nptII Gene Research Articles

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.

Co-overexpression of chitinase and β-1,3-glucanase significantly enhanced the resistance of Iranian wheat cultivars to Fusarium

Fusarium head blight (FHB) is a devastating fungal disease affecting different cereals, particularly wheat, and poses a serious threat to global wheat production. Chitinases and β-glucanases are two important proteins involved in lysing fungal cell walls by targeting essential macromolecular components, including chitin and β-glucan micro fibrils. In our experiment, a transgenic wheat (Triticum aestivum) was generated by introducing chitinase and glucanase genes using Biolistic technique and Recombinant pBI121 plasmid (pBI-ChiGlu (-)). This plasmid contained chitinase and glucanase genes as well as nptII gene as a selectable marker. The expression of chitinase and glucanase was individually controlled by CaMV35S promoter and Nos terminator. Immature embryo explants from five Iranian cultivars (Arta, Moghan, Sisun, Gascogen and A-Line) were excised from seeds and cultured on callus induction medium to generate embryonic calluses. Embryogenic calluses with light cream color and brittle texture were selected and bombarded using gold nanoparticles coated with the recombinant pBI-ChiGlu plasmid. Bombarded calluses initially were transferred to selective callus induction medium, and later, they were transfferd to selective regeneration medium. The selective agent was kanamycin at a concentration of 25 mg/l in both media. Among five studied cultivars, A-Line showed the highest transformation percentage (4.8%), followed by the Sisun, Gascogen and Arta in descending order. PCR and Southern blot analysis confirmed the integration of genes into the genome of wheat cultivars. Furthermore, in an in-vitro assay, the growth of Fusarium graminearum was significantly inhibited by using 200 μg of leaf protein extract from transgenic plants. According to our results, the transgenic plants (T1) showed the resistance against Fusarium when were compared to the non-transgenic plants. All transgenic plants showed normal fertility and no abnormal response was observed in their growth and development.

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.

STRATEGIES FOR IMPROVEMENT FOR CHARACTERIZATION AND QUALITY OF BT COTTON SEEDS

The study highlights diverse methodologies employed for seed quality assessment and characterization, including genetic, biochemical, and physiological analyses. In our present study, PCR and ELISA based techniques was adopted to identify the specific Cry1Ac gene and proteins in MECH 162 and RCH2 transgenic cotton plants. MECH 162 of 21.9ug/g and RCH2 of 17.37ug/g of seed protein was extracted. PCR analysis confirmed that gene specific CRY1Ac F/R primer as amplicon size of 550 bp for Bt gene, 600bp for NptII gene and 200bp for 35s promoter gene. The ELISA method was used for the validation of the developed assay, also ELISA needed simple equipment and took less time. Insights into the integration of these strategies into breeding programs are provided, emphasizing their crucial role in ensuring the production of high-quality Bt cotton seeds with improved agronomic performance and yield potential.

Knockdown of orthotospovirus-derived silencing suppressor gene by plant-mediated RNAi approach induces viral resistance in tomato

