Neomycin Phosphotransferase Research Articles

Genetic Variability and Evolutionary Dynamics of Papaya Ringspot Virus and Papaya Leaf Distortion Mosaic Virus Infecting Feral Papaya in Hainan Island.

The commercialized genetically modified (GM) papaya cultivars have protected papaya from the devastating disease caused by papaya ringspot virus (PRSV). However, papaya leaf distortion mosaic virus (PLDMV), which causes similar infection symptoms but is serologically distinct from PRSV, was found as a competitive threat to the papaya industry. Our study surveyed the occurrence of PRSV and PLDMV as well as the transgenic markers of the 35S promoter from cauliflower mosaic virus (CaMV 35S) and the neomycin phosphotransferase II (NPT II) gene in feral papaya plants, which were found frequently growing outside of cultivated papaya fields on Hainan Island. In total, 123 feral papayas, comprising 62 (50.4%) GM plants and 61 (49.6%) non-GM ones, were sampled. Among them, 23 (18.7%) were positive for PRSV, 49 (39.8%) were positive for PLDMV, including 5 plants co-infected by PRSV and PLDMV, and 56 (45.5%) plants were free of either virus. In traditional papaya growing regions, we detected fewer PRSV-infected plants (2 in 33, 6%) than in other regions (21 in 90, 23%). But overall, whether transgenic or not made no significance in PRSV incidence (P=0.230), with 9 PRSV-infected plants among 62 GM papayas and 14 among 61 non-GM papayas. Phylogenetic and genetic differentiation analysis showed a clear correlation between PRSV and PLDMV populations and their geographical origins. Negative selection was estimated for the selected gene regions of both viruses. Notably, PLDMV has deviated from neutral evolution and experienced population expansion, exhibiting increased genetic diversity and is becoming the predominant threat to papaya in Hainan.

An efficient gene targeting system using Δku80 and functional analysis of Cyp51A in Trichophyton rubrum

Trichophyton rubrum is one of the most frequently isolated fungi in patients with dermatophytosis. Despite its clinical significance, the molecular mechanisms of drug resistance and pathogenicity of T. rubrum remain to be elucidated because of the lack of genetic tools, such as efficient gene targeting systems. In this study, we generated a T. rubrum strain that lacks the nonhomologous end-joining-related gene ku80 (Δku80) and then developed a highly efficient genetic recombination system with gene targeting efficiency that was 46 times higher than that using the wild-type strain. Cyp51A and Cyp51B are 14-α-lanosterol demethylase isozymes in T. rubrum that promote ergosterol biosynthesis and are the targets of azole antifungal drugs. The expression of cyp51A mRNA was induced by the addition of the azole antifungal drug efinaconazole, whereas no such induction was detected for cyp51B, suggesting that Cyp51A functions as an azole-responsive Cyp51 isozyme. To explore the contribution of Cyp51A to susceptibility to azole drugs, the neomycin phosphotransferase (nptII) gene cassette was inserted into the cyp51A 3′-untranslated region of Δku80 to destabilize the mRNA of cyp51A. In this mutant, the induction of cyp51A mRNA expression by efinaconazole was diminished. The minimum inhibitory concentration for several azole drugs of this strain was reduced, suggesting that dermatophyte Cyp51A contributes to the tolerance for azole drugs. These findings suggest that an efficient gene targeting system using Δku80 in T. rubrum is applicable for analyzing genes encoding drug targets.

Impact of Exogenous dsRNA on miRNA Composition in Arabidopsis thaliana.

