• Journal of Life Science
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• v.12 no.1
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• pp.67-76
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• 2002

The effect of transgene inactivation in T2, T3 and F2 generations was analyzed in progeny seedlings which had been generated by Agrobacterium (LBA4404/pBI121)-mediated transformation in Arabidopsis thaliana. In a system which investigated in the expression of $\beta$-glucuronidase(GUS)gene in kanamycin-resistant (ke $n^{R}$)seedlings, GUS inactivated seedlings were observed in 5 of 12 tested lines of T2 generation and the frequency of GUS inactivation was approximately 2.3%. Lines with multi-copies of T-DNA exhibited severe GUS gene inactivation with the frequency of 5.8% in T2 generation. In T3 generation lines exhibited GUS gene inactivation with the frequency of 1.3%. In contrast, inactivation increased dramatically up to 12.6% in multi-copy T-DNA line. A similar phenomenon was also found in F2 progeny from a transgenic line which had been crossed with wild-type Arabidopsis plant, WS-O (GUS gene inactivation frequency 9.9%). These results indicate that the foreign gene introduced into the plant was inactivated progressively in its transmission during subsequent generations and the transgenic line with multi-copies of T-DNA tended to show more increased inactivation.

• KSBB Journal
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• v.6 no.4
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• pp.385-388
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• 1991

Embryogenic cell suspension cultures of B. striata were established as transferred selected embryogenic callus into liquid medium. Protoplasts isolated from embryogenic cell suspensions were electroporated in buffered solutions containing plasmid DNA of pBI121. Transient GUS (beta-glucuronidase) activity measurement and selection for kanamycin resistent showed that expression of foreign genes and stable transformation were achieved. GUS transient gene expression was increased by increasing DNA concentration of pBI121 plasmid and affected by the level of the applied voltage. An optimal level of GUS activity was obtained after electroporation with a pulse of 200-300 voltage/1180 uF. Protoplast viability was up to the 80% at the optimal voltage.

• Journal of Plant Biotechnology
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• v.29 no.4
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• pp.223-228
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• 2002

We have developed a homemade particle inflow gun (PIG) system which has simple operation method, low price and high gene introduction efficiency into rice callus. Rice callus were inflowed with gold particles containing DNA of a plasmid, pIG121Hm, harboring intron GUS ($\beta$-glucuronidase) gene, NPTII gene and HPT gene. For optimal GUS transient expression, the effects of parameters on DNA delivery efficiency of the PIG system was investigated by scoring transient GUS expression. The highest number of blue spots was observed at 16 mM of spemidine and 1.5 M of calcium chloride, respectively. And the amount of gold particles required for the best GUD expression was 2 mg. Optimum GUS transient expression was observed at target distance of 12 cm and helium pressure of 3.5 bar (50 psi). Gene introduction efficiency of the PIG system was observed almost similar to that of the Biolistic Gun (Bio-Rad Company). Since PIG system is simple to operate and one doesn't need disposable accessaries, the PIG system can be easily applied to various replication experiments.

• Journal of Plant Biology
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• v.37 no.1
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• pp.27-36
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• 1994

For establishing a transformation system of rice, an efficient introduction of foreign genes into embryogenic cell suspension by particle bombardment was conducted. The particle inflow gun based on the acceleration of DNA-coated tungsten particles using pressurized helium was constructed for delivery of DNA into rice cells. Several bombardment parameters were optimized using the transient expression of GUS gene. The conditions that gave the highest GUS gene expression of about 1000 blue spots per g fresh weight of bombarded cells include treatment of the cells with 0.5 M osmotic pressure, and use of the 410 kPa helium, 110 mm target distance, 13 mm syringe filter holder and 5 $\mu$L DNA/tungsten mixtures. It was also confirmed that rice actin promoter-intron construct gave the highest expression of all promoter-sequences studied. Eight weeks after the bombardment, stably transformed calluses were obtained on the selection medium containing 100 mg/L G418 and showed the strong activity in in situ GUS assay.

• Journal of Plant Biotechnology
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• v.35 no.4
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• pp.275-280
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• 2008

One of the limitation for Agrobacterium-mediated transformation via organogenesis from cotyledon explants routinely in cucumber is the production of chimeric plants. To overcome the limitation, Agrobacterium-mediated transformation system via somatic embryogenesis from hypocotyl explants of cucumber (c.v., Eunsung) on the selection medium with paromomycin as antibiotics was developed. The hypocotyl explants were inoculated with Agrobacterium tumefaciens strain EHA101 carrying binary vector pPTN290; then were subsequently cultured on the following media: co-cultivation medium for 2 days, selection medium for $5{\times}14$ days, and regeneration medium. The T-DNA of the vector (pPTN290) carried two cassettes, Ubi promoter-gus gene as reporter and 35S promoter-nptll gene conferring resistance to paromomycin as selectable agent. The confirmation of stable transformation and the efficiency of transformation was based on the resistance to paromomycin indicated by the growth of putative transgenic calli on selection medium amended with 100mg/L paromomycin, and GUS gene expression. Forty eight clones (5.2%) with GUS gene expressed of 56 callus clones with resistance to paromomycin were independently obtained from 928 explants inoculated. Of 48 clones, transgenic plants were only regenerated from 5 clones (0.5%) at low frequency. The histochemical GUS assay in the transgenic seeds ($T_1$) also revealed that the gus gene was successfully integrated and segregated into each genome of transgenic cucumber.

