• The Korean Journal of Mycology
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• v.14 no.4
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• pp.273-280
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• 1986

The degree of autolysis and lytic enzyme production in the culture filtrate of Rhizopus oryzae was investigated. The formation of protoplast by using autolytic enzymes from Rh. oryzae was also attempted. Protoplasts were liberated from Rh. oryzae mycelium by lytic enzymes present in autolytic-phase culture filtrate. Maximum release of chitosanase and proteolytic enzyme into culture filtrate during autolysis was corresponded to maximum protoplast-liberating activity. High yields of protoplasts were obtained from 10 hr-age of Rh. oryzae mycelium with 0.5 M mannitol as osmotic stabilizer. The optimum temperature and pH for mycelium digestion were $25{\sim}30^{\circ}C$ and $6.0{\sim}6.5$ respectively. The mycelium of the 18 hours cultures were treated with autolytic enzyme in same volume of osmotic stabilizer at $30^{\circ}C$ for 5 hours and then it was confirmed by scanning electoron microscope that protoplast were produced beside the digesting cell wall of the fungi.

• The Korean Journal of Mycology
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• v.20 no.1
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• pp.58-64
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• 1992

This research was focused on investigation of the general condition for protoplast formation of Trichoderma speues. for protoplast formation, the mycelia cultured in YM medium were collected from each growth phase and were treated with the Iytic enzymes. This procedure was carried out by all strains. The most optimal conditions of NOVOZYM 234 and DRISELASE were determined by T. saturnisporum IAM 12535 and T. longibruchiatum IBM 13107, respectively. The effect of osmotic stabilizers appeared ${KCI}>(NH_4)_2{SO_4}>NaCl>mannitol>{MgSO}_4$ and the optimal concentration of each osmotic stabilizer wns determined by 0.6-0.9 M. The optimal condition of DRISELASE for protoplast formation ; optimal pH 5.0, optimal concentration, 2%, optimal reaction time, 4 hours, and optimal temperature, $30^{\circ}C$. The optimal condition of NOVOZYM 234 for protoplast formation ; optimal pH 5.5, optimal concentration 1%, optimal reaction time 3 hours, and optimal temperature $30^{\circ}C$. The optimal culture period of mycelia for protoplast formation was between the initial and the middle exponential phase. Generally, DUSELASE was more effective than NOVOZYM 234 on protoplast formation except for T. longibruchiatum IAM 13107 and T. viride IAM 5141.

• Korean Journal of Plant Resources
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• v.19 no.3
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• pp.422-426
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• 2006

This research was conducted to get the basic materials necessary to obtain the somatic hybrid plant between Solanum sisymbriifolium and other Solanum species (S. integrifolium and S. toxicarium). Regarding the formation of colony from the protoplast in S. sisymbriifolium, S. integrifolium and the fused protoplast mixture; for the S. sisymbriifolium, a colony was observed in F medium(Kao medium containing $5.0mg{\cdot}L^{-1}\;NAA,\;1.0mg{\cdot}L^{-1}$ 2,4-D and $1.0mg{\cdot}L^{-1}$ BA); and for the S. integrifolium, in G medium (a half strength MS medium containing 0.03 M sucrose, 0.4 M mannitol, $1.0mg{\cdot}L^{-1}\;NAA,\;1.0mg{\cdot}L^{-1}$ kinetin) respectively. In mixed cultured protoplast after electriofusion treatment, the cell division and colony formation were observed in both media F and G. For the shoot and root formation rate, there was no difference between the parent of each breed and mixed protoplast regardless of the medium. In the fused protoplast mixture of S. sisymbriifolium and S. toxicarium, a colony formation was also observed in both media F and H(a half strength MS medium containing 0.03 M sucrose, 0.4 M mannitol, $1.0mg{\cdot}L^{-1}\;NAA,\;1.0mg{\cdot}L^{-1}$ kinetin); and there was no difference in the shoot and root formation rate between the parent and the mixed protoplast.

• Korean Journal of Plant Tissue Culture
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• v.24 no.5
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• pp.295-303
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• 1997

To investigate the response on feeder cell cultures, protoplasts isolated from cell suspensions initiated from mature seed scutellum-derived callus of the Japonica rice variety Taipei 309, were cultured on filter membranes under various conditions. The effects of various factors, such as gelling agents, feeder cell and protoplast densities, species of feeder cells and heat shock treatment, have been investigated to improve protoplast plating efficiencies on filter membranes. Maximum protoplast plating efficiencies were obtained when protoplasts were cultured on KPR medium semi-solidified with Sea Plaque agarose at a density of $5\;\times\;10^{5}\;ml^{-1}$ protoplasts in the presence of Lolium multiflorum as feeder cells (0.5 ml pcv per 10 ml of protoplast culture medium). Pre-culture heat shock treatments for 1 min. and 5 min. to the protoplasts did not give any appreciable increase on the plating efficiency of protoplasts in the presence of feeder cells. Maltose-supplemented medium was superior for plant regeneration from protoplast-derived colonies compared with medium containing only sucrose. The plants were transferred to the glasshouse, flowered and were fertile.