The application of double-stranded RNAs (dsRNAs) to plant surfaces has emerged as a promising tool for manipulating gene expression in plants and pathogens, offering new opportunities for crop improvement. While research has shown the capability of exogenous dsRNAs to silence genes, the full spectrum of their impact, particularly on the intricate network of microRNAs (miRNAs), remains largely unexplored. Here, we show that the exogenous application of chalcone synthase (CHS)-encoding dsRNA to the rosette leaves of Arabidopsis thaliana induced extensive alterations in the miRNA profile, while non-specific bacterial neomycin phosphotransferase II (NPTII) dsRNA had a minimal effect. Two days after treatment, we detected 60 differentially expressed miRNAs among the 428 miRNAs found in the A. thaliana genome. A total of 59 miRNAs were significantly changed after AtCHS-dsRNA treatment compared with water and NPTII-dsRNA, and 1 miRNA was significantly changed after AtCHS-dsRNA and NPTII-dsRNA compared with the water control. A comprehensive functional enrichment analysis revealed 17 major GO categories enriched among the genes potentially targeted by the up- and downregulated miRNAs. These categories included processes such as aromatic compound biosynthesis (a pathway directly related to CHS activity), heterocycle biosynthesis, RNA metabolism and biosynthesis, DNA transcription, and plant development. Several predicted targets of upregulated and downregulated miRNAs, including APETALA2, SCL27, SOD1, GRF1, AGO2, PHB, and PHV, were verified by qRT-PCR. The analysis showed a negative correlation between the expression of miRNAs and the expression of their predicted targets. Thus, exogenous plant gene-specific dsRNAs induce substantial changes in the plant miRNA composition, ultimately affecting the expression of a wide range of genes. These findings have profound implications for our understanding of the effects of exogenously induced RNA interference, which can have broader effects beyond targeted mRNA degradation, affecting the expression of other genes through miRNA regulation.

Multiplex Real-Time Loop-Mediated Isothermal Amplification (LAMP) Based on the Annealing Curve Analysis: Toward an On-Site Multiplex Detection of Transgenic Sequences in Seeds and Food Products.

Monitoring of the introduction of unapproved genetically modified (GM) events is required because the approval status of a GM event may differ from country to country. The on-site methods such as loop-mediated isothermal amplification (LAMP) offer a technological answer for the rapid GM detection beyond the laboratories. Real-time LAMP assays detecting one GM target were reported earlier. To increase the efficiency of the assay, a multiplex real-time LAMP simultaneously targeting Figwort Mosaic Virus promoter (P-FMV) that constructs region between the Cauliflower Mosaic Virus 35S promoter and cry1Ac gene (p35S-cry1Ac) and neomycin phosphotransferase II (nptII) marker gene was developed. The assay could detect as low as 0.1% for each GM target within 45 min. To the best of our knowledge, multiplexing in real-time LAMP using the Genie II system with applicability in GM detection has been reported herein for the first time. The developed method provides rapid, on-site, and real-time GM detection in seeds and food products.

Segregation analysis and floral phenotype of T2 transgenic yellow cosmos (Cosmos sulphureus Cav.) carrying neomycin phosphotransferase II gene in second generation

Abstract. Muchyiddin MGAA, Sawitri WD, Irsyadi MB, Swandari T, Purwantoro A. 2024. Segregation analysis and floral phenotype of T2 transgenic yellow cosmos (Cosmos sulphureus Cav.) carrying neomycin phosphotransferase II gene in second generation. Biodiversitas 25: 1711-1717. The seed of transgenic yellow cosmos (Cosmos sulphureus Cav.) lines harbouring neomycin phosphotranferase II (nptII) gene have been obtained from our previous studies. The nptII selectable marker gene is commonly used for developing a method to estimate the number of targeted gene integrated into the genome. In order to produce successful clones, it is important to ensure that transgenes are inherited sexually as the dominant trait in T2 progeny. This study aimed to obtain the segregation patterns of nptII gene in two populations derived from open- and self-pollinated and evaluated floral phenotypic of T2 transgenic yellow cosmos. The molecular analysis of transgenic yellow cosmos was conducted by PCR and the obtained data was analyzed using a Chi-Square test. The results showed that transgenes were inherited in 1:1 for transgenic and wild-type in both self- and open-pollinated populations. Therefore, it indicated that T2 transgenic plants were still unstable and evolved continuously to segregate independently in the over generation. Surprisingly, the morphological changes were detected in transgenic plants. The mixture of ligulate and tubular types in ray floret was found in some of the flower types in T2 yellow cosmos. In this case, nptII gene may diverge in apparently random genome insertion. This insertion would induce morphological changes in the ray floret type of flower since recombination estimates varied among transformation events.

