Transgenic potato plants with enhanced resistance to the tomato moth,Lacanobia oleracea: growth room trials

Insecticidal effects of three plant-derived genes, those encoding snowdrop lectin (GNA), bean (Phaseolus vulgaris) chitinase (BCH) and wheat -amylase (WAI), were investigated and compared with effects of the cowpea trypsin inhibitor gene (CpTI). Transgenic potato plants containing each of the three genes singly, and in pairwise combinations were produced. All the introduced genes were driven by the CaMV 35S promoter; expression was readily detectable at the RNA level in transformants, but not detectable accumulation of WAI could be detected in transgenic potatoes containing its encoding gene. GNA and BCH were accumulated at levels up to 2.0% of total soluble protein; both proteins were expressed in a functional form, and GNA was shown to undergo 'correct' N-terminal processing. Accumulation levels of individual proteins were higher in plants containing a single foreign gene than in plants containing two foreign genes.Resistance of the transgenic plants to insect attack was assayed by exposing the plants to larvae of the tomato moth, Lacanobia oleracea. All the plants tested which were expressing GNA showed an enhanced level of resistance. Leaf damage was reduced by more than 50% compared to controls; total insect biomass per plant was reduced by 45-65%, but larval survival was only slightly reduced (20%). These results support the hypothesis that GNA has a significant antifeedant effect on insects. Expression of BCH had no protective effect against this insect. Expression of CpTI in transgenic potatoes had similar effects to expression of GNA on total insect biomass and survival, but did not afford protection against insect damage to the plant.     

Expression of the insecticidal lectin from snowdrop (Galanthus nivalis agglutinin; GNA) in transgenic wheat plants: effects on predation by the grain aphid Sitobion avenae

Transgenic wheat plants containing the gene encoding snowdrop lectin (Galanthus nivalis agglutinin; GNA) under the control of constitutive and phloem-specific promoters were generated through the particle bombardment method. Thirty-two independently derived plants were subjected to molecular and biochemical analyses. Transgene integration varied from one to twelve estimated copies per haploid genome, and levels of GNA expression from 0 to ca. 0.2% of total soluble protein were observed in different transgenic plants. Seven transgenic plants were selected for further study. Progeny plants from these parental transformants were selected for transgene expression, and tested for enhanced resistance to the grain aphid (Sitobion avenae) by exposing the plants to nymphal insects under glasshouse conditions. Bioassay results show that transgenic wheat plants from lines expressing GNA at levels greater than ca. 0.04% of total soluble protein decrease the fecundity, but not the survival, of grain aphids. We propose that transgenic approaches using insecticidal genes such as gna in combination with integrated pest management present promising opportunities for the control of damaging wheat pests.     

Influence of plant development and environment on transgene expression in potato and consequences for insect resistance

Clonal replicates of different transformed potato plants expressing transgene constructs containing the constitutive Cauliflower Mosaic Virus (CaMV) 35S promoter, and sequences encoding the plant defensive proteins snowdrop lectin (Galanthus nivalis agglutinin; GNA), and bean chitinase (BCH) were propagated in tissue culture. Plants were grown to maturity, at first under controlled environmental conditions, and later in the glasshouse. For a given transgene product, protein accumulation was found to vary between the different lines of clonal replicates (where each line was derived from a single primary transformant plant), as expected. However, variability was also found to exist within each line of clonal replicates, comparable to the variation of mean expression levels observed between the different clonal lines. Levels of GNA, accumulated in different parts of a transgenic potato plant, also showed variation but to a lesser extent than plant–plant variation in expression. With the majority of the clonal lines investigated, accumulation of the transgene product was found to increase as the potato plant developed, with maximum levels found in mature plants. The variation in accumulation of GNA among transgenic plants within a line of clonal replicates was exploited to demonstrate that the enhanced resistance towards larvae of the tomato moth, Lacanobia oleracea L., caused by expression of this protein in potato, was directly correlated with the level of GNA present in the plants, and that conditions under which the plants were grown affect the levels of GNA expression and subsequent levels of insect resistance.     

