• Korean Journal of Plant Tissue Culture
• /
• v.22 no.6
• /
• pp.339-343
• /
• 1995

This study was performed to investigate the effect of iron ion on the mesophyll protoplast culture of Arabidopsis thaliana. Mesophyll protoplasts were isolated and cultured in a modified IMH medium supplemented with various concentrations of Fe-EDTA. Relatively low concentration of Fe-EDTA (<0.02 mM) induced the low level (4.8%) of cell division. In addition the cell division and microcallus growth were dose-dependently stimulated by 0 to 1 mM of Fe-EDTA. In 0.5 to 1 mM concentration range of Fe-EDTA, microcolonies were readily formed and the plating deficiency (8.5%) also showed maximal rate. However more than 1 mM of Fe-EDTA inhibited the initial growth of protoplase. The overall results suggest that Fe2+ion concentration plays an important role at the early developmental stage of protoplast regeneration.

• Journal of Korean Society of Forest Science
• /
• v.74 no.1
• /
• pp.29-36
• /
• 1986

A method isolating Populus euramericana cv. I-214 mesophyll protoplasts was developed to facilitate application of genetic engineering techniques to this species. The suitable medium for shoot multiplication in vitro was MS basal medium with $0.1mg/{\ell}$ BAP. The effects of several factors influencing protoplast isolation could be evaluated quickly by using leaf in vitro and known volumes of maceration and washing media. The best yields of mesophyll protoplasts were obtained using leaves in vitro in 2.0% Cellulase R-10, 0.8% Macerozyme R-10, 1.2% Hemicellulase, 2.0% Driselase, 0.05% Pectolyase Y-23, and O.6M Mannitol in addition to DTT and MES buffer adjusted to pH 5.6. Over $2.4{\times}10^6$ protoplasts per gram of leaf were produced using these conditions. For protoplast purification, the most favorable sucrose concentration of floating solution was 0.6M after washing them with CPW solution. This method of screening factors affecting protoplast isolation could be applicable to other species.

• Journal of Plant Biotechnology
• /
• v.49 no.4
• /
• pp.331-338
• /
• 2022

Sunflower (Helianthus annuus L.) protoplasts were isolated from seven-day-old etiolated hypocotyls of 10 A line and four-week-old fully expanded young leaves of PI 441983 line in vitro seedlings using an enzymatic method. Purified protoplasts were collected by filtration and floatation in sucrose solution. Semi-solid protoplast culture was performed using the L4 regeneration protocol with various culture media and plating densities to achieve the highest efficiencies for protoplast culture of hypocotyl and mesophyll protoplasts of 10 A and PI 441983 lines, respectively. The concentrations in liquid L'4M medium and different plating densities were evaluated in two types of cytokinins, the adenine-type 6-benzyladenine (BA) and the phenylurea-type thidiazuron (TDZ). The highest colony formation was achieved in both sunflower lines when 0.5 mgL^-1 BA and 0.5 mgL^-1 TDZ were applied with a high plating density (3 × 10⁵ protoplasts mL^-1). These conditions led to 38.45% and 39.40% colony formation for hypocotyl protoplasts of the 10 A line and mesophyll protoplasts of the PI 441983 line, respectively. Moreover, many hypocotyl protoplast-derived colonies developed into micro-calli. In addition, superior development of both sunflower protoplasts was observed with all plating densities when BA was used in combination with TDZ. This finding will be applicable to future sunflower hybrid production via somatic hybridization.

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

• Journal of Environmental Science International
• /
• v.17 no.2
• /
• pp.163-170
• /
• 2008

Protoplasts of Panax ginseng C. A. Meyer and Aralia continentalis K. (Araliaceae) were isolated from callus cells and mesophyll cells, respectively. The maximum yield of protoplasts isolated from callus cells of P. ginseng were obtained by incubation for 3 hrs in the enzyme mixture of 0.5% macerozyme, 1.5% cellulase, and 0.5 M mannitol as an osmoticum. In the case of mesophyll cells of A. continentalis, the highest yield of protoplasts were obtained by incubation for 5 hrs in the enzyme mixture of 1% macerozyme, 2% cellulase, and 0.6 M mannitol. A polyethylene glycol (PEG) treatment induced an intergeneric fusion of the protoplasts. The fusion products, that is, heterokaryocytes were obtained by treatment of 50% PEG containing 0.05 M Ca salts.

