Transient Β-glucuronidase Expression Research Articles

Co-treatment with surfactant and sonication significantly improves Agrobacterium-mediated resistant bud formation and transient expression efficiency in soybean

Soybean is a widely planted genetically modified crop around the world. However, it is still one of the most recalcitrant crops for genetic transformation due to the difficulty of regeneration via organogenesis and some factors that affect the transformation efficiency. The percentages of resistant bud formation and transient expression efficiency are important indexes reflecting the regeneration and transformation efficiency of soybean. In this study, the percentages of resistant bud formation and transient expression of β-glucuronidase (GUS) were compared after treatment with sonication or surfactant and co-treatment with both. The results showed that treatment with either sonication or surfactant increased the percentage of resistant bud formation and transient expression efficiency. The highest percentages were acquired and significantly improved when cotyledon node explants were co-treated with sonication for 2 s and surfactant at 0.02% (v:v) using two different soybean genotypes, Jack and Zhonghuang 10. The improved transformation efficiency of this combination was also evaluated by development of herbicide-tolerant soybeans with transformation efficiency at 2.5–5.7% for different genotypes, which was significantly higher than traditional cotyledonary node method in this study. These results suggested that co-treatment with surfactant and sonication significantly improved the percentages of resistance bud formation, transient expression efficiency and stable transformation efficiency in soybean.

Determining the GmRIN4 requirements of the soybean disease resistance proteins Rpg1b and Rpg1r using a nicotiana glutinosa-based agroinfiltration system.

Rpg1b and Rpg1r are soybean disease resistance (R) genes responsible for conferring resistance to Pseudomonas syringae strains expressing the effectors AvrB and AvrRpm1, respectively. The study of these cloned genes would be greatly facilitated by the availability of a suitable transient expression system. The commonly used Niciotiana benthamiana-based system is not suitable for studying Rpg1b and Rpg1r function, however, because expression of AvrB or AvrRpm1 alone induces a hypersensitive response (HR), indicating that N. benthamiana contains endogenous R genes that recognize these effectors. To identify a suitable alternative host for transient expression assays, we screened 13 species of Nicotiana along with 11 accessions of N. tabacum for lack of response to transient expression of AvrB and AvrRpm1. We found that N. glutinosa did not respond to either effector and was readily transformable as determined by transient expression of β-glucuronidase. Using this system, we determined that Rpg1b-mediated HR in N. glutinosa required co-expression of avrB and a soybean ortholog of the Arabidopsis RIN4 gene. All four soybean RIN4 orthologs tested worked in the assay. In contrast, Rpg1r did not require co-expression of a soybean RIN4 ortholog to recognize AvrRpm1, but recognition was suppressed by co-expression with AvrRpt2. These observations suggest that an endogenous RIN4 gene in N. glutinosa can substitute for the soybean RIN4 ortholog in the recognition of AvrRpm1 by Rpg1r.

Biolistic transformation of wheat using the HMW-GS 1Dx5 gene without selectable markers.

Genetic manipulation using linear DNA was applied to the common wheat variety Xindong No. 26 via particle bombardment with the aim to improve bread-making quality of flour. Initially, 2 biolistic parameters, helium pressure and target distance, were optimized using plasmid pAHC25. We transformed wheat immature embryo scutella with the linear 1Dx5 gene without selectable markers. The highest transient β-glucuronidase expression was obtained when scuttles were bombarded at 1100 psi with a 9-cm target distance. Using the optimized parameters, the transformation of the common wheat variety Xindong No. 26 was carried out using the linear 1Dx5. Three transgenic plants were identified from 1003 transgenic plants, yielding a transformation frequency of 0.4%. A sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis confirmed that the 1Dx5 gene was expressed in 4 T1 seeds of the transgenic plants. These experiments indicate the possibility of obtaining marker-free transgenic wheat plants via particle bombardment using the minimal gene cassettes with the particle bombardment parameters.

