Transgenic Sugarcane with a cry1Ac Gene Exhibited Better Phenotypic Traits and Enhanced Resistance against Sugarcane Borer

We developed sugarcane plants with improved resistance to the sugarcane borer, Diatraea saccharalis (F). An expression vector pGcry1Ac0229, harboring the cry1Ac gene and the selectable marker gene, bar, was constructed. This construct was introduced into the sugarcane cultivar FN15 by particle bombardment. Transformed plantlets were identified after selection with Phosphinothricin (PPT) and Basta. Plantlets were then screened by PCR based on the presence of cry1Ac and 14 cry1Ac positive plantlets were identified. Real-time quantitative PCR (RT-qPCR) revealed that the copy number of cry1Ac gene in the transgenic lines varied from 1 to 148. ELISA analysis showed that Cry1Ac protein levels in 7 transgenic lines ranged from 0.85 μg/FWg to 70.92 μg/FWg in leaves and 0.04 μg/FWg to 7.22 μg/FWg in stems, and negatively correlated to the rate of insect damage that ranged from 36.67% to 13.33%, respectively. Agronomic traits of six transgenic sugarcane lines with medium copy numbers were similar to the non-transgenic parental line. However, phenotype was poor in lines with high or low copy numbers. Compared to the non-transgenic control plants, all transgenic lines with medium copy numbers had relatively equal or lower sucrose yield and significantly improved sugarcane borer resistance, which lowered susceptibility to damage by insects. This suggests that the transgenic sugarcane lines harboring medium copy numbers of the cry1Ac gene may have significantly higher resistance to sugarcane borer but the sugarcane yield in these lines is similar to the non-transgenic control thus making them superior to the control lines.     

Resistance to Tecia solanivora (Lepidoptera: Gelechiidae) in three transgenic Andean varieties of potato expressing Bacillus thuringiensis CrylAc protein

Transgenic potato, Solanum tuberosum L., plants containing a synthetic cry1Ac gene coding for the Bacillus thuringiensis (Bt) crystalline insecticidal protein were produced and evaluated for resistance to Tecia solanivora Povolny (Lepidoptera: Gelechiidae), the larvae of which attack potato tubers. In total, 43 transgenic lines of commercial Andean potato varieties Diacol Capiro, Pardo Pastusa, and Pandeazúcar were obtained. These transgenic lines were found to have one to four copies of cry1Ac per genome and expression levels of Cry1Ac protein varying from 0.02 to 17 microg/g fresh tuber tissue. Bioassays of T. solanivora larvae on these transgenic potato tubers showed 83.7-100% mortality, whereas the mortality levels on nontransgenic lines were 0-2.67%. Our data indicate the capability of Bt transgenic technology to control the T. solanivora while reducing the use of chemical insecticides. Further studies under controlled field conditions will be helpful in exploring the potential of CrylAc potatoes in the insect pest management strategies.     

Advances in Transgenic Research for Insect Resistance in Sugarcane

The first phase of transgenic research in sugarcane concentrated on the development and evaluation of transgenic lines transformed for resistance to biotic stresses, particularly diseases and insect pests. Sugarcane is attacked by a range of insects including tissue borers, sucking pests and canegrubs. Losses due to these pests are estimated to be around 10%. Although chemical control and integrated pest management are regularly practiced for the control of insect pests, success is often limited due to practical difficulties. The genetic complexity of sugarcane coupled with the non-availability of resistance genes in the germplasm has made conventional breeding for insect resistance difficult. In this context, transgenic technology has become a handy tool for imparting insect resistance to an elite variety which is otherwise superior for most other agronomic traits. A number of transgenic sugarcane lines have been developed with genes expressing Cry proteins, proteinase inhibitors or lectins resistant to borers, sucking insects or grubs. While commercializing transgenic lines, issues such as higher and stable transgene expression, preparedness for resistance management and non-target effects need to be addressed. To manage the constant threat of resistance development in target insects, it is imperative to deploy field-level strategies taking clues from other crops coupled with the search for new potent replacement molecules for transformation.     

