Neomycin Phosphotransferase Research Articles

Magnetic nanoparticles mediate the transformation of antimicrobial peptides HeM into Chlorella ellipsoidea

Antimicrobial peptides are short peptides with broad-spectrum antimicrobial properties. Heliomicin, a peptide from Heliothis virescens, was re-engineered to have both antifungal and bacterial activities and renamed as HeM. Chlorella is a perfect bioreactor candidate due to fast growth, high photosynthetic efficiency, and easy large-scale culture. This study describes the successful transformation of HeM into Chlorella ellipsoidea F962. A binary expression vector pGcp4 was constructed that carried the antimicrobial peptide gene HeM and the gene encoding neomycin phosphotransferase II (NPT II). The pGcp4 was introduced into C. ellipsoidea F962 by magnetic nanoparticle-mediated transformation method. The positive transformants were obtained by G418 resistance screening. Molecular and genetic analyses on transformants revealed that the antimicrobial peptide gene and the NPT II gene had successfully entered C. ellipsoidea and expressed antimicrobial activity in transgenic cells. This method can provide a new way to produce transgenic Chlorella as well as explore wide utilization of Chlorella through magnetic transformation methods to mediate the production of antimicrobial peptides by C. ellipsoidea.

Expression of Tcchitinase-I gene in transgenic peanut (Arachis hypogaea L.) confers enhanced resistance against leaf spot and rust diseases

The disease-resistant transgenic peanut cv ICG 13942 plants were developed by using Tcchitinase-I gene. Agrobacterium tumefaciens strain LBA4404 harboring the binary vector (pBinAR) contains the chitinase (Tcchitinase-I) gene and neomycin phosphotransferase resistance (nptII) gene. The transformed shoots were developed on selection medium (MMS + 0.5 mg/L IAA + 15 mg/L TDZ + 100 mg/L Kan + 250 mg/L Cefotaxime) from deembryonated cotyledon (DC) explants. Established plantlets were screened for the presence of Tcchitinase-I and nptII genes. Stable integration and expression of the transgenes (T0) were confirmed by using PCR, RT-PCR and Southern blot analyses. The transformation frequency 63.34% was recorded. All the transformed (T0) plants were found normal, flowered and set seeds. After selfing the T0 plants, a Mendelian inheritance pattern (3:1) for the transgene in T1 progeny is revealed. T1 transgenic peanut plants were evaluated for resistance against Cercospora arachidicola, C. personatum and Puccinia arachidis by infection with the microspores using detached leaf assay. These T1 plants have shown longer incubation, latent period and lower infection frequencies in comparison to non-transformed (WT) plants. The Tcchitinase-I gene expression in resistant transgenic plants was compared to that of a susceptible control. A significant negative correlation was recorded between chitinase activity and the frequency of infection to the three tested disease causing agents.

Improvement of sugarcane for borer resistance using Agrobacterium mediated transformation of cry1Ac gene

ABSTRACT The sugarcane (Saccharum X officinarum) is one of the most important crops used to produce sugar and raw material for biofuel in the world. One of the main causes for sucrose content and yield losses is the attack by insect. In this investigation, cry1Ac gene was introduced into sugarcane variety GT54-9(C9) using the Agrobacterium tumefaciens transformation method for transgenic sugarcane production presenting insect-resistance. The A. tumefaciens strain GV1303 including pARTcry1Ac vector was used for the production of transformed sugarcane. The Bacillus thuringiensis cry gene were successfully used to produce transgenic plants used for the improvement of both agronomic efficiency and product quality by acquiring insect resistance. PCR and Southern hybridization techniques were used to confirm the cry1Ac gene incorporation into sugarcane genome. Transformation percentage was 22.2% using PCR analysis with specific primers for cry1Ac and npt-II (Neomycin phosphotransferase) genes. The expression of cry1Ac gene was determined using reverse transcriptase polymerase chain reaction (RT-PCR), QuickStix test, and insect bioassays. Bioassays for transformed sugarcane plants showed high level of toxicity to Sesamia cretica giving 100% mortality of the larvae. Sugarcane insect resistance was improved significantly by using cry1Ac gene transformation.

