• Journal of the Korean Society of Tobacco Science
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• v.13 no.2
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• pp.5-20
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• 1991

For the quality enhancement of harvested-year leaf tobacco to the quality of 2-year naturally aged leaf tobacco, cellulose and nicotine degradative bacteria were isolated and identified. Effects of artificial fermentation treated cellulase and nicotine degradative bacteria on the quality of leaf tobacco were investigated from the chemical and sensory points of view. 1, Changes in chemical composition of leaf tobacco resulted from the addition of cellulase extracted from Cellulomonas sp. [3ml(${\mu}{\textrm}{m}$ D-glucose/ml. mil-1) of enzymes solution 11009 of leaf tobacco] and nicotine degradative bacteria, Pseudomonas sp. 2ml(IX109 cells$\div$ 100g of leaf tobacco), and subsequently fermented at 40${\mu}{\textrm}{m}$$^{\circ}C$, 65% R. H. for 40 days are as follows : 1) Content of crude fiber decreased 12% It took 9 min, 53 sec. to reach full combustion in control group but took only 7 min. 47 sec. in the treated group, taking almost equal time to 2-year naturally aged leaf tobacco(7 min. 35sec.). 2) Light intensity of control group was 60.96% with bright lemon color but that of treated leaf tobacco accounted for 47.69 with orange to dark brown color series, which was almost equal to the value, 45.69, of 2-year naturally aged leaf tobacco. 3) Linoleic acid, serving mild taste among organic acids, amounted to 1.llmg/g in control group but increased to 1.35m9/9 in the treated leaf tobacco, identical to the content(1.35mg/g) of 2-year naturally aged leaf tobacco. 4) Content of solanone, on of the typical leaf tobacco flavor compounds, accounted for 2.95% in control group but increased to 2.87% in treated group. 5) Methyl furan, useful flavor compound in smoke composition, accounted for 17.6$\mu\textrm{g}$/cig. in control group but increased to 25.9$\mu\textrm{g}$/cig. in treated group. However, acroleine decreased from 69.3$\mu\textrm{g}$/cig. in control group to 58.6$\mu\textrm{g}$/cig. in treated group 2. In sonsory test, mild taste evaluation of control group scored 5.47 and treated group 7.93 which was evaluted almost equal to the value(8.00) of 2-year naturally aged leaf tobacco. Aroma evaluation of control group scored 5.60, treated group 8.20, and 2-year naturally aged leaf tobacco 8.33. In addition, total harmony taste of control group showed 5.67, treated group 8.07 (p<0.01), and 2-year naturally aged leaf tobacco 8.00. From these results, it can be said that quality of treated leaf tobacco is not inferior to that 2-year naturally aged leaf tobacco.

• Korean Journal of Plant Tissue Culture
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• v.26 no.3
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• pp.183-187
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• 1999

To establish an efficient screening system for new herbicides using plant cultured cells, responses of tobacco photomixotrophic cultured (PH) cells to various herbicides with different modes of action were surveyed by measuring the cell growth and ion conductivity in medium. The cells were cultured in Murashige and Skoog (MS) medium containing 0.7mg/L 2,4-D, 0.3mg/L kinetin and 30 g/L sucrose at $25^{\circ}C$ in the light (100 rpm). Chemicals were treated to suspension cultures of tobacco PH cells at the time of subculture. The cell growth and ion conductivity in the medium were investigated on 12 days after chemical treatment. The ion conductivity assay gave well correlated results to the cell growth inhibition data. The responses of tobacco PM cells were dependent on the modes of action of chemicals tested. Atrazine, an inhibitor of photosynthetic electron transport (PET), strongly inhibited both the cell membrane and cell growth ($IC_{50}$/, about 1 $\mu$M). Butachlor (an inhibitor of cell division), glufosinate (an inhibitor of amino acid biosynthesis), and fluridone (an inhibitor of carotenoid biosynthesis) showed a dose-dependent inhibition. However, Quinclorac, a herbicide with an auxin activity, did not affect the cell growth and ion leakage. These results suggested that tobacco PM cells is suitable materials for the simple screening of new herbicides such as PET, amino acid biosynthesis, ceil division inhibitors by measuring the cell growth and ion conductivity.

