• Journal of Plant Biology
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• v.31 no.4
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• pp.267-275
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• 1988

Effects of polyamines on ethylene biosynthesis were studied in synchronized suspension cultured cells from leaf segments of Nicotiana tabacum L. Putrescine, spermidine and spermine inhibited the endogenous production of both ACC and ethylene. Those production was more remarkably inhibited by spermidine and spermine than putrescine. These results were the same tendency with those obtained from exogenous application of SAM and ACC. Polyamines had more inhibitory effect on hte conversion of ACC to ethylene than that of SAM to ACC, but ACC was not accumulated. The inhibition rate of exogenously applied ACC conversion to ethylene was well coincident with that of exogenously applied SAM conversion to ethyene via ACC by polyamines. However, polyamines inhibited more the activity of ACC synthase than that of EFE. From these results we can suggest that polyamines inhibit both steps of SAM to ACC and ACC to ethylene, and more effectively the latter than the former.

• Journal of Plant Biology
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• v.33 no.2
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• pp.87-96
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• 1990

The role of S-adenosylmethionine (SAM) as an intermediate in interrelation between polyamine and ethylene biosynthesis was studied in suspension cultures of Nicotiana tabacum L. Exogenous SAM stimulated the polyamine and ethylene biosynthesis in 4 day-cultured cells, which were in active cell divisions, and 10 day cultured cells, which went on with active cell elongation and senescence. SAM-induced ethylene production was more effective in 10 day-cultured cells than in 4 day-cultured cells, but SAM-induced polyamine biosynthesis was more effective in 4 day-cultured cells than in 10 day-cultured cells. Polyamine contents were increased by the blockage of ethylene biosynthetic pathway in the conversion of SAM to ethylene via 1-aminocyclopropane-1-carboxylinc acid (ACC) with aminooxyacetic acid (AOA). Also, ethylene production was increased by the inhibitors of polyamine biosynthesis such as methylglyoxal bis-(guanylhydrazone) (MGBG), dicyclohexylamine (DCHA), $\alpha$-difluoromethylarginine (DFMA) and $\alpha$-difluoromethylorinithine (DFMO). These results suggest that there may be interrelations between polyamine and ethylene biosynthesis for the competition of SAM and the inherent mechanism of switch on-off in polyamine and ethylene biosynthetic activity with the progress of cell growth and senescence.

• KSBB Journal
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• v.22 no.4
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• pp.185-190
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• 2007

Productivity of secreted recombinant protein depends largely on its stability in the extracellular environment with protease. Most hGM-CSF produced by transgenic tobacco cell cultures and secreted to the medium was confirmed to be rapidly degraded by protease in medium. To increase the productivity, therefore, various protein stabilizers such as gelling agents such as carrageenan and alginate, polymers, polyols, and amino acids have been tested. The stability of hGM-CSF in spent medium without cells was improved by the presence of gelling agents. However, the reason for the enhanced production by the addition of gelling agents may be due to the increased expression level and permeability rather than stability. The addition of DMSO inhibited the cell growth, but improved specific yield. The others were not effective for stability as well as hGM-CSF production.

• Journal of Plant Biology
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• v.36 no.1
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• pp.19-27
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• 1993

Effects of polyamines on DNA synthesis were studied in synchronized culture of Nicotiana tabacum L. When DFMO and DFMA, inhibitors of ornithine decarboxylase and arginine decarboxylase, respectively were initially applied to the cells, the polyamine contents were rapidly dropped and [methyl-3H] thymidine incorporation into DNA was markedly reduced during the early stage of culture period. Inhibition of DNA synthesis, however, was partially reversed when these inhibitors were applied simultaneously with putrescine. In addition, exogenous administration of putrescine also increased the DNA synthesis during the all over the culture period. In vitro activity of DNA polymerase from Nicotiana tabacum L. was promoted by increasing concentrations of polyamines in the reaction mixture. Maximal activity was shown at 5 mM putrscine, 0.5 mM spermidine and spermine, respectively. Lack of Mg2+ ion in the reaction buffer resulted in an inhibition of the enzyme activity by about 30%. The inhibition could not be completely reversed by application of polyamines at optimal concentrations. These results suggest that polyamines promote the DNA synthesis in vivo and in vitro by stabilizing the DNA-helix upon binding to negatively charged groups on DNA or increasing the activity of DNA polymerase in Nicotiana tabacum L.