An Efficient Method for Agrobacterium-Mediated Genetic Transformation and Plant Regeneration in Cumin (Cuminum cyminum L.)

Cumin is an annual herbaceous medicinally important plant having diverse applications. An efficient and reproducible method of Agrobacterium-mediated genetic transformation was herein established for the first time. A direct regeneration method without callus induction was optimised using embryos as explant material in Gamborg's B5 medium supplemented with 0.5-μM 6-benzyladenine and 2.0-μM α-naphthalene acetic acid. About 1,020 embryos (a mean of 255 embryos per batch) were used for the optimisation of transformation conditions. These conditions were an Agrobacterium cell suspension of 0.6 OD600, a co-cultivation time of 72 h, 300-μM acetosyringone and wounding of explants using a razor blade. Pre-cultured elongated embryos were treated using optimised conditions. About 720 embryos (a mean of 180 embryos per batch) were used for transformation and 95 % embryos showed transient β-glucuronidase expression after co-cultivation. Putative transformed embryos were cultured on B5 medium for shoot proliferation and 21 regenerated plants were obtained after selection and allowed to root. T0 plantlets showed β-glucuronidase expression and gene integration was confirmed via PCR amplification of 0.96 and 1.28 kb fragments of the hygromycin-phosphotransferase II and β-glucuronidase genes, respectively. In this study, a transformation efficiency of 1.5 % was demonstrated and a total of 11 transgenic plants were obtained at the hardening stage, however, only four plants acclimatised during hardening. Gene copy number was analysed by Southern blot analysis of hardened plants and single-copy gene integration was observed. This is the first successful attempt of Agrobacterium-mediated genetic transformation of cumin.

Identification of an Internal RNA Element Essential for Replication and Translational Enhancement of Tobacco Necrosis Virus AC

Different regulatory elements function are involved in plant virus gene expression and replication by long-distance RNA-RNA interactions. A cap-independent functional element of the Barley yellow dwarf virus (BYDV) – like translational enhancer (BTE) is present in Tobacco necrosis virus A (TNV-A), a Necrovirus member in the Tombusviridae family. In this paper, an RNA stretch flanking the 5′ proximal end of the TNV-AC coat protein (CP) gene was shown to be essential for viral replication in Chenopodium amaranticolor plants and tobacco cells. This internal sequence functioned in transient expression of β-glucuronidase (GUS) when present at either the 5′ or 3′ sides of the GUS open reading frame. Serial deletion analyses revealed that nine nucleotides from nt 2609 to 2617 (−3 to +6 of the CP initiation site) within TNV-AC RNA are indispensable for viral replication in whole plants and tobacco cells. Fusion of this RNA element in mRNAs translated in tobacco cells resulted in a remarkable enhancement of luciferase expression from in vitro synthesised chimaeric RNAs or DNA expression vectors. Interestingly, the element also exhibited increased translational activity when fused downstream of the reporter genes, although the efficiency was lower than with upstream fusions. These results provide evidence that an internal RNA element in the genomic (g) RNA of TNV-AC, ranging approximately from nt 2543 to 2617, plays a bifunctional role in viral replication and translation enhancement during infection, and that this element may use novel strategies differing from those previously reported for other viruses.

Optimization of an Agrobacterium-mediated Transient Assay for Gene Expression Studies in Anthurium andraeanum

