Transgenic Resistance Research Articles

Overexpression of VpPR10.1 by an efficient transformation method enhances downy mildew resistance in V. vinifera.

Putrescine and spermidine increase the transformation efficiency of Vitis vinifera L. cv. Thompson seedless. Accumulation of VpPR10.1 in transgenic V. vinifera Thompson seedless, likely increases its resistance to downy mildew. A more efficient method is described for facilitating Agrobacterium-mediated transformation of Vitis vinifera L. cv. Thompson Seedless somatic embryogenesis using polyamines (PAs). The efficacies of putrescine, spermidine and spermine are identified at a range of concentrations (10µM, 100µM and 1mM) added to the culture medium during somatic embryo growth. Putrescine (PUT) and spermidine (SPD) promote the recovery of proembryonic masses (PEM) and the development of somatic embryos (SE) after co-cultivation. Judging from the importance of the time-frame in genetic transformation, PAs added at the co-cultivation stage have a stronger effect than delayed selection treatments, which are superior to antibiotic treatments in the selection stage. Best embryogenic responses are with 1mM PUT and 100µM SPD added to the co-culture medium. Using the above method, a pathogenesis-related gene (VpPR10.1) from Chinese wild Vitis pseudoreticulata was transferred into Thompson Seedless for functional evaluation. The transgenic line, confirmed by western blot analysis, was inoculated with Plasmopara viticola to test for downy mildew resistance. Based on observed restrictions of hyphal growth and increases in H2O2 accumulation in the transgenic plants, the accumulation of VpPR10.1 likely enhanced the transgenic plants resistance to downy mildew.

Transformation of Arabidopsis thaliana ESB1 Gene into Agrobacterium tumefaciens and Its Identification

Botrytis cinerea is a major disease in crop production. Chrysanthemum gray mold is easy to occur during chrysanthemum growing season. It can cause a lot of defoliation when serious, affecting plant flowering and reducing ornamental value. In this study, Arabidopsis thaliana ESB1 gene introduced into Agrobacterium tumefaciens was introduced into Agrobacterium tumefaciens and its identification after induction, and the sensitivity of chrysanthemum to antibiotics. It is expected that Agrobacterium tumefaciens will be transformed into chrysanthemum, result in transgenic chrysanthemum new lines resistance to disease, high quality, and high yield to lay the technical foundation. In this study, Arabidopsis thaliana ESB1 gene was introduced into Agrobacterium tumefaciens by freeze-thawing method. The PCR reaction was used to determine whether the ESB1 gene was successfully transferred into Agrobacterium tumefaciens and the susceptibility test of chrysanthemum to hygromycin was carried out to determine the critical tolerance concentration, Establishment of Chrysanthemum Transformation Receptor System. The result of PCR showed that the ESB1 gene was successfully transferred into Agrobacterium tumefaciens. The critical concentration of chrysanthemum to hygromycin was 30mg / L.rapid diffusion ability.

Transgene-mediated resistance to Papaya ringspot virus: challenges and solutions

Global papaya production is severely affected by papaya ringspot disease caused by Papaya ringspot virus (PRSV). Management of this potyvirus is challenging, due to 1) its non-persistent transmission by numerous aphid species and 2) the diversity of PRSV strains that exists within a country or between different geographical regions. Papaya cultivars with transgenic resistance have reduced the impact of the disease. There are no effective alternatives to transgenic resistance available in areas where disease pressure is high. In Hawaii, transgenic papayas such as “SunUp” and “Rainbow” have remained resistant to PRSV more than two decades saving the commercial papaya industry. Following the success in Hawaii, researchers from other countries have focused on developing PRSV-resistant transgenic papaya. These transgenic cultivars often demonstrated an initial transitory resistance that was ultimately overcome by the virus. For other cases, resistance was inconsistent. That is, some transgenic lines were resistant while others were not. Transgenic cultivars are now losing PRSV-resistance for various reasons in China and Taiwan. In this review, we present an update on work with transgenic papaya with resistance to PRSV. The focus is on factors affecting transgenic resistance in papaya and our attempt to explain why the Hawaiian scenario of complete and durable resistance has not been replicated in other regions. The utilization of more recent technologies to the development of virus resistance in papaya is also discussed.