RNA interference (RNAi) shows significant effectiveness in conferring resistance to viral infections in various plants. The current study was conducted to develop transgenic tomato (Solanum lycopersicum cv. Rio Grande) lines expressing a 320 bp conserved inverted region of the integral 35S promoter region. The molecular cloning was done for the construction of expression vectors in a hairpin structure by the Tomato spotted wilt orthotospovirus (TSWV) species Orthotospovirus tomatomaculae, silencing suppressor (NSs) gene. It contains both sense and antisense orientation in a binary vector pFGC5941. Short hairpin RNA (shRNA) structures were employed to reduce the possibility of inducing endogenous non-specific antiviral responses, which usually target longer double-stranded RNAs (dsRNAs). To confirm the transgenic plants, molecular analysis was performed using TSWV gene-specific primers (TSWV-NSs). Based on the EHA105 Agrobacterium strain and the pFGC5941 vector carrying NSs and nptII genes in the T-DNA region, the results validate the insertion of kanamycin resistance into the regenerated transgenic plants. The virus source harboring TSWV was used to inoculate putative transgenic plants for evaluating the confirmation of successful transformation strategy for inducing virus resistance. Quantitative real-time polymerase chain reaction (qRT-PCR) assays were performed by using qRT-NSs primer pairs to determine the relative gene expression of all constructs (TSWV-NSs both sense and antisense orientation) after inoculations. In comparison to the control groups, which contained wild type control (non-transgenic tomato plant), empty pFGC5941 vector and the pFGC5941+sense construct, the expression of the NSs gene was noticeably reduced in the full clone (pFGC5941+sense + antisense) construct. For the validation of RNAi effectiveness, Nicotiana tabacum plants were also inoculated with viral constructs. Specifically, they displayed severe symptoms of TSWV in plants containing the pFGC5941+sense, pFGC5941 empty vector, and in wild-type tomato plants that are correlated with bioassay results. The lack of TSWV symptoms validated the hypothesis after careful observation of the expression of NSs genes in terms of pathogenicity. This research demonstrates the RNA interference effectiveness by targeting NSs gene as a reliable technique for achieving long lasting antiviral protection in tomatoes. The findings have practical relevance and the potential to be a tool for limiting tomato crop losses caused by TSWV worldwide.

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.

Destruction of transgenic potato plants (Solanum tuberosum L.) remnants under the influence of soil microbiome

Scientific community has been considering various hypotheses regarding the possible impact of GM plants on non-target soil microorganisms for decades, distinguishing direct and indirect effects. Direct effects are usually easier to detect, as transgenic proteins, characterized by a range of elimination against certain types of insects, pathogenic fungi and bacteria, can affect non-target beneficial symbionts and soil microbial groups involved in the transformation of organic substances. Indirect effects are difficult to assess, as many different factors can affect the composition of root exudates and the metabolic activity of plants. Changes in metabolic pathways can affect the composition of soil exudates and gene expression in plant tissues. At the same time, these changes can affect the decomposition of organic matter and the course of microbiological processes in the soil. Based on experimental studies, an assessment of the rate of destruction of plant residues and DNA degradation in transgenic potato plants in soddy-podzolic soil of potato crop rotation was carried out. The results of the studies have established a significant role of the microbiome of soddy-podzolic soil of potato crop rotation in the destruction of residues of transgenic potato plants and DNA degradation. Destruction of residues of transgenic plants under the influence of microflora of soddy-podzolic soil of potato crop rotation was 30 days with a loss of raw mass 87.14±1.11%. DNA degradation over this period is 88% (186 ng/mg) from the initial amount of total DNA, and degradation of nptII and 35S genes — about 90% from their initial amount in PCR products. It was found that about 90% of nptII and 35S genes in DNA residues of transgenic potato plants under the influence of microbiome soddy-podzolic soil occurs on day 30.

Survivorship and food consumption of immatures and adults of Apis mellifera and Scaptotrigona bipunctata exposed to genetically modified eucalyptus pollen

Eucalyptus comprises the largest planted area of cultivated production forest in Brazil. Genetic modification (GM) of eucalyptus can provide additional characteristics for increasing productivity and protecting wood yield, as well as potentially altering fiber for a diversity of industrial uses. However, prior to releasing a new GM plant, risk assessments studies with non-target organisms must be undertaken. Bees are prominent biological models since they play an important role in varied ecosystems, including for Eucalyptus pollination. The main goal of this study was to evaluate whether a novel event (Eucalyptus 751K032), which carries the cp4-epsps gene that encodes the protein CP4-EPSPS and nptII gene that encodes the protein NPTII, might adversely affect honey bees (Apis mellifera) and stingless bees (Scaptotrigona bipunctata). The experiments were performed in southern Brazil, as follows: (i) larvae and adults were separately investigated, (ii) three or four different pollen diets were offered to bees, depending on larval or adult status, and (iii) two biological attributes, i.e., survivorship of larvae and adults and food intake by adults were evaluated. The diets were prepared with pollen from GM Eucalyptus 751K032; pollen from conventional Eucalyptus clone FGN-K, multifloral pollen or pure larval food. The insecticide dimethoate was used to evaluate the sensitivity of bees to toxic substances. Datasets were analyzed with Chi-square test, survival curves and repeated measures ANOVA. Results indicated no evidence of adverse effects of Eucalyptus pollen 751K032 on either honey bees or stingless bees assessed here. Therefore, the main findings suggest that the novel event may be considered harmless to these organisms since neither survivorship nor food consumption by bees were affected by it.

