35S Promoter Research Articles

Morpho-physiological investigations in transgenic tomato (Solanum lycopersicum L.) over expressing OsRGLP1 gene

The OsRGLP1 gene was overexpressed under the control of CaMV 35S promoter in tomato (Solanum lycopersicum L.) plants using Agrobacterium-mediated transformation. The transgene expression was validated using RT-qPCR (real-time quantitative polymerase chain reaction) in the transgenic plants. Noticeable morpho-physiological traits, higher SOD activity, and elevated histological H2O2 were manifested by transgenic tomato lines. An increase in total chlorophyll and relative water content (RWC) in the leaves of transgenic lines was noted. The electrolytic leakage/conductivity (EC) in leaves of transgenic lines was lower than the control. The transgenic tomato lines showed dwarf phenotypic features in vegetative and floral traits. Interestingly, the vascular cambium regions in cross sections of stem showed secondary growth in radial symmetry. The transgenic lines also produced higher density and diversity of trichomes. The differences observed in transgenic lines are quite interesting which need to be further explored referring to the functional aspects of the transgene with reference to stress.

The expansin gene NtEXPA5 increases stress tolerance of tobacco hairy roots through an effect on the antioxidant system

BACKGROUND: Expansins are non-enzymatic proteins involved in the softening of cell walls, the mechanism of action of which is associated with the weakening and breaking of hydrogen bonds between xyloglucans and cellulose microfibrils and is aimed at ensuring cell expansion.
 THE AIM of our work was to obtain hairy roots of tobacco with constitutive expression of the NtEXPA5 expansin gene, their morphometric analysis and assessment of the state of their antioxidant system in response to stress factors.
 MATERIALS AND METHODS: The hairy roots were obtained from transgenic tobacco plants expressing the NtEXPA5 gene under the control of the 35S promoter.
 RESULTS: Constitutive expression of the NtEXPA5 gene promoted an increase in the length and dry weight of hairy roots both under normal conditions and under the action of salinity, copper sulfate, cadmium acetate, and mannitol. Both under normal conditions and under the action of stress factors in transgenic hairy roots, an increase in the activity of superoxide dismutase and the total antioxidate activity was recorded.
 CONCLUSION: Expansins exert their positive effect on the productivity and stress tolerance of plants not only through their influence on cell expansion, but also through the effect on the antioxidant system.

Development of a Systematic qPCR Array for Screening GM Soybeans.

A screening method using the 35S promoter and nos terminator for genetically modified organisms (GMOs) is not sufficient to cover all GM soybean events. In this study, a real-time polymerase chain reaction (also known as quantitative polymerase chain reaction, qPCR) array targeting eight screening assays combined with a prediction system was developed for the rapid tracking of GM soybeans. Each assay’s specificity was tested and confirmed using 17 GM soybean events that have been approved in Korea. The sensitivity of each assay was determined to range from 0.01% to 0.05% using DNA mixtures with different GM ratios, and it was validated by the results of three experimenters. The applicability of this study was tested by monitoring 23 processed foods containing soybeans. It was figured out that 13 of the 23 samples included GM soybeans. The prediction system combined with screening results will be helpful to trace the absence/presence of GM soybean events. This new qPCR array and prediction system for GM soybean detection provides rapid, convenient and reliable results to users.

A valine residue deletion in ZmSig2A, a sigma factor, accounts for a revertible leaf-color mutation in maize