Glucanase and Chitinase from Some Isolates of Endophytic Fungus Trichoderma spp.

Endophytic fungi are those fungi that are able to grow in plant tissue without causing symptoms of disease. It is thought that these fungi may confer on the host plants degree of resistance to parasitic invasion. Endophytic fungi have been isolated from stem tissue and these fungi are known to be antagonistic to pathogenic fungi. These endophytes produce chitinase and β-1,3-glucanase enzymes. Based on the fact that chitin and β-1,3-glucan are the main skeletal polysaccharides of the cell walls of fungal patogen. The aim of this research is to do potential test on some of isolates of Trichoderma’s endophytic (L-1, L-2, Is-1, Is-2 and Is-7) in the chitinase and β-1,3-glucanase activity in effort to determine endophytic which be chossen to be gene resource for the next research. The gene will be transformed to citrus plant japanese citroen in effort to make citrus plant transgenic resistance to phytopatogenic invasion. The result of this research is endofit namely L-1 is the most potential endophytic fungi with chitinase activities is 4,8 10−2 Unit and glucanase 24,2. 1012 Unit. The addition of chitin and cell wall of phytophtora causes chitinase activity significantly increase, and also addition of laminarin and cell wall of phytophtora makes glucanase activity increase.

In planta silencing of NSs and Hc-Pro through RNAi constructs: to develop durable resistance

Groundnut bud necrosis disease and Papaya ring spot disease are devastating disease of tomato and papaya/cucumber. In order to develop transgenic resistance against these viruses, gene silencing approach was followed to target the conserved viral sequences for obtaining wide spectrum of resistance. Viral derived small non coding RNA was used to silence viral gene. To have efficient processing of hairpins, microRNA backbone has been used to harbour viral derived siRNA. An artificial microRNA having miRNA159a backbone harbouring 21-nt siRNA sequences from RNAi suppressor (GBNV–NSs and PRSV–Hc-Pro) genes were constructed. To establish the efficacy of these amiRNA constructs, transient expression assay were performed and attenuation of viral symptoms were observed on expression of artificial microRNA construct in infected plants. Our data demonstrate that expression of virus-specific siRNAs using artificial-miRNA is an effective and predictable approach to silence Groundnut bud necrosis virus (GBNV) and Papaya ringspot virus (PRSV).

RNAi-derived transgenic resistance to Mungbean yellow mosaic India virus in cowpea.

Cowpea is an important grain legume crop of Africa, Latin America, and Southeast Asia. Leaf curl and golden mosaic diseases caused by Mungbean yellow mosaic India virus (MYMIV) have emerged as most devastating viral diseases of cowpea in Southeast Asia. In this study, we employed RNA interference (RNAi) strategy to control cowpea-infecting MYMIV. For this, we generated transgenic cowpea plants harbouring three different intron hairpin RNAi constructs, containing the AC2, AC4 and fusion of AC2 and AC4 (AC2+AC4) of seven cowpea-infecting begomoviruses. The T0 and T1 transgenic cowpea lines of all the three constructs accumulated transgene-specific siRNAs. Transgenic plants were further assayed up to T1 generations, for resistance to MYMIV using agro-infectious clones. Nearly 100% resistance against MYMIV infection was observed in transgenic lines, expressing AC2-hp and AC2+AC4-hp RNA, when compared with untransformed controls and plants transformed with empty vectors, which developed severe viral disease symptoms within 3 weeks. The AC4-hp RNA expressing lines displayed appearance of milder symptoms after 5 weeks of MYMIV-inoculation. Northern blots revealed a positive correlation between the level of transgene-specific siRNAs accumulation and virus resistance. The MYMIV-resistant transgenic lines accumulated nearly zero or very low titres of viral DNA. The transgenic cowpea plants had normal phenotype with no yield penalty in greenhouse conditions. This is the first demonstration of RNAi-derived resistance to MYMIV in cowpea.