• Journal of Plant Biotechnology
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• v.2 no.2
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• pp.55-60
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• 2000

Experiments initiated 30 years ago to obtain selectable markers have led to a series of studies of Trp biosynthesis and anthranilate synthase (AS) the control enzyme using largely plant tissue cultures since they have experimental properties that can be readily exploited. Enzymological and compound feeding studies provided evidence that AS is the control point in the Trp biosynthesis branch and that altering the AS feedback control by the selection of mutants resistant to the Trp analog 5-methyl-tryptophan (5MT) can lead to the overproduction of this important amino acid. Plants regenerated from these Trp overproducing lines of most species also had high free Trp levels but Nicotiana tabaum (tobacco) plants expressed the feedback altered AS only in cultured cells and not in the regenerated plants. further tests by transient and stable expression of the cloned promoter for the naturally occurring tobacco feedback-insensitive AS, denoted ASA2, confirmed the tissue culture specific nature of the expression control. The 5MT caused by the expression of a feedback-insensitive AS from tobacco has been used to select protoplast fusion hybrids with several species since the resistance is expressed dominantly. Recently the ASA2 gene has been used successfully as a selectable marker to select transformed Astragalus sinicus and Glycine max hairy roots induced by Agrobactetium rhizogenes. These results show that the ASA2y-subunit can interact with the y-subunit of another species to form active feedback-insensitive enzyme that may be useful for selecting transformed cells. Plastid DNA transformation of tobacco has also effectively expressed ASA2 in the compartment in which Trp biosynthesis is localized in the cell.

• Journal of the Korean Society of Tobacco Science
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• v.15 no.2
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• pp.130-136
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• 1993

Protoplasts of palisade cells were aseptically isolated from leaves of Nicotiono apicona Merxm. by the one step enzymatic method. Efficiency of colony formation were depended on cell density and light condition during incubation, but an intensity of 47 ft-c during a period of 2 weeks after isolation of the protoplasts in the Nagata and Takebe's medium promoted the planting efficiency. Protoplast - derived calli of N. africana can be differentiated into shoot when cultured on Murashige and skoog's medium containing IAA(0.Smg/$\ell$) and zeatin(5.0mg/$\ell$).

• Journal of Ginseng Research
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• v.19 no.1
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• pp.39-44
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• 1995

Total saponin from Korean red ginseng changed thermodynamic parameters of membranes from dipalmitoylphosphatidylcholine (DPPC) and ghost erythrocytes of human. In liposomes from DPPC, temperature of the main transition (Lb'-La) in liquid-crystalline phase increases by 0.2$^{\circ}C$ in average, but enthalpy does not change. Total saponin at a concentration of smaller than $10^5$% "stabilizes" the timid bilayers. At larger than 0.07 of saponin/DPPC ratio, saponin leads to an exclusion of the bound lipid molecules from the main phase transition into lamella liquid crystalline La-phase. Total saponin influences specifically all erythrocyte membrane transitions in a concentration-dependent manner, i.e. on the structures of all the main membrane skeleton proteins. A high structural specificity of saponin with membrane proteins, could be a base of specificity of physiological response of not only erythrocytes, but also other cells.her cells.

• Molecules and Cells
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• v.20 no.1
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• pp.136-141
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• 2005

Mild stresses such as high temperature ($30^{\circ}C$) or a low $H_2O_2$ concentration induced transient cell cycle arrest at G1/S or G2/M depending on the cell cycle stage at which the stress was applied. When stresses were introduced during G0 or G1, the G1/S checkpoint was mainly used; when stresses were introduced after S phase, G2/M was the primary checkpoint. The slowing of cell cycle progression was associated with transient delays in expression of A-, B-, and D-type cyclins. The delay in expression of NtcycA13, one of the A-type cyclins, was most pronounced. The levels of expression of Ntcyc29 (a cyclin B gene) and of CycD3-1 differed most depending on the applied stress, suggesting that different cellular adjustments to mild heat and a low concentration of $H_2O_2$ are reflected in the expression of these two cyclins.