A Sensitive Bioassay for the Detection of Cry1A Toxin Expression in Transgenic Cotton

In the process of development of insect resistant transgenic plants and also to evaluate the consistency in expression of the toxin under greenhouse and field conditions, immunological and bioassays are commonly used. The assay being described in this report, is based on the high levels of sensitivity of a cotton leaf feeding insect, the semilooper, Anomis flava (Fabricius) to Cry toxins (Cry1Aa, Cry1Ab and Cry1Ac). The assay is sensitive, quick and reproducible. Cry1Ac was the most toxic followed by Cry1Ab and Cry1Aa. LC 50 s of the three toxins on first instar larvae ranged from 0.79-6.08 ng cm -2 of leaf. LC 50 s of Cry1Ac for the fourth instar larvae ranged from 12.91-21.14 ng cm -2 while LC 50 s for Cry1Aa and Cry1Ab were in the range 53.0-138 ng cm -2 . The fiducial limits (at 95% probability) of the probit assay data indicated that there was no difference in response between the three different populations to each of the three toxins. The data from all assays were pooled for each of the three toxins separately and subjected to regression analysis to obtain a cumulative log dose response for first and fourth instar larvae. These can be used as standard curves to quantify toxin expression in plants based on mortality response of either first or fourth instar A. flava larvae. Apart from being used to detect expression in putative Bt cotton transgenic plants, the assay can also be used to follow the activity of Cry toxins in transgenic cotton plants in the field during the growing season.     

Control of resistant pink bollworm (Pectinophora gossypiella) by transgenic cotton that produces Bacillus thuringiensis toxin Cry2Ab

Crops genetically engineered to produce Bacillus thuringiensis toxins for insect control can reduce use of conventional insecticides, but insect resistance could limit the success of this technology. The first generation of transgenic cotton with B. thuringiensis produces a single toxin, Cry1Ac, that is highly effective against susceptible larvae of pink bollworm (Pectinophora gossypiella), a major cotton pest. To counter potential problems with resistance, second-generation transgenic cotton that produces B. thuringiensis toxin Cry2Ab alone or in combination with Cry1Ac has been developed. In greenhouse bioassays, a pink bollworm strain selected in the laboratory for resistance to Cry1Ac survived equally well on transgenic cotton with Cry1Ac and on cotton without Cry1Ac. In contrast, Cry1Ac-resistant pink bollworm had little or no survival on second-generation transgenic cotton with Cry2Ab alone or with Cry1Ac plus Cry2Ab. Artificial diet bioassays showed that resistance to Cry1Ac did not confer strong cross-resistance to Cry2Aa. Strains with >90% larval survival on diet with 10 microg of Cry1Ac per ml showed 0% survival on diet with 3.2 or 10 microg of Cry2Aa per ml. However, the average survival of larvae fed a diet with 1 microg of Cry2Aa per ml was higher for Cry1Ac-resistant strains (2 to 10%) than for susceptible strains (0%). If plants with Cry1Ac plus Cry2Ab are deployed while genes that confer resistance to each of these toxins are rare, and if the inheritance of resistance to both toxins is recessive, the efficacy of transgenic cotton might be greatly extended.     

