Single-copy Transgenic Lines Research Articles

Arsenate reductase gene from Pityrogramma calomelanos L. enhances tolerance to arsenic in tobacco

Arsenic (As) contamination in soil, water and air is an alarming issue worldwide and has serious effects on human health and environment. Arsenic is a naturally occurring element found in rocks, soil, and water, and exposure to high levels of arsenic can lead to a range of health problems. The effects of arsenic contamination can also be felt in the environment, as it can harm plants and animals and disrupt ecological systems. The major purpose of this study was to produce transgenic plants with improved tolerance to and accumulation of arsenic via transformation of arsenate reductase gene (ArsC) into tobacco genome. Transgenic plants were screen by PCR and southern blot. Further, their tolerance and accumulation to arsenic were evaluated. In the result, we have cloned, characterized, and transformed the ArsC gene from Pityrogramma calomelanos L. (PcArsC). Its phylogenetic analysis revealed 99% homology to ArsC gene in GenBank (accession number X80057.1). Moreover, Southern blot analysis showed that ArsC gene was integrated into the tobacco genome as a single-copy. These single-copy transgenic lines showed much higher tolerance to and accumulation of As than wild type, with no other phenotypes observed. These results demonstrated that Pityrogramma calomelanos ArsC gene can improve arsenic tolerance and accumulation in transgenic tobacco lines. Thus, using Pityrogramma calomelanos L. ArsC gene for genetic engineering has potential implications in the decontamination of arsenic-containing soil.

Expression of Arabidopsis NPR1 (AtNPR1) in Brassica juncea var Varuna confers significant resistance against Sclerotinia sclerotiorum and Alternaria brassicae

The productivity of Brassica juncea, a major oilseed crop grown widely in South Asia, is limited by two major fungal pathogens: Sclerotinia sclerotiorum, and Alternaria brassicae. Currently, no effective resistance against these pathogens has been identified in the B. juncea germplasm. To improve the resistance against these fungal pathogens, Non-expressor of PR1 (NPR1) from Arabidopsis was expressed in the mega variety Varuna via Agrobacterium-mediated transformation. A total of 129, independent transgenic events were generated. Segregation analysis for Basta resistance and subsequent Southern hybridization identified six single-copy transgenic lines (PN23, 42, 60, 72, HN30, and HN32) which were taken forward for disease assays over two generations. Transgenic lines challenged with S. sclerotiorum exhibited a significant reduction in stem lesions ranging from 13 to 42% in the T3 generation and 4 to 31% in the T4 generation. We also observed a reduced infection against A. brassicae in the transgenic lines which developed smaller and fewer necrotic lesions as compared to untransformed lines. A correlation between the upregulation of defense marker genes and enhanced tolerance was observed in the transgenic lines upon S. sclerotiorum infection.

Expression of barley oxalate oxidase confers resistance against Sclerotinia sclerotiorum in transgenic Brassica juncea cv Varuna.

Sclerotinia Stem Rot (SSR) caused by the oxalic acid (OA)-secreting necrotrophic fungal pathogen Sclerotinia sclerotiorum, causes significant yields losses in the crop Brassica sps. Oxalate oxidase (OxO) can metabolize OA to CO2 and H2O2. Degradation of OA during the early phase of fungal-host interaction can interfere with the fungal infection and establishment processes. The present study demonstrates the potential of barley oxalate oxidase (BOxO) gene in conferring stable resistance against stem rot in a productive and highly susceptible Brassica juncea cv Varuna under field conditions. Four stable, independent, single-copy transgenic lines (B16, B17, B18, and B53) exhibited a significant reduction in the rate of lesion expansion i.e. 11-26%, 39-47%, and 24-35% reproducibly over the three-generation i.e. T2, T3, and T4 respectively. The enhanced resistance in the transgenic lines correlated with high OxO activity, accumulation of higher levels of H2O2, and robust activation of defense responsive genes upon infection by S. sclerotiorum.

Constitutive overexpression of rice metallothionein-like gene OsMT-3a enhances growth and tolerance of Arabidopsis plants to a combination of various abiotic stresses.