• Journal of Plant Biotechnology
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• v.40 no.3
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• pp.169-177
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• 2013

Recently, the number of people with diabetes is rapidly increasing, coupled with the fact that the insulin market is remarkably increasing. Therefore, molecular farming for plant-derived pharmaceutical protein production is reported as becoming more attractive than ever. In this study, we carried out experiments step by step for development of recombinant insulin constructs, which were transformed into E. coli system, in vitro transcription and translation system, and tobacco cells. At first, recombinant proinsulin protein was successfully produced in in vitro transcription and translation system with wheat germ extract. After which, recombinant construct of prominiinsulin encoded a fusion protein of 7.8 kDa with trypsin cleavage sites at N terminus and C terminus of minimized C-peptide was tried to in vitro expression using E.coli culture. After purification with His-tag column, the resulting recombinant prominiinsulin protein was processed with trypsin, and then checked insulin biosynthesis by SDS-PAGE and western blot analysis with anti-insulin monoclonal antibody. The immunoreactive product of trypsin-treated miniinsulin was identical to the predicted insulin hexamer. The construct of 35S promoter-driven preprominiinsulin recombinant gene with signal peptide region for ER-targeting and red fluorescence protein gene [N terminus ${\rightarrow}$ tobacco E2 signal peptide ${\rightarrow}$ B-peptide (1-29 AA) ${\rightarrow}$ AAK ${\rightarrow}$ A-peptide (1-21 AA) ${\rightarrow}$ RR ${\rightarrow}$ His6 ${\rightarrow}$ KDEL ${\rightarrow}$ C terminus] was transformed into BY-2 tobacco cells. A polypeptide corresponding to the 38-kDa molecular mass predicted for fusion protein was detected in total protein profiles from transgenic BY-2 cells by western analysis. Therefore, this recombinant preprominiinsulin construct can be used for generation of transgenic tobacco plants producing therapeutic recombinant insulin.

• KSBB Journal
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• v.5 no.2
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• pp.151-158
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• 1990

Investigations on the liability of nitrogen usuage by Nostoc muscorum that has nitrogen fixing ability, and cultured tobacco cells as they were associately cultured on nitrogen-free media and effects of polyamine on the associated culture condition were carried out. In addition, measurement on the activity of nitrate reductase, glutamine synthetase, glutamate dehydrogenase and glutamate synthase that take part in the metabolic pathway of nitrogen fixation product were performed. Among enzymes participating in the metabolic pathway of nitrogen fixation products, the activity of nitrogen reductase stimulated five times in associated culture, and that of glutamine synthetase of N. muscorum increased two times after heterocyst differentiated. Activity of glutamate dehydrogenase increased markedly when cultured tobacco cells were solely incubated on nitrogen-free media, but inhibited when cultured associately. And, glutamate synthase was showed the highest activity in 0.1 mM of spermine treated group.

• Journal of the Korean Society of Tobacco Science
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• v.13 no.1
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• pp.43-52
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• 1991

These studies were carred out to obtain the transformant from tobacco cells by Agrobacterium spp. from crown gall and soil at the natural field in Korea, and identified their virulence. Kodo's and Clark's selective media were used for isolation of Agrobacterium spp. In these media, total of 99 strains were characterized based on the morphological characteristics of colonies. Among them 34 strains were able to induce on carrot discs. And hypervirulent strains C23-1 and K29-1 were identified as Agrobacterium tumefaciens biotype 1 and biotype 2, respectively. These strains formed fast growing, larger gall as compared to those induced by other strains on the carrot discs. Transformed tobacco callus was initiated on the phytohormone free MS medium with 250$\mu\textrm{g}$/ml carbenicillin after co-cultivation of tobacco stem explants and Agrobacteria. On the phytohormone free media, shoot was rarely formed from transformed callus. However, these shoot were teratoma shoots which were not grown as normal shoot, and teratoma shoot from transformant by C23-1 was smaller than that of K29-1.

• Journal of the Korean Society of Tobacco Science
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• v.23 no.2
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• pp.133-140
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• 2001

PAP-I cDNA was synthesized from total RNA of Phytolacca americana leaves by RT-PCR, and then subcloned to recombinant vector pBluescript II SK-. Using PCR with primers designed in our laboratory, we could get the 9 deletion mutant PAP-I cDNA fragments. The first of the fragments was deleted by 66bp from immature N-terminal and then the rest were deleted by 90bp sequentially. Sequentially deletion mutant PAP-I cDNAs were inserted to pAc55M, on down-stream of gall promoter. Recombinant pAc55M was transformed to yeast cells, psy1 and the cells were spreaded on SC_urn-/glucose plate media. Colonies on SC_ura-/glucose plate were streaked on the same position of SC_ura-/glucose and SC_ura-/galactose plate, and we selected colonies growing on both plates, which carry non-cytotoxic deleted mutant PAP-I cDNA. We selected 4 deletion mutant PAP-I cDNAs which have not cytotoxicity.