转单、双Bt基因741杨外源基因表达和抗虫性比较

【目的】研究联合使用两种或两种以上的抗虫基因的抗虫效果, 同时鉴定并筛选出转双Bt基因741杨对鳞翅目和鞘翅目害虫有较强抗性的株系。【方法】选取转三基因（Cry3Aa+Cry1Ac+API）741杨8个株系、 转双基因（Cry1Ac+API）741杨1个株系和转单基因（Cry3Aa）741杨3个株系为试材, 从外源基因PCR检测、 毒蛋白表达和抗虫性三方面对转基因株系进行对比分析。【结果】经PCR扩增后各转基因株系出现了预期的电泳条带。ELISA蛋白检测显示转基因株系中都有与所含基因相应的Bt杀虫蛋白表达。用转基因株系新鲜叶片进行柳蓝叶甲Plagiodera versicolora和美国白蛾Hyphantria cunea室内饲虫实验表明： 转入不同抗虫基因的杨树对昆虫的抗性具有选择性, 对非靶标昆虫没有毒杀作用。转双Bt基因741杨具有双抗性, 不同转基因株系表现出高中低的抗性水平： 在对柳蓝叶甲的抗性上, 筛选出的其中5个高抗株系（pCCA1, pCCA2, pCCA5, pCCA6和pCCA9）的抗性水平明显比含Cry3Aa单Bt基因的3个高抗株系（pCC11, pCC53和pCC84）高; 在对美国白蛾的抗性上, 有7个株系（pCCA2~pCCA7和pCCA9）的抗性水平与含Cry1Ac单Bt基因株系（pB29）表现一致, 只有1个株系（pCCA1）对美国白蛾表现出了极低的抗性。【结论】多个抗虫基因在741杨上的联合使用, 不仅扩大了抗虫谱, 其中的高抗株系还具有了更高的抗虫能力, 有效地发挥了基因的叠加效应。     

Enhanced expression of Arabidopsis rubisco small subunit gene promoter regulated Cry1Ac gene in chickpea conferred complete resistance to Helicoverpa armigera

A major pest of chickpea, Helicoverpa armigera, can be controlled by expressing genes from the bacterium Bacillus thuringiensis as an environmentally compatible option. Here we show that transgenic chickpeas containing a cry1Ac gene conferred a high degree of resistance to H. armigera. The Agrobacterium binary vector contained the nptII gene as the selectable marker and cry1Ac gene driven by the Arabidopsis rubisco small subunit gene (ats1A) promoter. We generated 54 and 47 independent transgenic lines using truncated (trcry1Ac) and full-length versions of the cry1Ac (flcry1Ac) gene, respectively. Of these lines, twelve transmitted the trcry1Ac transgene to the next generation at a 3:1 ratio, while only 8 flcry1Ac lines segregated in a 3:1 ratio. Five lines expressed trCry1Ac protein > 50 μg/g fresh weight, however, only one line accumulated about 30 μg/g flCry1Ac protein. Such high levels of trCry1Ac protein have not been reported before in chickpea. When trCry1Ac lines were challenged to whole plant bioassays in the greenhouse, lowest pod damage was observed in BS100B (1.4%) followed by BS81P (4.4%), and BS100E (6.2%) compared to the parental line (49.9%). The phenotypes of the lines expressing high levels of Cry1Ac protein were indistinguishable from their null segregants and controls. Thus, trCry1Ac lines could be suitable for crossing with our existing Cry2Aa lines for generation of a pyramided Bt chickpea for enhanced insect resistance management in the field.

     

Control of Resistant Pink Bollworm (Pectinophora gossypiella) by Transgenic Cotton That Produces Bacillus thuringiensis Toxin Cry2Ab

Crops genetically engineered to produce Bacillus thuringiensis toxins for insect control can reduce use of conventional insecticides, but insect resistance could limit the success of this technology. The first generation of transgenic cotton with B. thuringiensis produces a single toxin, Cry1Ac, that is highly effective against susceptible larvae of pink bollworm (Pectinophora gossypiella), a major cotton pest. To counter potential problems with resistance, second-generation transgenic cotton that produces B. thuringiensis toxin Cry2Ab alone or in combination with Cry1Ac has been developed. In greenhouse bioassays, a pink bollworm strain selected in the laboratory for resistance to Cry1Ac survived equally well on transgenic cotton with Cry1Ac and on cotton without Cry1Ac. In contrast, Cry1Ac-resistant pink bollworm had little or no survival on second-generation transgenic cotton with Cry2Ab alone or with Cry1Ac plus Cry2Ab. Artificial diet bioassays showed that resistance to Cry1Ac did not confer strong cross-resistance to Cry2Aa. Strains with >90% larval survival on diet with 10 μg of Cry1Ac per ml showed 0% survival on diet with 3.2 or 10 μg of Cry2Aa per ml. However, the average survival of larvae fed a diet with 1 μg of Cry2Aa per ml was higher for Cry1Ac-resistant strains (2 to 10%) than for susceptible strains (0%). If plants with Cry1Ac plus Cry2Ab are deployed while genes that confer resistance to each of these toxins are rare, and if the inheritance of resistance to both toxins is recessive, the efficacy of transgenic cotton might be greatly extended.     

