• Journal of Plant Biology
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• v.39 no.4
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• pp.249-256
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• 1996

Beet curly top virus is the DNA virus that is providing useful for basic studies of the infection of Arabidopsis thaliana with viral host and provides a system for studying both resistance and the molecular basis of symptom development. An importnat aspect of symptom development observed in BCTV-infected A. thaliana (ecotype Sei-O) was the induction of cell division on phloem and surrounding cortex cells. Analysis of the expression of GUS reporter gene activity in transgenic plants containing constructs with promoter of the auxin-inducible saur gene showed that saur promoter activity was induced concomitantly in symptomatic tissues at the inflorescence shoot tips of the transgenic lines. The auxin sensitivity tests showed that hypersusceptible ecotype, Sei-O produced more amounts of callus than susceptible ecotype, Col-O. These studies indicated that changes in auxin concentration were involved in the induction of cell division in BCTV-infected plants and clearly demonstrated that there was a strong correlation between auxin-induced gene expression and the activation of cell division.

• Journal of Plant Biotechnology
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• v.4 no.4
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• pp.151-154
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• 2002

Conditions for lily pollen growth in vitro and transformation were optimized. Active pollen tube development was achieved effectively in a medium containing 7% sucrose with pH adjusted to 5.7 at the temperature of 27$^{\circ}C$ for about 16-24 hours. Pollen growth was little impaired by the presence of kanamycin at concentration up to 100 mg/L. Pollen rains near the beginning of germination stage were more reliable for Agrobacterium-mediated GUS DNA transformation via vacuum infiltration lasted for 20-40 minutes. GUS DNA integration and its expression in fully developed pollen tubes could be confirmed by Southern blot hybridization, RT-PCR and histochemical staining.

• Applied Biological Chemistry
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• v.47 no.4
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• pp.430-433
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• 2004

Lilium cv. Casablanca pollen grains stored at $-70^{\circ}C$ were grown in pollen germination medium with Agrobacterium tumefaciens LBA4404 cells harboring pBI121 for 18 hr at $27^{\circ}C$. Following this, cefotaxime (250 mg/L) was treated for 6 hr to eradicate the bacterial cells. Histochemical GUS analysis revealed that the transgenic pollen displayed deep blue color mostly from 12 hr after the co-cultivation. Presence of $200\;{\mu}M$ acetosyringone was determined not to be more effective for GUS transformation than its absence. GUS DNA integration in the transgenic pollen genomic DNA was clearly demonstrated by Southern blot analysis.

• Korean Journal of Medicinal Crop Science
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• v.12 no.5
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• pp.424-427
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• 2004

Microsomal ${\omega}-3$ fatty acid desaturase (FAD3) is an essential enzyme in the production of the n-3 polyunsaturated fatty acid ${\alpha}-linolenic$ acid during the seed developing stage. To understand the regulatory mechanism of the gene encoding the ${\omega}-3$ fatty acid desaturase, a genomic fragment corresponding to the previously isolated perilla seed PfFAD3 cDNA was amplified from perilla (Perilla frutescens Britt) by GenomeWalker PCR. Sequence analysis of the fragment provided with identification of a 1485-bp 5'-upstream region and a 241-bp intron in the open reading frame. To determine the tissue-specificity of the PfFAD3 gene expression, the 5'-upstream region was fused to the ${\beta}-glucuronidase$ (GUS) gene and incorporated into Arabidopsis thaliana. Histochemical assay of the transgenic plants showed that GUS expression was restricted to seed and pollen, showing that PfFAD3 gene was exclusively expressed in those tissues.

• Journal of Plant Biotechnology
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• v.31 no.4
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• pp.279-283
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• 2004

To lily (Lilium longflorum cv. Georgia) pollen, impacts by some physical, chemical and biological factors were examined in respects of its growth and transient gene expression via agro-infiltration. Rolling movement in liquid medium or vacuum pressure during Agro-infiltration was regarded as a impact that should be minimized for normal pollen growth. Pollen growth was maintained well in relatively broad range of temperature (19 to 27$^{\circ}C$) or pH (5.0 to 8.0). Chemical factors such as cefotaxime (up to 300mg/L), acetosyringone (up to 800 $\mu$M) and syringealdehyde (up to 800 $\mu$M) did not show any harmful effects but kanamycin severely did even at concentration as low as 25mg/L in some cases. For GUS gene expression, acetosyringone at 200 to 400 $\mu$M slightly improved the efficiency while syringealdehyde did not. Brief agro-infiltration followed by 18 hr of co-incubation of pollen along with Agrobacterium was suggested as a condition basically required for the transient expression system using lily pollen regardless of the presence of acetosyringone.