A nuclear-encoded sigma (σ) factor is essential for the transcriptional regulation of plant chloroplast-encoded genes. Five putative maize σ factors have been identified by database searches, but their functions are unknown. We report a maize leaf color mutant etiolated/albino leaf 1 (eal1) that was derived from space mutation breeding. The eal1 mutant displays etiolated or albino leaves that then gradually turn to normal green at the seedling stage. The changes in eal1 leaf color are associated with changes in photosynthetic pigment content and chloroplast development. Map-based cloning revealed that a single amino-acid deletion changing Val480-Val481-Val482 to Val480-Val481, in the C-terminal domain σ4 of the putative σ factor ZmSig2A, is responsible for the eal1 mutation. In comparison with the expression level of the wild-type (WT) allele ZmSig2A+ in WT plants, much higher expression of the mutant allele ZmSig2AΔV in eal1 plants was detected before the eal1 plants turned to normal green. ZmSig2A shows the highest similarity to rice OsSig2A and Arabidopsis SIG2. Ectopic expression of ZmSig2A+ or ZmSig2AΔV driven by the cauliflower mosaic virus 35S promoter rescued the pale green leaf of the sig2 mutant, but ectopic expression of ZmSig2AΔV driven by the SIG2 promoter did not. We propose that the Val deletion generated a new weak allele of ZmSig2A that cannot completely abolish the ZmSig2A function. Some genes involved in chloroplast development and photosynthesis-associated nuclear genes showed significant expression differences between eal1 and WT plants. We conclude that ZmSig2A encoding a σ factor is essential for maize chloroplast development. The eal1 mutant with a weak allele of ZmSig2A represents a valuable genetic resource for investigating the regulation of ZmSig2A-mediated chloroplast development in maize. The eal1 mutation may be useful as a marker for early identification and elimination of false hybrids or transgene transmission in the application of genetic male sterility to commercial hybrid seed production.

The effects of PROTEOLYSIS6 (PRT6) gene suppression on the expression patterns of tomato ethylene response factors

The ubiquitin-mediated proteolysis via Arg N-degron branch regulates various plant developmental processes and responds to multiple stresses. Nevertheless, limited information is available on the significance of the pathway during fruit ripening. This study reports that the suppression of PROTEOLYSIS6 (PRT6) E3 ligase in tomato through RNAi altered the gene expression of four Ethylene Response Factors (ERFs) (SIERF77, SIERF2, JERF1, and JERF3). The transient expression of 35S::PRT6_RNAi and PG::PRT6_RNAi constructs successfully down-regulated the PRT6 expression in leaves and mature green fruits through 35S promoter, and in breaker-stage fruits via polygalacturonase (PG) promoter, respectively. Reverse transcription-quantitative PCR (RT-qPCR) analysis showed an increased expression of SIERF77, SIERF2, and JERF1 but decreased JERF3 expression in the leaves and mature green fruits agroinfiltrated with 35S::PRT6_RNAi. Ripening-specific SIERF2 showed an increased expression in the breaker-stage fruits agroinfiltrated with PG::PRT6_RNAi, supporting the hypothesis that a low expression level of PRT6 affected the expression patterns of ERFs in specific tissues of tomato. Conversely, the expression of pectin methylesterase (PME) decreased in the breaker fruits. The four orthologues of tomato ERFs had been found to be the Arg N-degron substrates in Arabidopsis, with physiological functions being regulated at the protein level. This study showed that the suppression of PRT6 influenced the expression of these four transcription factors and PME.

35S promoter-driven transgenes are variably expressed in different organs of Arabidopsis thaliana and in response to abiotic stress.

The cauliflower mosaic virus (CaMV) 35S promoter is known as the most frequently used promoter in plant biotechnology. Although it is widely considered to be a strong constitutive promoter exhibiting high transcriptional activity, the transcriptional stability of CaMV 35S has not been extensively studied. Using the model plant species Arabidopsis thaliana, this study aimed for a comprehensive expression analysis of two widely used plant transgenes, neomycin phosphotransferase II (NPTII) and enhanced green fluorescent protein (EGFP), regulated by a double CaMV 35S promoter depending on the organ type, time of day, plant age, and in response to abiotic stress conditions. Quantitative real-time PCR (qRT-PCR) analysis revealed that the NPTII and EGFP transcript levels were markedly higher in the cotyledons, young leaves, and roots than in the inflorescences, stems, and adult leaves of three independent transgenic A. thaliana lines. The expression of NPTII and EGFP varied during the day and was elevated with the plant age. Drought and cold stress considerably affected the expression of the transgenes, while heat, high salinity, and wounding had no significant effect. This study shows that transgenes driven by a common constitutive promoter can exhibit marked variations in transcriptional activity depending on plant organ, physiological conditions, and in response to abiotic stress. Therefore, to ensure high and stable transgene activity, considerable attention should be given to the transgenic plant material and incubation conditions before harvesting the plant material.

Phloem loading in rice leaves depends strongly on the apoplastic pathway.

