• Molecules and Cells
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• v.27 no.4
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• pp.443-447
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• 2009

This paper reports on the structural rearrangement of satellite DNA type I repeats and heterochromatin during the dedifferentiation and cell cycling of mesophyll protoplasts of cucumber (Cucumis sativus). These repeats were localized in the telomeric heterochromatin of cucumber chromosomes and in the chromocenters of interphase nuclei. The dramatic reduction of heterochromatin involves decondensation of subtelomeric repeats in freshly isolated protoplasts; however, there are not a great many remarkable changes in the expression profile. In spite of that, reformation of the chromocenters, occurring 48 h after protoplast isolation, is accompanied by recondensation of satellite DNA type I; however, only partial reassembly of these repeats was revealed. In this study, FISH and a flow cytometry assay show a correlation between the partial chromocenter and the repeats reassembly, and with the reentry of cultivated protoplasts into the cell cycle and first cell division. After that, divided cells displayed a higher variability in the expression profile than did leaves' mesophyll cells and protoplasts.

• Journal of Plant Biology
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• v.23 no.2
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• pp.49-54
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• 1980

The optimal conditions for the protoplast isolation from the leaves of pea (Pisum sativum L. cv. Sparkle) and barley (Hordeum vulgare L. cv. Baecdong) were determined in order to achieve a somatic hybridization between two species. It was revealed that the use of 0.5M sorbitol as an osmoticum was appropriate for pea. The yield of intact protoplasts was the highest (40%) when pea leaves were incubated in the enzyme solution for 4 hours. In case of barley, the optimal concentrations of cellulase, pectinase and mannitol as the enzyme solution were 2%, 1% and 0.35M, respectively. And the yield of barley protoplasts was the highest(87%) when leaves were incubated in this enzyme solution for 3.5 hours. A fusion of protoplasts from pea and barley was induced by PEG treatment enriched with calcium salts within 60 minutes.

• Journal of the Korean Society of Tobacco Science
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• v.15 no.2
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• pp.123-129
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• 1993

Mesophyll protoplasts derived from young leaves of Nicotiana rustica and N. tabacum cv Burley 21 were fused with the aid of polyethylene glycol(PEG). Cytological examination of protoplasts after PEG treatment revealed 12.8 % heterokaryocytes. After 7 weeks culture, the hybrid calli showing greenish white with a compact appearance were selected in contrast to parental type calli tinged with white or green color. The somatic hybrid plants were verified by morphological, biochemical and cyclological analysis. A heterosis effect for plant vigor and height was observed but the shape of leaves and flower characteristics were intermediate between N. tabacum and N. rutstica. The isozyme banding patterns for peroxidase of somatic hybrid lines were compared with the parent species. A number of isozyme bands derived from both parental species were found in the hybrids. Somatic hybrid plants have been successfully backcrossed to the parental N. tabacum particularly with somatic hybrid plants as female parents. These hybrid plants yielded small seeds, only few which were germinable.

• KSBB Journal
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• v.13 no.4
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• pp.399-403
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• 1998

This study was carried out to optimize the concentration of Ca2+ and pH of fusion medium which affected electrofusion frequency of protoplasts isolated from Nicotiana tabacum L. (cv. BY4) mesophyll cells and callus. The protoplasts were electrofused in the fusion media containing two different Ca2+ concentrations and three different pH regions. Fusion frequency was lower in the fusion medium containing only 13% mannitol as osmotic stabilizer. However, higher degree of fusion frequency (47.3%) was observed in the fusion medium containing 50mM CaCl2 at pH 10.5 than any other conditions. Cell viability was decreased by Ca2+ and high pH treatment in the fusion media, while fusion frequency was increased. It is concluded that Ca2+ is involved in electrofusion of protoplasts.

• Current Research on Agriculture and Life Sciences
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• v.6
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• pp.13-18
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• 1988

This experiment was conducted to identify the enzyme treatment time, calcium ion effect, enzyme concentration and leaf position for protoplast isolation. It was also performed to determine the adequate molarity on protoplasts, and to investigate the incubation time, pH, PEG concentration and DMSO effect for protoplast fusion. The results obtained were summarized as follows ; The optimal time of incubation in enzyme solution was 4 hours. And the protoplast releasing time was delayed by $CaCl_2{\cdot}2H_2O$ addition to the enzyme solution compared with no added one. The viability had kept up to above 95% until the 4 hours after digestion. The high viability of the protoplast was preserved more than 16 hours by adding $CaCl_2{\cdot}2H_2O$ to digestion solution. The enzyme concentration had no effect on protoplast yield in range from 1% to 5% and the first or second leaf from the top of the plant produced the highest protoplast yield among the leaf position tested. The purity of healthy protoplast was better in 0.4M and 0.5M sucrose than in others, and the percentage of protoplast aggregation was more 20% to 50% in PEG 6,000 compared with 4,000 and PEG 1,500. Even though the percentage of protoplast aggregation was less increased by 3% to 7% than without DMSO, its treatment was effective to induce binucleated protoplasts.

