Bacillus thuringiensis cry1Ab gene confers resistance to potato againstHelicoverpa armigera (Hubner)

Summary Helicoverpa armigera is one of the important insect pests adversely affecting the yield of potatoes in India. A synthetic gene encoding the insecticidal crystal protein (Cry1Ab) ofBacillus thuringiensis (Bt) has been introduced into five genotypes of potato usingAgrobacterium tumefaciens. Southern analysis of DNA from transgenic plants confirmed the integration and copy number of the transgene. Double-antibody quantitative sandwich ELISA analysis demonstrated high levels of Cry1Ab protein expression in transgenic plants. Insect bioassays on the leaves of transgenic plants showed considerable protection against the larvae ofH. armigera in terms of leaf area consumed and larval weight reduction.     

Development and Evaluation of Codon-modified cry V Constructs in Cultivated Potato (Solanum tuberosum L. ) for Control of Potato Tuber Moth

A cryV gene,specifically toxic to Lepidoptera and Coleoptera,was incorporated into binary vectors with different promoters and the presence or absence of the β-glucuronidase(gus) reporter gene.These constructs were integrated into potato cv.Spunta by Agrobacterium tumefaciens-mediated transformation.Highest expression of cryV gene,determined by mRNA levels and insect mortality,was obtained using the CaMV 35S promoter without the gus gene configuration.Detached leaf and tuber bioassays showed a mortality rate of up to 83% and 100%,respectively,for potato tuber moth(Phthorimaea operculella Zeller) in the transgenic lines.Our results demonstrated that the presence of the gus gene negatively affects the expression level of the cryV gene.Bt expression was also facilitated by using the(ocs)3 mas super promoter,whereas the Bt expression regulated by the patatin promoter(tuber-specific) was too low to have any effect upon the mortality of potato tuber moth.These results represent significant improvement in the level of host plant resistance for the control of potato tuber moth via Bt transgenes.     

Integrated pest management of potato tuber moth in field and storage

Summary The potato tuber moth (PTM),Phthorimaea operculella (Zeller), is one of the most damaging pests of potatoes in field and storage. Tuber moth is active throughout the year in North Africa but is most active from late April to early August. Many cultural practices that are used by farmers to improve the yield and quality of potato can also limit the development of the potato tuber moth and minimise damage to tubers. As the farmers gain more experience with both agronomic practices and chemical insecticides, they are likely to rely more heavily on cultural practices and less on insecticides to manage PTM. Measures which could keep PTM damages in rustic and diffuse light stores to a minimum are discussed. Ideally, there is need to integrate pest management in potato fields and stores. PTM control in stores was found to be more effective if infestation in fields was kept to a minimum. Low infestation at harvest and rapid handling of the potatoes going into store established good initial storage conditions and decreased the likelihood of post-harvest losses. Biological insecticides in particular were more effective in preventing losses by insects in stores in cases where the initial level of infestation was relatively low.     

人工改造基因Cry1Abm的合成、原核表达及其抗虫鉴定

根据植物密码子的偏好性及使用频率,对苏云金芽孢杆菌Cry1Ab野生型基因的编码区序列进行优化和改造,改造后的Cry1Abm基因序列与原始序列同源性为66.2%,G+C含量由37.3%提高到62.7%。人工合成Cry1Abm基因,并将人工合成的改造后的Cry1Abm基因构建到原核表达载体pET28b中,构建原核表达载体pETAbm。将原核表达载体转入大肠杆菌BL21（DE3）中进行诱导表达,用诱导表达的蛋白进行饲虫（玉米螟）实验。结果表明,该蛋白对幼虫具有很强毒性,幼虫的死亡率高达86.63%,同时,存活幼虫的生长发育也受到明显抑制。该基因可以作为杀虫工程及培育转基因抗虫作物的候选基因。     

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.     

Elucidation of the sex pheromone system of the potato tuberworm moth,Phthorimaea operculella (Zeller) (Lepidoptera,Gelechiidae): a short review

Summary Recent research, reviewed here, of the pheromone system of the potato tuberworm moth has led to the isolation, identification, and synthesis of its components.trans-4,cis-7-tridecadien-l-ol acetate (PTM 1) andtrans-4,cis-7,cis-10-tridecatrien-l-ol acetate (PTM 2), mixtures of which are much more attractive in the field than 2 virgin female moths or each compound on its own.     