An improved method for transformation of Actinidia arguta utilized to demonstrate a central role for MYB110 in regulating anthocyanin accumulation in kiwiberry

Actinidia arguta and related species produce small edible kiwifruit (kiwiberry) with attractive consumer features and high nutritional value. As a recently developed crop, kiwiberry can be improved further by classical breeding or genetic manipulation. The currently available Agrobacterium-mediated transformation protocol is only applicable to a limited number of kiwiberry genotypes such as the female cultivar ‘Hortgem Tahi’. We developed protocols for regeneration and Agrobacterium-mediated transformation of two A. arguta genotypes, female AA06-01 and male AA05-06, also applicable to ‘Hortgem Tahi’. Altered composition of basal salts in the callus-induction media was sufficient to prevent callus browning in AA06-01 and ‘Hortgem Tahi’, but not in AA05-06. Altering the hormone composition to avoid a prolonged callus stage was successfully used to prevent browning and induce shoot development in all three genotypes. Using our improved protocols, Agrobacterium-mediated transformation of a binary vector carrying the neomycin phosphotransferase II (nptII) expression cassette gave rise to kanamycin-resistant plants of both AA06-01 and AA05-06 and the presence of the nptII transgene was confirmed by genomic PCR. The improved protocols were also used to ectopically overexpress the anthocyanin-related transcription factor AcMYB110 in ‘Hortgem Tahi’ giving rise to purple callus with elevated anthocyanin accumulation. AcMYB110 expression and increased levels of anthocyanins were detected in mature leaves of established transgenic plants compared to controls, clearly demonstrating the important role for MYB110 in regulation of anthocyanin accumulation in kiwiberry. The protocols developed during this study provide tools for further functional analyses and genetic manipulation of kiwiberry genotypes. Using a newly developed transformation method we were able to generate transgenic lines of three Actinidia arguta genotypes and demonstrate an important role for MYB110 in anthocyanin accumulation in kiwiberry.

LINE-1 ORF1p does not determine substrate preference for human/orangutan SVA and gibbon LAVA

BackgroundNon-autonomous VNTR (Variable Number of Tandem Repeats) composite retrotransposons – SVA (SINE-R-VNTR-Alu) and LAVA (L1-Alu-VNTR-Alu) – are specific to hominoid primates. SVA expanded in great apes, LAVA in gibbon. Both SVA and LAVA have been shown to be mobilized by the autonomous LINE-1 (L1)-encoded protein machinery in a cell-based assay in trans. The efficiency of human SVA retrotransposition in vitro has, however, been considerably lower than would be expected based on recent pedigree-based in vivo estimates. The VNTR composite elements across hominoids – gibbon LAVA, orangutan SVA_A descendants and hominine SVA_D descendants – display characteristic structures of the 5′ Alu-like domain and the VNTR. Different partner L1 subfamilies are currently active in each of the lineages. The possibility that the lineage-specific types of VNTR composites evolved in response to evolutionary changes in their autonomous partners, particularly in the nucleic acid binding L1 ORF1-encoded protein, has not been addressed.ResultsHere I report the identification and functional characterization of a highly active human SVA element using an improved mneo retrotransposition reporter cassette. The modified cassette (mneoM) minimizes splicing between the VNTR of human SVAs and the neomycin phosphotransferase stop codon. SVA deletion analysis provides evidence that key elements determining its mobilization efficiency reside in the VNTR and 5′ hexameric repeats. Simultaneous removal of the 5′ hexameric repeats and part of the VNTR has an additive negative effect on mobilization rates. Taking advantage of the modified reporter cassette that facilitates robust cross-species comparison of SVA/LAVA retrotransposition, I show that the ORF1-encoded proteins of the L1 subfamilies currently active in gibbon, orangutan and human do not display substrate preference for gibbon LAVA versus orangutan SVA versus human SVA. Finally, I demonstrate that an orangutan-derived ORF1p supports only limited retrotransposition of SVA/LAVA in trans, despite being fully functional in L1 mobilization in cis.ConclusionsOverall, the analysis confirms SVA as a highly active human retrotransposon and preferred substrate of the L1-encoded protein machinery. Based on the results obtained in human cells coevolution of L1 ORF1p and VNTR composites does not appear very likely. The changes in orangutan L1 ORF1p that markedly reduce its mobilization capacity in trans might explain the different SVA insertion rates in the orangutan and hominine lineages, respectively.

Construction of an expression vector mediated by the dual promoter for prokaryotic and mammalian cell expression system.