Use of RNAi technology to develop a PRSV-resistant transgenic papaya

Papaya ringspot virus (PRSV) seriously limits papaya (Carica papaya L.) production in tropical and subtropical areas throughout the world. Coat protein (CP)- transgenic papaya lines resistant to PRSV isolates in the sequence-homology-dependent manner have been developed in the U.S.A. and Taiwan. A previous investigation revealed that genetic divergence among Hainan isolates of PRSV has allowed the virus to overcome the CP-mediated transgenic resistance. In this study, we designed a comprehensive RNAi strategy targeting the conserved domain of the PRSV CP gene to develop a broader-spectrum transgenic resistance to the Hainan PRSV isolates. We used an optimized particle-bombardment transformation system to produce RNAi-CP-transgenic papaya lines. Southern blot analysis and Droplet Digital PCR revealed that line 474 contained a single transgene insert. Challenging this line with different viruses (PRSV I, II and III subgroup) under greenhouse conditions validated the transgenic resistance of line 474 to the Hainan isolates. Northern blot analysis detected the siRNAs products in virus-free transgenic papaya tissue culture seedlings. The siRNAs also accumulated in PRSV infected transgenic papaya lines. Our results indicated that this transgenic papaya line has a useful application against PRSV in the major growing area of Hainan, China.

Xanthomonas campestris pv. musacearum: a major constraint to banana, plantain and enset production in central and east Africa over the past decade.

Bacteria; Phylum Proteobacteria; Class Gammaproteobacteria; Order Xanthomonadales; Family Xanthomonadaceae; Genus Xanthomonas; currently classified as X. campestris pv. musacearum (Xcm). However, fatty acid methyl ester analysis and genetic and genomic evidence suggest that this pathogen is X. vasicola and resides in a separate pathovar. Xcm can be isolated on yeast extract peptone glucose agar (YPGA), cellobiose cephalexin agar and yeast extract tryptone sucrose agar (YTSA) complemented with 5-fluorouracil, cephalexin and cycloheximide to confer semi-selectivity. Xcm can also be identified using direct antigen coating enzyme-linked immunosorbent assay (DAC-ELISA), species-specific polymerase chain reaction (PCR) using GspDm primers and lateral flow devices that detect latent infections. Causes Xanthomonas wilt on plants belonging to the Musaceae, primarily banana (Musa acuminata), plantain (M. acuminata × balbisiana) and enset (Ensete ventricosum). There is a high level of genetic homogeneity within Xcm, although genome sequencing has revealed two major sublineages. Yellowing and wilting of leaves, premature fruit ripening and dry rot, bacterial exudate from cut stems. Xcm has only been found in African countries, namely Burundi, Ethiopia, Democratic Republic of the Congo, Kenya, Rwanda, Tanzania and Uganda. Xcm is transmitted by insects, bats, birds and farming implements. Long-distance dispersal of the pathogen is by the transportation of latently infected plants into new areas. The management of Xcm has relied on cultural practices that keep the pathogen population at tolerable levels. Biotechnology programmes have been successful in producing resistant banana plants. However, the deployment of such genetic material has not as yet been achieved in farmers' fields, and the sustainability of transgenic resistance remains to be addressed.

Genome-Wide Analysis of the RAV Family in Soybean and Functional Identification of GmRAV-03 Involvement in Salt and Drought Stresses and Exogenous ABA Treatment.