• Korean Journal of Agricultural Science
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• v.12 no.1
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• pp.22-30
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• 1985

Protoplasts of Pisum sativum L. were isolated and cultured from leaf mesophyll tissue. The successful yield of protoplast was obtained in an enzyme solution of 2% 'Onozuka R-10' and 2 % 'Macerozyme R-10' contained 6mM $CaCl_2{\cdot}2H_2O$ within 4 hours. They were divided in B5 culture medium supplemented with 2mg/l kinetin, 1mg/l 2, 4-D and 0.2% Difcobacto agar. Divisions of the protoplasts were continued and led to colony formation for 1 months. The colony from protoplasts of pea mesophyll tissue was formed to callus after subculture in a medium contained macronutrients and amino acids of BII medium and micronutrients and vitamins of B5 medium, and also supplemented with 2mg/l kinetin 2mg/l NAA.

• Korean Journal of Plant Tissue Culture
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• v.28 no.3
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• pp.141-146
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• 2001

Protoplasts were isolated from cotyledons, hypocotyls, and mesophyll tissues of three species of Capsicum species (C. anuumm, C. bacatuum, and C. chacoense). Combination of Cellulysin (1%) and Macero-zyme (0.25%) in 0.65 M sorbitol was found to be the most effective for the digestion of cell wall, regardless of the Capsicum species. Antioxidant MES (2-［N-Morpholino］ethanesulfonic acid) in the enzyme solution helped protoplasts overcome browning. After 5 days of initial culture, Cell division occurred in modified K8p medium containing 1~5 mg/L zeatin, 0.5 mg/L IAA, 0.1~0.5 mg/L TDZ, and 1 mg/L 2,4-D under continuous dark condition at $25^{\circ}C$. Semi-solid agarose culture method was more effective than liquid culture, and it also protected the cells from browning caused by polyphenolic compound released during protoplast culture. A total of 4000 calli were obtained from protoplast culture of different capsicum species. All of these calli were transferred to the 100 combinations of regeneration media using various plant growth regulators; TDZ, IAA, 2ip, BAP, NAA, and zeatin. These calli derived from protoplast of three species of capsicum were, however, not differentiated into shoots.

• Journal of Plant Biotechnology
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• v.47 no.4
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• pp.283-288
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• 2020

Although the evolution of Arabidopsis thaliana and humans diverged approximately 1.6 billion years ago, recent studies have demonstrated that protein function and cellular processes involved in disease response remain remarkably conserved. Particularly, γ-secretase, a multisubunit protein complex that participates in intramembrane proteolysis (RIP) regulation, is also known to mediate the cleavage of more than 80 substrates including the amyloid precursor protein (APP) and the Notch receptor. Although the genes (PS1/2, APH-1, PEN-2, and NCT) coding for the γ-secretase complex components are present in plant genomes, their function remains largely uncharacterized. Given that the deposition of 42 amino acid long amyloid-β peptides (hAβ₄₂) is thought to be one of the main causes of Alzheimer's disease, we aimed to examine the physiological effects of hAβ₄₂ peptides on plants. Interestingly, we found that Arabidopsis protoplast death increased after 24 h of exposure to 3 or 5 µM hAβ₄₂ peptides. Furthermore, transgenic Arabidopsis plants overexpressing the hAβ₄₂ gene exhibited changes in primary root length and silique phyllotaxy. Taken together, our results demonstrate that hAβ₄₂ peptides, a metazoan protein, significantly affect Arabidopsis protoplast viability and plant morphology.

• 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.

• Plant Biotechnology Reports
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• v.2 no.3
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• pp.219-226
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• 2008

Transgenic plants with the herbicide-resistance gene (bar gene) were obtained via organogenesis from isolated mesophyll protoplasts of Nierembergia repens after applying electroporation. Transient ${\beta}-glucuronidase$ (GUS) activity of electroporated protoplasts assayed 2 days after applying an electric pulse showed that optimum condition (transient GUS activity 319 pmol 4 MU/mg per min and plating efficiency 2.43%) for electroporation was 0.5 kV/cm in field strength and $100{\mu}F$ in capacitance. The protoplasts electroporated with the bar gene at this condition initiated formation of microcolonies on medium after 2 weeks. After 4 weeks of culture, equal volume of fresh 1/2-strength Murashige and Skoog (MS) medium containing 0.2 mg/l bialaphos was added for selection of transformed colonies. After 6 weeks of culture, growing colonies were transferred onto regeneration medium containing 1.0 mg/l bialaphos, on which they formed adventitious shoots 1-2 months after electroporation. The adventitious shoots rooted easily after transfer onto MS medium with bialaphos lacking plant-growth regulators. Transformation of these regenerants with the bar gene was confirmed by Southern analysis. Some of the transformants showed strong resistance to the application of bialaphos solution at 10.0 mg/l.