Gene function studies in anthurium (Anthurium andraeanum) have been hindered by the low efficiency of stable transformation, the long regeneration time required as well as the genotype-dependent nature of Agrobacterium (Agrobacterium tumemaciens)-mediated transformation protocols. Agrobacterium-mediated transient expression assays can serve as an attractive alternative for investigating gene function once such assays are optimized. The effects of host factors (genotype, explant type, and developmental maturity of explants), Agrobacterium factors (strain, growth phase, and its concentration), media conditions (infiltration medium used, acetosyringone concentration, type of surfactant, and its concentration), and other experimental factors (infiltration time, cocultivation time, and vacuum infiltration) were investigated on the efficiency of Agrobacterium transient transformation, with replications, using transient expression of β-glucuronidase as an indicator. Although the efficiency of transient transformation was initially found to be highly host genotype-dependent, the genotypic differences in transient transformation efficiency diminished as the protocol was optimized. Agrobacterium strain GV3101 grown to an optical density at 600 nM (OD600) of 1.5 and resuspended to a final OD600 of 0.8 in infiltration medium [0.5% glucose, 10 mm 2-(N-morpholino) ethanesulfonic acid] supplemented with 100 μM acetosyringone and 0.05% of a non-ionic surfactant (S240), for an infiltration period of 16 hours and a cocultivation timeframe of 2 days yielded transient transformation efficiencies as high as 100%.

A Simple and Efficient Protocol for Plant Regeneration and Genetic Transformation of Tomato cv. Micro-Tom from Leaf Explants

A simplified protocol to obtain transgenic tomato plants was established. The effects of culture media composition and Agrobacterium concentration were evaluated. The highest shoot-forming capacity index (5.6) was observed when leaf explants were cultured for 6 weeks with 2 mg·L−1 zeatin, 0.1 mg·L−1 indoleacetic acid, and 300 mg·L−1 timentin. Shoot elongation and root formation were performed in one step on growth regulator-free media. The highest percentage (82%) of fully developed plantlets was obtained when shoots were cultured for 4 weeks with 0.5× Murashige and Skoog (MS) media and 15 g·L−1 sucrose. A 100% of plant survival rate was observed after 4 weeks of being transplanted to ex vitro conditions followed by fruit production (15 fruits/plant) after 2 more weeks. Transient expression of β-glucuronidase was visualized in 100% of the leaf explants infected with Agrobacterium at an OD600 = 0.5 and cocultured for 48 h with 2 mg·L−1 benzylaminopurine, 0.1 mg·L−1 naphthaleneacetic acid, and 100 μM acetosyringone. Stable transformation was confirmed by histochemical glucuronidase assay and polymerase chain reaction (PCR) analysis with a total efficiency of 19.1%. The complete protocol, from shoot induction to fruit production of soil-adapted transgenic plants can be accomplished in only 4 months, and it seems to be very useful for both micropropagation and genetic transformation purposes.

Analysis of banana fruit-specific promoters using transient expression in embryogenic cells of banana cultivar Robusta (AAA Group)

The study reports the transient expression of gusA gene in embryogenic cells using three banana derived fruit-specific promoters. Banana embryogenic cells were transformed with a pCAMBIA-1301 derived plasmid construct harboring gusA gene driven by either chitinase, glucanase or expansin promoters derived from banana. The transient expression of β-glucuronidase was studied 5 days after co-cultivation with Agrobacterium harboring the expression plasmids. The transformed embryogenic cells were treated with different inducers of ethylene such as ethephon, methyl jasmonate, methyl salicylate, abscisic acid and indole acetic acid. The maximum expression of 64099.78 pmoles 4-MU/h/mg total protein was noted with expansin promoter when the cells were treated with the combination of ethephon (0.25 mM) and MJ (10 mM). The results suggest that these promoters can be used to achieve fruit-specific expression of useful transgenes in banana. The results should prove to be an important guide for short term expression studies for promoter validation and gene screening.