• Plant Biotechnology Reports
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• v.3 no.4
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• pp.325-331
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• 2009

To efficiently express a gene of interest in transgenic plants, the choice of promoter is a crucial factor as it directly affects the expression of the transgene that will yield the desired phenotype. The Arabidopsis ${\beta}-carotene$ hydroxylase 1 gene (AtBch1) shows constitutive and ubiquitous expression and was thus selected as one of best candidates for constitutive promoter analysis by both in silico northern blotting and semi-quantitative RT-PCR analysis. To investigate AtBch1 promoter activity, the 1,981-bp 5'-upstream region of this gene was fused with ${\beta}-glucuronidase$ (GUS) and transformed into Arabidopsis. Through the molecular characterization of transgenic leaf tissues, the AtBch1 promoter generated strong activity that drives 1.8- and 2-fold higher GUS expression than the cauliflower mosaic virus 35S (35S) promoter at the transcriptional and translational levels, respectively. Furthermore, the GUS enzyme activity driven by the AtBch1 promoter was 2.8-fold higher than that produced by the 35S promoter. By histochemical GUS staining, the ubiquitous expression of the AtBch1 promoter was observed in all tissues of Arabidopsis. Semi-quantitative RT-PCR analysis with different tissues further showed that this promoter serves as a strong constitutive driver of transgene expression in dicot plants.

• BMB Reports
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• v.43 no.1
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• pp.9-16
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• 2010

The promoters of OsCaM1 and OsCaM3 were characterized after sequencing and fused to the reporter gene, GUS. The constructs were then transformed into the tobacco plant. Histochemical analysis of GUS showed different expression patterns in pOsCaM1::GUS and pOsCaM3:: GUS transgenic plants. The expression of pOsCaM1::GUS in 4- to 15-day-old seedlings in particular was observed only in the root, while the expression of pOsCaM3::GUS was detected in both the cotyledons and root. Also, pRCaM1::GUS was detected in all the tissues surrounding the root system, while the presence of pOsCaM3::GUS was observed in the root, except in the root meristem. However, in mature transgenic plants, the expression of pOsCaM1::GUS and OsRCaM3::GUS was scarcely detected. Under wounding stress, the GUS activity of pOsCaM1 and pOsCaM3 was strongly induced, and the activity of pOsCaM3 especially, was retained for long periods. In the phloem, pOsCaM3 activity induced by hormone treatments and abiotic stresses was also identified.

• BMB Reports
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• v.32 no.1
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• pp.51-59
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• 1999

The Korean radish cationic peroxidase (KRCP) promoter, comprising nucleotides -471 to +704 relative to the transcriptional initiation site, was fused to the GUS gene and transformed to tobacco BY-2 cells. We examined how auxin (2,4-dichlorophenoxyacetic acid, 2,4-D), cytokinin (6-benzylaminopurine, BAP), gibberellic acid ($GA_3$), abscisic acid (ABA), methyl jasmonate (MeJA), and phosphatidic acid (PA) affect the GUS expression in the presence or absence of 2,4-D in a modified LS medium. Exogenous 2,4-D or BAP greatly decreased the GUS expression regulated by the KRCP promoter in a modified LS medium containing 0.2 mg/l 2,4-D. $GA_3$ increased the GUS expression and ABA completely reduced the inductive effect of $GA_3$. The GUS expression was also increased dose-dependently by plant defense regulators, MeJA and PA. In contrast to the above results, auxin deprivation from the modified LS medium increased the GUS expression after treatment with exogenous 2,4-D whereas BAP still greatly decreased the GUS expression dose-dependently. $GA_3$ or MeJA slightly decreased the GUS expression. The data suggest that auxin deprivation changes the sensitivity of the suspension cells to exogenous chemicals and that the regulation of the KRCP promoter by 2,4-D, $GA_3$, and MeJA is dependent on auxin, whereas the regulation by BAP is not. This study will be valuable for understanding the function and expression mode of the Korean radish cationic peroxidase in Korean radish.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.49 no.5
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• pp.434-439
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• 2004

Medicago truncatula is a model plant for molecular genetic studies of legumes and plant-microbe interactions. To accelerate finding of genes that play roles in the early stages of nodulation and stress responses, a trans-genic plant was developed that contains a promoter­reporter fusion. The promoter of rip], a Rhizobium-induced peroxidase gene, was fused to the coding region of $\beta-glucuronidase (GUS)$ gene and inserted into a modified plant transformation vector, pSLJ525YN, in which the bar gene was preserved from the original plasmid but the neomycin phosphotransferase gene was replaced by a polylinker. Transformation of M. truncatula was carried out by vacuum infiltration of young seedlings with Agrobacterium. Despite low survival rates of infiltrated seedlings, three independent transformants were obtained from repeated experiments. Southern blot analyses revealed that 7 of 8 transgenic plants of the T 1 generation contained the bar gene whereas 6 $T_1$ plants contained the GUS gene. These results indicate that vacuum infiltration is an effective method for transformation of M. truncatula. The progeny seeds of the transgenic plants will be useful for mutagenesis and identification of genes that are placed upstream and may influence the expression of rip] in cellular signaling processes including nodulation.

• KSBB Journal
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• v.5 no.4
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• pp.323-327
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• 1990

Protoplasts isolated from embryogenic cell suspensions were electroporated in buffered solutions containing plasmid DNA of pBI121. Transient GUS (beta-glucuronidase) activity measurement and selection for kanamycin resistent showed that expression of foreign genes and stable loransformation were achieved. GUS transient gene expression was increased by increasing DNA concentration of pBI121 plasmid and affected by the level of the applied voltage. An optimal level of GUS activity was obtained after electroporation with a pulse of 200 voltage/1180 uF. Protoplast viability was up to the 60% at the optimal voltage. Cell colonies resistent to 200$\mu\textrm{g}$/ml kanamycin were selected in agar medium and identified by histochemical GUS assay.