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.

Horizontal transfer of antibiotic resistance genes into microflora and blood cells in rats fed on GM-diet

BackgroundWith regard to the extensive production of genetically modified organisms, scientists focused on the safety of antibiotic resistance marker (ARM) genes present in GM-crops produced for the consumption of human or animal. We investigated the possible transfer of these genes to the microflora present in the gastrointestinal tract and blood cells of two groups of Male Wistar Albino rats fed on a transgenic diet containing the ARM genes nptII and aadA for 90 days. The conventional PCR was employed to screen for the presence of nptII and aadA genes in the experimental diets, and animals’ samples.ResultsThe occurrence of DNA transfer of nptII and aadA genes from GM-diet to blood and bacterial cells has been unambiguously demonstrated. Results were further confirmed by sequencing and blast analysis, indicating that both transferred segments shared significant alignment with number of Binary and Cloning Vectors with variable similarities. The frequency of transfer of DNA segments from the GM-diet into blood DNA was unexpectedly higher than its transfer to bacterial DNA which may be attributed to a number of factors.ConclusionsOur results unambiguously demonstrated the occurrence of DNA transfer of ARM genes (nptII and aadA) from GM plant diet to blood cells and enteric microflora in rats. Results may draw attentions to the importance of exploring the possible effects of transfer of ARM genes horizontally from GM products to consumers and to extend our attentions to the importance of a better understanding of the factors influencing HGT in the intestine of the GM-food consumers.

Expression of radish defensin (RsAFP2) gene in chickpea (Cicer arietinum L.) confers resistance to Fusarium wilt disease.

Chickpea (Cicer arietinum L.), a major nutritional source cultivated worldwide, is vulnerable to several abiotic and biotic stresses, including different types of soil-borne pathogens like Fusarium oxysporum f. sp. ciceri, which causes root rot disease and severely affects productivity. In this study, putative transgenic plants were obtained with the Radish defensin (Rs-AFP2) gene through Agrobacterium tumefaciens mediated transformation using the embryo axis explants. Transgenes were confirmed in 18 putative transgenic plants with PCR-specific primers for nptII and Rs-AFP2 genes. Twelve transgenic plants were established successfully under greenhouse conditions. The T0 plants were allowed for self-pollination to obtain T1 seeds. The T1 plants, selected for Fusarium wilt assay using Fusarium oxysporum f. sp. Cicero, showed different resistance levels, from moderate to high levels in comparison to control plants (wild-type) which exhibited severe wilt symptoms. Our results suggest the application of Radish defensins (RsAFP1/RsAFP2 genes) for improving pathogen resistance in chickpea.

PECULIARITIES OF THE GROWTH OF Artemisia tilesii Ledeb. “HAIRY” ROOTS WITH DIFFERENT FOREIGN GENES

Aim. To compare Artemisia tilesii “hairy” root lines with different transferred genes in terms of the relationship between the total content of flavonoids, the levels of antioxidant activity (AOA) and reducing power (RP), as well as the activity of phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), rolB and rolC genes. Methods. We compared the root lines Nos. 10 and 16, obtained by transformation with the wild Agrobacterium rhizogenes strain A4, lines Nos. 2 and 4, obtained using A. rhizogenes carrying pSV124 vector with ifn-α2b and nptII genes, as well as the roots of non-transformed plants that were cultivated in vitro. The presence and activity of rolB, rolC, PAL, and CHS genes were determined by PCR and real-time PCR, respectively. The content of flavonoids, AOA and RP were determined by standard tests with AlCl3, DPPH (2,2-diphenyl-1-picrylhydrazyl) and K3[Fe(CN)6] accordingly. Results. The content of flavonoids in most of the lines was higher than in the control, and correlated with AOA and RP. Roots No. 10 were characterized by the fastest growth, which coincided with higher activity of rolB and rolC genes. The activities of PAL and CHS in “hairy” roots were lower than those in non-transformed ones. Conclusions. Root lines carrying only rolB and rolC and lines with additional ifn-α2b and nptII genes had similar ranges of flavonoids concentration, AOA and RP levels that exceeded those in the control. The dependence of the root growth rate, and lack of the dependence of the flavonoids content with the activity of the rol genes were demonstrated. PAL activity inversely correlated with flavonoids content in all experimental lines, which may be the result of overproduction of compounds in transgenic roots.

