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

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

• Proceedings of the Korean Society of Crop Science Conference
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• 2022.10a
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• pp.221-221
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• 2022

Chromosome breakage occurred by DNA methylation inhibitor. Zebularine is known as DNA methylation inhibitor and suitable for water solubility among different DNA methylation inhibitors as 5-Azacytidine and 5-aza-2'-deoxycytidine. We used zebularine as mutagen according to different methods by roots absorption and seed imbibition. After zebularine treatment, DNA methylation inhibitor, we observed mitotic chromosome behavior what is different according to two different treatment methods. First, seed imbibition treatment in 1,000 μM of zebularine solution for 72 hours in dark conditions. The second treatment to seedlings of Keumkang was also treated in 1,000 μM of zebularine solution for 72 hours after germination. Root and shoot showed different elongations in each treatment. Root absorption treatment(3.01±0.48, 2.00±0.26) showed the shortest elongation in root and shoot than control(8.16±0.61, 4.03±0.48) and seed imbibition treatment(4.33±0.80, 2.48±0.36). It can be explained root tip meristematic cell activity was damaged by DNA methylation inhibitor. Primary root tips were collected in DW for 24 hours at low temperature(0℃) and fixed in fixation solution for 3 days to chromosome observation in mitosis. Mitotic index, chromosome structure and chromosome aberration were observed by phase-contrast microscope. Mitotic index of the control(0.29) showed twice mitotic cells as the treated groups(imbibition 0.15, absorption 0.14). Observation of chromosomes showed some short chromosomes and loosen chromosomes affected by zebularine. It is considered because of zebularine damage DNA in mitosis. We observed "gap by chromosome breakage" in chromosomes that have loose parts between centromere and telomere. It seems demethylation of zebularine occurs chromosome breakage.

• Journal of Life Science
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• v.25 no.2
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• pp.158-163
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• 2015

Rice embryo is more abundant than endosperms in nutrients such as proteins, lipids, and vitamin B1. In this study, we constructed p53 plasmid that could be expressed in a plant system, and investigated optimal germination conditions in a variety of media. For construction of p53 plasmid, we performed p53 amplification from pCDNA-p53, subcloned to TA cloning vector, and then reconstructed into pGEM-CaMV plant expression vector. On the other hand, we prepared a variety of imbibition buffers and complete media for efficient germination of the rice embryo. Imbibition buffers prepared with different concentrations of salt or detergent showed no significant effect on germination efficiency. We prepared further culture media, such as solid agar, liquid media, and paper towel to establish the optimal conditions. Rice embryo showed germination rates of more than 70% in the solid medium, more than 60% in the paper towel medium, but less than 25% in liquid media, although germination rate did not differ with varying concentrations of salt and sucrose in culture media. Under the optimal germination conditions, we introduced the p53 plasmid using imbibition method, and finally detected human p53 gene expression in the germinated rice embryo. This method might present a novel, practical approach for evaluating efficient gene expression utilizing imbibition method in rice embryo.

• Journal of Microbiology and Biotechnology
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• v.10 no.3
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• pp.415-418
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• 2000

Productivity of a rice plant is greatly influenced by salt stress. One of the ways to achieve tolerance to salinity is to transfer genes encoding protective enzymes from other organisms, such as microorganisms. The bacterial gene, mtlD, which encodes mannitol-1-phosphate dehydrogenase (Mtl-DH), was introduced to the cytosol of a rice plant by an imbibition technique to overproduce mannitol. The germination and survival rate of the imbibed rice seeds were markedly increased by transferring the mtlD gene when it was delivered in either a pBIN19 or pBmin binary vector. When a polymerase chain reaction was performed with the genomic DNAs of the imbibed rice leaves as a template and with mtlD-specific primers, several lines were shown to contain an exogenous mtlD DNA. However, a reverse transcription (RT)-PCR analysis revealed that not all of them showed an expression of this foreign gene. This paper demonstrates that the growth and germination of rice plants transiently transformed with the bacterial gene, mtlD, are enhanced and these enhancements may have resulted from the experssion of the mtlD gene. The imbibition method empolyed in this study fulfills the requirements for testing the function of such a putative gene in vivo prior to the production of a stable transgenic plant.

• Proceedings of the Botanical Society of Korea Conference
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• 1999.07a
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• pp.63-67
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• 1999

We have isolated more than a dozen cDNA clones corresponding to genes that were expressed in Arabidopsis leaves when they were kept in the dark. The nucleotide sequence analysis showed that some of the clones encoded proteins with significant homology to $\beta$-glucosidase (din2), branched-chain $\alpha$-keto acid dehydrogenase subunit E1$\beta$(din3), and another subunit E2 (din4), yeast RAD23 (din5), asparagine synthetase (din6), pre-mRNA splicing factor SRp35 (din7), phosphomannose isomerase (din9), seed imbibition protein (din10), and 2-oxoacid-dependent oxidase (din11). Accumulation of transcripts from din3,4,6 and 10 occurred rapidly after the plants were transferred to darkness. Transcripts from din2,9, and 11 could be detected only after 24 h of dark treatment. Inhibition of photo-synthesis by DCMU strongly induced the accumulation of transcripts from those genes, and application of sucrose to detached leaves suppressed the accumulation both in the dark and by DCMU. These observations indicate that expression of the genes is caused by sugar starvation resulted from the cessation of photosynthesis. We further showed that din2-encoded protein also accumulated in senescing leaves. Given these results, possible roles of din genes in leaves in the dark and senescing leaves are discussed.

• Journal of Plant Biotechnology
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• v.36 no.3
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• pp.281-288
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• 2009

Seed germination is the important stage to express many genes for regulation of energy metabolism, starch degradation and cell division from seed dormancy state. For the functional analysis of seed germination mechanisms, we were analyzed the rice cDNA clones (Oryzasativa cultivar Ilpum) obtained from seed imbibition during 48 hours. Total number of 18,101 Expressed Sequence Tags (ESTs) were clustered using SeqMan program. Among them, 8,836 clones were identified as unique clones. We identified the chitinase gene specifically expressed in seed germination and amylase gene involved to starch degradation from the full length cDNA analysis, and several genes were registered to NCBI GeneBank. To analyzed the commonly expressed genes between inmature seed and germinated seed, 25,66 inmature ESTs and 18,101 germinated ESTs were clustered using SeqMan program and identified 2,514 clones as commonly expressed unigene. Among them, alpha-glubulin and alcohol dehydrogenase I were supposed to LEA genes only expressed in the immature and germinated seed stages. For the clustering of orthologous group genes, we further analyzed the 8,836 EST clones from germinating seeds using NCBI clusters of orthologous groups database. Among the clones, 5,076 clones were categorized into information storage and processing, cellular processes and signaling, metabolism and poorly characterized genes, proportioning 783 (14.29%), 1,484 (27%), 1,363 (24.8%) and 1,869 (34%) clones to the previous four categories, respectively.