Genetic transformation and pyramiding of aprotinin-expressing sugarcane with cry1Ab for shoot borer (Chilo infuscatellus) resistance

We evaluated the insecticidal toxicity of Cry1Aa, Cry1Ab and Cry1Ac toxins against neonate larvae of sugarcane shoot borer Chilo infuscatellus Snellen (Lepidoptera: Crambidae) in vitro on diet surface. With the lowest LC₅₀ value, Cry1Ab emerged as the most effective among the three toxins. Sugarcane cultivars Co 86032 and CoJ 64 were transformed with cry1Ab gene driven by maize ubiquitin promoter through particle bombardment and Agrobacterium-mediated transformation systems. Gene pyramiding was also attempted by retransforming sugarcane plants carrying bovine pancreatic trypsin inhibitor (aprotinin) gene, with cry1Ab. Southern analysis confirmed multiple integration of the transgene in case of particle bombardment and single site integration in Agrobacterium-mediated transformants. The expression of cry1Ab was demonstrated through Western analysis and the toxin was quantified using ELISA. The amount of Cry1Ab protein in different events varied from 0.007 to 1.73% of the total soluble leaf protein; the events transformed by Agrobacterium method showed significantly higher values. In in vivo bioassay with neonate larvae of shoot borer, transgenics produced considerably lower percentage of deadhearts despite suffering feeding damage by the borer compared with the untransformed control plants. Expressed Cry1Ab content was negatively related to deadheart damage. Aprotinin-expressing sugarcane pyramided with cry1Ab also showed reduction in damage. The potential of producing sugarcane transgenics with cry1Ab and aprotinin genes resistant to early shoot borer was discussed in the light of the results obtained.     

Transgenic plants expressing two Bacillus thuringiensis toxins delay insect resistance evolution

Preventing insect pests from developing resistance to Bacillus thuringiensis (Bt) toxins produced by transgenic crops is a major challenge for agriculture. Theoretical models suggest that plants containing two dissimilar Bt toxin genes ('pyramided' plants) have the potential to delay resistance more effectively than single-toxin plants used sequentially or in mosaics. To test these predictions, we developed a unique model system consisting of Bt transgenic broccoli plants and the diamondback moth, Plutella xylostella. We conducted a greenhouse study using an artificial population of diamondback moths carrying genes for resistance to the Bt toxins Cry1Ac and Cry1C at frequencies of about 0.10 and 0.20, respectively. After 24 generations of selection, resistance to pyramided two-gene plants was significantly delayed as compared with resistance to single-gene plants deployed in mosaics, and to Cry1Ac toxin when it was the first used in a sequence. These results have important implications for the development and regulation of transgenic insecticidal plants.     

Expression of Cry1Ac in Transgenic Tobacco Plants Under the Control of a Wound-Inducible Promoter (AoPR1) Isolated from Asparagus officinalis to Control Heliothis virescens and Manduca sexta

Expression of cry1Ac gene from Bacillus thuringiensis (Bt) was evaluated under the control of a wound-inducible AoPR1 promoter from Asparagus officinalis in transgenic tobacco plants. The leaves of transgenic plants were mechanically wounded to evaluate the activity of the AoPR1 promoter in driving the expression of Cry1Ac protein at the wound site. Our results indicate that mechanical wounding of transgenic plants was effective in inducing the expression of Cry1Ac protein. As a result of this induction, the accumulated levels of Cry1Ac protein increased during 6–72 h post-wounding period. The leaves of transgenic tobacco plants were evaluated for resistance against Heliothis virescens and Manduca sexta in insect bioassays in two different ways. The detached tobacco leaves were either fed directly to the insect larvae or they were first mechanically wounded followed by a 72 h post-wounding feeding period. Complete protection of mechanically wounded leaves of transgenic plants was observed within 24 h of the bioassay. The leaves of transgenic plants fed directly (without pre-wounding) to the larvae achieved the same level of protection between 24 and 72 h of the bioassay.     