Agrobacterium rhizogenes-mediated genetic transformation of Musa acuminata cv. Vaibalhla (AAA)

Agrobacterium rhizogenes-mediated genetic transformation of Musa acuminata cv. Vaibalhla (AAA) was successfully carried out using A. rhizogenes strain A4 harboring the binary vector pCAMBIA2301with VrNHX1gene. In the study, male flowers of M. acuminata cv. Vaibalhla were used as explants to obtain white bud-like structures by culturing on MS basal medium supplemented with 2 mg/l 6-Benzylaminopurine (BAP) and 0.5 mg/l 1-Naphthaleneacetic acid (NAA). The subsequent shoot induction and plantlet regeneration were carried out in MS medium supplemented with Kinetin (2 mg/l) and NAA (0.5 mg/l). For the genetic transformation, in vitro raised plantlets were inoculated with A. rhizogenes strain A4 harboring pCAMBIA2301VrNHX1 for 30 min followed by 2 days of co-cultivation in the dark in a semi solid MS basal medium. The treated plants were then transferred and maintained in MS basal medium supplemented with ascorbic acid (75 mg/l), kanamycin (150 mg/l) and cefotaxime (400 mg/l). Initiation of hairy roots were observed within 2 days of transfer which were evaluated for GUS activity and the subsequent GUS+ roots were assayed for transient transformation by PCR using nptII and NHX1 gene specific primers. The transfer of the plasmid as well as the gene was confirmed with positive bands observed at 540 bp and 1.6 kb respectively.

Infl uence of interference silencing of the gene of translation initiation factors <i>eIF(iso)G</i> and <i>eIF(iso)E</i> on the resistance of the clone stone fruits rootstock 146-2 and commercial plum variety Startovaya to the Plum pox virus

The method of RNA interference gene expression silencing was used to obtain Plum pox virus (PPV) resistant rootstock and commercial variety Startovaya.For this purpose, a vector with self-complementary sequences of the 578 bp eIF(iso)4G and eIF(iso)E genes fragment was created. The eIF(iso)4G and eIF(iso)E genes encodes factors of initiation of translation involved in the life cycle of a Plum pox virus. A strong promoter of the ribulose-1.5-bisphosphate carboxylase/oxygenase (RuBisCo) gene was chosen to drive the expression of RNA interference hairpin in full and truncated variants. Successful genetic transformation of the 146-2 rootstock and variety Startovaya were carried out by A. tumefaciens CBE21 strain. Whole leaves from in vitro cultured shoots were used as an explant source. The nptII and hpt genes coding for neomycin II and hygromycin phosphotransferase were used as a plant-selectable markers. In our experiments, 5 independent transgenic lines of clonal rootstock and variety were obtained and acclimatized to greenhouse conditions. Th eir status was confirmed by PCR and Southern blot analyses. The transformation efficiency was 0.3-0.4 %. One of these lines was grafted with PPV-infected plum buds and its resistance was verified by ELISA. The use of a full-length gene promoter of the small subunit of ribulosobiephosphate carboxylase (RBCS) in the transformation of plants of the Starter variety led to a decrease in plant viability in the case of suppression of the eIF(iso)4E gene and ensured stability at least in the first year after inoculation in the case of suppression of the eIF(iso)4G gene.