• The Journal of Natural Sciences
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• v.4
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• pp.103-142
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• 1991

These studies were carried out to select somatic hybrid using selectable marker genes of Nicotiana glauca transformed by NPTII gene and Solanum tuberosum transformed by T- DNA, and to study characteristics of transformant. The results are summarized as follows. 1. Crown gall tumors and hairy roots were formed on potato tuber disc infected by A. tumefaciens Ach5 and A. rhizogenes ATCC15834. These tumors and roots could be grown on the phytohormone free media. 2. Callus formation from hairy root was prompted on the medium containing 2, 4 D 2mg/I with casein hydrolysate lg/l. 3. The survival ratio of crown gall tumor callus derived from potato increased on the medium containing the activated charcoal 0. 5-2. 0mg/I because of the preventions on the other hand, hairy roots were necrosis on the same medium. 4. Callus derived from hairy root were excellently grown for a short time by suspension culture on liquid medium containing 2, 4-D 2mg/I and casein hydrolysate lg/l. 5. The binary vector pGA643 was mobilized from E. coli MC1000 into wild type Agrobacteriurn tumefaciens Ach5, A. tumefaciens $A_4T$ and disarmed A. tuniefaciens LBA4404 using a triparental mating method with E. ccli HB1O1/pRK2013. Transconjugants were obtained on the minimal media containing tetracycline and kanamycin. pGA643 vectors were confirmed by electrophoresis on 0.7% agarose gel. 6. Kanamycin resistant calli were selected on the media supplemented with 2, 4-D 0.5mg/1 and kanamycin $100\mug$/ml after co- cultivating with tobacco stem explants and A. tumefaciens LBA4404/pGA643, and selected calli propagated on the same medium. 7. The multiple shoots were regenerated from kanamycin resistant calli on the MS medium containing BA 2mg/l. 8. Leaf segments of transformed shoot were able to grow vigorusly on the medium supplemented with high concentration of kanamycin $1000\mug$/ml. 9. Kanamycin resistant shoots were rooting and elongated on medium containing kanamycin $100\mug$/ml, but normal shoot were not. 10. For the production of protoplast from potato calli transformed by T-DNA and mesophyll tissue transformed by NPTII gene, the former was isolated in the enzyme mixture of 2.0% celluase Onozuka R-10, 1.0% dricelase, 1.0% macerozyme. and 0.5M mannitol, the latter was isolated in the enzyme mixture 1.0% Celluase Onozuka R-10, 0.3% macerozyme, and 0.7M mannitol. 11. The optimal concentrationn of mannitol in the enzyme mixture for high protoplast yield was 0.8M at both transformed tobacco mesophyll and potato callus. The viabilities of protoplast were shown above 90%, respectively. 12. Both tobacco mesophyll and potato callus protoplasts were fused by using PEG solution. Cell walls were regenerated on hormone free media supplemented with kanamycin after 5 days, and colonies were observed after 4 weeks culture.

• Applied Biological Chemistry
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• v.39 no.5
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• pp.355-360
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• 1996

In vitro-tested ribozyme against synthesized cucumber mosaic virus (CMV) RNA (Agric. Chem. & Biotech. 37:56-63(1994)) was expressed in tobacco plant to develop virus resistant plants. The ribozyme sequence was linked to cauliflower mosaic virus 35S promoter and nopaline synthase(nos) terminator and this chimeric 35S-ribozyme-nos gene was sequenced. The sequenced chimeric gene was transferred to Agrobacterium tumefaciens LBA4404 using tri-parental mating system. The E. coli HB101 containing chimeric gene was incubated with E. coli HB101(pRK2073) as a helper and Agrobacterium tumefaciens LBA4404. Then Agrobacterium cells containing the ribozyme construct was cocultivated with tobacco leaf pieces. Ten different plants were regenerated from kanamycin containing MS medium. The presence of the ribozyme construct in the transgenic tobacco plants was confirmed by polymerase chain reaction (PCR). Seven different transgenic plants in ten different kanamycin resistant plants showed the expected size (570 base pairs) of 35S-ribozyme-nos gene fragment. Total RNAs were isolated from four different transgenic plants and separated on a 1% agarose gel containing formamide. Northern hybridization with 35S-ribozyme-nos gene fragment as a probe indicated that ribozyme transcripts may be degraded tv nuclease. Therefore, nuclease-resistant ribozymes are needed for the development of virus-resistant transgenic plants using ribozymes.