Influence of bacterial density during preculture on Agrobacterium-mediated transformation of tomato

An improved bacterial preculture protocol for Agrobacterium-mediated genetic transformation was developed for an economic tomato cultivar (Solanum lycopersicum L. cv. Zhongshu No. 4). Frequencies of transient gene expression and stable transformation were influenced by the density of Agrobacterium preculture and not the density of Agrobacterium used for infection. The improved protocol presented in this study depends on the use of an overnight-grown Agrobacterium preculture density of OD600 nm = 1.0, diluted 1/10th with Luria-Bertani (LB) liquid medium, and grown for an additional 4 h. Cultures are collected and resuspended in a liquid cocultivation medium-I, adjusted to OD600 nm = 0.1. Using this modified Agrobacterium preparation, transient β-glucuronidase expression was higher than 90%, and transformation efficiency reached 44.7%. This improved transformation is simple, repeatable, does not require a feeder layer, and most notably, the transformation frequency is stable and highly efficient.

Transient β-glucuronidase expression in lily (Lilium longflorum L.) pollen via wounding-assisted Agrobacterium-mediated transformation

A pollen-based transient expression system has been developed. Lily pollen grains, wounded by vigorous shaking in the presence of aluminum oxide particles, were transformed by infiltration with Agrobacterium tumefaciens LBA4404 cells harboring the beta-glucuronidase (GUS) gene construct, pBI121. In histochemical and fluorometric GUS analysis, the wounding processes allowed efficient transformation and, in cDNA blot hybridization, GUS mRNA synthesis was clearly detected. Lily pollen with appropriate wounds, therefore, can be used conveniently for the rapid production of recombinant proteins.

Factors Affecting the Agrobacterium-Mediated Transient Transformation of the Wetland Monocot, Typha latifolia

An Agrobacterium-mediated transformation system, using transient transformation assays, was used to evaluate conditions influencing transformation for the wetland monocot Typha latifolia. These studies were aimed at the long-term objective of evaluating candidate genes for phytoremediation. The binary plasmid vector pCAMBIA1301/EHA105, containing the β-glucuronidase coding sequence, was used in combination with factors known to affect transformation. These included callus age at the time of cocultivation with Agrobacterium tumefaciens, type and concentration of auxin for explant growth, light or dark culture environment, the presence or absence of acetosyringone (AS), explant type, explant wounding and the number of days used for cocultivation. The number of days needed for the first detection of transient expression of the β-glucuronidase gene was also examined. Three days of Agrobacterium cocultivation of 50-day-old seedling-derived calluses, grown on 20.7 µM (5 mg l−1) picloram supplemented medium, in the dark, resulted in higher levels of transient β-glucuronidase expression than were seen in calluses cultured on 4.5 or 22.6 µM (1 or 5 mg l−1) 2,4-dichlorphenoxyacetic acid containing media. The addition of 100 µM acetosyringone significantly enhanced transient β-glucuronidase activity. Wounding of explants, by cutting into two or three pieces, 3 days before cocultivation, increased expression of β-glucuronidase only in calluses cultured under light conditions. Transient β-glucuronidase expression was observed as early as 24 h after cocultivation and increased as the days post cultivation increased. The developed transient system will be used for stable transformation of Typha species.

Transient Expression of β-Glucuronidase in Embryo Axes of Cotton by Agrobacterium and Particle Bombardment Methods

Transient expression of β-glucuronidase (GUS) in zygotic embryo axes of two cotton (Gossypium hirsutum L.) cultivars NHH-44 and DCH-32 was induced by Agrobacterium mediated transformation or by particle bombardment. For Agrobacterium transformation, disarmed A. tumefaciens strain GV 2260/p35SGUSINT was used. In cv. NHH-44, the maximum frequency of transient expression (14.28 %) was achieved on spotting Agrobacterium paste on the apical regions of the split embryo axes. The method resulted in a transformed callus line, which showed strong GUS activity. Integration of NPTII gene was confirmed by Southern analysis. Transgene expression by particle bombardment was achieved with p35SGUSINT and pIBGUS plasmids independently. The maximum frequency of GUS expression in 29.16 % explants was observed in cultivar NHH-44 with gold microcarriers (1.1 µm) when bombarded once with rupture disc of 7586 kPa at target cell distance of 6 cm. A transformed callus line was obtained when explants were bombarded with p35SGUSINT and cultured on Murashige and Skoog's medium supplemented with B5 vitamins, 0.1 mg dm-3 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea, 0.01 mg dm-3 α-naphthaleneacetic acid, 3 % glucose + 50 mg dm-3 kanamycin. High GUS activity was observed in callus tissue as well as in somatic embryo like structures achieved in liquid shake cultures.