Phloem loading is the first step in sucrose transport from source leaves to sink organs. The phloem loading strategy in rice remains unclear. To determine the potential phloem loading mechanism in rice, yeast invertase (INV) was overexpressed by a 35S promoter specifically in the cell wall to block sugar transmembrane loading in rice. The transgenic lines exhibited obvious phloem loading suppression characteristics accompanied by the accumulation of sucrose and starch, restricted vegetative growth and decreased grain yields. The decreased sucrose exudation rate with p-chloromercuribenzenesulfonic acid (PCMBS) treatment also indicated that rice actively transported sucrose into the phloem. OsSUT1 (SUCROSE TRANSPORTER 1) showed the highest mRNA levels of the plasma membrane-localized OsSUTs in source leaves. Cross sections of the OsSUT::GUS transgenic plants showed that the expression of OsSUT1 and OsSUT5 occurred in the phloem companion cells. Rice ossut1 mutants showed reduced growth and grain yield, supporting the hypothesis of OsSUT1 acting in phloem loading. Based on these results, we conclude that apoplastic phloem loading plays a major role in the export of sugar from rice leaves.

Gene expression patterns in shoots of Camelina sativa with enhanced salinity tolerance provided by plant growth promoting bacteria producing 1-aminocyclopropane-1-carboxylate deaminase or expression of the corresponding acdS gene

Growth of plants in soil inoculated with plant growth promoting bacteria (PGPB) producing 1-aminocyclopropane-1-carboxylate (ACC) deaminase or expression of the corresponding acdS gene in transgenic lines reduces the decline in shoot length, shoot weight and photosynthetic capacity triggered by salt stress in Camelina sativa. Reducing the levels of ethylene attenuated the salt stress response as inferred from decreases in the expression of genes involved in development, senescence, chlorosis and leaf abscission that are highly induced by salt to levels that may otherwise have a negative effect on plant growth and productivity. Growing plants in soil treated with Pseudomonas migulae 8R6 negatively affected ethylene signaling, auxin and JA biosynthesis and signalling, but had a positive effect on the regulation of genes involved in GA signaling. In plants expressing acdS, the expression of the genes involved in auxin signalling was positively affected, while the expression of genes involved in cytokinin degradation and ethylene biosynthesis were negatively affected. Moreover, fine-tuning of ABA signaling appears to result from the application of ACC deaminase in response to salt treatment. Moderate expression of acdS under the control of the root specific rolD promoter or growing plants in soil treated with P. migulae 8R6 were more effective in reducing the expression of the genes involved in ethylene production and/or signaling than expression of acdS under the more active Cauliflower Mosaic Virus 35S promoter.

Development of a Virus-Based Reporter System for Functional Analysis of Plant rRNA Gene Promoter.

Reporter gene-based expression systems have been intensively used in plants for monitoring the activity of gene promoters. However, rRNA transcripts are unable to efficiently express a reporter gene due to a lack of a 5' cap. Because of this obstacle, plant rRNA gene promoters are less well characterized to this day. We developed a virus-based reporter system to characterize the Nicotiana benthamiana rRNA (NbrRNA) gene promoter. The system utilizes the cap-independent translation strategy of viral genomic mRNA and uses the virus-expressed green fluorescent protein (GFP) as an indicator of the rRNA gene promoter activity in virus-infected plants. Based on the reporter system, some characteristics of the N. benthamiana rRNA gene promoter were revealed. The results showed that the strength of the NbrRNA gene promoter was lower than that of the cauliflower mosaic virus (CaMV) 35S promoter, a well-characterized polymerase II promoter. The sequences between −77 and +42 are sufficient for the NbrRNA gene promoter-mediated transcription and the NbrRNA gene promoter may lack the functional upstream control element (UCE). Interestingly, NbrRNA gene promoter activity was increased when the 35S enhancer was introduced. An intron-excision mediated assay revealed that the NbrRNA gene promoter can be inefficiently used by RNA polymerase II in N. benthamiana cells. This virus-based reporter system is easier to operate and more convenient when compared with the previously Pol I promoter assays. And it offers a promising solution to analyzing the functional architecture of plant rRNA gene promoter.