• Journal of Plant Biotechnology
• /
• v.7 no.3
• /
• pp.169-174
• /
• 2005

The optimal culture conditions were studied for plant regeneration from mesophyll protoplasts of Solanum sisymbriifolium. Axenic seedlings of S. sisymbriifolium were used as a explant for protoplast culture. Many viable protoplasts were isolated by incubating leaf slices in an enzyme solution containing 0.25% Meicerase and 0.05% Macerozyme for 16 hr at $25^{\circ}C$ without shaking. Protoplast density of $5.0{\times}10^4\;ml^{-1}$ in Kao medium containing 5.0 mg/L NAA, 1.0 mg/L 2,4-D and 1.0 mg/L BA was optimal for colony formation. Most colonies were formed when protoplasts were cultured at $25^{\circ}C$ after initial culture at $30^{\circ}C$ for one week. On the MS agar medium with 1.0 mg/L zeatin, 38.4% of protoplast-derived calli differentiated shoots. These shoots rooted on 1/2MS medium with 5.0 g/L sucrose and 2.5 g/L gellan gum, and developed into whole plants.

• Journal of Plant Biology
• /
• v.29 no.3
• /
• pp.197-205
• /
• 1986

Leaf mesophyll protoplasts from five cultivars of tobacco (N. tabacum L.) were cultured. The protoplasts did not survive in culture medium containing Murashige and Skoog inorganic salts for over 6 days. NH4NO3 and FeSO4.Na2EDTA concentration of Murashige and Skoog medium were toxic in tobacco leaf mesophyll protoplast culture. Therefore we investigated optimum condition in Murashige and Skoog medium. High plating efficiency was obtained by reducing the concentrations of NH4NO3 and FeSO4.Na2EDTA to 1/3 and 1/10, respectively, on the supplemented with 5$\mu$M IAA, 0.5 $\mu$M 2.4-D 5 $\mu$M BAP. Plants were regenerated from protoplast-derived calluses.

• Journal of the Korean Society of Tobacco Science
• /
• v.20 no.1
• /
• pp.40-49
• /
• 1998

Protoplasts were isolated from mesophyll of tobacco(Nicotiana glauca) transformed with kanamycin-resistant gene (NPT II gene) and potato hairy root callus containing Ri plasmid of Agrobacterium rhiEogenes, and protoplasm fusion was made between the isolated protoplasts. The transgenic tobacco leaf tissue could grow on the media containing high concentrations of kanamycin, but not on the phytohormone-free media. On the other hand, the potato hairy root calli could be cultured on the phytohormone-free media but not on media containing more than 40 ㎍/ml kanamycin. In these conditions, the viability of both protoplasts were above 90%, These selection markers were used for the selection of protoplasts fused between the two, i.e. protoplast fusion was detected using selection media containing 100㎍/ml kanamycin and with no phytohormone. The mixture of 1.0% cellulase, 0.3% macerozyme, and 0.7M mannitol was best for the maximum protoplast production for tobacco, and that of 2.0% cellulase, 2.0% macerozyme, 1.0% dricelase, and 0.5M mannitol for potato. Both tobacco mesophyll and potato callus protoplasts were fused by using PEG solution on the selectable medium. Cell walls were regenerated after 5 days in this medium, and colonies were alive until 4 weeks after cultural, but died after 6 weeks.

• Korean Journal of Plant Tissue Culture
• /
• v.22 no.5
• /
• pp.277-281
• /
• 1995

The present study was performed to investigate the effect of calcium ion on the mesophyll protoplast culture of Arabidopsis thaliana. The mesophyll protoplase were isolated and cultured on an IMH medium supplemented with CaCl$_2$of various concentrations. When the protoplasts were cultured on the medium containing 0 to 12.5 mM CaCl$_2$, extreme vacuolization occurred without cell division. When the protoplasts were cultured with higher levels of CaCl$_2$ up to 50 mM, vacuolization decreased dose-dependent): and the number of plasma-rich cells increased. Cell division was induced when the protoplast were cultured on the medium with CaCl$_2$ higher than 25 mM. The highest plating efficiency (5-6%) was obtained with 50 mM CaCl$_2$. However the plating efficiency was markedly inhibited by 100mM CaCl$_2$ or above. These resole suggest that the relatively high concentration (50 mM) of calcium ion may be required for the culture of protoplasts.

• KOREAN JOURNAL OF CROP SCIENCE
• /
• v.27 no.2
• /
• pp.147-154
• /
• 1982

This study was conducted to investigate an effective method of protoplast isolation, the plating efficiency for cell division, and fusion of plant protoplasts by polyethylene glycol for somatic hybridzation. The effectiveness of protoplast isolation was different with the various enzyme concentrations, but, in the protoplast isolation from tobacco mesophyll, the enzyme solution with 0.5% macerozyme and 2.0% cellulase was very effective. The protoplast isolation from callus cultured in vitro for a long period was not obtained in any of the enzyme solution used. Protoplasts divided actively at cell densities above $10^44/ml and at $25^{\circ}C$ under 12hr illumination by inflorecient light (l50 Lux), regardless of presence of agar. The highest frequency of protoplast fusion was obtained after treatment with a solution of 0.33 M polyethylene glycol 1500.