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.     

转Cry1A.401基因抗虫玉米CM8302和CM8303对亚洲玉米螟的抗性研究

采用农杆菌介导法获得转Cry1A.401基因抗虫玉米CM8302和CM8303,通过多代回交与自交获得BC₄F₁、BC₄F₂和BC₄F₃世代的转基因玉米材料。利用qRT-PCR和ELISA方法明确转基因抗虫玉米叶片、花丝和子粒均有Cry1A.401基因表达,不同世代间目的基因表达量无显著差异。田间接虫鉴定结果表明,CM8302和CM8303三个世代心叶期和吐丝期的抗性等级均为1级,抗性水平为高抗。室内生测结果表明,亚洲玉米螟幼虫取食CM8302心叶、花丝和子粒第6天存活率分别为0、4.0%和4.0%;取食CM8303心叶、花丝和子粒第6天存活率分别为0、4.0%和2.0%。分析显示转基因抗虫玉米CM8302和CM8303的心叶、花丝和子粒均具有显著杀虫效果且遗传稳定。     

Development of Bt transgenic potatoes for effective control of potato tuber moth by using cry1Ab gene regulated by GBSS promoter

Transgenic of Indian potato cultivar Kufri Badshah expressing synthetic, modified cry1Ab gene were developed against potato tuber moth (Phthorimaea opercullela Z.) a destructive pest. The cry1Ab gene was in spatial and temporal expression under the control of tuber-specific GBSSi promoter. The transformation vector pBinCG1 was developed harbouring transgene expression cassette comprising cry1Ab gene under the control of potato GBSSi promoter, castor bean catalase intron (5′UTR) and OCS termination signals. Selected kanamycin-resistant putative transformed potato lines were evaluated by positive PCR amplification of 713 bp, 1206 bp and 700 bp with npt II, GBSSi promoter and cry1Ab gene specific primers, respectively. Gene integration in transformed potato plants was elucidated through Southern hybridization and in planta transgene expression analysis. Transgene expression was highest in stolons, followed by tubers matured leaves and young leaves. Insect bioassay of stored transgenic tubers resulted in significant retardation and mortality in neonate tuber moth larvae. RT-PCR and northern hybridization confirmed stable expression in stored tubers. These transgenic lines; thus can represent an effective resistance management strategy which can significantly reduce the selection pressure on target and non-target insect pests to a threshold level.     

Studies on natural repellents against potato tuber moth (Phthorimaea operculella Zeller) in country stores

Summary The effects of the leaves of five plant species, one from each of the generaAmbrosia, Anemone, Eupatorium, Eucalyptus andLantana, on potato tuber moth were investigated under indigenous storage conditions at the Central Potato Research Station, Shillong (1800 m above sea level). Their action was compared with that of a biological insecticide (spores ofBacillus thuringiensis), a chemical insecticide (carbaryl), and an untreated control. The data collected after six months storage on tuber damage, sprout damage and the rotting indicated that the leaves ofLantana aculeata provided most protection to the tubers, reducing damage from over 70% in the check to below 5%, and sprout damage from over 45% to below 3%. Next best wasEucalyptus globulus followed byB. thuringiensis. They may be used on tubers stored for table use or for seed as they had no adverse effect on germination or on the yield of a subsequent crop.     

Transgenic potatoes expressing anErwinia pectate lyase gene — results of a 4-year field experiment

Summary Four potato lines of cv. Désirée that express the pectate lyase (PL) isoenzyme 3 ofErwinia carotovora subsp.atroseptica were examined in a 4-year field experiment with respect to plant development, tuber yield and resistance of tuber tissue toErwinia soft rot. The PL3 degrades plant cell wall pectin into unsaturated oligogalacturonates eliciting plant defence responses. In one line, enzyme expression was controlled by the CaMV 35S promoter (C) and in three lines it was driven by the potato patatin B33 promoter (D). Plant development of the D-lines in field plots was not distinguishable from that of the non-transgenic counterpart. Also tuber yield was not too different. By contrast, plants of the C-line were smaller than those of the nontransformed counterpart and also showed reduced tuber yield. There were no significant differences in dry mass, starch and protein content of tuber tissue between PL transgenic and non-transgenic potatoes. But compared with the latter, field-grown tubers expressing the PL displayed an enhanced resistance toErwinia soft rot. Thus, average rotting caused byEc-bacteria was diminished in tubers of PL-transgenic lines by 34.1%. The resistance of tubers toEc soft rot was significantly correlated with the PPO activity in tuber tissue.     