雪花莲凝集素基因（gna）的改造及其抗蚜性

用定点突变方法对编码雪花莲凝集素（Galanthus nivalis agglutinin,GNA)前体蛋白的DNA序列进行了改造和转基因烟草9Nicotana tabacum L.）抗蚜性的研究。结果表明,将GNA编码序列中含有的稀有密码子改造后,GNA的表达水平从占总可溶性蛋白的0.17％增加到0.25％,转基因烟草的抗蚜性也随之增强,从平均抑制桃蚜（Myzus per-sicae(Sulzer））虫口密度63.7％显地提高到71.0％。     

Enhancement of resistance to aphids by introducing the snowdrop lectin genegna into maize plants

In order to enhance the resistance to pests, transgenic maize (Zea mays L.) plants from elite inbred lines containing the gene encoding snowdrop lectin (Galanthus nivalis L. agglutinin; GNA) under control of a phloem-specific promoter were generated through theAgrobacterium tumefaciens- mediated method. The toxicity of GNA-expressing plants to aphids has also been studied. The independently derived plants were subjected to molecular analyses. Polymerase chain reaction (PCR) and Southern blot analyses confirmed that thegna gene was integrated into maize genome and inherited to the following generations. The typical Mendelian patterns of inheritance occurred in most cases. The level of GNA expression at 0.13%-0.28% of total soluble protein was observed in different transgenic plants. The progeny of nine GNA-expressing independent transformants that were derived separately from the elite inbred lines DH4866, DH9942, and 8902, were selected for examination of resistance to aphids. These plants synthesized GNA at levels above 0.22% total soluble protein, and enhanced resistance to aphids was demonstrated by exposing the plants to corn leaf aphid (Rhopalosiphum maidis Fitch) under greenhouse conditions. The nymph production was significantly reduced by 46.9% on GNA-expressing plants. Field evaluation of the transgenic plants supported the results from the inoculation trial. After a series of artificial self-crosses, some homozygous transgenic maize lines expressing GNA were obtained. In the present study, we have obtained new insect-resistant maize material for further breeding work.     

Effects of artificial diet containing GNA and GNA-expressing potatoes on the development of the aphid parasitoid Aphidius ervi Haliday (Hymenoptera: Aphidiidae)

Aphid parasitoids are important biological control agents. The possibility arises that whilst foraging on insect-resistant transgenic plants, they are themselves at risk from direct and indirect effects of the expression of a transgene used to control the pest species. A liquid artificial diet was successfully used to deliver the snowdrop lectin (Galanthus nivalis agglutinin; GNA) to the peach-potato aphid, Myzus persicae. Bioassays utilising artificial diet incorporating GNA, and excised leaves of the GNA-expressing transgenic potato line, GNA2#28, were performed to assess the potential effects of GNA on the development of the aphid parasitoid Aphidius ervi. The results indicate that GNA delivered via artificial diet to the aphids can be transferred through the trophic levels and has a dose-dependent effect on parasitoid development. Parasitoid larvae excreted most of the ingested GNA in the meconium but some of it was detected in the pupae. Although A. ervi development was not affected when developing within hosts feeding on transgenic potato leaves, this probably reflected sub-optimal expression of the toxin in the transgenic potato line used     

Tritrophic Interactions Between Transgenic Potato Expressing Snowdrop Lectin (GNA), an Aphid Pest (Peach–Potato Aphid; Myzus Persicae (Sulz.) and a Beneficial Predator (2-Spot Ladybird; Adalia Bipunctata L.)

Tritrophic interactions between transgenic potato expressing the insecticidal lectin from snowdrop (Galanthus nivalis agglutinin; GNA), an aphid pest, Myzus persicae (Sulz.), and a beneficial predator, the 2-spot ladybird (Adalia bipunctata L.) were investigated. Clonal plants expressing GNA at 0.1–0.2% total soluble protein in leaves were used. No significant effects on development and survival of ladybird larvae fed on aphids from these transgenic plants were observed, with larval survival in the experimental group being 90% compared to 89% for controls. There were also no effects on subsequent female or male longevity. Female fecundity was also investigated. Although no significant differences (p > 0.05) were observed in egg production between control and experimental groups, a 10%, reduction (p < 0.01) in egg viability (determined by % hatch) occurred in ladybirds fed aphids reared on transgenic plants. Additional studies were carried out using aphids fed on artificial diet containing GNA, to deliver quantified levels of the protein to ladybird adults. GNA had no deleterious effects upon adult longevity, but resulted in a consistent trend for improved fecundity. Egg production was increased by up to 70% and egg viability also increased significantly. The results suggest that GNA is not deleterious to ladybirds. Results from these studies highlight the need to discriminate between direct and indirect effects when studying tritrophic interactions between plants/pests/natural enemies. Furthermore, it emphasises the importance of demonstrating cause and effect.     