Agrobacterium tumefaciens-mediated transformation of maize embryos using a standard binary vector system.

We have achieved routine transformation of maize (Zea mays) using an Agrobacterium tumefaciens standard binary (non-super binary) vector system. Immature zygotic embryos of the hybrid line Hi II were infected with A. tumefaciens strain EHA101 harboring a standard binary vector and cocultivated in the presence of 400 mg L-1 L-cysteine. Inclusion of L-cysteine in cocultivation medium lead to an improvement in transient beta-glucuronidase expression observed in targeted cells and a significant increase in stable transformation efficiency, but was associated with a decrease in embryo response after cocultivation. The average stable transformation efficiency (no. of bialaphos-resistant events recovered per 100 embryos infected) of the present protocol was 5.5%. Southern-blot and progeny analyses confirmed the integration, expression, and inheritance of the bar and gus transgenes in R0, R1, and R2 generations of transgenic events. To our knowledge, this represents the first report in which fertile, stable transgenic maize has been routinely produced using an A. tumefaciens standard binary vector system.

Electroporation of intact embryonic leaflets of peanut: Gene transfer and stimulation of regeneration capacity

Direct gene transfer into peanut intact embryonic leaflets was performed through electroporation. In transient β-glucuronidase expression assays, maximal expression was obtained by using pulses of 625 V cm−1 in EPRm (modified electroporation) buffer supplemented with 75 μM NaCl. Kanamycin-resistant plants were obtained, and the presence of the nptII gene was demonstrated by PCR analysis. The positive effect of electroporation on the efficiency of in vitro regeneration was demonstrated. Explants submitted to field strengths between 500 and 625 V cm−1 displayed a significantly increased number of shoots and originated faster growing calluses relative to control explants. Whereas in control explants callus formation occurred only at the petiolule, electroporated leaflets developed additional organogenic calluses on the foliar lamina.

Aluminium Borate Whisker-Mediated DNA Delivery into Callus of Rice and Production of Transgenic Rice Plant

Japonica rice ( Oryza sativa L., cv. Notohikari) was transformed by the use of aluminium borate whiskers (ABW) to deliver DNA into the scutellar tissues of embryos isolated from mature seeds. DNA delivery was mediated by mixing ABW and plasmid DNA. Some parameters of DNA delivery employing ABW were determined in the tissues based on the transient expression of β-glucuronidase (GUS) reporter gene. The optimum condition was established as the mixing of 290 μL buffer containing 64 mg ABW and 25 μg DNA and 20 tissues for 30 min. Under this condition, we tried to introduce the phosphinothricin acetyl transferase (bar) gene whose product confers resistance to the bialaphos into the tissues, together with the GUS gene, and obtained bialaphos-resistant calli. Transgenic rice plants were regenerated from one of them. The integration of GUS gene and bar gene were confirmed by GUS histochemical assay and polymerase chain reaction (PCR) analysis, respectively. Southern hybridization analysis also confirmed the integration of bar gene. These results suggest that a simple and inexpensive DNA delivery procedure via ABW can be useful to produce transgenic rice plants.

Transgenic caraway, Carum carvi L.: a model species for metabolic engineering.