Metallothioneins (MT) are primarily involved in metal chelation. Recent studies have shown that MT proteins are also involved in the responses of plants to various environmental stresses. The rice metallothionein-like gene OsMT-3a is upregulated by salinity and various abiotic stressors. A DNA construct containing the complete OsMT-3a coding sequence cloned downstream to the CaMV35S promoter was transformed into Arabidopsis and homozygous single-copy transgenic lines were produced. Compared to wild-type plants, transgenic plants showed substantially increased salinity tolerance (NaCl), drought tolerance (PEG), and heavy metal tolerance (CdCl2) as individual stresses, as well as different combinations of these stresses. Relevantly, under unstressed control conditions, vegetative growth of transgenic plants was also improved. The shoot Na+ concentration and hydrogen peroxide in transgenic plants were lower than those in wild-type plants. OsMT-3a-overexpressing Arabidopsis lines accumulated higher levels of Cd2+ in both shoots and roots following CdCl2 treatment. In the transgenic MT-3a lines, increased activity of two major antioxidant enzymes, catalase and ascorbate peroxidase, was observed. Thus, rice OsMT-3a is a valuable target gene for plant genetic improvement against multiple abiotic stresses.

Generation of a selectable marker free, highly expressed single copy locus as landing pad for transgene stacking in sugarcane.

A selectable marker free, highly expressed single copy locus flanked by insulators was created as landing pad for transgene stacking in sugarcane. These events displayed superior transgene expression compared to single-copy transgenic lines lacking insulators. Excision of the selectable marker gene from transgenic sugarcane lines was supported by FLPe/FRT site-specific recombination. Sugarcane, a tropical C4 grass in the genus Saccharum (Poaceae), accounts for nearly 80% of sugar produced worldwide and is also an important feedstock for biofuel production. Generating transgenic sugarcane with predictable and stable transgene expression is critical for crop improvement. In this study, we generated a highly expressed single copy locus as landing pad for transgene stacking. Transgenic sugarcane lines with stable integration of a single copy nptII expression cassette flanked by insulators supported higher transgene expression along with reduced line to line variation when compared to single copy events without insulators by NPTII ELISA analysis. Subsequently, the nptII selectable marker gene was efficiently excised from the sugarcane genome by the FLPe/FRT site-specific recombination system to create selectable marker free plants. This study provides valuable resources for future gene stacking using site-specific recombination or genome editing tools.

Development and Characterization of Transgenic Sugarcane with Insect Resistance and Herbicide Tolerance.

Genetically modified crops which had been commercial applied extensively majorly are the insect resistance and herbicide tolerance events. In this study, the Bt insecticidal gene Cry1Ab, the glyphosate-tolerant gene EPSPS, and the selection marker gene PMI were combined into a single transferred DNA fragment and introduced into sugarcane by Agrobacterium-mediated transformation. Thirty-three resistant plantlets were obtained after selection using a PMI/mannose selection system. Thirty of these resistant plantlets were PCR positive for the three target genes. Southern blot assay revealed that the copy number of the integrated fragment in the transformed plantlets varied from 1 to 7. ELISA analysis showed that 23 of the 33 resistant plantlets expressed Cry1Ab and EPSPS protein. Five single-copy and ELISA-positive transgenic lines were tested under laboratory and field conditions to determine their resistance to insects and herbicides, and also evaluated their agronomic characteristics and industrial traits. Results showed that larvae fed with fodder mixture containing stem tissues from single-copy transgenic lines were weak and small, moreover, pupation and eclosion were delayed significantly during voluntary feeding bioassays. None of transgenic sugarcane was destroyed by cane borer while more than 30% of wild type sugarcane was destroyed by cane borer. For herbicide resistance, the transgenic plantlets grew healthy even when treated with up to 0.5% roundup while wild type plantlets would die off when treated with 0.1% roundup. Thus demonstrate that these transgenic lines showed strong insect resistance and glyphosate tolerance under both laboratory and field conditions. But in the field most of the transgenic plants were shorter and more slender than non-transformed control plants. So they presented poor agronomic characteristics and industrial traits than non-transformed control plants. Thus, a considerable number of embryogenic calli should be infected to obtain transgenic lines with potential for commercial use.