Expression of the Galanthus nivalis agglutinin (GNA) gene in transgenic potato plants confers resistance to aphids

Aphids, the largest group of sap-sucking pests, cause significant yield losses in agricultural crops worldwide every year. The massive use of pesticides to combat this pest causes severe damage to the environment, putting in risk the human health. In this study, transgenic potato plants expressing Galanthus nivalis agglutinin (GNA) gene were developed using CaMV 35S and ST-LS1 promoters generating six transgenic lines (35S1-35S3 and ST1-ST3 corresponding to the first and second promoter, respectively). Quantitative real-time polymerase chain reaction (qRT-PCR) analysis indicated that the GNA gene was expressed in leaves, stems and roots of transgenic plants under the control of the CaMV 35S promoter, while it was only expressed in leaves and stems under the control of the ST-LS1 promoter. The levels of aphid mortality after 5 days of the inoculation in the assessed transgenic lines ranged from 20 to 53.3%. The range of the aphid population in transgenic plants 15 days after inoculation was between 17.0 ± 1.43 (ST2) and 36.6 ± 0.99 (35S3) aphids per plant, which corresponds to 24.9–53.5% of the aphid population in non-transformed plants. The results of our study suggest that GNA expressed in transgenic potato plants confers a potential tolerance to aphid attack, which appears to be an alternative against the use of pesticides in the future.     

Enhanced Resistance of Snowdrop Lectin (Galanthus nivalis L. Agglutinin)-Expressing Maize to Asian Corn Borer (Ostrinia furnacalis Guenée)

In order to enhance the resistance to pests, transgenic maize (Zea mays L.) plants from elite inbred lines containing the gene encoding snowdrop lectin (Galanthus nivalis L. agglutinin; GNA) under control of a phloem-specific promoter were generated through the Agrobacterium tumefaciens-mediated also studied. Thirty-six independently derived plants were subjected to molecular analyses. The level of GNA expression at 0.13%-0.28% of total soluble protein was observed in different transgenic plants. The progeny of three GNA-expressing independent transformants that were derived separately from the elite inbred lines DH4866, DH9942, and 8902, were selected for examination of resistance to ACB. These plants synthesized GNA at levels above 0.24% total soluble protein and enhanced resistance to ACB was demonstrated by exposing the plants to insects under greenhouse conditions. Semi-artificial diet bioassays also showed the toxic effect of GNA on ACB. Field evaluation of the transgenic plants supported the results from the artificial trial. In the present study, we have obtained new insect-resistant maize material for further breeding work and have found that GNA-expressing plants not only gained significant resistance to homopterans, but also showed toxicity to ACB, which is a type of Lepidoptera.     

Expression of snowdrop lectin in transgenic tobacco plants results in added protection against aphids

The range of sap-sucking insect pests to which GNA, (the mannose specific lectin from snowdrops (Galanthus nivalis) has been shown to be insecticidal in artificial diets has been extended to include the peach potato aphid (Myzus persicae). A gene construct for constitutive expression of GNA from the CaMV35S gene promoter has been introduced into tobacco plants. A transgenic tobacco line which expresses high levels of GNA has been shown to have enhanced resistance toM. persicae in leaf disc and whole plant bioassays,demonstrating the potential for extending transgenic plant technology to the control of sap-sucking insect pests.     