Seasonal expression of Bt proteins in transgenic rice lines and the resistance against Asiatic rice borer Chilo suppressalis (Walker)

Laboratory bioassays and field surveys were carried out to compare the resistance of three transgenic rice (Oryza sativa L.) lines including Bt-DL expressing a single gene cry1Ab, Bt-KF6 expressing stacked genes cry1Ac and CpTI genes and Bt-SY63 expressing a fusion gene cry1Ab/cry1Ac, respectively, to an important rice pest Chilo suppressalis (Walker). In addition, enzyme-linked immunosorbent assays (ELISA) were conducted to monitor the Bt protein expressions in rice leaves and stems at different rice growth stages. Results showed that all the transgenic rice lines exhibited significantly high resistance to the pest compared with their corresponding nontransformed isolines. Among the transgenic rice lines, Bt-SY63 and Bt-KF6 had higher resistance to C. suppressalis at early growth stage, but lower resistance at late stages, while the pest resistance of Bt-DL was relatively stable throughout the growing season. The results were consistent with ELISA results showing that Bt protein levels in Bt-SY63 or Bt-KF6 leaves decreased in late growth stages, but were relatively stable in Bt-DL at all growth stages. This demonstrates that the resistance to a pest by Bt plants is positively correlated with Cry protein expression levels in plant tissues. Compared with Bt-SY63 and Bt-KF6, the Bt protein expression levels were significantly lower in Bt-DL, while its resistance to C. suppressalis was the highest. This may suggest that C. suppressalis is more susceptible to Cry1Ab than to Cry1Ac. The data from the current study are valuable for decision-making for commercial use of Bt rice lines and development of appropriate pest control and resistance management strategies for the transgenic rice lines.     

Fitness of Cry1A-resistant and -susceptible Helicoverpa armigera (Lepidoptera: Noctuidae) on transgenic cotton with reduced levels of Cry1Ac

The performance of Helicoverpa armigera (Hübner) on 15-wk-old cotton plants was compared for a susceptible strain, a near-isogenic laboratory-selected strain, and F1 progeny of the two strains. Glasshouse experiments were conducted to test the three insect types on conventional plants and transgenic plants that produced the Bacillus thuringiensis (Bt) toxin Cry1Ac. At the time of testing (15 wk), the Cry1Ac concentration in cotton leaves was 75% lower than at 4 wk. On these plants, < 10% of susceptible larvae reached the fifth instar, and none survived to pupation. In contrast, survival to adulthood on Cry1Ac cotton was 62% for resistant larvae and 39% for F1 larvae. These results show that inheritance of resistance to 15-wk-old Cry1Ac cotton is partially dominant, in contrast to results previously obtained on 4-wk-old Cry1Ac cotton. Growth and survival of resistant insects were similar on Cry1Ac cotton and on non-Bt cotton, but F1 insects developed more slowly on Cry1Ac cotton than on non-Bt cotton. Survival was lower and development was slower for resistant larvae than for susceptible and F1 larvae on non-Bt cotton. These results show recessive fitness costs are associated with resistance to Cry1Ac.     