A 64-bp sequence containing the GAAGA motif is essential for CaMV-35S promoter methylation in gentian

This study investigated sequence specificity and perenniality of DNA methylation in the cauliflower mosaic virus (CaMV) 35S promoter of transgenic gentian (Gentiana triflora×G. scabra) plants. Unlike conventional transgene silencing models, 35S promoter hypermethylation in gentian is species-specific and occurs irrespective of the T-DNA copy number and genomic location. Modified 35S promoters were introduced into gentian, and single-copy transgenic lines were selected for methylation analysis. Modified 35S promoter lacking a core (−90) region [35S(Δcore)] in gentian conferred hypermethylation and high levels of de novo methylation of the CpHpH/CpCpG sites in the 35S enhancer regions (−298 to −241 and −148 to −85). Therefore, promoter transcription may not be an absolute requirement for the methylation machinery. In vitro, de novo methylation persisted for more than eight years. In another modified 35S promoter, two “GAAGA” motifs (−268 to −264 and −135 to −131) were replaced by “GTTCA” in the two highly de novo methylated regions. It did not support hypermethylation and showed transgene expression. A 64-bp fragment of the 35S enhancer region (−148 to −85) was introduced into gentian and the resultant transgenic lines analyzed. The 64-bp region exhibited hypermethylation at the CpG/CpWpG sites, but the CpHpH/CpCpG methylation frequency was lower than those of the unmodified 35S- and 35S(Δcore) promoters. Nevertheless, a distinct CpHpH/CpCpG methylation peak was found in the 64-bp region of all single-copy transgenic lines. These results suggest that the 64-bp region may contain an element required for 35S methylation but insufficient for high de novo methylation compared with those in the unmodified 35S and 35S(Δcore) promoters.

Enhancement of sheath blight tolerance in transgenic rice by combined expression of tobacco osmotin (ap24) and rice chitinase (chi11) genes

Agrobacterium-mediated transformation was used to introduce pathogenesis-related protein genes into scutellum-derived callus of rice (Oryza sativa L. subsp. indica ‘White Ponni’) as a means of increasing resistance to sheath blight disease caused by Rhizoctonia solani Kuhn. Transformation with a tobacco osmotin (ap24) gene driven by the Cauliflower mosaic virus 35S promoter (P35S) yielded six single-copy transgenic lines. Homozygous T2 plants of all lines accumulated high levels of the ap24 transcript and the osmotin protein and reduced the percent disease index (PDI) of sheath blight disease from 100% down to 49–77%. Transformation with the binary plasmid pNSP2, which harbored the P35S-ap24 gene and a maize ubiquitin promoter (PUbi1)-driven rice chitinase (chi11) gene in the same T-DNA, yielded three single-copy transgenic lines: CO1, CO2, and CO3. Homozygous T2 plants of all three lines accumulated high levels of the chi11 transcript and the chitinase protein. Accumulation of ap24 transcript and osmotin protein was high in homozygous CO1 and CO2, but very low in CO3. At 7 d post infection with R. solani, the transgenic rice lines C18a (with chi11 alone), O8 (with ap24 alone), and CO1, CO2, and CO3 (with chi11 + ap24) reduced the sheath blight PDI from 100% to 64.2, 57.2, 43.3, 50.2, and 58.6%, respectively. At 15 d post infection, the sheath blight disease PDI was significantly lower in the CO1 and CO2 transgenic rice lines, which expressed both chi11 and ap24, than in those which expressed either chi11 or ap24 individually.

The activities of four constitutively expressed promoters in single-copy transgenic rice plants for two homozygous generations.

