commencement of efficacy testing through to the present situation, where the Cry1Ab toxin has lost its efficacy against B. fusca at many localities throughout the maize producing region, and single-gene Bt maize events often require insecticide treatments for which farmers are compensated. Significant levels of pest survival on Bt maize was observed in the first season after commercial release in 1998 and confirmed seven years later. Reduced selection pressure on the target pest is the objective of insect resistance management (IRM), and strategies to accomplish this should receive highest priority. Where resistance is prevalent, the only viable options to reduce selection pressure are withdrawal of the product and/or enforcement of high-dose/refuge requirements. The latter action may however be of no value under conditions where resistance is prevalent, since the value of refugia to an IRM strategy may be compromised. Remedial actions taken in South Africa included the propagation and enforcement of refuge compliance followed by the release of pyramided maize hybrids in 2011. These pyramids combine Cry1A.105 and Cry2Ab2 toxin-producing transgenes, replacing the ineffective single-transgene. However, it remains uncertain if cross-resistance occurs between Cry1A.105/Cry2Ab2 and the closely related Cry1Ab toxin, and for how long this pyramided event will endure. Cultivation of Cry1Ab-expressing hybrids continues in areas where resistance levels have been confirmed to be high. In retrospect, this case provides lessons regarding IRM, not only in South Africa, but wherever Bt crops are being introduced.     

Influences of Cry1Ac Broccoli on Larval Survival and Oviposition of Diamondback Moth

Larval survival and oviposition behavior of three genotypes of diamondback moth, Plutella xylostella L. (Lepidoptera: Plutellidae), (homozygous Cry1Ac-susceptibile, Cry1Ac-resistant, and their F₁ hybrids), on transgenic Bacillus thuringiensis (Bt) broccoli expressing different levels of Cry1Ac protein were evaluated in laboratory. These Bt broccoli lines were designated as relative low, medium, and high, respectively, according to the Cry1Ac content. Untransformed brocccoli plants were used as control. Larval survival of diamondback moth on non-Bt leaves was not significantly different among the three genotypes. The Cry1Ac-resistant larvae could survive on the low level of Bt broccoli plants, while Cry1Ac-susceptible and F₁ larvae could not survive on them. The three genotypes of P. xylostella larvae could not survive on medium and high levels of Bt broccoli. In oviposition choice tests, there was no significant difference in the number of eggs laid by the three P. xylostella genotypes among different Bt broccoli plants. The development of Cry1Ac-susceptible and Cry1Ac-resistant P. xylostella on intact Bt plants was also tested in greenhouse. All susceptible P. xylostella larvae died on all Bt plants, while resistant larvae could survive on broccoli, which expresses low Cry1Ac protein under greenhouse conditions. The results of the greenhouse trials were similar to that of laboratory tests. This study indicated that high dose of Bt toxins in broccoli cultivars or germplasm lines is required for effective resistance management.     

转基因玉米Bt蛋白降解规律及其与土壤动物互作研究

以转Cry11Ac基因玉米及其非转基因对照为试验材料,利用大、中、小不同孔径的凋落物分解袋,研究黑土区转基因玉米外源基因表达Bt蛋白的降解规律及其与土壤动物的互作关系。结果表明,第二年春天大孔径凋落物分解袋内转基因玉米残体Bt蛋白含量为初始含量的25.98%,至8月份Bt蛋白只剩下初始含量的0.35%。分解袋孔径对转基因玉米及其对照玉米凋落物分解率和Bt蛋白降解率均有显著影响,表现为大孔径中孔径小孔径,说明大型和中型土壤动物都可以促进凋落物分解和Bt蛋白的降解。与对照玉米相比,转基因玉米凋落物分解率和凋落物内土壤动物群落结构参数都没有显著差异(P0.05,t-test),说明导入Bt基因对玉米残体分解速率和土壤动物均无显著影响。     

Characteristics of water balance in a rainfed paddy field in Northeast Thailand

Rice productivity in rainfed paddy fields varies with seasonal changes of water availability in which the conditions of flooding are affected by the water balance. Hydrometeorological measurements were performed in a rainfed paddy field in Northeast Thailand from July 2004 to December 2006 to analyze the water balance. As a result of our measurements, climatologically conditions were classified as semi-humid with an annual precipitation of 1,100 mm/year and annual potential evaporation of 1,660 mm/year in both the year. The surface layer of the paddy soil was clayey and the hydraulic conductivity was very low, so groundwater levels remained below the soil surface even under flooded conditions during the rainy season. Seasonal changes in the amount of soil water were very small, comprising only less than 16% of the total precipitation during the rainy season. Consequently, an effective precipitation of less than 180 mm was enough to establish standing water in the rainfed paddy field. Shinkichi Goto, Tsuneo Kuwagata and Pisarn Konghakote contributed equally to the paper.     

