• Journal of Plant Biotechnology
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• v.31 no.1
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• pp.13-17
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• 2004

To improve transformation efficiency, the callus of Lilium lancifolium Thunb. were bombarded by particles coated with pIG 121 Hm which include NPT II and GUS genes, and then cocultivated with Agrobacterium tumefaciens EHA101 which contain pIG121Hm binary vector, carrying neomycin phosphotransferase (NPT II) and $\beta$-Glucuronidase (GUS) genes. Three days after cocultivation with Agrobacterium tumefaciens and particle bombardment, the callus clusters were transferred to MS medium containing 1mg/L 2,4-D, 0.1mg/L BAP, 100mg/L kanamycin and 200mg/L carbenicillin. Four weeks after transfer to the selection medium, GUS expression was detected and PCR analysis revealed the presence of NPT II fragment of the expected size (700 bp) in the transformed callus. The GUS expression from Agrobacterium-mediated transformants after particle bombardment increased to over 3-folds compared with that of callus cocultivated with Agrobacterium tumefaciens without particle bombardment. The callus clusters containing kanamycin resistant gene were transferred to MS medium containing 1mg/L NAA and 1mg/L BAP. Somatic embryos were developed in four weeks and microbulbs expressing GUS were formed.

• Plant Biotechnology Reports
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• v.2 no.4
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• pp.267-270
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• 2008

We have successfully used the low-pressure BioWare gene gun, developed for gene transfer in animal cells, for plant tissues. The BioWare device is easy to manipulate. Just 50 psi helium pressure was sufficient to transfer foreign genes into the aleurone layer and embryo of maize without causing tissue damage in the impact area. As shown by expression signals from invasive histochemical ${\beta}-glucuronidase$ (GUS) activity, the foreign reporter gene expressed well in bombarded tissues. This successful GUS-transient expression extends the application of this low-pressure gene gun from animal cells to plant tissues.

• 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.7 no.4
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• pp.211-218
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• 2005

Genetic transformation was carried out by using biolistic gun method. The hypocotyl derived embryogenic calli (explants) of cotton (Gossypium hirsutum L.) cv. Cocker-312 were transformed with a recombinant pGreen II plasmid, in which both, bar (selection marker) and GUS (${\beta}$-glucuronidase) reporter genes were incorporated. Explants were arranged on osmoticum-containing medium (0.5M mannitol) 4 hours prior to and 16 hours after bombardment that was resulted into an increase about >80% for GUS stable expression. 3 days after bombardment, GUS assay was performed, which exhibited, $18.36{\pm}1.00$ calli showed blue spots. The transformed embryogenic calli were cultured on selection medium (@ 6 mg/L basta) for 3 months. The putative transgenic plants were developed via selective somatic embryogenesis (@1.50 mg/L basta); maximum $27.58{\pm}1.25$ somatic embryos were obtained while $17.47{\pm}1.00$ embryos developed into plantlets (@ 0.75mg/L basta). In five independent experiments, up to 7.24% transformation efficiency was recorded. The presence of the transgenes was analyzed by using PCR and southern hybridization analysis. The transgenic plants were developed with in 6-7 months, but mostly transformants were abnormal in morphology.

• 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.

• The Plant Pathology Journal
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• v.23 no.2
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• pp.76-89
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• 2007

CaCDPK4, a full-length cDNA clone encoding Capsicum annuum calcium-dependent protein kinase 4, was isolated from chili pepper (Capsicum annuum L.). Deduced amino acid sequence of CaCDPK4 shares the highest homology with tobacco NpCDPK8 and chickpea CaCDPK2 with 79% identity. Genomic blot analyses revealed that CaCDPK4 is present as a single copy in pepper genome, but it belongs to a multigene family. CaCDPK4 was highly induced when pepper plants were inoculated with an incompatible bacterial pathogen. Induced levels of CaCDPK4 transcripts were also detected in pepper leaves by the treatment of ethephon, an ethylene-inducing agent, and high-salt stress condition. The bacterial-expressed GST-CaCDPK4 protein showed to retain the autophosphorylation activity in vitro. GUS expression driven by CaCDPK4 promoter was examined in transgenic Arabidopsis containing transcriptional fusion of CaCDPK4 promoter. GUS expression under CaCDPK4 promoter was strong in the root and veins of the seedlings. GW (-1965) and D3 (-1377) promoters conferred on GUS expression in response to inoculation of an incompatible bacterial pathogen, but D4-GUS (-913) and DS-GUS (-833) did not. Taken together, our results suggest that CaCDPK4 can be implicated on signal transduction pathway of defense response against an incompatible bacterial pathogen in pepper.

• Journal of Plant Biotechnology
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• v.32 no.4
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• pp.275-279
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• 2005

Tah tasai chinese cabbage (Brassica campestris var. narinosa), a vegetable plant popularly consumed as several-days-old seedlings in oriental countries, can be easily cultivated using a simple appliance. We demonstrated that Agrobacterium-mediated transformation via vacuum infiltration (agroinfiltration) resulted in a successful transient GUS gene expression in tah tasai chinese cabbage seedlings. Pre-germinated seeds were found to be more susceptible to Agrobacterium infection than one-day-old or two-days-old seedlings. We also demonstrated that hydrogen peroxide (HPO) treatment increased GUS expression especially for two-days-old seedlings. In ELISA using seedlings transformed with hepatitis B surface antigen (HBsAg) DNA by agroinfiltration, HBsAg protein synthesis increased more than two folds by HPO treatment to two-days-old seedlings in comparison to the mock-treated pre-germinated seeds.

• 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 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.

• 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.