Direct and indirect sublethal effects of Galanthus nivalis agglutinin (GNA) on the development of a potato-aphid parasitoid, Aphelinus abdominalis (Hymenoptera: Aphelinidae)

Snowdrop lectin (Galanthus nivalis agglutinin, GNA), has been shown to confer partial resistance to two potato aphids Myzus persicae and Aulacorthum solani, when incorporated in artificial diet and/or expressed in transgenic potato. First-tier laboratory-scale experiments were conducted to assess the potential effect of GNA on the aphid parasitoid Aphelinus abdominalis. GNA (0.1% w/v) was successfully delivered to Macrosiphum euphorbiae via artificial diet and induced a reduced growth rate and increased mortality compared to aphids fed a control diet. As aphid parasitoid larvae are endophagous, they may be exposed to GNA during their larval development and potential "chronic toxicity" on A. abdominalis was investigated. The amounts of GNA present in aphid and parasitoid tissues were estimated by western blotting. Results suggest that parasitoids excrete most of the GNA ingested. Sublethal effects of GNA on several parasitoid fitness parameters (parasitism success, parasitoid development and size, emergence success, progeny survival and sex ratio) were studied. No direct detrimental effect of GNA on A. abdominalis was observed. However, GNA had an indirect host-size-mediated effect on the sex ratio and the size of parasitoids developing in GNA-fed aphids. This work highlights the need to determine the exact "causes and effects" when assessing the ecological impact of transgenic plants on non-target beneficial insects. Such bioassays form the basis of a tiered risk assessment moving from laboratory studies assessing individuals towards field-scale experiments assessing populations.     

Enhanced Resistance of Snowdrop Lectin (Galanthus nivalis L. agglutinin)-Expressing Maize to Asian Corn Borer (Ostrinia furnacalis Guenée)

Abstract: In order to enhance the resistance to pests, transgenic maize ( Zea mays L.) plants from elite inbred lines containing the gene encoding snowdrop lectin ( Galanthus nivalis L. agglutinin; GNA) under control of a phloem-specific promoter were generated through the Agrobacterium tumefaciens-mediated method. The toxicity of GNA-expressing plants to Asian corn borer ( Ostrinia furnacalis Guenée; ACB) was also studied. Thirty-six independently derived plants were subjected to molecular analyses. The level of GNA expression at 0.13%–0.28% of total soluble protein was observed in different transgenic plants. The progeny of three GNA-expressing independent transformants that were derived separately from the elite inbred lines DH4866, DH9942, and 8902, were selected for examination of resistance to ACB. These plants synthesized GNA at levels above 0.24% total soluble protein and enhanced resistance to ACB was demonstrated by exposing the plants to insects under greenhouse conditions. Semi-artificial diet bioassays also showed the toxic effect of GNA on ACB. Field evaluation of the transgenic plants supported the results from the artificial trial. In the present study, we have obtained new insect-resistant maize material for further breeding work and have found that GNA-expressing plants not only gained significant resistance to homopterans, but also showed toxicity to ACB, which is a type of Lepidoptera.
(Managing editor: Li-Hui ZHAO)     

Papaya transformed with the Galanthus nivalis GNA gene produces a biologically active lectin with spider mite control activity

Snowdrop (Galanthus nivalis) lectin has previously been shown to have anti-feedant and insecticidal activity towards sap-sucking insects. However, its effectiveness against plant-parasitic mites has not been demonstrated. In this study, the commercial papaya (Carica papaya L.) cultivar Kapoho, which is highly susceptible to mites, was transformed with the snowdrop lectin (G. nivalis agglutin [GNA]) gene. Polymerase chain reaction confirmed the presence of the transgene and six independent transformed lines were selected for expression analysis. Western blot analysis showed that the lines expressed a recombinant protein with a molecular weight similar to that of the native snowdrop lectin. Leaf extracts containing the recombinant GNA protein agglutinated trypsinized rabbit erythrocytes thus, showing the GNA protein to be biologically active. ELISA and indirect measurement from the agglutination assay showed there to be variation in GNA expression among the lines produced. A laboratory bioassay using carmine spider mites (Tetranychus cinnabarinus) suggested improved pest resistance in the transgenic papaya plants. This is the first report that a transgenic plant expressing the GNA gene possesses enhanced resistance to a plant-parasitic mite.     

