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

• Journal of Plant Biology
• /
• v.35 no.3
• /
• pp.237-245
• /
• 1992

To understand the properties of the cauliflower mosaic virus (CaMV) 35S promoter and the effect of the deleted maize alcohol dehydrogenase I-S (Adhl-S) intron 1 on the expression of the CaMV $35S{\beta}-glucuronidase$ (GUS) gene in potato (Solanum tuberosum L. cv. Superior), we constructed a chimeric gene and transferred it into potato with Agrobacterium tumefaciens mediated method. The pLS201, a gene transfer vector of 17.7 kilobase pairs, was composed of the CaMV 35S promoter, the 249 base pairs of deleted maize Adhl-S intron 1, the GUS reporter gene, and the kanamycin resistance gene as a selectable marker for transformation. The GUS activity was examined by histochemical and spectrophotometric assay in transformed potato plants. The GUS activity was found primarily around the vascular tissue cells in stem and root. In the spectorophotometric assay, the level of GUS activity of transgenic potato transformed with CaMV 35S/249 bp of intron 1 fragment-GUS (pLS201) was compared with that of potato transformed with CaMV 35S-GUS (pBI121). The quantitative spectrophotometric assay showed that the level of GUS activity in potato transformed with pLS201 was higher in leaf, stem and root by 30-, 34- and 42-fold, respectively than those in potato transformed with pBI121. This results indicate that the inclusion of the deleted maize Adhl-S intron 1 resulted in increament of the GUS gene expression in transgenic potato.potato.

• Horticultural Science & Technology
• /
• v.18 no.5
• /
• pp.594-598
• /
• 2000

This study was carried out to investigate the tissue-specific expression of ${\beta}$-glucuronidase (gus) gene driven by endosperm-specific promoter (Z4 promoter) in the transgenic tobacco and to find out inheritance pattern of transgene to the next generation. Tobacco (Nicotiana tabaccum cv. Havana SR1) was transformed with Agrobacterium tumerfaciens LBA4404 harboring BV3 construct containing gus gene driven by Z4 promoter and a kanamycin resistant gene. Seven hundred bp PCR products, indicating the presence of npt II gene, were found in the all eight transformants by PCR analysis using nptII primers. To study the expression pattern of the two different kind of promoters, leaf disks of the Z4pro-gus-transformed plants and 35Spro-gus-transformed plants were analyzed histochemically for gus activity. As a result, leaf disks of Z4pro-gus-transformed plants showed very weak and partial positive gus activity. In contrast, leaf disks of 35Spro-gus-transformed plants showed relatively strong positive gus activity. To investigate the expressed position of Z4 promoter, seeds from Z4pro-gus-transformed plants and 35Spro-gus-transformed plants were analyzed histochemically for gus activity. Z4pro-gus-transformed seeds showed positive gus activity restricted to the endosperm. However, the blue-colored product in 35Spro-gus-transformed seeds was observed in all the area including endosperm. Kanamycin resistance assay showed that transgenes were stably inherited to next generation in all lines.

• Journal of Plant Biology
• /
• v.37 no.1
• /
• pp.77-83
• /
• 1994

DNA uptake in dry embryos of rice by DNA imbibition was detected by monitoring the expression of chimeric vectors. The selective markers of expression vectors used were ${\beta}-glucuronidase$ ronidase (GUS) and hygromycin phosphotransferase (HPT) genes under the control of CaMV35 S promoter. Frequency of transient expression of the foreign gene was generally 30-50% varying according to the types of vectors and rice cultivars. Dot blot analysis and DNA sequence analysis of inverse polymerase chain reaction products showed that selected rice in hygromycin B (HmB) medium had HPT gene and CaMV35S promoter DNA sequence in genomic DNA of rice. To investigate what ratio of rice having two marker genes simultaneously as rice embryos imbibed the vector DNA having two HPT and GUS gene, transform ants selected in lImB medium were subjected to PCR for GUS gene. It was shown that about 90 percentage of surviving ones in HmB medium had GUS gene.S gene.

• Journal of Plant Biotechnology
• /
• v.7 no.1
• /
• pp.37-43
• /
• 2005

Explants of button daisy were screened for their regeneration potential and transient GUS gene expression. Medium containing MS salts minerals and $B_5$ vitamins supplemented with $0.1\;\cal{mg/L}$ BA and $0.1\;\cal{mg/L}$ TDZ showed the best regeneration. Disc florets and receptacles were the most responsive explants in regeneration and transient gene expression respectively. Regenerated plants were successfully rooted and established in the green-house conditions. Infection and co-cultivation of explants with Agrobacterium tumefaciens containing pCAMBIA 1301 resulted in transient GUS foci. Among the different explants, receptacles showed the highest percentage of transient GUS gene expression. Enzymatic and molecular analyses of transformed calli confirmed the integration of GUS gene.

• Journal of Life Science
• /
• v.15 no.2 s.69
• /
• pp.176-181
• /
• 2005

To study calmodulin (CaM) gene expression and its regulation, rice CaM promoter (RCaM-2) was isolated and fused to $\beta-glucuronidase$ (GUS), reporter gene. X-Glue staining patterns revealed that GUS localization is high in meristemic tissues such as the stem apex, stolen tip, and vascular regions. GUS staining in the transverse sections of stem and petiole was restricted to the inside of the vascular system, and cortex and epidermis located outside of the vascular system usually did not show GUS staining even a plant that expressed strong activity. GUS activity was found to be tissue specific expressed and exhibited a dramatic transient increase in response to wounding. These results suggest that the 5'-flanking region of RCaM gene regulates wound-inducible expression.