转Cry1Ab-Ma基因玉米CM8101对亚洲玉米螟抗性研究

利用q RT-PCR和ELISA等方法测定转Cry1Ab-Ma基因玉米CM8101不同世代、不同组织目标基因表达量,开展玉米螟室内和田间生测鉴定,评价转Cry1Ab-Ma基因玉米CM8101对亚洲玉米螟的抗性。结果表明,Cry1Ab-Ma基因在CM8101各组织中均有表达,不同世代间目的基因的表达量无显著性差异,遗传稳定。室内生测结果表明,转基因玉米CM8101各组织均对亚洲玉米螟具有显著的杀虫效果,不同世代间玉米螟的存活率均无显著性差异。田间接虫鉴定结果表明,CM8101在T3、T4、T5世代的抗性等级均为1级,抗性水平为高抗。     

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%.     

TaCYP78A5基因过表达小麦的遗传和功能初步分析

为了验证小麦籽粒大小相关基因TaCYP78A5在小麦籽粒发育中的功能,对pINO启动子驱动的TaCYP78A5基因过表达的转基因小麦后代株系进行了鉴定,检测了T_0代植株目标基因拷贝数,定量分析了7个T_1代阳性植株的目标基因表达,并对其籽粒大小进行了统计。结果表明,利用Bar试纸条和目标基因特异PCR检测相结合的方法对21株转基因T_0代再生苗进行检测,共鉴定出14个阳性植株,除2个植株的目标基因拷贝数为3和1个植株为7外,其余11个T_0代转基因植株目标基因插入拷贝数均为1~2个,其中有6个单拷贝植株。与野生型相比,7个T_1代阳性植株目标基因表达量均极显著增加,粒厚和粒宽均有不同程度增加,粒重极显著增加。     

Genetic basis of Cry1F resistance in two Brazilian populations of fall armyworm,Spodoptera frugiperda

Large-scale adoption of transgenic crops expressing genes from Bacillus thuringiensis (Bt) imposes high selection pressure for evolution of field-relevant resistance that can reduce pest control efficacy, such as reported for Cry1F maize (Zea mays L.) in populations of fall armyworm, Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae), of Puerto Rico, Brazil, and the United States. As part of our effort to improve fall armyworm resistance management to Bt crops, here we determined the genetic basis of Cry1F resistance in two S. frugiperda strains originated from field collections in different regions of Brazil and further selected in the laboratory for high levels of resistance to Cry1F maize. Continuous exposure to the TC1507 event for 11 generations resulted in more than 183-fold resistance to Cry1F in the two strains studied, and such a high resistance level enabled the insects to complete larval development on the Bt maize plants. Genetic analyses using concentration-response bioassays with progenies from reciprocal crosses between resistant and susceptible insects indicated that the inheritance of the resistance is autosomal, recessive and without maternal effects. Backcross of the F₁ progeny with the parental resistant strains revealed that the resistance in the two selected strains is conferred by a single locus or set of tightly linked loci. These results support some of the assumptions of the strategy in use for fall armyworm resistance management to Bt Cry1F maize, but survival rates of heterozygotes on the Bt plants were higher than 5%, showing that the Cry1F maize does not produce a high dose of the insecticidal protein for S. frugiperda. Additionally, we detected a delay in larval development time that may favor assortative mating of individuals carrying resistance alleles. These findings are consistent with the rapid evolution of Cry1F resistance in certain field populations of fall armyworm. Implications for resistance management of S. frugiperda to Bt maize are discussed.     