Evaluation of Chilo partellus and Busseola fusca susceptibility to δ-endotoxins in Bt maize

Susceptibility of Chilo partellus (Lepidoptera, Crambidae) and Busseola fusca (Lepidoptera, Noctuidae) populations to Cry proteins from the bacterium, Bacillus thuringiensis (Bt), the δ-endotoxins Cry1Ab and Cry1Ba in Bt-maize, were evaluated under biosafety greenhouse conditions. Larval feeding on Bt-maize was adjusted to deliver sub-lethal doses of δ-endotoxins from the two events; survivors were reared on artificial diet to obtain successive generations. Eight generations of three C. partellus populations and five generations of a B. fusca population were screened for susceptibility on each event. Mean proportion of surviving larvae from Bt-maize plants, and the corresponding pupal weights of survivors for each population, were lower for individuals exposed to δ-endotoxins. Both Bt Cry proteins expressed in maize leaves controlled C. partellus and showed stability in control, with no indication of a change in susceptibility among generations. Neither toxin, however, provided complete control of B. fusca, but no changes in susceptibility were observed after five generations of selection. Implications for development of future transgenic Bt maize events, and research for East Africa are discussed.     

Relationships between Cry1Ac and Cry2Ab protein expression in field-grown Bollgard II® cotton and efficacy against Helicoverpa armigera and Helicoverpa punctigera (Lepidoptera: Noctuidae)

Seven-day laboratory bioassays with first-instar Helicoverpa armigera and Helicoverpa punctigera larvae were conducted using leaves from field-grown Bollgard II^® cotton during the 2009/10 season and larval mortality and development recorded. Leaves were from three farms in the St George region and two fields on one farm at Emerald in eastern Australia. The Cry1Ac and Cry2Ab content of leaves from the same samples was determined (see Knight et al., 2013) and the relationships between Cry protein content and larval mortality and development were examined. The Cry1Ac protein content was between 3.98 and 12.08 μg/g during the growing season, while Cry2Ab content ranged between 300.6 and 953.3 μg/g. Cry1Ac and Cry2Ab content of leaves were highly correlated (r = 0.8276, P < 0.001). Seven-day mortality of H. punctigera larvae was close to 100% throughout the season. H. armigera mortality was close to 100% early in the season, but fell to ∼65% by mid-to late February in the laboratory bioassays. Fitting three-dimensional non-linear models associating Cry1Ac and Cry2Ab content with H. armigera and H. punctigera mortality elucidated the relative importance of the two proteins in determining larval mortality; for this analysis, data were pooled with data from an isoline study to provide better sampling of the three-dimensional surface being modelled. For both Helicoverpa species, the fitted mortality response to the Cry1Ac protein was close to its maximum at protein concentrations above ∼3 μg/g Cry1Ac. For H. punctigera, response to the Cry2Ab protein was close to maximal once Cry2Ab was greater than ∼200 μg/g. In contrast, the fitted H. armigera mortality response to Cry2Ab increased steadily with concentration up to ∼1200 μg/g Cry2Ab. These responses led to markedly different response surfaces for the two species; H. punctigera mortality was close to 100% at most places on the response surface, while for H. armigera the response surface showed stronger increases in mortality with concentration for Cry2Ab than for Cry1Ac. These results can be interpreted as meaning that at the plant-expressed range of concentrations in Bollgard II cotton the two proteins are approximately equally important for H. punctigera but that changes in Cry2Ab content more strongly influences changes in larval mortality in H. armigera than does Cry1Ac, with Cry1Ac contributing a consistent 40–45% mortality for concentrations above 3 μg/g. For H. armigera, there was no evidence of either synergism or antagonism between Cry1Ac and Cry2Ab proteins (P > 0.05) but this aspect was not testable for H. punctigera because mortality was mostly close to 100%.