Construction and biological characterization of an infectious full-length cDNA clone of a Chinese isolate of Wheat yellow mosaic virus

Wheat yellow mosaic virus (family Potyviridae; genus Bymovirus), is an important soil-borne virus that causes serious economic losses in wheat. In this study, we constructed infectious cDNA clones of WYMV genomic RNAs under the control of 35S or SP6 promoter for versatile usage (agroinfiltration or in vitro RNA transcription). Our results showed that an Agrobacterium-mediated inoculation system enabled WYMV to infect the leaves of Nicotiana benthamiana without causing WYMV systemic infection. However, in vitro transcripts from infectious cDNA clones using the SP6 promoter promoted WYMV systemic infection of wheat plants, which was then developed for further assays. The optimal temperature for virus multiplication and systemic infection of wheat was 8 °C. Additionally, a synergistic effect between WYMV and Chinese wheat mosaic virus (CWMV) was also detected. This is the first report of the construction of a Chinese isolate of WYMV and should facilitate the investigation of viral pathogenesis.

SiFBA5, a cold-responsive factor from Saussurea involucrata promotes cold resilience and biomass increase in transgenic tomato plants under cold stress

BackgroundSaussurea involucrata survives in extreme arctic conditions and is very cold-resistant. This species grows in rocky, mountainous areas with elevations of 2400–4100 m, which are snow-covered year-round and are subject to freezing temperatures. S. involucrata’s ability to survive in an extreme low-temperature environment suggests that it has particularly high photosynthetic efficiency, providing a magnificent model, and rich gene pool, for the analysis of plant cold stress response. Fructose-1, 6-bisphosphate aldolase (FBA) is a key enzyme in the photosynthesis process and also mediates the conversion of fructose 1, 6-bisphosphate (FBP) into dihydroxyacetone phosphate (DHAP) and glycerol triphosphate (GAP) during glycolysis and gluconeogenesis. The molecular mechanisms underlying S. involucrata’s cold tolerance are still unclear; therefore, our work aims to investigate the role of FBA in plant cold-stress response.ResultsIn this study, we identified a cold-responsive gene, SiFBA5, based on a preliminary low-temperature, genome-wide transcriptional profiling of S. involucrata. Expression analysis indicated that cold temperatures rapidly induced transcriptional expression of SiFBA5, suggesting that SiFBA5 participates in the initial stress response. Subcellular localization analysis revealed that SiFBA5 is localized to the chloroplast. Transgenic tomato plants that overexpressed SiFBA5 were generated using a CaMV 35S promoter. Phenotypic observation suggested that the transgenic plants displayed increased cold tolerance and photosynthetic efficiency in comparison with wild-type plants.ConclusionCold stress has a detrimental impact on crop yield. Our results demonstrated that SiFBA5 positively regulates plant response to cold stress, which is of great significance for increasing crop yield under cold stress conditions.

Overexpression of miR319 in petunia (Petunia × hybrida) promotes de novo shoot organogenesis from leaf explants

MicroRNA319 (miR319) plays an important role in leaf development. Although leaf discs are frequently used for plant transformation, little is known about the function of miR319 in shoot regeneration from leaf explants. To investigate the role of miR319 in de novo shoot organogenesis from leaf explants, the MIR319 gene was overexpressed in petunia (Petunia × hybrida), under the control of the 35S promoter. Petunia leaves overexpressing miR319 (miR319-OX) showed several morphological abnormalities, such as rounded, uneven, and curled leaf blade, larger leaf size, and smaller and rounded abaxial surface cells, which were differences compared to wild-type (WT) leaves. Additionally, miR319-OX leaf explants demonstrated enhanced shoot organogenesis on a growth medium containing 6-benzylaminopurine (BA) at shoot-inducing concentrations, and cytokinin-independent shoot regeneration on hormone-free medium compared with WT explants. Quantitative real-time PCR (qRT-PCR) analysis indicated that the expression of petunia NO APICAL MERISTEM (PhNAM) and SHOOT MERISTEMLESS (PhSTM) genes was markedly increased in miR319-OX explants compared with WT explants under shoot induction conditions, whereas the expression of petunia cytokinin-responsive type-A response regulator genes, PhRR2 and PhRR3, showed no differences in expression between WT and miR319-OX leaf explants. These results suggest that miR319 regulates the formation of shoot meristems from leaf explants in petunia during in vitro culture.