We have characterized four novel constitutive promoters ARP1, H3F3, HSP and H2BF3 that are active in all tissues/stages of transgenic plants and stable over two homozygous generations. Gene promoters that are active and stable over several generations in transgenic plants are valuable tools for plant research and biotechnology. In this study, we characterized four putative constitutive promoters (ARP1, H3F3, HSP and H2BF3) in transgenic rice plants. Promoter regions were fused to the green fluorescence protein (GFP) reporter gene and transformed into rice. Single-copy transgenic lines were then selected and promoter activity was analyzed in various organs and tissues of two successive homozygous generations. All four promoters showed a broad expression profile in most tissues and developmental stages, and indeed the expression of the ARP1 and H3F3 promoters was even greater than that of the PGD1 promoter, a previously described constitutive promoter that has been used in transgenic rice. This observation was based on expression levels in leaves, roots, dry seeds and flowers in both the T2 and T3 generations. Each promoter exhibited comparable levels of activity over two homozygous generations with no sign of transgene silencing, which is an important characteristic of promoters to be used in crop biotechnology applications. These promoters therefore have considerable potential value for the stable and constitutive expression of transgenes in monocotyledonous crops.

Transgenic rice lines constitutively co-expressing tlp-D34 and chi11 display enhancement of sheath blight resistance

Transgenic rice (Oryza sativa L. subsp. indica cv. White Ponni) constitutively expressing the rice thaumatin-like protein gene (tlp-D34, PR-5) individually or in combination with the rice chitinase gene (chi11, PR-3) was generated using an Agrobacterium vir helper strain with multiple copies of pTiBo542 virB and virG. Transformation with the tlp-D34 gene alone and tlp-D34 + chi11 genes yielded five and seven single-copy transgenic lines, respectively. Southern blot analysis with two probes, one flanking the right T-DNA border and the second flanking the left T-DNA border, confirmed that all transgenic plants harboured single and complete T-DNA copies. Homozygous transgenic lines were first identified in the T1 generation by Southern blot analysis and were subsequently confirmed by segregation analysis of T2 plants. Accumulation of transcripts encoded by the transgenes was confirmed in T0 plants and homozygous T2 plants by Northern blot analysis. The homozygous T2 plants harbouring tlp-D34 + chi11 genes showed 2.8- to 4.2-fold higher chitinase activity. Western blot analysis revealed the accumulation of thaumatin-like protein and chitinase in the respective transgenic plants. Upon infection with Rhizoctonia solani, the disease index reduced from 100 % in control plants to 65 % in a T3 homozygous transgenic line T4 expressing the tlp-D34 gene alone. In a T2 homozygous transgenic line CT22 co-expressing tlp-D34 and chi11 genes, the disease index reduced to 39 %.

Characterization of the stress-inducible OsNCED3 promoter in different transgenic rice organs and over three homozygous generations

To be effective in crop biotechnology applications, gene promoters need to be stably active over sequential generations in a population of single-copy transgenic lines. Most of the stress-inducible promoters characterized in plants thus far have been analyzed at early (T₀, T₁ or T₂) generations and/or by testing only a small number of transgenic lines. In our current study, we report our analysis of OsNCED3, a stress-inducible rice promoter involved in ABA biosynthesis, in various organs and tissues of transgenic rice plants over the T(2-4) homozygous generations. The transgene copy numbers in the lines harboring the OsNCED3:gfp construct were determined and six single- and two double-copy transgenic lines were analyzed for promoter activity in comparison with the Wsi18, a stress-inducible promoter previously characterized. The exogenous promoter activities were found to be significantly enhanced in the roots and leaves, whereas zero or low levels of activity were evident in grains and flowers, under drought and high-salinity conditions. The highest induction levels of gfp transcripts in the OsNCED3:gfp plants upon drought treatments were 161- and 93-fold in leaves and roots, respectively, and these levels were comparable with those of gfp transcripts in the Wsi18:gfp plants. A comparison of the promoter activities between the T₂-T₄ plants revealed that comparable activity levels were maintained over these three homozygous generations with no evidence of silencing. Thus, our results provide the OsNCED3 promoter that is stress-inducible in a whole rice plant except for in the aleurones and endosperm and stably active over three generations.

