• Proceedings of the Plant Resources Society of Korea Conference
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• 2003.10a
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• pp.50-58
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• 2003

Isolation and culture of leaf protoplasts from two chilli cultivars (Capsicum annuum var. accumnatum and Bird chilli) were developed to enhance selection process in the somatic hybridization programmes. In order to isolate the protoplasts from leaves of these two chilli cultivars different incubation periods (3, 5 and 10 hours) were tested with combinations of enzyme mixtures containing cellulase and macerozyme. Leaves were incubated on three enzyme mixtures (2% cellulase + 0.4% macerozyme, 1% cellulase + 0.2% macerozyme and 0.5% cellulase + 0.1 % macerozyme in 13% mannitol) at 251oC in the dark. Three hours of incubation using 2% cellulase and 0.4% macerozyme was the best for the protoplast isolation of both chilli cultivars tested. The yield was 5 ${\times}$ 108protoplasts/ml/ g leaf tissue in both chilli varieties. It was found that in the mixed nurse method using Nagata and Takebe (NT) medium supplemented with 1.0mg/12,4-D, NAA and BAP with 0.5M mannitol and 1.2% Sea Plaque agarose is the best medium for protoplast culture. Protoplasts of Capsicum annum var. accumnatum were alive for 14 days forming cell walls and initiating cell division.

• Journal of Korean Society of Forest Science
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• v.73 no.1
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• pp.33-42
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• 1986

This study was carried out to investigate the optimum conditions for isolation and culture of mesophyll protoplasts from Populus alba ${\times}$ P. glandulosa. The results obtained from the experiments are as follows; 1) The suitable concentration of BAP for shoot multiplication was 0.4 mg/l. 2) High yield and viability of isolated protoplasts were obtained by our high enzyme-short time incubation method. 3) Optimum enzyme concentrations for mesophyll protoplast isolation were Cellulase 2%, Macerozyme 0.8%, Hemicellulase 1.2%, Driselase 2%, and Pectolyase Y-23 0.05%. 4) 0.6M mannitol in enzyme solution was the most effective for protoplast isolation and viability. 5) The most adequate pH level of enzyme solution was pH 5.6. 6) The effect of DTT and MES buffer was significant. 7) For protoplast purification, 0.6M sucrose was the most proper concentration. 8) The adding effect of Dextran T40 in floating solution was important. 9) The mesophyll protoplasts isolated through our high enzyme-short time incubation method revealed successful response to culture condition over 3 weeks of culture.

• YAKHAK HOEJI
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• v.38 no.6
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• pp.696-702
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• 1994

An interspecific fusant strain, MS1, was obtained by protoplast fusion between S. peucetius subsp. caesius and S. platensis. We studied on the microbiological and cultural characteristics of the fusant MS1. In liquid culture, the viscosity of culture broth of S. peucetius increased during incubation However the fusant MS1 formed pellet like S. platensis without viscosity change. On agar medium the colony morphology of MSI resembled S. platensis but the color was similar to S. peucetius. The fermentation products of the fusant MS1 was identical with S. peucetius.

• Journal of the Korean Society of Tobacco Science
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• v.1 no.2
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• pp.138-149
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• 1979

For the preliminary study on tobacco cell fusion as one of new breeding techniques, the conditions that would be most effective in isolation, fusion, and culture of tobacco protoplasts were examined ; 1. The enzyme solution of 0.5% macerozyme and 2% cellulase( or meicellase) was the most economic and efficient in isolating protoplasts from tobacco leaves. 2. The proper incubation period of tobacco leaves in cell wall digesting solution was 4 hours. 3. As an osmotic stabilizer, sorbitol or mannitol solutions were employed. The concentration of 0.5~0.7 M of either hexitol gave satisfying results as the osmotic stabilizer. 4. The calcium concentration appeared to be an important factor in protoplast fusion. The adhesion of protoplasts was enhanced by enrichment of calcium ion in PEG solution. The highest frequency of protoplast fusion was obtained when tobacco protoplasts were incubated in PEG solution. containing 9mM CaCl2. 5. Cell divisions of the isolated protoplasts were continued and have generated colonies when they were grown on B-5 medium at 28$^{\circ}C$.

• Korean Journal of Plant Tissue Culture
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• v.22 no.6
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• pp.311-316
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• 1995

In an attempt to obtain intersubgeneric somatic hybrids of Pelargonium aridum and P.zonale, protoplast isolated from the two species were fused by using polyethylene glycol(PEG) and electorfusion methods. Protoplast were isolated from cotyledon and leaf tissues using MS medium containing 550 mM sucrose, 0.7% cellulase (Onozuka R-10) and 0.4% Macerozyme. The optimum number of protoplasts per mL of culture medium was 6 x 10$^4$. Protoplast fused by the electrofusion method were more active than by PEG method. Heterokaryotically fused protoplasts formed calli when cultured in MS medium containing 550 mM glucose, 1 to 2 mg/L NAA and 0.5 to 1 mg/L BA.