IMPERFECTIVE EXINE FORMATION (IEF) is required for exine formation and male fertility in Arabidopsis

IEF, a novel plasma plasma membrane protein, is important for exine formation in Arabidopsis. Exine, an important part of pollen wall, is crucial for male fertility. The major component of exine is sporopollenin which are synthesized and secreted by tapetum. Although sporopollenin synthesis has been well studied, the transportation of it remains elusive. To understand it, we analyzed the gene expression pattern in tapetal microdissection data, and investigated the potential transporter genes that are putatively regulated by ABORTED MICROSPORES (AMS). Among these genes, we identified IMPERFECTIVE EXINE FORMATION (IEF) that is important for exine formation. Compared to the wild type, ief mutants exhibit severe male sterility and pollen abortion, suggesting IEF is crucial for pollen development and male fertility. Using both scanning and transmission electron microscopes, we showed that exine structure was not well defined in ief mutant. The transient expression of IEF-GFP driven by the 35S promoter indicated that IEF-GFP was localized in plasma membrane. Furthermore, AMS can specifically activate the expression of promoterIEF:LUC in vitro, which suggesting AMS regulates IEF for exine formation. The expression of ATP-BINDING CASSETTE TRANSPORTER G26 (AGCB26) was not affected in ief mutants. In addition, SEM and TEM data showed that the sporopollenin deposition is more defective in abcg26/ief-2 than that of in abcg26, which suggesting that IEF is involved in an independent sporopollenin transportation pathway. This work reveal a novel gene, IEF regulated by AMS that is essential for exine formation.

Heterologous expression of Arabidopsis thaliana rty gene in strawberry (Fragaria \xd7 ananassa Duch.) improves drought tolerance

BackgroundStrawberry (Fragaria × ananassa Duch.) is an important fruit crop worldwide. It was particularly sensitive to drought stress because of their fibrous and shallow root systems. Mutant rty of Arabidopsis thaliana ROOTY (RTY) results in increased endogenous auxin levels, more roots, and shoot growth. It is still unclear whether the rty gene improves stress tolerance in strawberry.Resultsrty gene was isolated from Arabidopsis thaliana and placed under the control of the cauliflower mosaic virus (CaMV) 35S promoter in the pBI121-rty binary vector carrying the selectable marker of neomycin phosphotransferase II (NPT II). Seven transgenic lines were confirmed by PCR and western blot analysis. Accumulations of IAA and ABA were significantly increased in the transgenic plants. The endogenous IAA contents were 46.5 ng g− 1 and 66.0 ng g− 1in control and transgenic plants respectively. The endogenous ABA contents in the control plant were 236.3 ng g− 1 and in transgenic plants were 543.8 ng g− 1. The production of adventitious roots and trichomes were enhanced in the transgenic plants. Furthermore, transcript levels of the genes including IAA and ABA biosynthetic, and stress-responsive genes, were higher in the transgenic plants than in the control plants under drought conditions. Water use efficiency and a reduced water loss rate were enhanced in the transgenic strawberry plants. Additionally, peroxidase and catalase activities were significantly higher in the transgenic plants than in the control plants. The experiment results revealed a novel function for rty related to ABA and drought responses.ConclusionsThe rty gene improved hormone-mediated drought tolerance in transgenic strawberry. The heterologous expression of rty in strawberry improved drought tolerance by promoting auxin and ABA accumulation. These phytohormones together brought about various physiological changes that improved drought tolerance via increased root production, trichome density, and stomatal closure. Our results suggested that a transgenic approach can be used to overcome the inherent trade-off between plant growth and drought tolerance by enhancing water use efficiency and reducing water loss rate under water shortage conditions.

Overexpression of the Golden SNP-Carrying Orange Gene Enhances Carotenoid Accumulation and Heat Stress Tolerance in Sweetpotato Plants.