Inheritance of a pectate lyase mediated soft rot resistance in crosses between transgenic potato lines of cv. désirée andS. tuberosum cultivars

Summary Expression of the pectate lyase (PL) isoenzyme 3 in transgenic potato lines of cv. Désirée mediates an enhanced resistance of tuber tissue toErwinia carotovora (Ec) soft rot due to a pre-activation of plant defence mechanisms. Therefore, theSolanum tuberosum cvs Agave and Adretta with a moderate level of soft rot resistance were crossed with such PL-expressing potato lines. The resulting progenies were assessed with respect to plant/tuber characteristics over a period of four years and then tested for PL3-expression as well as for soft rot resistance. 71% of the selected progeny lines exhibited a stable production of the PL3 enzyme. Statistical analysis revealed differences between the transgenic and the non-transgenic progeny concerning the soft rot resistance of tuber tissue. Compared with the PL-inactive progeny, extension of Ec-rotting on the wound surface of PL-transgenic potatoes was diminished on average by 51.8%. Similarly, the degree of cell lysis caused by bacterial maceration was significantly (P<0.05) reduced in tuber tissue of PL-expressing progeny lines. The latter also revealed an enhanced PPO and PAL activity in their tuber tissue indicating an active plant defence. It is concluded therefore that the PL-mediated soft rot resistance introduced into potatoes by means of molecular techniques is heritable.     

Development of Marker-Free Transgenic Cry1Ab Rice with Lepidopteran Pest Resistance by Agrobacterium Mixture-Mediated Co-transformation

Cry1Ab gene was transformed into four rice varieties, Zhejing 22, Zhejing 27, Jiahua 1 and Xiushui 63 mediated by Agrobacterium-mixture co-transformation. Rice genotype had an important effect on callus induction and transformation efficiency. Different mixtures of Agrobacterium strains (EHA105 and EHA101) contained Hpt and Cry1Ab genes resulted in different frequencies of resistant calli. There was no correlation between the frequency of transformants with the ratio of the Agrobacterium strain mixture contained Hpt and Cry1Ab genes. A total of 509 transgenic plants were obtained from the four rice varieties, and 272 T₂ progenies were analyzed for Cry1Ab and Hpt genes. PCR analysis revealed that 412 regenerated plants were Hpt positive (80.94%), 62 plants were also Cry1Ab co-transformants (15.05% in total frequency), and 42 plants among the 272 T₂ progenies were Cry1Ab positive but Hpt negative. This suggests that marker-free transgenic plants could be produced by co-transformation mediated by mixed Agrobacterium strains with the selectable marker gene and target gene. Southern blot analysis of five independent marker-free T₂ transgenic lines co-transformed from Zhejing 22 showed that Cry1Ab gene had been inserted into rice genome with a single copy. The transgenic plants showed significantly stronger resistance to lepidopteron than the non-transgenic plants under no application of insecticides against lepidopteron.     

Toxicity of several δ-endotoxins of Bacillus thuringiensis against the cotton pest Earias insulana (Lepidoptera: Noctuidae)

The toxicity of nine Bacillus thuringiensis Cry proteins against neonate Earias insulana larvae was tested using a mixture of crystals and spores. The mean lethal concentration (LC₅₀) of Cry1Ac was 1.99 μg/ml. Cry1Fa, Cry1Ca, Cry1Ja and Cry2Aa were more active than Cry1Ac, with LC₅₀ values of 0.22, 0.24, 0.29, 0.43 μg/ml, respectively. Cry1Da and Cry1Aa were considerably less active than Cry1Ac. The remaining proteins, Cry1Ba and Cry1Ab, displayed no activity. Relative potencies were also calculated. Cry1Ja and Cry1Fa were significantly more active (7.72 and 5.71 times, respectively) than Cry1Ac, while Cry1Ca was significantly (1.95 times) more active than Cry2Aa.     

Marker-assisted Breeding for Disease Resistance in Potato

Sub-project 5 of BIOEXPLOIT aims to design durable disease resistance through marker-assisted breeding by converting existing markers for high-throughput application, developing and validating high-throughput marker technologies and pyramiding major R genes and/or quantitative trait loci into elite material. Activities include (1) the fine mapping of the quantitative trait locus PiXspg which accounts for a large proportion of the variation in late blight resistance, (2) converting SNP-based markers and an AFLP marker to easy-to-use-markers, (3) testing of progenies with combined sources of late blight resistance for presence of R genes and agronomic features, (4) backcrossing new sources of resistance to S. tuberosum and molecular screening of breeding materials with marker GP94 linked with gene Rpi-phu1 conferring late blight resistance, (5) evaluating potato clones with enhanced resistance against Phytophthora infestans under field conditions of Toluca (México), and (6) developing populations and marker-assisted breeding for disease resistance.