Use of the rice sucrose synthase-1 promoter to direct phloem-specific expression of {beta}-glucuronidase and snowdrop lectin genes in transgenic tobacco plants

The promoter region from the rice sucrose synthase-1 gene (RSs1)was fused with coding sequences for ß-glucuronidase(GUS) and snowdrop (Galanthus nivalis) lectin (GNA). Tobaccoplants were transformed with these chimaenc genes in order todetermine the expression pattern directed by the RSs1 promoter.Histochemical and immunochemical assays demonstrated that theexpression of both GUS and GNA was restricted to phloem tissue,and was not observed in any other tissues. This phloem-specificexpression pattern was consistent in stem, leaf and root, andin different transgenic plants. Chimaeric genes of RSs 1-GUSand RSs1 GNA were stably inherited in T1 plants. In addition,GNA was detected by immunological assay in the honeydew producedby peach potato aphids (Myzus persicae) feeding on RSs1-GNAtransgenic tobacco plants. This provided direct evidence thatGNA was not only expressed in the phloem tissue, but was alsopresent in the phloem sap of transgenic tobacco plants. TheRSs1 promoter can thus be used to direct expression of an insecticidalprotein, such as GNA, in transgenic plants to control phloemsap-feeding insect pests. Key words: Rice sucrose synthase-1 promoter, phloemspecific, transgenic plants, ß-glucuronidase, Galanthus nivalis agglutinin, gene expression     

Improvement of Freezing Tolerance in Transgenic Tobacco Leaves by Expressing the hiC6 Gene

A cryoprotective protein, HIC6, was expressed transgenically in tobacco, a cold-sensitive plant, and the localization of the protein within the cell as well as freezing tolerance of the transgenic tobacco was investigated. For constitutive expression of HIC6 in tobacco, its corresponding gene was subcloned into pBI121. Through the transformation with pBI121/hiC6, fifteen transgenic tobacco lines were acquired, out of which twelve lines expressed the HIC6 protein. None of the transgenic tobacco lines, however, showed significant differences in freezing tolerance from the control plants (wild-type and transformed with pBI121) at ?1, ?3, and ?4°C, with the exception that their freezing temperature was ?2°C. In order to increase the accumulation level of HIC6, pBE2113 with a stronger promoter was used. Eight lines expressed the protein out of thirteen lines transformed with pBE2113/hiC6. The accumulation levels of the protein were clearly higher in the tobacco plants transformed with pBE2113/hiC6 than in those with pBI121/hiC6. The HIC6 protein seemed to be localized in mitochondria of the transgenic tobacco plants. Freezing-tolerance test at ?1 - ?4°C showed that the degree of electrolyte leakage was significantly lower in the plants with pBE2113/hiC6 than in the control plants. A leaf browning observation also showed that high accumulation of HIC6 significantly suppressed injury caused by freezing to the transgenic tobacco at ?3°C.     

农杆菌介导的雪花莲凝集素基因转入玉米骨干自交系

以农杆菌AGL0介导,将雪花莲凝集素基因转入玉米骨干自交系齐319和掖515胚性愈伤组织细胞,从筛选后的抗性愈伤组织获得再生植株。农杆菌浓度和共培养时间均能显著影响侵染后玉米愈伤组织的抗性频率。在农杆菌浓度OD600 0．2～0．3,共培养时间3d时,侵染后玉米愈伤组织的抗性频率最高,平均约4％。对再生植株及其子代基因组DNA的PCR及Southern杂交分析表明雪花莲凝集素基因已经整合到玉米基因组中,并遗传给后代。在蚜虫人工接种试验中,转基因植株上蚜虫的繁殖力为非转基因对照植株上的50％,这表明转基因植株抗蚜性显著增强。     

Virus Movement Protein Gene Mediated Resistance Against Cucumber Mosaic Virus Infection

Tobacco (Nicotiana tabactum L. ) "NC89" plants were transformed with deletion mutant of cucumber mosaic virus (CMV) movement protein (MP) gene and full-length CMV MP gene, respectively. The transformed plants were analyzed with polymerase chain reaction (PCR), PCR-Southem, Southern and Western blots. R0 generation of the transgenic plants were inoculated with CMV. Five out of 10 lines of tobacco plants (BMPK) transformed with CMV MP deletion mutant gene showed high resistance to CMV infection and remained symptomless for up to 50 days post-inoculation. In contrast, tobacco plants (BMPR) transformed with full-length CMV MP gene did not show resistance to CMV infection. However, most of the infected full-length CMV MP gene transgenic plants recovered by showing none or very mild mosaic symptoms in 40 days post-inoculation. The results of R1 generation of the BMPK transgenic plants tested under field conditions showed that all 5 lines of transgenic plants could delay the virus disease development.     