Evaluation of antibacterial properties of nisin peptide expressed in carrots

Nisin, derived from Lactococcus lactis, is a well-known natural food preservative. In the present study, the gene of nisin was transformed to carrot by Agrobacterium tumefaciens strain LBA4404 harboring the recombinant binary vector pBI121 containing neomycin phosphotransferase II (nptII) gene, peptide signal KDEL, and Kozak sequence. The integration of nisin and nptII transgenes into the plant genome was confirmed by polymerase chain reaction (PCR) and dot blot analysis. The gene expression was also performed by RT-PCR and Enzyme-Linked Immunosorbent Assay. The level of nisin expressed in one gram of transgenic plant ranged from 0.05 to 0.08 μg/ml. The stability of nisin varied in orange and peach juices depending on the temperature on the 70th day. The leaf protein extracted from the transgenic plant showed a significant preservative effect of nisin in peach and orange juice. A complete inhibition activity against Staphylococcus aureus and Escherichia coli in orange juice was observed within 24 h. After 24 h, log 1 and log 2 were obtained in a peach juice containing Staphylococcus aureus and Escherichia coli, respectively. Results of HPLC indicated that Chlorogenic and Chicoric acid compounds were increased in transgenic plants, but this increase was not significant. The study of determining the genetic stability of transgenic plants in comparison with non-transgenic plants showed high genetic stability between non-transgenic plants and transgenic plants. This study confirmed the significant inhibitory effect of nisin protein on gram-positive and gram-negative bacteria.

A facile Agrobacterium-mediated transformation method for the model unicellular green algae Chlamydomonas reinhardtii

A reliable and simple Agrobacterium-mediated transformation system for the unicellular green algae model organism Chlamydomonas reinhardtii has been developed. The protocol has been successfully employed with both neomycin phosphotransferase II (nptII) and the phleomycin resistance (bleI) genes coupled with the selective agents paromomycin and zeocin, respectively. A set of binary vectors were assembled that carry the selectable marker cassettes under control either of the Rbcs2 alone or fused to the HSP270A leader sequence, PsaD, or ß-tubulin2 promoters. The corresponding T-DNA elements also harbored a cassette with a codon-optimized version of yellow fluorescence protein (YFP) under control of the Rbcs2 promoter in which the YFP open reading frame was interrupted with the first intron of Rbcs2 to prevent expression in Agrobacterium tumefaciens. The resultant binary vectors were introduced into A. tumefaciens strain C58C1/pMP90, and the derived transconjugants were used for transformation studies with the walled C. reinhardtii strain CC124. Estimated transformation frequencies ranged from 0.09 to 2.86 colonies per 106 cells inoculated. Molecular characterizations on a subset of the transgenic lineages revealed that most of the transgenic events harbored single locus insertions. Moreover, sequencing of captured junction fragments about the T-DNA insertion site showed that minimal disruption of the C. reinhardtii genome occurred. However, the transgenic lineages often harbored truncated T-DNA regions within the non-selectable marker gene cassettes.

Agrobacterium tumefaciens-mediated transformation of the white-rot fungus Dichomitus squalens

Dichomitus squalens is an efficient white-rot fungus that generates a wide range of extracellular enzymes to degrade lignocellulose in nature. Although a protoplast-mediated transformation method for D. squalens has been developed, the transformation efficiency remains low. Here, we established a highly efficient Agrobacterium tumefaciens-mediated transformation (ATMT) procedure for D. squalens by transferring a binary vector harboring the neomycin phosphotransferase II (nptII) resistance gene fused with DsRed-Express2, under the control of the native glyceraldehyde-3-phosphate dehydrogenase (GPD) gene promoter. Key factors affecting the efficiency of transformation were tested. A. tumefaciens EHA105 strain with a cell density of 0.4 OD600nm and 96 h co-cultivation resulted in the highest transformation efficiency, with an average of 98 ± 11 transformants per co-cultivation plate. Besides, the strong expression of DsRed-Express2 indicates the effectiveness of the DsGPD promoter in driving gene expression in D. squalens. This ATMT system of D. squalens would be beneficial for its molecular genetic studies.

Safety Assessment of the CP4 EPSPS and NPTII Proteins in Eucalyptus

ABSTRACT Glyphosate herbicide treatment is essential to sustainable Eucalyptus plantation management in Brazil. Eucalyptus is highly sensitive to glyphosate, and Suzano/FuturaGene has genetically modified eucalyptus to tolerate glyphosate, with the aim of both protecting eucalyptus trees from glyphosate application damage and improving weed management. This study presents the biosafety results of the glyphosate-tolerant eucalyptus event 751K032, which expresses the selection marker neomycin phosphotransferase II (NPTII) enzyme and CP4-EPSPS, a glyphosate-tolerant variant of plant 5-enolpyruvyl-shikimate−3-phosphate synthase enzyme. The transgenic genetically modified (GM) event 751K032 behaved in the plantations like conventional non-transgenic eucalyptus clone, FGN-K, and had no effects on arthropods and soil microorganisms. The engineered NPTII and CP4 EPSPS proteins were heat-labile, readily digestible, and according to the bioinformatics analyses, unlikely to cause an allergenic or toxic reaction in humans or animals. This assessment of the biosafety of the glyphosate-tolerant eucalyptus event 751K032 concludes that it is safe to be used for wood production.