• The Korean Journal of Mycology
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• v.21 no.1
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• pp.1-8
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• 1993

ABSTRACT: Factors responsible for protoplast formation and regeneration of Phytophthora capsici were examined. Protoplasts were successfully liberated from the mycelial culture by digestion for 6-9 hrs with Novozym 234 in 0.35 M $CaCl_2$, (pH 5.7) as osmotic stabilizer. Young rapidly-growing mycelium (24 hrs old) showed highest protoplast yields. High concentrations of Novozym 234 were effective in releasing protoplasts from the mycelium. The combination of 0.4 M mannitol and 0.1 M $CaCl_2$ was optimal osmotic stabilizers for protoplast regeneration. The synthetic Henninger media containing all nutritional elements gave the best regeneration rate. The protoplast regeneration was greatly inhibited in the media which were not supplement with amino acids or ${\beta}-sitosterol$. Certain amino acids such as L-aspartic acid and L-glutamic acid remarkably enhanced protoplast regeneration. However, the addition of microelements did not affect protoplast regeneration.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.42 no.1
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• pp.33-41
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• 1997

This study was carried out to establish simple and easy methods to judge the survival, senescence and death of the protoplasts in culture system by identifying the marker substance related to metabolic status of the cells. When rice and tobacco protoplasts were cultured in MS and KM-8P media containing 2,4-D or coconut milk ABA decreased especially in the media containing coconut milk, but GA$_3$, IAA and zeatin increased as the cultures progressed. The decrease of ABA and increase of zeatin was especially remarkable. When the supraoptimal amount of osmoticum (mannitol) was added to the culture media ABA decreased after a momentary increase, but other growth hormones slowly increased as the concentration of the osmoticum increased. Contents of individual hormones were contrasted when protoplasts rice and tobacco were cultured on the same medium containing 10mM super mine or NaCl. Tobacco protoplasts were more sensitive to NaCl stress and stopped protoplast division at the late stage of culture. Protoplast viability decreased greatly in 48 hours when the protoplast were at 32$^{\circ}C$ on a medium lacking several components. ABA content increased up to 10 days from incubation in negative proportion to the protoplast viability. On the other hand contents of other growth hormones, especially zeatin, decreased. The present results clearly showed that the contents of individual growth hormones in the plant protoplasts in culture varied sensitively in response to environmental factors that they are faced with. This indicates that the physiological states of the protoplast, such as survival, senescence or death can be simply judged based on the quantitative analysis of those hormones by ELISA.

• Proceedings of the Microbiological Society of Korea Conference
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• 2000.10a
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• pp.85-91
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• 2000

Aspergillus oryzae is a filamentous fungus classified in the group Aspergillaceae Ascomycetes. It is an important microorganism for industrial production of enzymes and fermented food productions. It secrets large quantities of proteins or enzymes into the culture medium which makes this organism appealing for the production of heterologous proteins. Recently Electric field-mediated transformation method, electroporation, has been applied to fungal transformation. In this study, fungal transformation was carried out by bypassing the protoplast isolation step, decreasing the culturing time and non-protoplast transformation for the increment of transformation efficiency. Transformants were obtained with electroporation in optimal condition 2,500 voltage, 1,540 ohm and 0.50 capacitance. More than 1,000 transform ants were obtained with 6-10 hrs cultured mycelia without enzyme treatment, called non-protoplast transformation.

• Journal of Plant Biology
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• v.34 no.1
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• pp.19-23
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• 1991

Culture of embryogenic callus and suspension were induced from rice seeds in MS2.5 medium. In hormone free N6 medium, whole plantlets were regenerated from embryogenic callus. We observed cell division and reformation of embryogenic callus on culture of protoplast isolated from embryogenic cell suspensions. In addition, we studied the influencing factors on viability of protoplast treated with electroporation. Viability was decreased according to the increase of voltage and capacitance during electroporation. An optimal level of viability was obtained after treatment with $200-300\;V/1180\;\mu\textrm{F}$ in HEM buffer at $4^{\circ}C$..

• Journal of Plant Biotechnology
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• v.38 no.1
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• pp.69-76
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• 2011

This study describes the effect of myo-inositol on sustained cell division and plant regeneration from cotyledon-derived protoplast of cabbage (Brassica oleracea var. capitata). Freshly isolated protoplasts were cultured in modified Murashige and Skoog (MS) medium removed ammonia ions and containing $0.4\;mg\;l^{-1}$ thiamine HCl, $100\;mg\;l^{-1}$ myo-inositol, $2\;mgl^{-1}$ 2,4-D, $0.5\;mgl^{-1}$ BA, $30\;gl^{-1}$ sucrose and several concentrations of myo-inositol (2, 4, 6, 8, 10% (w/v)) as an osmotic stabilizer. After 3 weeks of culture in the dark at $25^{\circ}C$, the plating efficiency of cabbage protoplasts reached to $22.5{\pm}2.9%$ when cultured in modified MS medium supplemented with $2\;mgl^{-1}$ 2,4-D, $0.5\;mgl^{-1}$ BA, $30\;gl^{-1}$ sucrose and 8% (w/v) of myo-inositol at a density of $2{\times}10^5$ protoplasts/ml. Rapidly growing cell colonies after 3 weeks of culture were transferred to the same culture medium removed osmoticum. To induce shoot regeneration from calluses, calluses with about 2 mm in diameter were transferred to the MS medium containing $2\;mgl^{-1}$ BA and $0.5\;mgl^{-1}$ NAA. After further three weeks of incubation onto the medium in the light, green shoots were formed on the surface of calluses at a frequency of 30%. Upon transfer to half-strength MS basal medium, roots were formed onto the bottom of regenerated shoots without auxin treatments. These regenerated plantlets were successfully acclimatized to soil transfer, grown to normal mature plants. The cabbage protoplast culture system established in this study could be applied for production of somatic hybrids or cybrids by asymmetric protoplast fusion and mass proliferation of elite somatic clones of cabbage.