Transcription factors play vital roles in plant growth and in plant responses to abiotic stresses. The RAV transcription factors contain a B3 DNA binding domain and/or an APETALA2 (AP2) DNA binding domain. Although genome-wide analyses of RAV family genes have been performed in several species, little is known about the family in soybean (Glycine max L.). In this study, a total of 13 RAV genes, named as GmRAVs, were identified in the soybean genome. We predicted and analyzed the amino acid compositions, phylogenetic relationships, and folding states of conserved domain sequences of soybean RAV transcription factors. These soybean RAV transcription factors were phylogenetically clustered into three classes based on their amino acid sequences. Subcellular localization analysis revealed that the soybean RAV proteins were located in the nucleus. The expression patterns of 13 RAV genes were analyzed by quantitative real-time PCR. Under drought stresses, the RAV genes expressed diversely, up- or down-regulated. Following NaCl treatments, all RAV genes were down-regulated excepting GmRAV-03 which was up-regulated. Under abscisic acid (ABA) treatment, the expression of all of the soybean RAV genes increased dramatically. These results suggested that the soybean RAV genes may be involved in diverse signaling pathways and may be responsive to abiotic stresses and exogenous ABA. Further analysis indicated that GmRAV-03 could increase the transgenic lines resistance to high salt and drought and result in the transgenic plants insensitive to exogenous ABA. This present study provides valuable information for understanding the classification and putative functions of the RAV transcription factors in soybean.

The Impact of "Coat Protein-Mediated Virus Resistance" in Applied Plant Pathology and Basic Research.

Worldwide, plant viruses cause serious reductions in marketable crop yield and in some cases even plant death. In most cases, the most effective way to control virus diseases is through genetically controlled resistance. However, developing virus-resistant (VR) crops through traditional breeding can take many years, and in some cases is not even possible. Because of this, the demonstration of the first VR transgenic plants in 1985 generated much attention. This seminal report served as an inflection point for research in both basic and applied plant pathology, the results of which have dramatically changed both basic research and in a few cases, commercial crop production. The typical review article on this topic has focused on only basic or only applied research results stemming from this seminal discovery. This can make it difficult for the reader to appreciate the full impact of research on transgenic virus resistance, and the contributions from fundamental research that led to translational applications of this technology. In this review, we take a global view of this topic highlighting the significant changes to both basic and applied plant pathology research and commercial food production that have accumulated in the last 30 plus years. We present these milestones in the historical context of some of the scientific, economic, and environmental drivers for developing specific VR crops. The intent of this review is to provide a single document that adequately records the significant accomplishments of researchers in both basic and applied plant pathology research on this topic and how they relate to each other. We hope this review therefore serves as both an instructional tool for students new to the topic, as well as a source of conversation and discussion for how the technology of engineered virus resistance could be applied in the future.

Interaction of transgenic and natural insect resistance mechanisms against Spodoptera littoralis in cotton.

Insect-resistant transgenic plants that express insecticidal Cry proteins from Bacillus thuringiensis (Bt) are grown on millions of hectares worldwide. While these proteins are efficient in controlling key lepidopteran pests, not all pests are affected and the development of resistance in target pests is always a concern. These shortcomings could be addressed by exploiting the natural insect resistance of cotton, especially inducible terpenoids such as gossypol. To assess the potential of gossypol in supplementing Cry proteins as a resistance trait, we conducted a range of feeding assays with Spodoptera littoralis using artificial diet with defined amounts of Cry proteins and gossypol. This was supplemented by assays with leaf discs of induced and uninduced non-Bt and Bt cotton (expressing Cry1Ac and Cry2Ab). Additionally, we quantified Cry proteins and cotton terpenoids to describe the interactions in planta. We found that gossypol can increase the efficacy of Cry proteins in artificial diet in an additive way. Induced production of gossypol and other cotton terpenoids, however, did not increase the efficacy of Bt cotton owing to the strong impact of the Bt trait. Cotton terpenoids may offer the chance to supplement the insect resistance of Bt cotton in cases were the pest is not strongly affected by the Cry proteins. © 2016 Society of Chemical Industry.