• Journal of Korean Society of Forest Science
• /
• v.86 no.3
• /
• pp.261-269
• /
• 1997

Excised immature ovules and calli derived from the stems of cottonwood were bombarded with microprojectiles carrying plasmid DNA containing CaMV-35S promoter and ${\beta}$-glucuronidase(GUS) gene. After bombarded, the expression of GUS gene was detected by the assay of 5-bromo-4-chloro-3-indolyl-${\beta}$-gluconide(X-gluc). Transient gene expression was measured by counting the number of distinct regions of GUS activity per explant. As major parameters, the number of shots and the period of exposure to X-gluc after the bombardment were investigated for detecting GUS gene expression. In this experiment, the percents of GUS gene expression showing spots were 56.8 from immature ovules and 75.9 from micro-calli of cottonwood species. Among the treatments, two consecutive shots and 48 hour exposure produced about $25.75{\pm}2.77$(per ovule), $11.43{\pm}1.22$(per mini petridish) spots, respectively, Microprojectile particle bombardment provides a useful method to assay transient expression in both types of explants. Furthermore, our results represent that the excised ovule and/or the calli might be stably transformed by the biolistics.

• Journal of Crop Science and Biotechnology
• /
• v.10 no.1
• /
• pp.50-56
• /
• 2007

Barley S-adenosylmethionine synthetase1 gene, which was differentially expressed in seed development of extra early barley, was regulated by the phytohormones and abiotic stresses. In order to identify the regulation regions which were involved in transcriptional control of the phytohormones and abiotic stresses, we isolated 1459 bp fragment of HvSAMS1 gene promoter using genome walking strategy and deletion series were constructed. Deleted upstream fragments(-1459, -1223, -999, -766, -545, -301 bp) were fused to the GUS reporter gene and evaluated via Agrobacterium-mediated transient expression assay. Increased GUS activity of HvSMAS1 promoter -301/GUS construct under each of NaCl, $GA_3$, ABA and ethylene application was found. However, GUS activity was negligible in the leaves transformed with the HvSMAS1 promoter(-1459, -1223, -999, -766 and -545)/GUS constructs. No significant induction of GUS activity was observed for the ethionine and spermidine treatments. In order to locate promoter sequence of the HvSAMS1 gene that was critical for the activation of gene expression, deletion and addition promoter derivatives(+, includes 43 bp of 5' ORF) of the HvSAMS1 gene fused to the GUS reporter gene were applied. The tobacco leaves which harbored the additional HvSAMS1 promoter(-1459+, -1459 to -546, -545+ and -301+)/GUS construct did not significantly induce GUS activity as compared to the HvSAMS1 promoter(-1459, -545 and -301)/GUS constructs under each of NaCl, ABA and $GA_3$ treatment. However, the GUS activity was high in the tobacco leaves which harboring the -211 to -141 regions of the HvSAMS1 promoter. This result suggested that HvSAMS1 gene expression might be regulated by this region(from -211 to -141).

• Journal of Plant Biotechnology
• /
• v.37 no.4
• /
• pp.511-516
• /
• 2010

The process to acquire intron-GUS gene-expressed transformants from somatic embryos (including embryogenic calli) of Rosa hybrida cv. 'Sweet Yellow' using Agrobacterium-meditated transformation method was reported in this study. Somatic embryos including embryogenic calluses were infected with Agrobacterium tumefaciens AGL1 strain (O.D = 0.7~1.6) including intron-GUS gene for 30 min, and were co-cultured for 3 days. After co-cultivation, they were cultured on embryo germination medium (EGM) supplemented with $250\;mg{\cdot}L^{-1}$ cefotaxim at $4^{\circ}C$ for 7 days. Then, transient GUS gene expression was observed. Shoots were regenerated from the shoot primodia induced from the intron-GUS gene-transferred either somatic embryos or embryogenic calli cultured on EGM supplemented with both cefotaxim $250\;mg{\cdot}L^{-1}$ and ppt $2\;mg{\cdot}L^{-1}$. Before induction of rooting from shoots cultured on shoot growing medium supplemented with both cefotaxim $250\;mg{\cdot}L^{-1}$ and ppt $2\;mg{\cdot}L^{-1}$, the shoots were cultured on multi-shoot induction medium supplemented with both cefotaxim $250\;mg{\cdot}L^{-1}$ and ppt $2\;mg{\cdot}L^{-1}$ to induce multi-shoots. When expression of the gene from a part of the multi-shoots was identified by GUS transient assay, the putative transgenic multishoots were transferred to rooting medium supplemented with cefotaxim $250\;mg{\cdot}L^{-1}$. After the formation of healthy roots, transgenic plantlets were transferred to the greenhouse after acclimatization. The expression rate of the intron-GUS gene in the multi-shoots was 100%.

• KSBB Journal
• /
• v.8 no.2
• /
• pp.104-109
• /
• 1993

A vector for plant transformation which had two reporter genes(Gus and Hpt genes) in a single plasmid was constructed. After rice embryos imbibed DNA solution, DNA uptake and gene expression in rice were monitored. Main expression sites of the Gus gene were meristem of root and coleoptiles. There was no difference in Hpt gene expression between a single Hpt vector and the constructed vector in viability of rice in the hygromycin medium after DNA imbibition, The genomic DNA and total RNA extracted from rice transformant survived in the hygromycin medium were subjected to PCR and RT PCR analysis, respectively. As a result, we found the existence of the Hpt gene and its expression in rice.