• The Korean Journal of Pesticide Science
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• v.10 no.2
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• pp.131-137
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• 2006

This experiment was conducted to select prominent microorganisms with a good insecticidal activity among the ten species, which isolated from soil at the near of Chung-buk, Chung-nam, and Gang-won provinces and made protein crystal endotoxin. As a result, GB-413 strain was finally selected, which showed the high insecticidal activity against susceptible diamondback moth (Plutella xylostella), beet army worm (Spodoptera exigua) and tobacco cutworm (Spodoptera litura) as well as resistant diamondback moth strains. By modifying the cultivation process f.g. lowing the glucose concentration at early cultivation stage and adding the carbon after inducing the spores, the percentage of making spore as well as the number of active spore were increased and the time for cultivation and spore forming was reduced without a reduction of insecticidal activity. These results were not only applied successfully for the optimized cultivation process for a fermentation tank containing five tons capacity, but also improved the possibility of mass cultivation for commercial production.

• Journal of Life Science
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• v.8 no.5
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• pp.604-613
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• 1998

The last enzyme of the sesquiterpen phytoalexin capsidiol synthesis in tobacco cell, 5-epi-aristolochene hydro-xylase which convert 5-epi-aristolochene (EAS) to capsidiol, was cloned by a reverse transcription polymerase chain reaction strategy and cDNA library screening. Cloned CYP-B3 contained high probability amino acid matches to known plant cytochrome P450 sequences and open reading frame with the conserved FxxGxRxCxG heme-binding region. Transcripts of CYP-B3 were not detected in control cells, but induced in elicitor-treated cells. Furthermore, CYP-B3 transcripts were induced by fungal extracts and cellulase but not by other stimuli(chilling, heat shock and 2,4-D). Induction of CYP-B3 transcripts by elicitor treatment was not affected by ancymidol and ketoconazole treat-ments suggesting that an inhibition of hydroxylase activity by Cyt P450 inhibitors resulting from post translational processing event.

• Proceedings of the Korean Society of Plant Biotechnology Conference
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• 2002.04b
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• pp.49-58
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• 2002

Oxidative stress derived from reactive oxygen species (ROS) is one of the major damaging factors in plants exposed to environmental stress. In order to develop the platform technology to solve the global food and environmental problems in the 21st century, we focus on the understanding of the antioxidative mechanism in plant cells, the development of oxidative stress-inducible antioxidant genes, and the development of transgenic plants with enhanced tolerance to stress. In this report, we describe our recent results on industrial transgenic plants by the gene manipulation of antioxidant enzymes. Transgenic tobacco plants expressing both superoxide dismutase (SOD) and ascorbate peroxidase (APX) in chloroplasts were developed and were evaluated their protection effects against stresses, suggesting that simultaneous overexpression of both SOD and APX in chloroplasts has synergistic effects to overcome the oxidative stress under unfavorable environments. Transgenic tobacco plants expressing a human dehydroascorbate reductase gene in chloroplasts were showed the protection against the oxidative stress in plants. Transgenic cucumber plants expressing high level of SOD in fruits were successfully generated to use the functional cosmetic purpose as a plant bioreactor. In addition, we developed a strong oxidative stress-inducible peroxidase promoter, SWPA2 from sweetpotato (Ipomoea batatas). We anticipate that SWPA2 promoter will be biotechnologically useful for the development of transgenic plants with enhanced tolerance to environmental stress and particularly transgenic cell lines engineered to produce key pharmaceutical proteins.

• Journal of Plant Biotechnology
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• v.29 no.2
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• pp.69-77
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• 2002

Oxidative stress derived from reactive oxygen species (ROS) is one of the major damaging factors in plants exposed to environmental stress. In order to develop the platform technology to solve the global food and environmental problems in the 21st century, we focus on the understanding of the antioxidative mechanism in plant cells, the development of oxidative stress-inducible antioxidant genes, and the development of transgenic plants with enhanced tolerance to stress. In this report, we describe our recent results on industrial transgenic plants by the gene manipulation of antioxidant enzymes. Transgenic tobacco plants expressing both superoxide dismutase (SOD) and ascorbate peroxidase (APX) in chloroplasts were developed and were evaluated their protection effects against stresses, suggesting that simultaneous overexpression of both SOD and APX in chloroplasts has synergistic effects to overcome the oxidative stress under unfavorable environments. Transgenic tobacco plants expressing a human dehydroascorbate reductase gene in chloroplasts were showed the protection against the oxidative stress in plants. Transgenic cucumber plants expressing high level of SOD in fruits were successfully generated to use the functional cosmetic purpose as a plant bioreactor. In addition, we developed a strong oxidative stress-inducible peroxidase promoter, SWPA2 from sweetpotato (lpomoea batatas). We anticipate that SWPA2 promoter will be biotechnologically useful for the development of transgenic plants with enhanced tolerance to environmental stress and particularly transgenic cell lines engineered to produce key pharmaceutical proteins.