Carotenoids function as photosynthetic accessory pigments, antioxidants, and vitamin A precursors. We recently showed that transgenic sweetpotato calli overexpressing the mutant sweetpotato (Ipomoea batatas [L.] Lam) Orange gene (IbOr-R96H), which carries a single nucleotide polymorphism responsible for Arg to His substitution at amino acid position 96, exhibited dramatically higher carotenoid content and abiotic stress tolerance than calli overexpressing the wild-type IbOr gene (IbOr-WT). In this study, we generated transgenic sweetpotato plants overexpressing IbOr-R96H under the control of the cauliflower mosaic virus (CaMV) 35S promoter via Agrobacterium-mediated transformation. The total carotenoid contents of IbOr-R96H storage roots (light-orange flesh) and IbOr-WT storage roots (light-yellow flesh) were 5.4–19.6 and 3.2-fold higher, respectively, than those of non-transgenic (NT) storage roots (white flesh). The β-carotene content of IbOr-R96H storage roots was up to 186.2-fold higher than that of NT storage roots. In addition, IbOr-R96H plants showed greater tolerance to heat stress (47 °C) than NT and IbOr-WT plants, possibly because of higher DPPH radical scavenging activity and ABA contents. These results indicate that IbOr-R96H is a promising strategy for developing new sweetpotato cultivars with improved carotenoid contents and heat stress tolerance.

Transformation and evaluation of Broad-Spectrum insect and weedicide resistant genes in Gossypium arboreum (Desi Cotton)

ABSTRACT Gossypium arboreum (Desi Cotton) holds a special place in cotton industry because of its inherent ability to withstand drought, salinity, and remarkable resistance to sucking pests and cotton leaf curl virus. However, it suffers yield losses due to weeds and bollworm infestation. Genetic modification of G. arboreum variety FBD-1 was attempted in the current study to combat insect and weedicide resistance by incorporating cry1Ac, cry2A and cp4-EPSPS genes under control of 35S promoter in two different cassettes using kanamycin and GUS as markers through Agrobacterium-mediated shoot apex cut method of cotton transformation. The efficiency of transformation was found to be 1.57%. Amplification of 1700 bp for cry1Ac, 167 bp for cry2A and 111 bp for cp4-EPSPS confirmed the presence of transgenes in cotton plants. The maximum mRNA expression of cry1Ac and cp4-EPSPS was observed in transgenic cotton line L3 while minimum in transgenic cotton line L1. The maximum protein concentrations of Cry1Ac, Cry2A and Cp4-EPSPS of 3.534 µg g−1, 2.534 µg g−1 and 3.58 µg-g−1 respectively were observed for transgenic cotton line L3 as compared to control cotton line. On leaf-feed-based insect bioassay, almost 99% mortality was observed for Helicoverpa armigera on the transgenic cotton plant (L3). It completely survived the 1900 ml hectare−1 glyphosate spray assay as compared to non-transgenic cotton plants. The necrotic spots appeared on the third day, leading to the complete death of control plants on the fifth day of assay. The successful multiple gene-stacking in G. arboreum FBD-1 variety could be further used for qualitative improvement of cotton fiber through plant breeding techniques.

An Efficient <i>Agrobacterium</i>-Mediated Genetic Transformation Using <i>cry1F</i> Gene in Castor ( <i>Ricinus communis</i> L.) for Protection Against <i>Lepidopteran</i> Insects

Castor ( Ricinus communis L.) is an essential non-edible, pharmaceutical, and industrial oilseed. The vulnerability of this economically valuable crop to foliage feeders like Spodoptera litura has resulted in a loss of production. This report focuses on the development of an optimized protocol for the transformation of castor shoot apices by Agrobacterium tumefaciens strain LBA4404 containing plasmid construct pBIN1F harboring neomycin phosphotransferase (nptII), as selectable marker gene and Bacillus thuringiensis var. aizawai (Bt) cry1F gene controlled by cauliflower mosaic virus 35S promoter. Several parameters like O.D. (O.D600-0.6), concentration of kanamycin (50 mg/L) and acetosyringone (100 µM) were optimized and produced a significant difference in the transformation efficiency. Co-cultivation time and seedling age were factors, with overall transformation efficiency of 2.0% in fifteen-day-old seedlings and co-cultivated for three days. The surviving and actively developing shoot apices were validated for gene integration by molecular analysis after being preliminarily screened on 50 mg/L kanamycin and 300 mg/L cefotaxime. PCR, qRT-PCR and insect bioassay were used to confirm the putative primary transformants. When bioassayed against newly hatched Spodoptera litura hatchlings, these putative transgenics with cry1F gene caused significant (≤93%) insect mortality. Cry1F gene expressing transgenic plants had adequate defence against Spodoptera litura when exposed to castor. This convention protocol would succeed by using Agrobacterium-mediated transformation to apply agronomically appealing qualities to castor.