Expression Analysis of Hairpin RNA Carrying Sugarcane mosaic virus (SCMV) Derived Sequences and Transgenic Resistance Development in a Model Rice Plant.

Developing transgenic resistance in monocotyledonous crops against pathogens remains a challenging area of research. Sugarcane mosaic virus (SCMV) is a serious pathogen of many monocotyledonous crops including sugarcane. The objective of present study was to analyze transgenic expression of hairpin RNA (hpRNA), targeting simultaneously CP (Coat Protein) and Hc-Pro (helper component-proteinase) genes of SCMV, in a model rice plant. Conserved nucleotide sequences, exclusive for DAG (Aspartic acid-Alanine-Glycine) and KITC (Lycine-Isoleucine-Threonine-Cysteine) motifs, derived from SCMV CP and Hc-Pro genes, respectively, were fused together and assembled into the hpRNA cassette under maize ubiquitin promoter to form Ubi-hpCP:Hc-Pro construct. The same CP:Hc-Pro sequence was fused with the β-glucuronidase gene (GUS) at the 3′ end under CaMV 35S promoter to develop 35S-GUS:CP:Hc-Pro served as a target reporter gene construct. When delivered into rice callus tissues by particle bombardment, the Ubi-hpCP:Hc-Pro construct induced strong silencing of 35S-GUS:CP:Hc-Pro. Transgenic rice plants, containing Ubi-hpCP:Hc-Pro construct, expressed high level of 21–24 nt small interfering RNAs, which induced specific suppression against GUS:CP:Hc-Pro delivered by particle bombardment and conferred strong resistance to mechanically inoculated SCMV. It is concluded that fusion hpRNA approach is an affordable method for developing resistance against SCMV in model rice plant and it could confer SCMV resistance when transformed into sugarcane.

Introgression of sheath blight disease tolerance from the transgenic rice event Pusa Basmati1-CG27 to the variety White Ponni through backcross breeding

Sheath blight disease caused by Rhizoctonia solani is a major constraint for rice production. In order to obtain transgenic resistance against sheath blight disease, an indica rice variety Pusa Basmati1 (PB1) was transformed with constitutively express chitinase (chi11) and β-1,3-glucanase (GLU) genes. A single-copy transgenic PB1-CG27 showed a high level of tolerance to R. solani. Here, we report the introgression of sheath blight tolerance from PB1-CG27 into a non-basmati rice variety, White Ponni (WP) through backcross breeding. In each of the four backcross generations, the presence of the transgene was checked by hygromycin phosphotrans-ferase (hph) and βglucuronidase (gus) gene-specific foreground selection markers present in the T-DNA of the PB1-CG27 event. RAPD marker-based background selection was done to choose a BC4F1 plant that was similar to the WP recurrent parent. Morphological characters of the introgressed BC4F4 plants matched those of the recurrent WP parent. RT-PCR and western blot analyses confirmed the constitutive expression of the chi11 and GLU genes in the introgressed homozygous BC4F3 plants. Bioassay with R. solani a high level disease tolerance (43% disease reduction) revealed over the control WP plants.

Laboratory evaluation of transgenic Bt rice resistance against rice leaf roller, Cnaphalocrocis medinalis

Pest resistance of 8 transgenic Bt rice events with a synthetic cry1Ac gene linked to rice rbcS-tp sequence were assessed under laboratory conditions. Bioassays were conducted with neonates and third instar larvae of Cnaphalocrocis medinalis, which is a significant pest of rice in Asia. C. medinalis larvae were shown to be susceptible to all eight events, even though there were differences between the causes of death. The results differed between developmental stages of the larvae, despite the fact that all 8 events led to high mortalities. Neonates displayed feeding avoidance and death by starvation on six Bt rice events. In the case of third instar larvae however, only two events resulted in feeding avoidance. Nevertheless, all Bt rice events were shown to be highly effective against C. medinalis larvae in laboratory conditions. These results may be a significant foundation for the evaluation of improved transgenic Bt rice.