Melatonin strongly enhances the Agrobacterium- mediated transformation of carnation in nitrogen-depleted media

With the rising demand for new cultivars of carnation, efficient transformation protocols are needed to enable the bioengineering of new traits. Here, we established a novel and efficient Agrobacterium-mediated transformation system using callus as the target explant for four commercial carnation cultivars. Leaf-derived calli of all cultivars were inoculated with Agrobacterium tumefaciens strain LBA4404 containing the plasmid pCAMBIA 2301 harboring genes for β-glucuronidase (uidA) and neomycin phosphotransferase (nptII). Polymerase chain reaction (PCR) and histochemical assays confirmed the presence of uidA and β-glucuronidase (GUS), respectively in transgenic shoots. The effect on transformation efficiency of medium composition and the presence of antioxidants during inoculation and co-cultivation was investigated. The transformation efficiency was increased in Murashige and Skoog (MS) medium lacking KNO3 and NH4NO3, and also in MS medium lacking macro and micro elements and Fe to 5% and 3.1% respectively, compared to 0.6% in full-strength medium. Transformation efficiency was increased dramatically to 24.4% across all carnation cultivars by the addition of 2 mg/l melatonin to nitrogen-depleted MS medium. Shoot regeneration was also doubled in this treatment. The establishment of this efficient and reliable transformation protocol can advance the development of novel carnation cultivars through molecular breeding approaches.

Efficient floral dip transformation method using Agrobacterium tumefaciens on Cosmos sulphureus Cav.

Yellow cosmos (Cosmos sulphureus Cav.) is a specific flowering plant and considered a suitable genetic engineering model. Agrobacterium-mediated plant transformation is commonly used for plant genetic engineering. Floral dip transformation is one of the plant genetic transformation methods, and it involves dipping flower buds into an Agrobacterium suspension. Studies on floral dip transformation of yellow cosmos have never been reported. Therefore, an efficient method in plant genetic engineering must be established. This study developed an effective and efficient floral dip transformation method for yellow cosmos.In this study, flower buds with sizes of 5–7 mm were used. Several parameters have been observed to optimize the floral dip method. These parameters included the optical density (OD600) of Agrobacterium culture, concentration of surfactant, and duration of flower bud dipping into the Agrobacterium suspension.The results showed that the floral dip method was most efficient when the flower buds were dipped into Agrobacterium suspension with OD600 = 0.8 and containing 5% sucrose and 0.1% Silwet L-77 for 30 s. This method enhanced the transformation efficiency at a rate of 12.78 ± 1.53%. The neomycin phosphotransferase II and green fluorescent protein genes with sizes of 550 and 736 bp, respectively, were confirmed by polymerase chain reaction. In addition, the transgenic plants were kanamycin resistant and fluorescent under ultraviolet light observation. This finding suggests that the proposed floral dip transformation provides new insights into efficient plant genetic engineering methods for yellow cosmos.

An Efficient Agrobacterium-Mediated Genetic Transformation Method for Solanum betaceum Cav. Embryogenic Callus

Somatic embryogenesis in Solanum betaceum (tamarillo) has proven to be an effective model system for studying morphogenesis, since optimized plant regeneration protocols are available, and embryogenic competent cell lines can be induced from different explants. Nevertheless, an efficient genetic transformation system for embryogenic callus (EC) has not yet been implemented for this species. Here, an optimized faster protocol of genetic transformation using Agrobacterium tumefaciens is described for EC. The sensitivity of EC to three antibiotics was determined, and kanamycin proved to be the best selective agent for tamarillo callus. Two Agrobacterium strains, EHA105 and LBA4404, both harboring the p35SGUSINT plasmid, carrying the reporter gene for β-glucuronidase (gus) and the marker gene neomycin phosphotransferase (nptII), were used to test the efficiency of the process. To increase the success of the genetic transformation, a cold-shock treatment, coconut water, polyvinylpyrrolidone and an appropriate selection schedule based on antibiotic resistance were employed. The genetic transformation was evaluated by GUS assay and PCR-based techniques, and a 100% efficiency rate was confirmed in the kanamycin-resistant EC clumps. Genetic transformation with the EHA105 strain resulted in higher values for gus insertion in the genome. The protocol presented provides a useful tool for functional gene analysis and biotechnology approaches.