• Current Research on Agriculture and Life Sciences
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• v.11
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• pp.11-17
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• 1993

The transfer of genetic material into pea tissue was accomplished by using an avirulent strain of Agrobacterium tumefaciens containing the binary vector. The method used for transformation requires non-tissue culture steps as it involves the inoculation of the site of the shoot removed of germinating seeds. The identification of ${\beta}$-glucuronidase activity in the tissues of $T_0$ pea plants indicates that the plant expressible ${\beta}$-glucuronidase gene, contained the T-DNA region from pLPBO2, had been transferred at least into somatic tissues. Putative transformed $T_0$ pea plants were advanced to produce $T_1$ plants which were also assayed for the presence of the transferred ${\beta}$-glucuronidase gene. The presence of the ${\beta}$-glucuronidase gene in DNAs isolated from $T_1$ plant was demonstrated by DNA gel blot hybridization. This analysis revealed that the transformed plants contained ${\beta}$-glucuronidase gene.

• 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.

• Journal of Plant Biotechnology
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• v.30 no.3
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• pp.233-239
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• 2003

We have developed a reliable and high-frequency genetic transformation and regeneration system via somatic embryogensis of Eleutherococcus sessiliflorus. Embryogenic callus obtained from seed were co- cultivated with Agrobacterium tumefaciens strain EHA101/pIG121Hm harboring genes for intron-$\beta$-glucoronidase(GUS), kanamycin and hygromycin resistance. Following co-cultivation, two types of samples(fine embrogenic calli and early globular embryo clusters) were cultivated on Murashige and Skoog(MS) medium containing 1 mg/L2.4-D for 3day in dark. Transient expression of GUS gene was found to be higher in the early globular embryo clusters than in the embryogenic calli. Also, co-cultivated period affected expression of GUS gene; the best result was obtained when globular embryo clusters were co-cultivated with Agrobacterium for 3 days. Subsequently, this callus transferred to selective MS medium containing 1mg/L2.4-D, 50mg/L kanamycin or/and 30mg/L hygromycin and 300mg/L cefortaxime. These embryogenic calls were subcultured to the same selection medium at every 2 weeks intervals. Approximately 24.5% of the early globular embryos co-cultivated with Agrobacterium for 3days produced kanamycin or/and hygromycin-resistant calli. Transgenic somatic embryos were converted into plantlets in half strength MS medium supplemented with 3mg/L GA$_3$ kanamycin and were confirmed by GUS histochemical assay and polymerase chain reaction analysis. Genomic Southem blot hybridization confirmed the incorporation of NPT II gene into the host genome.

• Journal of Plant Biotechnology
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• v.50
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• pp.56-62
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• 2023

Transgenic lilies have been obtained using Agrobacterium tumefaciens (AGL1) with the plant scale explants, followed by DL-phosphinothricin (PPT) selection. In this study, scales of lily plants cv. "red flame" were transformed with the pCAMBIA3301 vector containing the gus gene as a reporter and the blpR gene as a selectable marker, as well as a gene of interest showing herbicide tolerance, both driven by the CaMV 35S promoter. Using a 20-minute infection time and a 5-day cultivation period, factors that optimized and demonstrated a high transformation efficiency were achieved. With these conditions, approximately 22-27% efficiency was observed for Agrobacterium-mediated transformation in lilies. After transformation with Agrobacterium, scales of lilies were transferred to MS medium without selective agents for 2 weeks. They were then placed on selection MS medium containing 5 mg/L PPT for a month of further selection and then cultured for another 4-8 weeks with a 4-week subculture regime on the same selection medium. PPT-resistant scales with shoots were successfully rooted and regenerated into plantlets after transferring into hormone-free MS medium. Also, most survived putatively transformed plantlets indicated the presence of the blpR gene by PCR analysis and showed a blue color indicating expression of the gus gene. In conclusion, when 100 scales of lily cv. "red flame" are transformed with Agrobacterium, approximately 22-27 transgenic plantlets can be produced following an optimized protocol. Therefore, this protocol can contribute to the lily breeding program in the future.

• Korean Journal of Plant Tissue Culture
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• v.25 no.1
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• pp.13-19
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• 1998

We have reported previously that intact embryogenic cells can be used instead of protoplasts for electroporation-mediated transformation of zoysiagrass and rice. In this study, conditions of the tissue electroporation were examined to optimize the procedures. Embryogenic cell suspensions were established in liquid MS medium containing 2 mg/L of 2,4-D with embryogenic calluses induced from mature embryos of Z. japonica. The suspension-cultured cell clumps were electroporated with 35S-gusA expression vector DNA, and degrees of DNA introduction into the cells were determined by histological expression rates of the gusA marker gene. DNA transfer into the cell clumps occurred in wide range of voltage (100-400 V) and capacitance (10-1980 $\mu\textrm{F}$), but more in the ranges of 200-300 V and 330-800 $\mu\textrm{F}$ DNA concentrations higher than 6 $\mu\textrm{g}$/mL were adequate for GUS expression of the electroporated cells. DNA transfers were confirmed in all three embryogenic cell lines but only in one out of eleven non-embryogenic lines. Positive GUS expressions occurred with DNAs added even 20-40 h after pulse treatments. As a promoter of gusA, Act1 and Ubi1 were effective 7 and 5 times than 35S respectively in number of GUS expression units on electroporated cell clumps. Embryogenic cell clumps survived and regenerated into plantlets after pulse treatments of wide range of conditions.