The aim of this study was to construct an expression vector mediated by the dual promoter that can simultaneously drive the recombinant protein production in eukaryotic and prokaryotic cells. The prokaryotic T7 promoter and ribosome binding site (RBS) was cloned downstream of CMV promoter in the eukaryotic expression vector pIRES-neo, and T7 termination sequence was inserted upstream of neomycin phosphotransferase gene to generate the dual promoter vector. The enhanced green fluorescent protein (eGFP) gene was used as reporter gene. Then, the resultant vector was transfected into Chinese hamster ovary (CHO) cells and transformed into Escherichia coli (E. coli) BL21, and the eGFP expression levels were analyzed by fluorescence microscopy, flow cytometry and Western blot, respectively. Fluorescence microscopy revealed that the eGFP was expressed in both CHO cells and E. coli BL21. Flow cytometry showed that the eGFP expression level had no significant difference between the dual promoter vector and control vector in transfected CHO cells. Western blot analysis indicated the eGFP expressed in transformed E. coli. In conclusion, a prokaryotic-eukaryotic double expression vector was successfully constructed, which has potential applications in rapid cloning and expression of recombinant proteins in both prokaryotic and eukaryotic expression systems.

Effects of viral promoters, the Woodchuck hepatitis post-transcriptional regulatory element, and weakened antibiotic resistance markers on transgene expression in Chinese hamster ovary cells

The product productivity of Chinese hamster ovary (CHO) cells need to be further increased and solved. Promoter and selection marker are essential elements for transgene expression. We investigated the effects of promoters, Woodchuck hepatitis post-transcriptional regulatory (WPRE) element, and selection marker on transgene expression in CHO cells. A weakened form of resistance gene neomycin phosphotransferase (NPT) and WPRE element were cloned into the expression vectors driven by the cytomegalovirus (CMV) and simian virus 40 (SV40) promoters, following by transfecting into CHO cells and screened under G418 pressure. qRT-PCR and Flow cytometry were used to detect the expression level of eGFP gene, and transgene copy number was determined using qPCR technology. Western blot and ELISA were utilized to analyze the expression levels of cetuximab. The weakened NPT gene enhanced eGFP expression both under the CMV or SV40 promoters. Moreover, WPRE, combined with the NPT gene considerably enhanced eGFP expression in CHO cells. Furthermore, the CMV promoter and WPRE combination resulted in higher cetuximab expression levels than CMV promoter alone. However, WPRE could not enhance the transgene expression under the circumstance of CMV promoter and weakened NPT gene combination, suggesting that the effects of the WPRE combined with selection markers were specificity.

Transformation of hybrid black poplar with selective and reporter genes affects leaf proteome, yet without indication of a considerable environmental hazard

Poplar is an economically important forest tree used for multiple applications, including bioenergy production. Although genetic engineering allows a quick introduction of desirable traits, simultaneously, it may deliver unintended side effects. Herein, we compared the protein composition in leaves of microclonally propagated fast-growing hybrid clone of Populus deltoides × Populus nigra (Populus × euroamericana), genetically modified with a reporter (GUS) and selective (NPTII) traits, and its non-transgenic parental genotype. By two-dimensional gel electrophoresis, we revealed that 5.5% of 330 detected protein spots were differentially accumulated. The vast majority of them showed lower abundance in the transgenic line. These proteins were mainly involved in primary metabolism, energy production, and protein synthesis/homeostasis; notably, none of them was directly linked to neomycin phosphotransferase or β-glucuronidase activities. Some of the differentially accumulated proteins could be associated with stress response caused by Agrobacterium-mediated transformation. Furthermore, the lower abundance of two cytosolic glutamine synthase isoforms and accumulation of bark storage protein A in the transgenic line, plausibly indicated impaired nitrogen metabolism and storage. The insertion of a model genetic construct did not alter the neighboring genetic loci, and neither toxins nor allergens were differentially abundant. In essence, we detected no protein indicating considerable health or environmental hazard in the transgenic hybrid black poplar.

An Efficient Agrobacterium-Mediated Genetic Transformation Using Embryonic Axis in Cotton (Gossypium hirsutum L.)