Regeneration in caraway was obtained via two different routes. Hypocotyls showed delayed shoot formation after a callus phase and at relatively low frequencies. In contrast, high-frequency, direct regeneration occurred when cotyledonary node explants were used. Transient expression of β-glucuronidase was monitored after inoculation of both explant types with Agrobacterium tumefaciens AGL0(pMOG410). Gene transfer was more efficient when using cotyledonary node explants. This explant type also proved to be the best for stable transformation resulting in transgenic plants. Several parameters determining regeneration and transformation efficiency were tested. The percentage of explants giving one to numerous transgenic plants could be as high as 13%. This system for the rapid production of many transgenic caraway plants opens up possibilities for studying metabolic engineering with this crop.

Direct regenerationin vitro and transient GUS expression inMentha xpiperita

Genetic transformation of peppermint is known to be very difficult essentially because of low efficiency regeneration. A regeneration protocol allowing 51% shooting frequency is proposed. Transient β-glucuronidase expression and adjustment of selection pressure with kanamycin are also reported. The final retained method to attempt peppermint transformation is:Agrobacterium inoculation or biolistic treatment of the first apical leaves ofin vitro clones, regeneration in the dark with kanamycin (1 mg l(-1)) and 6-benzylaminopurine (2 mg l(-1)), followed by selection of regenerated shoots with 200 mg 1(-1) kanamycin.

Transient expression of β-glucuronidase following biolistic delivery of foreign DNA into coffee tissues

Some conditions related to the transient expression of β-glucuronidase in biolistically-treated Coffea spp. tissues were investigated, and subsequently used in a promoter study. Bombardments were performed on different types of tissue (leaves, somatic embryos and suspension cultures) of genotypes of C. arabica, C. canephora and Arabusta, using 4 different promoter sequences. Tobacco leaves were used as a comparison. In general, similar large variation and mean values of transient expression were observed between coffee and tobacco leaves. With regard to the coffee tissues effect, transient expression was best detectable and most frequently observed with bombarded leaves of microcuttings. Disturbing endogenous light blue staining was found with control treatments of somatic embryos. For the three coffee species tested, the most effective promoter was the EF1α-A1 promoter of Arabidopsis thaliana.

Transient Expression of β-Glucuronidase (GUS) Activity in Embryogenic Callus Tissue of Alkaligrass (Puccinellia distans) using Particle Bombardment

Callus induction and regeneration of alkaligrass (Puccinellia distans) was developed in our laboratory for use in transformation studies of turfgrass. Particle bombardment of the embryogenic callus is being evaluated using a helium particle inflow gun constructed at Colorado State Univ., according to the design of Philippe et al. (Ohio State Univ., 1993). Its utility in delivering DNA to plant cells is being tested by measuring the frequency of transient gene expression of a reporter gene (GUS pBI121) in embryogenic callus of alkaligrass. Varying pressure of helium and the distance of the calli in the chamber are also being evaluated for efficiency in transformation.

Transient expression of β-glucuronidase in plastids of various plant cells and tissues delivered by a pneumatic particle gun

Chloroplast expression plasmids pTRBCL-GUS (tobaccorbcL promoter-gusA-tobaccorbcL terminator) and pHHU3004 (spinach ‘x gene’ promoter-gusA-spinachrbcL terminator) and a control nuclear expression plasmid pBI221 (CaMV 35S promoter-gusA-NOS terminator) were introduced separately into cultured cells and tissues of tobacco andArabidopsis thaliana, as well as into cultured cells of the lower land plants liverwort and hornwort by a pneumatic particle gun. The pTRBCL-GUS and pHHU3004 plasmids produced many blue spots in the BY-2 cells and the roots ofArabidopsis thaliana, but not in any of the green cells or tissues. The results suggest that the pTRBCL-GUS and pHHU3004 plasmids are expressed more in proplastids and amyloplasts than in chloroplasts. GUS activities of the BY-2 cells bombarded with pTRBCL-GUS and pHHU3004 were insensitive to α-amanitin treatment (10 and 50 μg/ml), while that of the cells with pBI221 greatly decreased by the same treatment. Hence, it is likely that the pTRBCL-GUS and pHHU3004 plasmids were substantially expressed in the proplastids.