Citrus psorosis virus coat protein-derived hairpin construct confers stable transgenic resistance in citrus against psorosis A and B syndromes.

Citrus psorosis virus (CPsV) is the causal agent of psorosis, a serious and widespread citrus disease. Two syndromes of psorosis, PsA and PsB, have been described. PsB is the most aggressive and rampant form. Previously, we obtained Pineapple sweet orange plants transformed with a hairpin construct derived from the CPsV coat protein gene (ihpCP). Some of these plants were resistant to CPsV 90-1-1, a PsA isolate homologous to the transgene. In this study, we found that expression of the ihpCP transgene and siRNA production in lines ihpCP-10 and -15 were stable with time and propagation. In particular, line ihpCP-15 has been resistant for more than 2years, even after re-inoculation. The ihpCP plants were also resistant against a heterologous CPsV isolate that causes severe PsB syndrome. Line ihpCP-15 manifested complete resistance while line ihpCP-10 was tolerant to the virus, although with variable behaviour, showing delay and attenuation in PsB symptoms. These lines are promising for a biotech product aimed at eradicating psorosis.

RNAi-mediated transgenic rice resistance to Rice stripe virus

Rice stripe virus (RSV) often causes severe rice yield loss in temperate regions of East Asia. Although the correlation of small interfering RNAs (siRNAs) with transgenic virus resistance of plants using RNA interference (RNAi) is known for decades, no systematical research has been done on the profiling of siRNAs from a genomic scale. Our research is aiming to systematically study the RNAi impact in RSV-resistant transgenic rice, which was generated by introducing an inverted repeat construct that targets RSV nucleocapsid protein (NCP) gene. In this paper, three independent RSV-re-sistant transgenic rice lines were generated, their stable integration of the T-DNA fragment and the expression of siRNAs were confirmed by Southern blotting and Northern blotting analyses, and the majority of siRNAs were in lengths of 21, 22, and 24 nucleotides (nt), which have validated a connection between the presence of the RSV NCP homologous siRNAs and the RSV resistance in those transgenic rice lines. In one of these transgenic lines (T4-B1), the T-DNA fragment was found to have been inserted at chromosome 1 of the rice genome, substituting the rice genome fragment from 32 158 773 to 32 158 787 nt. Bioinformatics analysis of small RNA-Seq data on the T4-B1 line also confirmed the large population of NCP-derived siRNAs in transgenic plants, and the RSV-infected library (T4-B1-V) possessed more siRNAs than its mock inoculated libraries (T4-B1-VF), these results indicating the inverted repeat construct and RSV could introduce abundance of siRNAs in transgenic rice. Moreover, a varied expression level of specific siRNAs was found among different segments of the NCP gene template, about 47% of NCP-derived siRNAs reads aligned with the fragment from 594 to 832 nt (239 nt in length) in NCP gene (969 nt in length) in the T4-B1-V, indicating a potential usage of hotspot regions for RNAi silencing in future research. In conclusion, as the first study to address the siRNA profile in RSV-resistant transgenic plant using next generation sequencing (NGS) technique, we confirmed that the massive abundance of siRNA derived from the inverted repeat of NCP is the major reason for RSV-resistance.

A novel strategy to enhance resistance to Cucumber mosaic virus in tomato by grafting to transgenic rootstocks

Cucumber mosaic virus (CMV) can infect a wide range of host species. For the lacking of CMV resistant varieties of tomato, RNA interference (RNAi) can be used as a fast and effective method for the generation of transgenic resistant varieties. In this current study, five intron-spliced hairpin RNA (ihpRNA) plant expression vectors aimed at five genes of CMV have been constructed. Transgenic tomatoes were obtained by Agrobacterium tumefaciens-mediated transformation with expression vectors. Highly resistant generations of transgenic plants were employed as rootstocks and grafted onto non-transgenic tomatoes that resulted in the successful transfer of resistance to the scions. Using a novel method of plant cuttings for rootstock propagation, we obtained large quantities of disease-resistant material. Further, this method produces scions that can remain undetectable for transgenic resistance marker genes that may provide novel approaches to evade collective concerns about genetically-modified organism (GMO) biosafety.

