• 식물조직배양학회지
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• 제25권5호
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• pp.335-339
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• 1998

미숙배주조직 유래의 배발생캘러스를 이용하여 원형질체의 유리 및 배양에 미치는 요인을 조사하였다. 배발생캘러스로부터 원형질체를 유리시키는데 적당한 배양시간은 16시간이었고, 건전한 원형질체를 유리하는데 적당한 효소용액의 농도는 0.7M $\textrm{BH}_{3}$ 용액과 cellulase 1.0%, macerozyme 1.0%, pctolyase 0.2%가 혼합된 효소용액을 동량으로 조합하였을 때가 가장 효과적이었다. 유리된 원형질체는 MT기본배지에 0.1 mg/L 2,4-D를 첨가한 배지로 배양하면 캘러스 형성이 양호하였다. 유도된 캘러스는 고체배지에 계대배양하고 있으나 생육이 징약한 상태이다. 본 실험결과 배발생캘러스유래의 원형질체와 엽육세포유래의 원형질체 융합에 의한 배양도 가능할 것으로 생각되었다.

• Plant Biotechnology Reports
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• 제2권3호
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• pp.171-177
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• 2008

This paper reports an improved protocol for isolation, culture and regeneration of Lotus corniculatus protoplasts. A range of parameters which influence the isolation of L. corniculatus protoplasts were investigated, i.e., enzyme combination, tissue type, incubation period and osmolarity level. Of three enzyme combinations tested, the highest yield of viable protoplasts was achieved with the combination of 2% Cellulase Onozuka RS, 1% Macerozyme R-10, 0.5% Driselase and 0.2% Pectolyase. The use of etiolated cotyledon tissue as a source for protoplast isolation proved vital in obtaining substantially higher protoplast yields than previously reported. Culture of the protoplasts on a nitrocellulose membrane with a Lolium perenne feeder-layer on the sequential series of PEL medium was highly successful in the formation of microcolonies with plating efficiencies 3-10 times greater than previous studies. Shoot regeneration and intact plants were achieved from 46% of protoplast-derived cell colonies.

• 한국자원식물학회지
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• 제3권1호
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• pp.1-30
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• 1990

The traditional plant imprownent methods consisted of pure line selection, cross breeding, heterosis breeding, polyploid breeding, mutati-onbreeding, ect.Biotechmoiogy is divided into gene spliclng , monocle-nal antibodies , protein engineering , agricultural research, and microbiological engineering. Of these , high plants deal with agricultural research, and the importent part of which is tissue culture and celLculture , Tissue .culture and cell culture are again divided into embryoculture, test tube fertilization, anther and pollen culture, somatichybridization , transformation, recombination, recombinant DNA moleculehybrid plasmid, ect For these haploid production, protoplast culture,protoplast fusion, selection and propagation, ect. , the technical sett-lement is needed.

• Journal of Plant Biology
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• 제15권3호
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• pp.14-18
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• 1972

This paper includes a review on recent development on protoplast culture, regeneraton of plant from protoplast, and fusion of isolated protoplasts, and also describes the possibility of obtaining interspecific hybrid plants through asexual fusion of protoplasts of cells from distantly related plants which are not crossed by the ordinary sexual method.

• Journal of Plant Biotechnology
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• 제49권4호
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• pp.331-338
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• 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.

• Archives of Pharmacal Research
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• 제20권3호
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• pp.206-211
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• 1997

The optimal conditions for the production and regeneration of the protoplasts from Lentinula edodes were studied. Protoplast formation from the mycelia of L. edodes which were cultured in liquid medium showed a significantly high yield compared with that of the mycelia which were cultured on cellophane covered agar media. A mixture of Novozyme 234 (15 mg/ml) and Cellulase Onozuka R10 (10 mg/ml) in 0.6 M mannitol (pH 4) was optimal lytic enzyme for the protoplast release. The optimal incubation time and mycelia age were 3.5-4 hours at $30^{\circ}C$ and 6-8 days, respectively. Regeneration frequency was 0.18% plated onto a medium containing 0.6 M sucrose, and 0.08% plated onto a medium containing mannitol. But hardly any regeneration was observed in the media containing NaCl, KCl, or $MgSO_{4}$ More than 90% of the protoplasts contained nuclei and the nucleus number per protoplast was 1.1. The DNA content per nucleus was 5.1 pg. The diameter of the protoplast was $3-5{\mu}m$ and it had a well defined cell structure.

• Journal of Plant Biotechnology
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• 제7권3호
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• pp.169-174
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• 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.

• 미생물학회지
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• 제22권1호
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• pp.35-40
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• 1984

Streptomyces속 균주개발의 수단으로서 이용할 목적으로 원형질체 융합방법의 확립을 시도하였다. 특히 융합빈도를 높이고 실험을 간편화하는데 역점을 두었다. 원형질체의 형성 및 재생빈도는 균의 배양시간에 따라 변하였는데 대수기에서 수확한 균체로부터 가장 높은 빈도의 수율을 얻었다. 원형질체의 형성은 다른 용균효소를 사용하지 않고 Lysozyme 단독처리 만으로도 충분히 가능하였고 원형질체의 세포막 재생은 Monolay법 보다는 Overlay법이 훨씬 좋은 결과를 주었다. Monolay법은 1.8%, Overly법은 14%의 재생빈도를 나타냈다. 본 실험에서 PEG1000 (50% W/V)를 사용한 원형질체 융합방법으로 얻은 S. coelicolor의 재조합체의 빈도는 $1.8 {\times} 10^{-2}$이었다.

• Journal of Plant Biology
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• 제29권3호
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• pp.197-205
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• 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 Plant Biotechnology
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• 제3권1호
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• pp.27-31
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• 2001

New types of cytoplasmic male sterility in Brassica species would be very useful for the production of F$_1$, hybrid seeds. Leaves and stems of rapid cycling stock of B.juncea (CrGC4-3) containing Anand CMS were used as experimental materials for plant regeneration from protoplast culture. Very high plant regeneration rate (85%) was found in the Kao & Michayluk medium supplemented with 2 mg/L zeatin, 0.5 mg/L BAP, and 1 mg/L NAA when only leaf, not stem, segments were cultured. Protoplasts were isolated from leaves using mixtures of enzymes (1% Cellulycin, 0.5% Macerozyme) in 0.4 M mannitol and 50 mM $CaCl_2$.$2H_2$O. Mcrocalli induced from protoplasts were transferred to the shoot regeneration medium containing 2 mg/L BAP, 2 mg/L zeatin, and 0.5 mg/L NAA. After 60 days of initial protoplast culture, regenerated plantlets were obtained, acclimatized, transplanted into the pots, and grown up to the flowering stage.