Performance of Transgenic Chrysanthemum Harbouring Wasabi Defensin Gene for White Rust Disease Resistance

This study was intended to obtain white rust (Puccinia horiana) disease resistance Chrysanthemum transformed with wasabi defensin gene through mediation of Agrobacterium tumefaciens from three explant sources, i.e., leaf, lateral shoot bud, and internode. Observations were made on transformation efficiency, PCR analysis, in vitro and ex vitro disease resistance tests. Results showed that efficiency of transgenic callus and shoot regeneration was found both highest from lateral shoot buds (57.5% and 50.0%, respectively). PCR analysis showed that three putative transgenic plantlets from lateral shoot buds and one from leaf explant were putative transgenic carrying the wasabi, hpt, and nptII genes. Rooting test showed that the highest number of rooted plants was found in treatment of hygromycin (Hg) 25 mg L-1 (81%) and lowest was in treatment combination of kanamycin (Km) 50 mg L-1 + Hg 25 mg L-1 (25%). In vitro disease resistance test with sorus inoculation of P. horiana, directly on the leaves, resulted in 20 resistant plants out of 30 putative transgenic plants (66.67%). Ex vitro testing on adult plants of the same samples in a confined closed greenhouse (CGH) resulted in average of 80% transgenic Chrysanthemum plants were resistant, whereas in control plants caused white rust disease symptom.

Development of methods for regeneration and genetic transformation of the commercial plum variety Startovaya from somatic tissues

An effi cient technique for regeneration of plants from somatic tissues of domestic plum (Prunus domestica L.) varieties, as well as selecting schemes using various reporter (uidA, gfp) and selective marker genes (nptII, hpt) have been developed. During the transformation of somatic tissues of the commercial plum variety, transgenic plants containing both reporter genes and genes of economically valuable traits were obtained. For the fi rst time, the eff ect of calcium pantothenate on the adventive regeneration of plum shoots was investigated. It was shown that the selective antibiotic hygromycin is more eff ective for the selection of plum transformants than kanamycin or geneticin, which was expressed in a higher frequency of transformation — 1.2-2.2 % versus 0.2-0.5 % in selection on kanamycin. When using geneticin, transgenic plants were not obtained. Optimization of conditions made it possible to increase the percentage of regeneration of shoots to 79.4 % for the variety Startovaya and up to 39.6 % for the variety Etude. Analysis of GFP expression in transgenic lines showed that the leader sequences of herbaceous plant species encoding signal peptides can be used to eff ectively cellular compartmentalization of heterologous gene expression products in the plum. Preliminary results also show a higher level of expression of the reticular form of GFP in comparison with the vacuolar and cytosolic forms. Nine transgenic lines containing the insertion of the target bar gene, which gives resistance to phosphinothricin-containing herbicides of the “Basta” type, were obtained, which was confi rmed by the presence in the genome of the sequence of the transferred gene and the resistance of the obtained transgenic plants to 3 times the fi eld concentration of the herbicide “Basta”.

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.

Transgenic Peanut (<i>Arachis hypogaea</i> L.) Plants Conferring Enhanced Protection Against Fungal Pathogens by Expressing <i>Tc chitinase-I</i> Gene

In this study, an efficient Agrobacterium-mediated genetic transformation method followed by Tc chitiI gene for enhanced expression in peanut (Arachis hypogaea L.) cv ICG 7827 is reported. A. tumefaciens strain LBA 4404 harboring the plant transformation plasmid pBinAR contains the Tc chitI gene and nptII gene as a selectable marker. The precultured leaflet (LL) explants were infected with A. tumefaciens containing Tc chitiI gene and cocultivated on SIM (MMS+10 mg/L BAP+1 mg/L NAA+2 mg/L AgNO3) for four days. After co-cultivation, these explants were transferred to selection medium (SIM+75 mg/L Kan+250 mg/L Cefotaxime). In T0 generation, 70 % of transformation efficiency was recorded. T1 generation derived from the primary transgenic (T0) events revealed a Mendelian inheritance pattern (3:1) for the Tc chit I transgene. T1 transgenic peanut plants were tested for resistance against C. arachidicola, C. personatum, and P. arachidis by infection with the microspores using detached leaf assay. The Tc chitI gene expressed in T1 transgenic plants have shown longer incubation, longer latent period, and lower infection frequencies than non-transformed (WT) plants. A significant negative correlation was recorded between chitinase activity and the frequency of infection to the C. arachidicola, C. personatum and P. arachidis pathogens.