Agrobacterium-mediated genetic transformation approach allows for introducing novel genes in cotton (Gossypium hirsutum L.). Development of efficient regeneration and transformation protocol is very important for recalcitrant plants like cotton. In the present study, five-day-old germinated mature embryo parts especially embryonic axis, hypocotyl and plumule of cotton ‘Lashata’ were wounded and inoculated with A. tumefaciens strain GV2260 harbouring plasmid p35S-GUS-INT. The binary plasmid p35S-GUS-INT contains neomycin phosphotransferase II (NPTII) gene driven by nopaline synthase (NOS) promoter and β-glucuronidase (GUS) gene controlled by cauliflower mosaic virus (CaMV) 35S promoter. After inoculation, explants were co-cultivated in liquid and agar-solidified MS medium for 48 h in dark condition. Agar-solidified co-cultivation medium increased transformation frequency as compared to liquid medium. The putative primary transformants were confirmed with histochemical GUS assay, PCR and RT-PCR. However, comparing the three culture explant, the embryonic axis explants had significant difference with embryonic hypocotyl and plumule explants in terms of number and percentage (%) of GUS, PCR and RT-PCR positive plant. The total transformation efficiency was recorded as 3% with varying GUS expression levels.

Obtaining Transgenic Potato Plants Expressing the Human Lactoferrin Gene and Analysis of Their Resistance to Phytopathogens

The human lactoferrin gene was transferred into genomes of several potato (Solanum tuberosum) cultivars of Ukrainian breeding using the Agrobacterium-mediated transformation. The plasmid vector pBIN35LF carrying the human lactoferrin gene hLf controlled by the 35S cauliflower mosaic virus promoter (CaMV35S) and the octopine synthase terminator, as well as the selective marker neomycin phosphotransferase II gene (nptII) conferring the resistance to kanamycin, was used. As a result of selection, 44 lines of Vernisage, 26 lines of cv. Levada, 25 lines of cv. Svitanok Kyivskyi, and 16 lines of cv. Zarevo cultivars resistant to 100 mg/L of kanamycin were obtained. PCR and Western blot analyses were carried out for transformed lines with primers specific to the hLf gene and a monoclonal antibody against lactoferrin to confirm the transgenic nature of selected tomato plants and hLf gene expression. The selected transgenic potato lines were tested for resistance to bacterial and fungal phytopathogens. The disk diffusion assay revealed that the juice of transgenic potato lines possesses an antibacterial effect against phytopathogenic bacteria Ralstonia solanacearum (causing potato brown rot) and Clavibacter michiganensis subsp. sepedonicus (causing potato ring rot). The resistance of transgenic potato plants to late blight was investigated by in vitro infection of plants with the Phytophthora infestans isolate. As a result, it was found that the obtained transgenic potato lines have enhanced resistance to P. infestans as compared to the control. Thus, the obtained data show that the transfer of the hLf gene into the potato genome enhances potato’s resistance to bacterial and fungal pathogens.

Transgenic tomato lines expressing human lactoferrin show increased resistance to bacterial and fungal pathogens

Abstract Agrobacterium-mediated transformation of tomato (Lycopersicon esculentum Mill.) plants was carried out to transfer the human lactoferrin gene (hLf) under control of the constitutive Cauliflower Mosaic Virus (CaMV) 35 S promoter to increase their resistance to phytopathogens. Since binary plasmid construct contained also neomycin phosphotransferase gene (nptII) conferring resistance to kanamycin, the selection of transgenic lines was carried out on nutrient medium supplemented with 100 mg/L kanamycin (as selective agent). PCR and Western blot analyses of transformed lines with primers specific to hLf gene and a monoclonal antibody against lactoferrin were performed to confirm the transgenic nature of selected tomato plants and hLf gene expression. The resistance of transgenic tomato lines expressing hLf to bacterial pathogens Clavibacter michiganensis subsp. michiganensis (causing bacterial wilt and canker of tomato), and Ralstonia solanacearum (causing bacterial wilt), and fungal pathogen Phytophthora infestans (causing late blight) was demonstrated. Although the partial resistance to bacterial wilt in transgenic tomatoes expressing modified lactoferrin has already been shown (Lee et al., 2002), here we report for the first time that the transfer and the expression of human lactoferrin gene in transgenic tomato plants leads to their enhanced resistance not only to R. solanacearum but also to another bacterial pathogen C. michiganensis and to fungal pathogen P. infestans. The present study demonstrates that the genetic transformation of plants with the human lactoferrin gene (hLf) is a promising technology to increase the resistance of plant species to certain phytopathogens.