Improvement of freezing tolerance in transgenic tobacco leaves by expressing the hiC6 gene

A cryoprotective protein, HIC6, was expressed transgenically in tobacco, a cold-sensitive plant, and the localization of the protein within the cell as well as freezing tolerance of the transgenic tobacco was investigated. For constitutive expression of HIC6 in tobacco, its corresponding gene was subcloned into pBI121. Through the transformation with pBI121/hiC6, fifteen transgenic tobacco lines were acquired, out of which twelve lines expressed the HIC6 protein. None of the transgenic tobacco lines, however, showed significant differences in freezing tolerance from the control plants (wild-type and transformed with pBI121) at -1, -3, and -4 degrees C, with the exception that their freezing temperature was -2 degrees C. In order to increase the accumulation level of HIC6, pBE2113 with a stronger promoter was used. Eight lines expressed the protein out of thirteen lines transformed with pBE2113/hiC6. The accumulation levels of the protein were clearly higher in the tobacco plants transformed with pBE2113/hiC6 than in those with pBI121/hiC6. The HIC6 protein seemed to be localized in mitochondria of the transgenic tobacco plants. Freezing-tolerance tests at -1 - -4 degrees C showed that the degree of electrolyte leakage was significantly lower in the plants with pBE2113/hiC6 than in the control plants. A leaf browning observation also showed that high accumulation of HIC6 significantly suppressed injury caused by freezing to the transgenic tobacco at -3 degrees C.     

两种凝集素基因在转基因烟草中表达的研究

构建了含尾穗苋凝集素基因(ACA)的cDNA序列和改造后的雪花莲凝集素基因(GNA)的植物表达载体pBACG。在此表达载体中,ACA和GNA基因的表达分别由35S启动子和CoYMV启动子控制。通过农杆菌介导,将ACA和GNA基因转化到烟草中,经卡那霉素筛选获得60株转化再生植株。对PCR检测呈阳性的50株植株进行接蚜虫实验,结果表明,其平均抑虫率达83．9％。Southern blotting分析表明,ACA和GNA基因都已整合到烟草基因组中。Western blotting结果显示这两个基因在不同植株中都可表达其相应的蛋白质,但表达水平不同。部分Western blotting分析呈阳性植株的抗蚜性与T0代相近,达85．3％,说明这两个基因的抗蚜功能可以稳定遗传。     

Expression of Sambucus nigra agglutinin (SNA-I′) from elderberry bark in transgenic tobacco plants results in enhanced resistance to different insect species

Tobacco plants (Nicotiana tabacum cv Samsun NN) have been transformed with the gene encoding the type-2 ribosome-inactivating protein (RIP) SNA-I′ from elderberry (Sambucus nigra) under the control of the Cauliflower Mosaic Virus 35S promoter. Previous research confirmed that these plants synthesize, correctly process and assemble a fully active RIP. Variability in protein expression was observed within the transgenic lines. The effects of the type-2 RIP SNA-I′ delivered through a leaf feeding assay were evaluated in the laboratory on two economically important pest insects belonging to the orders of Hemiptera, the tobacco aphid (Myzus nicotianae) and Lepidoptera, the beet armyworm (Spodoptera exigua). In the experiment with aphids, significant effects were observed on the life parameters, such as survival, intrinsic rate of increase, net reproductive rate, mean generation time and mean daily offspring, whereas with caterpillars significant reduction in fresh weight as well as retardation in development were observed. In addition, significant increases in mortality were noted for insects fed on the transgenic lines as compared to wild type plants. This information provides further support for RIPs having a role in plant resistance to insect pest species.