Development and utilization of analytical methods for rapid GM detection in processed food products: a case study for regulatory requirement

Genetically modified (GM) food crops for desired traits have been approved in some of the countries. The approval status of a GM event varies from country to country. In India, Bt cotton is the only approved GM crop. So far, no GM food crop has got regulatory approval in the country, which may be considered as unauthorized GM (UGM) event in the Indian context. The entry of UGM events of food crops needs to be checked in the imported consignments as well as in the marketplace. In the present study, screening elements were identified based on the genetically modified organism (GMO) matrix developed as decision support system for 22 GM food crops approved globally. Three sets of multiplex PCR assays were developed and validated for GM detection in food crops and products: triplex PCR targeting control elements [Cauliflower Mosaic Virus 35S promoter (P-35S), Figwort Mosaic Virus promoter (P-FMV), nopaline synthase terminator (T-nos)], triplex PCR targeting marker genes [aminoglycoside-3’-adenyltransferase (aadA), neomycin phosphotransferase (nptII), phosphinothricin-N-acetyltransferase (pat)] and duplex PCR targeting Bt genes (cry1Ab/Ac and cry2Ab2). Limit of detection (LOD) ranged from 0.5–0.01% for different targets, which is in compliance with the labelling threshold of many countries. The developed assays were utilized to check the GM status of apple and maize products along with an additional test for ctp2-cp4epsps for herbicide tolerance in maize. Based on the tests conducted, none of these samples were found positive for GM targets screened. These procedures could be efficiently employed as a part of GMO testing to trace GM contamination, if any, in both the imported as well as domestic food products.

A protocol for Agrobacterium-mediated genetic transformation of Lens culinaris Medik (lentil)

A reliable protocol for Agrobacterium-mediated genetic transformation of Lens culinaris Medik (lentil) was developed. Using cultivar Laird, the protocol yielded rooted shoots from an average of 6.8 independent events per hundred seeds. The protocol utilized longitudinal slices of embryo axes from imbibed mature seed as a starting explant and a plasmid containing a β-glucuronidase:neomycin phosphotransferase (gus:nptII) fusion gene in Agrobacterium strain EHA105. A series of four media, each with appropriate levels of kanamycin selection were identified and other factors tested included the optical density of the Agrobacterium suspension, and type and concentration of plant growth regulators. The expression of the gus reporter gene was visualized through histochemical staining, and further molecular analysis through PCR, qPCR, ddPCR and Southern hybridization confirmed transformation and provided copy number. The inserted genes were inherited into the T1 generation and chimaeras were not identified. The time from co-cultivation to the planting of rooted shoots ranged from 4 to 7 months, as transgenic clusters continue to produce additional clonal shoots.

DETECTION OF GENETICALLY MODIFIED RICE LOCALLY AVAILABLE IN PUNJAB, PAKISTAN

Rice (Oryza sativa L.) is an important cereal crop that provides food to half of the world's population. Pakistan's traditional and premium quality rice is mostly exported to Europe. Transgenic or genetically modified rice (GM RICE) has been developed in the USA, China, and other countries, like herbicide-tolerant Liberty-Link rice (LLRICE06, LLRICE62, LLRICE601) and insect-resistant Bt-63. International and national biosafety regulations allow countries to stop the import of any genetically modified organism (GMO) due to its possible safety concerns. PCR-based methods are recommended for the accurate detection of GM rice. Qualitative PCR is used to detect the presence or absence of GMOs in a rice sample. The objective of this study was to check the presence of GMOs in the rice samples collected from rice-growing areas in Punjab. Eighty-two rice samples were collected randomly from various markets/locations in rice-growing areas of Punjab for this study. Twelve bulks were prepared to represent eighty-two rice samples. The CTAB method was used to extract the DNA from rice samples to analyze it. PCR of rice bulk samples was performed using primers specific for SPS, CP, p35S, tNOS, nptII, Hpt and Bt 63 rice. Positive and negative controls were included in each PCR. Primers specific for chloroplast (CP) and Sucrose phosphate synthase (SPS) genes were used to confirm DNA quality for amplification. Then, primers specific for the cauliflower mosaic virus (CaMV) 35S promoter and the Agrobacterium nopaline synthase (NOS) terminator were used for the detection of GM rice through PCR, neomycin phosphotransferase II (nptII) and Hygromycin phosphotransferase (Hpt) were used as a selectable marker and Bt 63 rice was used in insect resistance. The gel electrophoresis of PCR was conducted using a DNA ladder and then taking of the image was through gel documentation system for visualization and analysis. According to the results, all the rice bulks representing the eighty-two rice samples were negative for p35S, tNOS, nptII, Hygro and Bt 63 rice event. These findings are encouraging and demonstrate that rice grown in kalar track of Punjab, Pakistan, is non-GMO. Such surveillance and monitoring would be largely beneficial to maintain the purity of our traditional non-GM rice and to avoid any possible mixing of GM rice seeds, especially at the time of import. All these efforts will ensure and protect our national share in the international trade of rice.