Agrobacterium-mediated transformation of brinjal (Solanum melongena L.) using fungal resistant gene

The investigation standardized the protocol for Agrobacterium-mediated transformation system in Brinjal (Solanum melongena L), cultivar ‘Arka Neelkanth’ . Initially the regeneration system was standardized with regeneration medium (MS basal medium supplemented with 8.88 µM BAP + 1.14 µM IAA) by using 10-12 days old cotyledon explants. Transformation of the hevein, a gene responsible for fungal resistance. into cotyledon explants was carried out with Agrobacterium tumefaciens strain LBA4404 harbouring binary plasmid pGPTV/hevein (Plasmid Gus Plant Transformation Vector) with marker gene neomycin phosphotransferase (npt-II). Transformation efficiency showed a higher percentage in one day pre-cultured explants, infected with bacterial suspension of optical density 0.4 at OD600 nm with 1:1 dilution for 10min and 48hrs co-cultivation on regeneration medium in the dark. Transformed explants were cultured on selection medium. The PCR molecular confirmation of amplified npt-II gene from the genomic DNA of the transformed plant indicates the integration of the hevein gene in the brinjal plant.

Genetic Transformation of Tribonema minus, a Eukaryotic Filamentous Oleaginous Yellow-Green Alga.

Eukaryotic filamentous yellow-green algae from the Tribonema genus are considered to be excellent candidates for biofuels and value-added products, owing to their ability to grow under autotrophic, mixotrophic, and heterotrophic conditions and synthesize large amounts of fatty acids, especially unsaturated fatty acids. To elucidate the molecular mechanism of fatty acids and/or establish the organism as a model strain, the development of genetic methods is important. Towards this goal, here, we constructed a genetic transformation method to introduce exogenous genes for the first time into the eukaryotic filamentous alga Tribonema minus via particle bombardment. In this study, we constructed pSimple-tub-eGFP and pEASY-tub-nptⅡ plasmids in which the green fluorescence protein (eGFP) gene and the neomycin phosphotransferase Ⅱ-encoding G418-resistant gene (nptⅡ) were flanked by the T. minus-derived tubulin gene (tub) promoter and terminator, respectively. The two plasmids were introduced into T. minus cells through particle-gun bombardment under various test conditions. By combining agar and liquid selecting methods to exclude the pseudotransformants under long-term antibiotic treatment, plasmids pSimple-tub-eGFP and pEASY-tub- nptⅡ were successfully transformed into the genome of T. minus, which was verified using green fluorescence detection and the polymerase chain reaction, respectively. These results suggest new possibilities for efficient genetic engineering of T. minus for future genetic improvement.

Monitoring adventitious presence of transgenes in brinjal (Solanum melongena L.) collections from the regions in India bordering Bangladesh: a case report

India has been predicted as a center of origin of brinjal (Solanum melongena L.) with large number of wild and weedy relatives distributed across the Indian subcontinent. Fruit and Shoot Borer (FSB) is one of the most devastating insect pests of brinjal causing production losses up to 80%. Bt technology has played a key role in imparting FSB resistance in brinjal. In 2013, Bt brinjal event EE1 was commercialized in Bangladesh. Since the neighbouring country shares porous borders with north-eastern states of India, it is important to check for any unapproved entry of genetically modified (GM) brinjal in India. Adventitious presence of transgenes was monitored in the samples collected from regions of Assam, Meghalaya, Mizoram, Tripura and West Bengal bordering Bangladesh. A total of 211 brinjal samples were collected from 32 locations of selected states. Conventional singleplex and multiplex PCR assays and real time PCR were employed to target the genetic elements of Bt Brinjal viz. cry1Ac transgene, Cauliflower Mosaic Virus 35S promoter (P-35S), neomycin phosphotransferase II (nptII) and aminoglycoside-3′-adenyltransferase (aadA) marker genes. Based on the tests conducted, adventitious presence of transgenes was not detected in any of the collected samples. This approach targeting common screening elements or specific GM target could be efficiently employed to check for unapproved GM events in food and supply chain to comply with the regulatory requirements.

Two dominant selectable markers for genetic manipulation in Neurospora crassa.