Clean gene technology to develop selectable marker-free pod borer-resistant transgenic pigeon pea events involving the constitutive expression of Cry1Ac.

The most crucial yield constraint of pigeon pea is susceptibility to the pod borer Helicoverpa armigera, which causes extensive damage and severe economic losses every year. The Agrobacterium-mediated plumular meristem transformation technique was applied for the development of cry1Ac transgenic pigeon pea. Bioactivity of the cry1Ac gene was compared based on integration and expression driven by two promoters, the constitutive CaMV35S promoter and the green-tissue-specific ats1A promoter, in those transgenic events. The transgenic events also contained the selectable marker gene nptII flanked by loxP sites. Independent transgenic events expressing the Cre recombinase gene along with a linked bar selection marker were also developed. Integration and expression patterns of both cry1Ac and cre were confirmed through Southern and western blot analysis of T1 events. The constitutive expression of the Cry1Ac protein was found to be more effective for conferring resistant activity against H. armigera larvae in comparison to green-tissue-specific expression. Constitutively expressing Cry1Ac T1 events were crossed with Cre recombinase expressing T1 events. The crossing-based Cre/lox-mediated marker gene elimination strategy was demonstrated to generate nptII-free Cry1Ac-expressing T2 events. These events were subsequently analyzed in the T3 generation for the segregation of cre and bar genes. Five Cry1Ac-expressing T3 transgenic pigeon pea events were devoid of the nptII marker as well as cre-bar genes. H. armigera larval mortality in those marker-free T3 events was found to be 80-100%. The development of such nptII selectable marker-free Cry1Ac-expressing pigeon pea transgenics for the first time would greatly support the sustainable biotechnological breeding program for pod borer resistance in pigeon pea. KEY POINTS: • Constitutive expression of Cry1Ac conferred complete resistance against Helicoverpa armigera • Green-tissue-specific expression of Cry1Ac conferred partial pest resistance • Cre/lox-mediated nptII elimination was successful in constitutively expressing Cry1Ac transgenic pigeon pea events.

Optimization of selective agents for In vitro regeneration of sugarcane transformants using NPTII gene

In the present study, selection media was optimized, for in vitro regenerated sugarcane transformants using nptII gene as negative selectable marker, that kills non-transformed cells and ensures proliferation and growth of only transformed cells. To identify a suitable agent and its optimal dose, the embryogenic calli of sugarcane were bombarded with plasmid vector containing nptII gene, along with DREB gene. After dose optimization on untransformed calli with different selective agents and the post-bombarded transgenic calli were selectively proliferated onto selection media, composing MS+2,4-D (2.5 mg/L)+Kinetin (0.5 mg/L)+Proline (560 mg/L)+Sucrose (30 g/L) +Agar 8g/L supplemented with three promising selective agents viz. paromomycin (100 mg/L), kanamycin (50 mg/L) and geneticin (50 mg/L) for selection of transgenic calli. After two selection cycles of fifteen days each, the selected transformed putative calli were transferred onto shoot regeneration media composed of MS + IAA (5.0 mg/L) + Kin (0.5 mg/L) + BAP (0.5 mg/L), supplemented with similar selection agents. Regeneration was successful in calli carrying the nptII gene on all the regeneration media supplemented with different selective agents. Green and albino shoots with variable frequencies ranging from 13.33% to 22.58% proliferated as regenerants on different selection media. PCR analysis revealed higher number of putative transformants selected on paromomycin @ 100 mg/L, as compared to other two selection agents. Among the three different selection agents, paromomycin was observed to be the best for selection of transformed calli, using nptII gene in sugarcane.