Establishment of homozygous transgenic rice lines after elimination of unlinked albino traits from transgenic events through segregation

In an analysis of 339 independent T 0 transgenic rice lines generated by Agrobacterium-mediated transformation, albino plants appeared in the T 1 generation in two single-copy transgenic lines, O54 and O36 and in one double-copy transgenic line, C18. While the T 0 plants of these three lines were green, albino and green plants emerged in a 1:3 ratio in the T 1 generation. The albino phenotype segregated as a monogenic recessive trait. Southern blot analysis of the green and albino plants in the T 1 generation confirmed that the albino trait and the T-DNA insertion events were unlinked. Segregation of the albino trait from the transgenic trait in the lines O54 and O36 was confirmed in T 2 and T 3 generations, respectively. Homozygous transgenic plants free from the albino trait were also identified. In the double-copy transgenic line C18, we genetically separated the two transgenic loci, out-segregated the albino locus from both transgene loci, and identified homozygous plants for each of the transgenic events by Southern blot analysis in the T 1 generation itself. Thus, we demonstrate that when an albino trait appears in the T 1 generation and is unlinked to a transgene locus, the albino locus can be segregated from the transgene locus and homozygous transgenic lines free from albinos can be established.

Combined expression of chitinase and β-1,3-glucanase genes in indica rice ( Oryza sativa L.) enhances resistance against Rhizoctonia solani

Agrobacterium-mediated transformation of rice was done using the binary vector pNSP3, harbouring the rice chitinase ( chi11) gene under maize ubiquitin promoter and the tobacco β-1,3-glucanase gene under CaMV 35S promoter in the same T-DNA. Four of the six T 0 plants had single copies of complete T-DNAs, while the other two had complex integration patterns. Three of the four single-copy lines showed a 3:1 segregation ratio in the T 1 generation. Northern and western blot analyses of T 1 plants revealed constitutive expression of chitinase and β-1,3-glucanase genes. Homozygous T 2 plants of the single-copy lines CG20, CG27 and CG53 showed 62-, 9.6- and 11-fold higher chitinase activity over the control plants. β-1,3-Glucanase activity was 1.1- to 2.5-fold higher in the transgenic plants. Bioassay of homozygous T 2 plants of the three single-copy transgenic lines against Rhizoctonia solani revealed a 60% reduction in sheath blight Disease Index in the first week. The Disease Index increased from 61.8 in the first week to 90.6 in the third week in control plants, while it remained low (26.8–34.2) in the transgenic T 3 plants in the corresponding period, reflecting the persistence of sheath blight resistance for a longer period.

Transgenic American elm shows reduced Dutch elm disease symptoms and normal mycorrhizal colonization

The American elm (Ulmus americana L.) was once one of the most common urban trees in eastern North America until Dutch-elm disease (DED), caused by the fungus Ophiostoma novo-ulmi, eliminated most of the mature trees. To enhance DED resistance, Agrobacterium was used to transform American elm with a transgene encoding the synthetic antimicrobial peptide ESF39A, driven by a vascular promoter from American chestnut. Four unique, single-copy transgenic lines were produced and regenerated into whole plants. These lines showed less wilting and significantly less sapwood staining than non-transformed controls after O. novo-ulmi inoculation. Preliminary observations indicated that mycorrhizal colonization was not significantly different between transgenic and wild-type trees. Although the trees tested were too young to ensure stable resistance was achieved, these results indicate that transgenes encoding antimicrobial peptides reduce DED symptoms and therefore hold promise for enhancing pathogen resistance in American elm.

Efficient in vivo manipulation of mouse genomic sequences at the zygote stage.

We describe a transgenic mouse line carrying the cre transgene under the control of the adenovirus EIIa promoter that targets expression of the Cre recombinase to the early mouse embryo. To assess the ability of this recombinase to excise loxP-flanked DNA sequences at early stages of development, we bred EIIa-cre transgenic mice to two different mouse lines carrying loxP-flanked target sequences: (i) a strain with a single gene-targeted neomycin resistance gene flanked by 1oxP sites and (ii) a transgenic line carrying multiple transgene copies with internal loxP sites. Mating either of these loxP-carrying mouse lines to EIIa-cre mice resulted in first generation progeny in which the loxP-flanked sequences had been efficiently deleted from all tissues tested, including the germ cells. Interbreeding of these first generation progeny resulted in efficient germ-line transmission of the deletion to subsequent generations. These results demonstrate a method by which loxP-flanked DNA sequences can be efficiently deleted in the early mouse embryo. Potential applications of this approach are discussed, including reduction of multicopy transgene loci to produce single-copy transgenic lines and introduction of a variety of subtle mutations into the line.