Dampak Tanaman Transgenik Bt terhadap Populasi Serangga Pengendali Hayati

<p class="p1">An alternative technique to improve plant resistance to insect pests is plant transformation using the genetic engineering technology. Several transgenic plants resistant to insect have been produced and commercially released to environment in some industrial and developing countries. Before release, transgenic plants need to be assessed for their potential risks to human health and environment. One of the environmental risk assessments is the potential risk to non-target insects, including the biocontrol insects. Laboratories, glasshouse, and field experiments have been conducting the study of the impact of transgenic plant resistance to insect, especially transgenic Bt plants to the population of predators and parasitoids. However the results were controversial. The objective of this review is to inform some of controversial results, and to suggest serial experiments need to be done to solve the problem. The impact of the transgenic plant resistance to insects depends on several factors, such as genes that are used to transform the plants, the kind of plant pests, and the kind and stages of the insect natural enemies. Results of the experiments were influenced by sites of the experiments (laboratory, glasshouse, or field) and contact of the natural enemies to the toxin. Some experiments showed that the transgenic Bt plants have no impact to the natural enemies population, and otherwise. Due to the controversial results, the experiment and assessment should be done in depth and carefully studied. A sequential experiments need to be adopted to avoid the misleading interpretation, and the assessment need to be based on a case by case study.</p>

Modification of Cry4Aa toward Improved Toxin Processing in the Gut of the Pea Aphid, Acyrthosiphon pisum.

Aphids are sap-sucking insects (order: Hemiptera) that cause extensive damage to a wide range of agricultural crops. Our goal was to optimize a naturally occurring insecticidal crystalline (Cry) toxins produced by the soil-dwelling bacterium Bacillus thuringiensis for use against the pea aphid, Acyrthosiphon pisum. On the basis that activation of the Cry4Aa toxin is a rate-limiting factor contributing to the relatively low aphicidal activity of this toxin, we introduced cathepsin L and cathepsin B cleavage sites into Cry4Aa for rapid activation in the aphid gut environment. Incubation of modified Cry4Aa and aphid proteases in vitro demonstrated enhanced processing of the toxin into the active form for some of the modified constructs relative to non-modified Cry4Aa. Aphids fed artificial diet with toxin at a final concentration of 125 μg/ml showed enhanced mortality after two days for one of the four modified constructs. Although only modest toxin improvement was achieved by use of this strategy, such specific toxin modifications designed to overcome factors that limit aphid toxicity could be applied toward managing aphid populations via transgenic plant resistance.

Generation of transgenic watermelon resistance to Cucumber mosaic virus facilitated by an effective Agrobacterium-mediated transformation method

Watermelon is an annual vegetable crop that suffers from various diseases during growth. Genetic engineering is an effective tool for improving plant disease resistance. This paper presents an effective transformation protocol designed for a female parent watermelon line mediated by Agrobacterium. In this protocol, 2 times MS Fe-EDTA were supplemented into Murashige and Skoog (MS) medium to suppress the chlorosis during watermelon explants culturing, and the optimal preculture time (5days) and coculture time (5days) were screened out to improve the frequency of shoots regeneration. Using our protocol, 17 putative transgenic plants expressing artificial microRNAs that target Cucumber mosaic virus (CMV) 2a/2b genes were recovered, and seven of them were succeeded in obtaining T1 generation seeds because of the technique difficulties of self-pollination of the transgenic watermelon lines. PCR and Southern blot analyses confirmed that the transferred fragment was successfully imported and integrated into the genome of three of these seven transgenic lines. DAS-ELISA assay indicated that the CMV can be detected in the nontransgenic line but not in the transgenic lines, and the transgenic lines displayed resistance to CMV infection.