Molecular and phenotypic characteristics of T1 transgenic yellow cosmos (Cosmos sulphureus) carrying neomycin phosphotransferase II gene

Abstract. Irsyadi MB, Sawitri WD, Purwantoro A. 2022. Molecular and phenotypic characteristics of T1 transgenic yellow cosmos (Cosmos sulphureus) carrying neomycin phosphotransferase II gene. Biodiversitas 23: 6097-6105. Yellow cosmos (Cosmos sulphureus) is a tropical ornamental flower that contains secondary metabolites useful fornatural pesticide application. Plant genetic transformation is one genetic engineering method used to increase secondary metabolite accumulation. However, published reports on this issue have yet to be made available. This is the first report of genetic transformation in transgenic yellow cosmos using neomycin phosphotransferase II (nptII) encoding gene. This study aimed to determine the efficiency transformation and phenotypic character of the T1 yellow cosmos transgenic nptII. This genetic transformation was carried out by the floral dip method mediated with Agrobacterium tumefaciens and characterized based on quantitative and qualitative observations, as well as a cluster analysis of transgenic plants. The T1 transgenic yellow cosmos was confirmed using PCR with a transformation efficiency of 73.33% based on the total plants resistant to Kanamycin and 10.57% based on the total seeds transformed. The presence of the nptII encoding gene was shown in transgenic plant samples with a size of 550 bp. In general, the introduction of the nptII gene generated no novel traits except the resistance to the kanamycin antibiotic. However, there was a decrease and increase in the number of stomata and the size of stomata in transgenic plants, respectively. In addition, there was a change in the type of ray floret to a mixture of ligulate and tubular, indicating that the nptII gene affected the phenotype of yellow cosmos. The result revealed that the nptII gene was inserted fairly randomly into the plant genome.

THE PRODUCTION OF TRANSGENIC TOBACCO PLANTS OVEREXPRESSING OAK DEHYDRIN GENE

This work is focused on the production of transgenic tobacco plants overexpressing the oak dehydrin gene AY607707.1. Transgenic tobacco plants were generated via Agrobacterium-mediated transformation. The T-DNA of the plant transformation vector pMK contained the dehydrin gene fused to the constitutive double dCaMV35S promoter and the selectable neomycin phosphotransferase gene. The sequence of the dehydrin gene was isolated from Quercus robur by PCR approach and cloned. The constructed binary vector pMK was introduced into Agrobacterium tumefaciens LBA4404 and used in the transformation experiments. Transgenic plants were generated with an efficiency of 32.7%. PCR analyses confirmed the transgenic nature of regenerated T0 plants. The expression of the oak dehydrin gene was proved by RT-PCR and quantified by qPCR analyses.

In vitro</em></strong><strong> direct regeneration and <em>Agrobacterium tumefaciens</em> mediated <em>in planta</em> transformation of <em>Ocimum sanctum L.