Bt-toxin uptake by the non-target herbivore, Myzus persicae (Hemiptera: Aphididae), feeding on transgenic oilseed rape in laboratory conditions

The potential non-target effects of genetically modified crops are some of the more debated topics within applied biotechnologies in agriculture and environmental risk assessment. The objective of the present research was to study the potential Bt-toxin uptake by the non-target herbivore Myzus persicae Sulzer (Hemiptera: Aphididae) feeding on transgenic oilseed rape plants (Brassica napus cv. 'Westar' lines GT 2-4) expressing the Cry1Ac endotoxin. A specific aim was to replicate our previous experiment in controlled laboratory conditions to avoid or minimize the risk of contamination leading to potential false positive results. The toxin levels in vernalized (V) and not-vernalized (not-V) transgenic oilseed rape plants was also monitored to better clarify the role of physiological processes on Bt-toxin expression. Cry1Ac expression in not-V plants (mean concentration±SE=167.8±5.7 μg kg-1 FW) showed a pattern of large variability, in comparison with V plants whose expression (mean concentration±SE=227.7±1.9 μg kg-1 FW) was significantly more stable. Cry1Ac toxin was detected in three aphid samples reared on V plants with a mean toxin concentration±SE of 4.8±0.6 μg Kg-1 FW and in three out of six samples of aphids reared on not-V plants (mean toxin concentration±SE=7.1±1.2 μg kg-1 FW). The mean Bt-toxin concentration of all the positive aphid samples was 5.9±1.0 μg kg-1 FW. Our results confirmed the findings of our previous experiment and highlighted the potential for Cry1Ac toxin uptake by aphids feeding on transgenic oilseed rape plants.     

Overexpression of a novel Cry1Ie gene confers resistance to Cry1Ac-resistant cotton bollworm in transgenic lines of maize

Although transgenic crops expressing either Cry1Ab or Cry1Ac, both derived from Bacillus thuringiensis (Bt), have been used commercially, the evolution of insects resistance to these CRY proteins has become a challenge. Thus, it has been proposed that co-expression of two Bt proteins with different modes of action may delay the development of resistance to Bt. However, few Bt proteins have been identified as having different modes of action from those of Cry1Ab or Cry1Ac. In this study, transgenic lines of maize over-expressing either Cry1Ie or Cry1Ac gene have been developed. Several independent transgenic lines with one copy of the foreign gene were identified by Southern blot analysis. Bioassays in the laboratory showed that the transgenic plants over-expressing Cry1Ie were highly toxic against the wild-type cotton bollworm (Heliothis armigera), producing mortality levels of 50 % after 6 days of exposure. However, the mortality caused by these plants was lower than that caused by the Cry1Ac transgenic plants (80 %) and MON810 plants expressing Cry1Ab (100 %), which both exhibited low toxicity toward the Cry1Ac-resistant cotton bollworm. In contrast, three transgenic maize lines expressing Cry1Ie induced higher mortality against this pest and were also highly toxic to the Asian corn borer (Ostrinia furnacalis) in the field. These results indicate that the Cry1Ie protein has a different mode of action than the Cry1Ab and Cry1Ac proteins. Therefore, the use of transgenic plants expressing Cry1Ie might delay the development of Bt-resistant insects in the field.     

Binding of Bacillus thuringiensis toxins in resistant and susceptible strains of pink bollworm (Pectinophora gossypiella)

Evolution of resistance by pests could cut short the success of transgenic plants producing toxins from Bacillus thuringiensis, such as Bt cotton. The most common mechanism of insect resistance to B. thuringiensis is reduced binding of toxins to target sites in the brush border membrane of the larval midgut. We compared toxin binding in resistant and susceptible strains of Pectinophora gossypiella, a major pest of cotton worldwide. Using Cry1Ab and Cry1Ac labeled with (125)I and brush border membrane vesicles (BBMV), competition experiments were performed with unlabeled Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ba, Cry1Ca, Cry1Ja, Cry2Aa, and Cry9Ca. In the susceptible strain, Cry1Aa, Cry1Ab, Cry1Ac, and Cry1Ja bound to a common binding site that was not shared by the other toxins tested. Reciprocal competition experiments with Cry1Ab, Cry1Ac, and Cry1Ja showed that these toxins do not bind to any additional binding sites. In the resistant strain, binding of (125)I-Cry1Ac was not significantly affected; however, (125)I-Cry1Ab did not bind to the BBMV. This result, along with previous data from this strain, shows that the resistance fits the "mode 1" pattern of resistance described previously in Plutella xylostella, Plodia interpunctella, and Heliothis virescens.