Neurospora crassa is an excellent model fungus for studies on molecular genetics, biochemistry, physiology, and molecular cell biology. Along with the rapid progress of Neurospora research, new tools facilitating more efficient and accurate genetic analysis are in high demand. Here, we tested whether the dominant selective makers widely used in yeasts are applicable in N. crassa. Among them, we found that the strains of N. crassa are sensitive to the aminoglycoside antibiotics, G418 and nourseothricin. 1000μg/mL of G418 or 50μg/mL of nourseothricin is sufficient to inhibit Neurospora growth completely. When the neomycin phosphotransferase gene (neo) used in mammalian cells is expressed, N. crassa shows potent resistance to G418. This establishes G418-resistant marker as a dominant selectable marker to use in N. crassa. Similarly, when the nourseothricin acetyltransferase gene (nat) from Streptomyces noursei is induced by qa-2 promoter in the presence of quinic acid (QA), N. crassa shows potent resistance to nourseothricin. When nat is constitutively expressed by full-length or truncated versions of the promoter from the N. crassa cfp gene (NCU02193), or by the trpC promoter of Aspergillus nidulans, the growth of N. crassa in the presence of nourseothricin is proportional to the expression levels of Nat. Finally, these two markers are used to knock-out wc-2 or al-1 gene from the N. crassa genome. The successful development of these two markers in this study expands the toolbox for N. crassa and very likely for other filamentous fungi as well.

Breeding of the transgenic chrysanthemum (Chrysanthemum morifolium Ramat.) carrying aphid-resistance gene, Pinellia ternata agglutinin (PTA)

Plasmid pCAMBIA2201-pta, containing the selectable marker neomycin phosphotransferase gene (npt II), Pinellia ternata agglutinin (PTA) gene and β-glucuronidase gene, were transformed into leaf discs of Chrysanthemum (Chrysanthemum morifolium Ramat.) via Agrobacterium tumefaciens-mediated transformation. It was confirmed by GUS (β-glucuronidase) and PCR analysis that the PTA gene had been successfully inserted into the plant genome. Hemagglutination assay was carried out and the expression of PTA gene in transgenic plants in vitro and the aphid-resistance assay result showed that the inhibition rates of the transformed plants were improved than the control. T1 progenies generated through the cuttage breeding from T0 generation had preserved the aphid-resistance and the inhibition rates of T1 generation had no significant difference with T0 generation (P > 0.05). The results suggest that PTA gene can improve aphid resistance of chrysanthemum and its aphid resistance can be generated to T1 generation stably.

An optimized double T-DNA binary vector system for improved production of marker-free transgenic tobacco plants.

In the plant transformation process, marker genes play a vital role in identifying transformed cells from non-transformed cells. However, once transgenic plants have been obtained, the presence of marker genes may provoke public concern about environmental or biosafety issues. In our previous study, a double T-DNA vector system has been developed to obtain marker-free transgenic plants, but the T-DNA left border (LB) and right border (RB) of the vector showed an RB-LB-RB-LB pattern and led to high linkage integration between the selectable marker gene (SMG) and the gene of interest (GOI). To improve this double T-DNA vector system, we inverted the first T-DNA direction such that a LB-RB-RB-LB pattern resulted to avoid transcriptional read-through at the LB and the subsequent linkage transfer of the SMG and GOI. We separately inserted the green fluorescent protein (GFP) gene as the GOI and the neomycin phosphotransferase II (NPTII) gene as the SMG in both optimized and original vectors and carried out Agrobacterium-mediated tobacco transformation. Statistical analysis revealed that the linkage frequency was 25.6% in T0 plants transformed with the optimized vector, which is a 42.1% decrease compared with that of the original vector (44.2%). The frequency of obtaining marker-free transgenic plants was 66.7% in T1 plants transformed with the optimized vector, showing a 33.4% increase compared with that of the original vector (50.0%). Our results demonstrate that the optimized double T-DNA binary vector system is a more effective, economical and time-saving approach for obtaining marker-free transgenic plants.

Transgene suppression in plants by foliar application of in vitro-synthesized small interfering RNAs.