An in vitro regeneration system for propagation has been successfully developed for a valuable medicinal and aromatic plant ‘ Ocimum sanctum L’. In the present study, petiole explants, from in-vitro grown cultures of O. sanctum, was used for direct regeneration. The developed protocol employed 98% of regeneration frequency in addition to 9.6 shoots per explant when cultured on Murashige and Skoog (MS) medium fortified with 3 mg/L benzylamino purine (BAP) and 1 mg/L Naphthalene acetic acid (NAA). Furthermore, Agrobacterium tumefaciens Mediated genetic Transformation (ATMT) protocol (transient and stable) was also developed using LBA4404 strain harboring pBI121 with uid-A and neomycin phosphotransferase genes. The regenerated transformants were shifted on MS with kanamycin (50 mg/L) and afterwards placed on the half-strength MS medium. The validation was done through polymerase chain reaction (PCR) with neomycin phosphotransferase-II ( npt-II) & β-glucoronidase ( uid-A) gene primers. The maximum stable transformation frequency of 70% ± 0.35 was achieved. Hence, it is apparent that the established protocols i.e. in vitro direct regeneration and ATMT are appropriate for integrating novel enzymes/genes through high throughput techniques such as gene tagging, and targeted gene replacement to modulate the primary as well as secondary metabolic flux towards desired agronomic product or trait in planta.

Genetic transformation of clone rootstock of stone fruits 146-2 using the green fluorescent protein reporter gene

For the form of dwarf winter-hardy clonal rootstocks of stone crops146-2 (Prunus pumilaL.xP.tomentosaThunb.), system of regeneration and genetic transformation using green fluorescent protein (GFP) has been developed. For eff ective regeneration of accessory shoots, no pre-treatment with 6-benzylamine-purine (BA) and auxin was required. Stimulation of the regeneration of shoots from leaf explants required 2-3 weeks of a dark period. Th e best percentage of regeneration (greater than 75 %) was observed with a combination of 3 mg/L BA and 0.75 mg/L IBA. The achieved regeneration effi ciency made it possible to develop a protocol for genetic transformation, mediated byAgrobacterium, for rootstock 146-2. Whole leaves from in vitro-cultured shoots were used as explants for transformation by theA. tumefaciensstrain CBE21, with the binary vector pBINmGFP5ER containing thenptIIencoding neomycin phosphotransferase II as a plant-selectable marker under the control of the NOS promoter (nopalin synthase) and the reportergfpgene encoding a green fluorescent protein under the control of the cauliflower mosaic virus (CaMV) promoter 35S. Th e integration ofnptIIandgfpinto transgenes was confirmed by PCR. Expression of the green fluorescent protein was observed using fluorescence microscopy. The efficiency of transformation based on PCR analysis of independent lines resistant to kanamycin was 0.41-0.83 %. All transgenic lines showed resistance to kanamycin at a concentration of 40 mg/L. They were rooted and acclimatized to greenhouse conditions. Th e developed protocols will be used to producePlum pox virus(PPV) resistant plants.

Exogenous dsRNA Induces RNA Interference of a Chalcone Synthase Gene in Arabidopsis thaliana.

Recent investigations have shown the possibility of artificial induction of RNA interference (RNAi) via plant foliar treatments with naked double-stranded RNA (dsRNA) to silence essential genes in plant fungal pathogens or to target viral RNAs. Furthermore, several studies have documented the downregulation of plant endogenous genes via external application of naked gene-specific dsRNAs and siRNAs to the plant surfaces. However, there are limited studies on the dsRNA processing and gene silencing mechanisms after external dsRNA application. Such studies would assist in the development of innovative tools for crop improvement and plant functional studies. In this study, we used exogenous gene-specific dsRNA to downregulate the gene of chalcone synthase (CHS), the key enzyme in the flavonoid/anthocyanin biosynthesis pathway, in Arabidopsis. The nonspecific NPTII-dsRNA encoding the nonrelated neomycin phosphotransferase II bacterial gene was used to treat plants in order to verify that any observed effects and processing of AtCHS mRNA were sequence specific. Using high-throughput small RNA (sRNA) sequencing, we obtained six sRNA-seq libraries for plants treated with water, AtCHS-dsRNA, or NPTII-dsRNA. After plant foliar treatments, we detected the emergence of a large number of AtCHS- and NPTII-encoding sRNAs, while there were no such sRNAs after control water treatment. Thus, the exogenous AtCHS-dsRNAs were processed into siRNAs and induced RNAi-mediated AtCHS gene silencing. The analysis showed that gene-specific sRNAs mapped to the AtCHS and NPTII genes unevenly with peak read counts at particular positions, involving primarily the sense strand, and documented a gradual decrease in read counts from 17-nt to 30-nt sRNAs. Results of the present study highlight a significant potential of exogenous dsRNAs as a promising strategy to induce RNAi-based downregulation of plant gene targets for plant management and gene functional studies.