• Plant Biotechnology Reports
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• v.2 no.3
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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.

• Korean Journal of Plant Tissue Culture
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• v.26 no.3
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• pp.157-161
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• 1999

This study was performed to enhance the proliferation rate of Gladiolus 'Topaz' callus. The callus was induced from the cermet tissue explants on MS solid medium with 10 mg/L 2,4-D. In the case of liquid shaking culture, proliferation of the callus was effective in MS medium with 0.05 mg/L 2,4-D at 2$0^{\circ}C$ under 16 hours daylength and in a 100 mL Erlenmeyer flask containing 20 mL of the liquid medium and at 75 rpm in rotation speed of the horizontal shaking culture. Furthermore the callus was also able to be subcultured in the same liquid medium.

• 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 Plant Biology
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• v.23 no.3_4
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• pp.91-98
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• 1980

In the present study effects of 2,4-D and kinetin on the callus tissue growth of Korean ginseng(Panax ginseng C. A. Meyer), in relation to the synthesis of saponin were investigated. The saponin synthesis in the callus culture of ginseng root was enhanced by 2,4-D and kinetin. The total saponin content of callus grown on the optima growth conditions, that is, 5mg/l of 2,4-D and 2mg/l of kinetin, was about three times as high as that of the 6 year-old ginseng roots commercially used as herbs. The kinetin specifically increased the synthesis of protopanaxadiol group ginsenoside and decreased the syntehsis of protopanaxatriol gropu in callus cultures, while 2,4-D caused to an increase in the synthesis of protopanaxatriol group ginsenoside and decrease the synthesis of protopanaxadiol group.

• Korean Journal of Pharmacognosy
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• v.22 no.2
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• pp.91-94
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• 1991

Callus was derived from the seedlings of Agastache rugosa(Labiatae). The growth rate of callus and the production of essential oil were studied with the variation of culturing conditions. 2, 4-D 2ppm in the medium was more effective for the production of essential oil than NAA 2ppm. The growth rate of callus and the production of essential oil were inhibited by the illumination of the light. The essential oils from Agastache rugosa and the callus cultivated on the medium containing 2, 4-D 2 ppm and kinetin 0.2 ppm were analysed by TLC, gas chromatography and mass spectrometry. These two oils showed different compositions. The main component of the plant oil, methyl chavicol was not contained in the callus oil.

• Korean Journal of Plant Tissue Culture
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• v.22 no.3
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• pp.131-135
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• 1995

Physiologically modified stem nodes derived from ex vitro and in vivo explants of hybrid aspen (Populus alba L.X P.grandidentata Michx. 'Crandon') were tested for their multiple shoot regeneration capacity using a broad spectrum dosage of cytokinins. Ex vitro derived stem nodes with excised axillary buds at the time of culture produced 11 to 13 multiple shoots on 20 to 30 $\mu$M zeatin containing Woody Plant Medium (WPM) after 6 weeks. Excision of axillary bud sprouts after 2 weeks of culture and culture of the remaining stem nodes on WPM with 1.0 to 2.0 $\mu$M BA or 10 to 30 $\mu$M zeatin produced 13 to 15 and 7 to 8 shoots per explant, respectively, Multiple tiny shoots were produced when in vivo derived stem nodes (on which all leaves were removed) were cultured on WPM with 30 to 50 $\mu$M 2iP or 20 to 50 $\mu$M zeatin. The greatest number of multiple tiny shoot proliferation (32 to 50 shoots per explant) were obtained when the explants were cultured on media containing 20 $\mu$M zeatin. Successful transplanting of these multiple shoots into the greenhouse and/or nursery was achieved.

• Plant Biotechnology Reports
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• v.3 no.3
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• pp.199-203
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• 2009

Rugosa rose (Rosa rugosa) is cultivated as a garden flower and an important genetic resource for the breeding of roses (R. hybrida). This study describes culture conditions for high frequency plant regeneration from zygotic embryo explants via somatic embryogenesis in rugosa rose. Mature zygotic embryo, cotyledon, and radicle explants formed embryogenic calluses at frequencies of 38, 6.7, and 8.8% when cultured on half-strength Murashige and Skoog medium (${\frac{1}{2}}MS$) supplemented with 2.26, 9.05, and $9.05{\mu}M$ 2,4-dichlorophenoxyacetic acid, respectively. Embryogenic calluses produced numerous somatic embryos, which then developed into plantlets on ${\frac{1}{2}}MS$ without growth regulators. Regenerated plantlets were grown to whole plants in a growth chamber.

• Korean Journal of Plant Resources
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• v.24 no.5
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• pp.591-598
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• 2011

This study was conducted to establish the tissue culture system for Atractylodes plant which is most frequently used in oriental medicine. Root and auxiliary bud of Dachul cv., which is Atractylodes hybrid (A. macrocephala x A. japonica), were used as target tissues for in vitro culture. In root culture, callus induction rate was higher in the treatment of BAP combined with NAA than others, however, 2-iP was more effective for callus proliferation and root induction. Although calli were effectively induced from the root and proliferated in lower concentration of cytokinin combined with higher auxin, root tissue was inappropriate for shoot regeneration. For plant regeneration with axillary bud, BAP combined with NAA was more effective than 2-iP with NAA or IBA. Number of regenerated plant per bud was 3.8, which was highest, and stem diameters was shown as 5.0mm under the conditions of 1 mg/L BAP combined with 1 mg/L NAA. Although, plant height was tend to be higher in 2-iP than BAP, number of the regenerated plant was lower via versus. Furthermore, root proliferation of regenerated plant was more effective in higher concentration of sucrose (7%) than in lower concentration (3%). In results, auxiliary bud was an efficient target tissue for producing regenerated plant of Atractylodes under the conditions of 1 mg/L BAP combined with 1 mg/L NAA and higher concentration of sucrose was effective for root proliferation of regenerated plants.

• Korean Journal of Plant Tissue Culture
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• v.28 no.3
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• pp.123-128
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• 2001

In order to develop the biotechnological methods for the mass production of anticancer compounds from tissue culture of Panax ginseng C.A. Mayer, these studies were carried out for the selection of ginseng cell lines containing higher concentration of polyacetylene compounds and optimal condition for their biosynthesis. Panaxynol, one of ginseng polyacetylene, was not detected in any callus induced from ginseng superior cell lines cultured on MS medium supplemented with $\beta$-chlorophenoxy acetic acid (CPA). Panaxydol, another one of polyacetylene and anticancer compounds, were detected in calli of 5 cell lines by thin layer chromatogram and gas chromatogram. Among the 18 ginseng superior lines, the cell line 30201 has higher content of panaxydol. Especially, panaxydol was not detected in the callus induced from cell line 10301 which cultured on the medium containing CPA only, however, it was detected on the same callus cultured on mixed medium containing CAP 2 mg/L and BA 0.05 mg/L. SH medium was better than MS medium for ginseng callus growth and biosynthesis of polyacetylene, and also found that it was not effected by NAA and sucrose concentration in the culture medium.

• Journal of Plant Biotechnology
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• v.3 no.1
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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.