• Korean Journal of Plant Tissue Culture
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• v.25 no.2
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• pp.81-88
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• 1998

The Southeast Asian javanica rice variety Tinawen was investigated for efficient protoplast culture and plant regeneration from cell suspension-derived protoplasts using a feeder cell culture method. Feeder cells of both Lolium multiflorum and Oryza ridleyi, either alone, or in combination, were employed and plants were regenerated from protoplast-derived colonies on several plant regeneration media. Dehydration of protoplast-derived colonies was also investigated as a means of enhancing plant regeneration. In the presence of L. multiflorum or O. ridleyi feeder cells, the protoplast plating efficiency ranged from 0.09% to 1.48%, depending on the feeder cell type and the age of the cell suspension. L. multiflorum feeder cells induced approximately 6-fold higher plating efficiency compared with those of O. ridleyi. The plant regeneration frequencies were 19.3-31.7% with L. multiflorum, 13.0-18.0% with O. ridleyi and 18.0-22.0% with a mixture of both in various plant regeneration media when protoplast-derived colonies were dehydrated, while for the non-dehydrated colonies, the values were 2.0-7.0%, 3.0-5.0% and 0-4.0%, respectively. Flow cytometric analysis of 34 protoplast-derived plants showed that the majority of plants were diploids and only 2 plants were tetraploids. The plants which were transferred to glasshouse were fertile.

• Journal of Plant Biology
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• v.32 no.4
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• pp.313-322
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• 1989

A system was established for induction of multi-shoots and plant regeneration from mesophyll protoplasts of alfalfa, Medicago sativa L. cv. Vernal. Different hormonal effects were tested at each step of protoplast culture, i.e. cell division in modified Kao's liquid medium (K566-7). calli formation on SH semi solid medium, and multi-shoot regeneration from calli on SHa and SHb solid media. Frequency of multi-shoots and plant regeneration was affected by various combinations of phytohormones in final step. The evaluation of multi-shoots induction systems via protoplast culture was discused.

• Journal of Plant Biology
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• v.30 no.4
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• pp.287-298
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• 1987

The regenerative capacities of protoplasts isolated from potato (Solamum tuberosum L.) tubers and tobacco (Nicotiana tabacum L.) mesophyll tissues were examined, and then their intergeneric protoplast fusion was carried out. The potato tuber-derived protoplasts proliferated into the calli some of which showed rudimentary shoot-like structures, which had not been attempted before from tubers, while the tobacco protoplasts were regenerated into the whole plants. Intergeneric protoplast fusion between potato and tobacco was carried out and the heteroplasmic fusion products were formed. The first cell division of some of them was observed after 5 days of culture.

• The Korean Journal of Mycology
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• v.21 no.4
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• pp.323-331
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• 1993

The protoplast formation and intergeneric protoplast fusion between Gliocladium virens and Trichoderma harzianum were attempted to obtain fusants. Protoplast formation was the most effective when the strains were treated with concentration of 5 mg/ml of Novozyme 234 and Cellulase at $25^{\circ}C$ for 3 hours in phosphate buffer, pH 6.5, supplemented with 0.6 M sorbitol as osmotic stabilizer. Auxotrophic mutants of G. virens G88 did not grow in minimal medium and benomyl resistant T. harzianum T95 from wild types, however, was selected by treatment with UV light as genetic marker to isolate fusants. When the intergeneric protoplast fusion between G. virens G88 and T. harzianum T95 was carried out using 30% PEG 4000 containing 10 mM $CaCl_{2}$, and 50 mM glycine (pH 8.5) as fusogenic agent at $25^{\circ}C$ for 10-15 min, the fusion frequency was $0.8{\times}10^{-4}$. Fusants obtained from intergeneric protoplast fusion were spontaneously segregated into va rious strains by continous culture on complete medium. Several intergeneric hybrids were classified into three types: parent-like hybrids, segregants, and recombinants.

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

• KOREAN JOURNAL OF CROP SCIENCE
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• v.42 no.3
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• pp.306-316
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• 1997

Embryogenic calli were induced from mature seed scutella of anther culture-derived rice variety Zhonghua 8. Cell suspension cultures were initiated from friable embryogenic calli and utilized as source material for protoplast isolation. Generally, the older and finer cell suspensions gave higher protoplast yields than younger suspension cultures. Protoplasts exhibited sustained cell division and formed microcalli when cultured in KPR medium supplemented with 0.5 mg $l^{-1}$ 2,4-D, 1.0 mg $l^{-1}$ NAA and 0.5 mg $l^{-1}$ zeatin using the agarose embedding procedure without feeder cells. Protoplast plating efficiencies ranged from 0.20 to 0.54％. Microcalli were transferred to MS medium supplemented with 2.0 mg $l^{-1}$ kinetin and 0.5mg $l^{-1}$ NAA for plant regeneration. The regeneration frequencies were 2 to 12％, depending on the cell suspension lines of Zhonghua 8. The plants were transferred to the glasshouse and were fertile.