Recent research has shown that plants can uptake long dsRNAs and dsRNA-derived siRNAs that target important genes of infecting fungi or viruses when applied on the surface of plant leaves. The external RNAs were capable of local and systemic movement inducing plant resistance against the pathogens. Few studies have been made for plant gene regulation by foliar application of RNAs. In this study, several types of ssRNA and siRNA duplexes targeting the neomycin phosphotransferase II (NPTII) transgene were in vitro-synthesized and externally applied to the leaf surface of 4-week-old transgenic Arabidopsis thaliana plants. External application of the synthetic NPTII-encoding siRNAs down-regulated NPTII transcript levels in transgenic A. thaliana 1 and 7days post-treatment with a higher and more consistent effect being observed for siRNAs methylated at 3' ends. We also analyzed the effects of external NPTII-encoding dsRNA precursors and a dsRNA-derived heterogenous siRNA mix. Digestion of the NPTII-dsRNA to the heterogeneous siRNAs did not improve efficiency of the transgene suppression effect. Key Points• Foliar application of siRNAs down-regulated a commonly used transgene in Arabidopsis. • A more consistent effect was observed for methylated siRNAs. • The findings are important for development of plant gene regulation approaches.

Monitoring Adventitious Presence of Transgenes in Cotton Collections from Genebank and Experimental Plots: Ensuring GM-Free Conservation and Cultivation of Genetic Resources

Bt cotton is the commercialized genetically modified (GM) crop in India with four approved GM events, namely MON531 (Bollgard® I), MON15985 (Bollgard® II), GFM and Event 1. Several GM events of cotton with insect-resistant or herbicide-tolerant GM traits were under field trials or imported for research purposes. With the increase in adoption of Bt cotton, unintentional introgression of transgenes in the conserved diversity-rich germplasm and in the field samples needs to be monitored in a systematic manner employing efficient GM diagnostics. Adventitious presence of transgenes was checked in 100 ex situ accessions of cotton being conserved in the National Genebank. These accessions belonging to Gossypium hirsutum, G. barbadense, G. arboreum, G. hirsutum × G. barbadense, represented seven Bt cotton growing states. Pentaplex PCR assay covering screening for over 90% of globally approved GM cotton events was employed, simultaneously targeting Cauliflower Mosaic Virus 35S promoter (P-35S), Agrobacterium tumefaciens nos terminator (T-nos), neomycin phosphotransferase (nptII) and phosphinothricin-N-acetyltransferase (pat) marker genes, and endogenous stearoyl acyl carrier protein desaturase (Sad1) gene as an internal control. Real-time PCR assays targeting P-35S and T-nos, with the limit of detection up to 0.01%, were employed for further confirmation. A study was also conducted on fifty cotton samples collected from the experimental plots, which were grown at distance of 50 and 10 metres from Bt cotton event MON15985. Adventitious presence of transgenes was checked using multiplex PCR assay targeting cry1Ac, cry2Ab2 and endogenous Sad1 gene to define a strategy for screening for specific GM events. Adventitious presence of transgenes was not detected in any of the tested samples based on the screening tests conducted. This strategy targeting specific GM traits would assist in GM-free cultivation of varieties in the experimental plots. The reported approach with wide GM coverage is reliable for checking adventitious presence of transgenes in genebanks, which could be applied to test conserved germplasm on a regular basis, for ensuring GM-free conservation of genetic resources.

High Kanamycin Concentration as Another Stress Factor Additional to Temperature to Increase pDNA Production in E. coli DH5α Batch and Fed-Batch Cultures.

Plasmid DNA (pDNA) vaccines require high supercoiled-pDNA doses (milligrams) to achieve an adequate immune response. Therefore, processes development to obtain high pDNA yields and productivity is crucial. pDNA production is affected by several factors including culture type, medium composition, and growth conditions. We evaluated the effect of kanamycin concentration and temperature on pDNA production, overflow metabolism (organic acids) and metabolic burden (neomycin phosphotransferase II) in batch and fed-batch cultures of Escherichia coli DH5α-pVAX1-NH36. Results indicated that high kanamycin concentration increases the volumetric productivity, volumetric and specific yields of pDNA when batch cultures were carried out at 42 °C, and overflow metabolism reduced but metabolic burden increased. Micrographs taken with a scanning electron microscope (SEM) were analyzed, showing important morphological changes. The high kanamycin concentration (300 mg/L) was evaluated in high cell density culture (50 gDCW/L), which was reached using a fed-batch culture with temperature increase by controlling heating and growth rates. The pDNA volumetric yield and productivity were 759 mg/L and 31.19 mg/L/h, respectively, two-fold greater than the control with a kanamycin concentration of 50 mg/L. A stress-based process simultaneously caused by temperature and high kanamycin concentration can be successfully applied to increase pDNA production.