Overexpression of

Sugarcane (Saccharum hybrid) is an important industrial crop worldwide. Its growth and sucrose contents are severely affected by drought stress. Genetic engineering offers a rapid solution to improve tolerance level of sugarcane against this stress. This study was designed to transform sugarcane with the Tomato ethylene responsive factor 1 (TERF1) gene through Agrobacterium. Embryogenic callus of sugarcane cv. XintaitangR22 was used for transformation with Agrobacterium strain LBA4404 harbouring the pROK2 vector containing the TERF1 gene driven by the CaMV 35S promoter. Highest regeneration efficiency (74%) was obtained with inoculum density (OD600) at 0.4 and co-cultivated for 4 days on MS-based medium; 5.4% transformation efficiency was acquired from the regenerated plants. Successful insertion of the TERF1 gene into sugarcane was indicated by PCR-positive plants (n = 4). Expression of TERF1 transcripts in transgenic lines at various levels was detected by reverse transcriptase-PCR. Under normal conditions, growth status of transgenic lines was similar to that of wild-type plants; by contrast, only transgenic lines were able to withstand water-deficit stress conditions, showing tolerance against drought stress. Physiological and biochemical assays revealed that TERF1-overexpressed plants showed not only increased accumulation of proline, soluble sugars and glycine betaine but also reduced malondialdehyde and H2O2 content in response to drought stress. Our results revealed that overexpression of TERF1 in sugarcane conferred drought tolerance through increased accumulation of osmo-protectant, decreasing reactive oxygen species and malondialdehyde content, which possibly resulted from activation of expression of stress-related genes by TERF1 under stress. These findings indicate that the gene might have a regulatory role in the response to drought stress in sugarcane.

Rapid Validation of Transcriptional Enhancers Using a Transient Reporter Assay.

Enhancers are one of the main classes of cis-regulatory elements (CREs) in the regulation of plant gene expression. Plant enhancers can be predicted based on genomic signatures associated with open chromatin. However, predicted enhancers need to be validated experimentally. We developed an experimental system for rapid enhancer validation. Predicted enhancer candidates are cloned into a vector containing a minimal 35S promoter and a luciferase reporter gene. The construct is then agroinfiltrated into Nicotiana benthamiana leaves followed by bioluminescence signal detection and analysis. Positive bioluminescence signals indicate the enhancer function of each candidate, and the relative signal strength from different enhancers can be quantitatively measured and compared. In summary, we have developed an efficient and rapid plant enhancer validation assay based on a bioluminescent luciferase reporter and agroinfiltration-based N. benthamiana leaf transient expression. This assay can be used for the initial screening of candidate enhancers that are active in leaf tissue. The system can potentially be used to examine the activity of candidate enhancers under different environmental conditions.

Transient assay for validating the efficacy of antifungal genes in plant cells rapidly against phytopathogens

The antifungal efcacy of 1, 3-glucanase and chitinase genes isolated from Trichoderma spp. was gauged in agroinfected rice callus cells following in vitro confrontation with the pathogen. The binary vector pBI121 containing 1, 3-glucanase or chitinase under the control of CaMV 35S promoter and nos terminator mobilized in Agrobacterium strain LBA 4404 was used to transform indica rice callus. The transformed calli were passed through three antibiotic selection sub-culture passages of 15 days each on MS medium supplemented with kanamycin (50 mg/l) and cefotaxime (250mg/l). The calli surviving after third selection passage were PCR analyzed for the presence of 3–1, 3-glucanase and chitinase genes using gene specific primers. The results revealed detection of-1, 3-glucanase gene in 28.97% transformed calli and amplifcation of chitinase in 32.67% calli. PCR verifed calli were confronted in vitro with Magnapor the oryzae the casual organism of rice blast, an important disease of the crop. The mycelial growth of M. Oryzae was inhibited (27.62%) by callus transformed with 1, 3 glucanase gene construct and the callus transformed with chitinase gene construct also exhibited the pathogen restraint (28.84%) as compared to the control. The results confrmed effectiveness of 1, 3 glucanase and chitinase against M. Oryzae, suggesting that rapid transient assay can be used to test antifungal effect of the transgenes in plant cells against plant pathogens before attempting to develop transgenic plants.