• 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.

• Journal of Plant Biology
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• v.27 no.3
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• pp.139-147
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• 1984

This in vitro study was employed to clarify the effects of several pollutants i.e. $SO_2$, fluoride, cadmium(Cd), aluminum(Al) and NaCl, on the organogenesis and growth responses of shoot-tip, stem and multiple-buds segments derived from hypocotyl or cotyledon culture of petunia seedlings. ${Na_2}{SO_3}$levels of more than 200$\mu{g}$/ml had significantly reduced organogenesis, growth, and chlorophyll content. The injuries caused by ${Na_2}{SO_3}$, concentration of more than 400$\mu{g}$/ml were alleviated by increasing hydrogenion concentration of medium, indicating some relationship between two factors. Organogenesis was not affected by the fluoride concentration up to 100ppm in the media, but the growth and chlorophyll content were greatly reduced by the fluoride. The effect of Cd depended on the explant sources used for the culture; 1.0ppm was effective for fresh weight increase in shoot tip culture, and 3.0ppm in stem segments culture. Organogenesis and growth were greatly reduced by more than 10.0 Cd treatment. Growth and formation of shoots were better with Na conc. of 0.3% compared to control, but those of roots were inhibited. Na concentration goes over 1.0%, organogenesis and subsequent growth were inhibited, and chlorophyll synthesis was drastically reduced. Chlorophyll content was increased on the medium supplemented with Al 50$\mu{g}$/ml compared to control. However the formation and growth of shoots were greatly inhibited with more than 400$\mu{g}$/ml and roots were not produced at all.

• Korean Journal of Plant Tissue Culture
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• v.22 no.4
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• pp.207-211
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• 1995

Immature zygotic embryos from immature seeds of Wasabia japonica (cv Dalma) were isolated and cultured on modified MS medium supplemented with 2,4-D, IAA, and BA. Immature zygotic embryos were classified into torpedo shape and cotyledon stage. The highest rates of callus formation were obtained of 1.0mg/L IAA(torpedo stage, 90.0%)and 1.0mg/L 2,4D plus 0.1mg/L BA(cotyledany stage,84.3%). Somatic embryos after 60 days of culture. These numerous somatic embryo could be seperated and subcultured on the same media for further propagation. After 90 days of culture, most somatic embryos were developed well organized embryos which were able to produce into whole plants.

• Journal of Plant Biotechnology
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• v.35 no.3
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• pp.235-243
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• 2008

Here we describe a procedure for Chinese cabbage protoplast culture and effects of various treatments. Chinese cabbage protoplasts were isolated from different parts of young seedlings as using an enzyme mixture, of which yield was maximized in seven hours around after digestion. The highest rate of initial cell division followed by micro-callus formation was obtained in the medium with 1.0 mg/L 2,4-D, 0.5 mg/L NAA, and 1.0 mg/L BA when the cotyledon-derived protoplasts were cultured. Initiation of cell division and micro-callus proliferation significantly depended upon Chinese cabbage genotype under a same culture circumstances. The micro-calli developed from cotyledon tissue of Norang-Bom cultivar successfully grew toward callus colonies on the solidified medium with 0.2 mg/L zeatin and 0.1 mM spermidine. The callus colonies generated de novo shoots at the maximum frequency of 4.3% on the medium with 5.0 mg/L BA and 1.0 mg/L NM. Our results might be helpful for further studies to enhance the regeneration efficiency in Chinese cabbage protoplast culture.

• Journal of Korean Society of Forest Science
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• v.100 no.4
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• pp.693-697
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• 2011

This study was conducted to examine effects of concentrations of abscisic acid (ABA) or /kinds of osmotica on induction of somatic embryos (SEs), germination and plantlet regeneration in Japanese larch (Larix kaempferi). In comparison of duration of culture, concentrations of ABA and osmoticum, the highest induction number (191/g tissue) of the SE was showed in $60{\mu}M$ ABA+0.2 M sucrose for 4 weeks culture. However, the lowest number (3.5~23.5) of SEs was induced from $4{\mu}M$ ABA+0.1 M sucrose, regardless of culture duration for SEs induction. In comparison of germination efficiency of SEs, the highest induction frequencies of cotyledon (90.9%), hypocotyl (95.8%) and root (96.5%), respectively, were obtained from the SEs that cultured from the treatment of $60{\mu}M$ ABA+0.2 M sucrose with 5 weeks culture. In contrast, the lowest germination response was showed in SEs that induced from the treatment of $4{\mu}M$ ABA+0.1 M sucrose. In comparison of effect of different kinds/concentrations of osmotica for germination and plantlet regeneration, the best response was obtained from the treatment of 0.2 M sucrose with induction of cotyledon (98.3%), hypocotyl (78.4%), root (57.5%) and plantlet regeneration (54.8%), respectively.

• Journal of Life Science
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• v.12 no.1
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• pp.19-21
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• 2002

The pathway of embryos formed anther culture in herbaceous peony was influenced by addition of 2,4-D. MS medium with 2,4-Dichlorophenoxy acetic acid (2,4-D) alone did not arise direct embryogenesis, but was proliferate callus. Embryos through calli were produced on medium containing 0.2 mg/1 zeatin or without growth regulators. Direct embryogenesis was obtained from MS basal medium. However, after the anthers were cultured on medium with 0.1 mg/1 2,4-D, 3 g/1 AC, 30 g/1 sucrose, 2 g/1 gelrite for 40 days. Its efficiency (32.3 %) was markedly improved when anthers cultured on medium without 2,4-D. Embryo morphology was also affected by the 2,4-D used in medium. The induction of normal embryos with two cotyledons was higher in the embryos formed through direct embryogenesis than those formed callus. The embryos formed from calli were mainly showed abnormal embryo with one, three, four cotyledons or hors and bowling pin type.

• Journal of Plant Biotechnology
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• v.46 no.2
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• pp.114-118
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• 2019

Efficient protocol for plant shoot regeneration of Brassica juncea L. CZERN was established by using organic media components and growth stimulating factors of the vermicompost and coelomic fluids. Formulated organic plant tissue culture media (Vermicompost (30%) extracts supplemented with 20 mL/L coelomic fluid) have shown maximum shoot regeneration when compared with the Murashige and Skoog (MS) medium, which were supplemented with 1 mg/L 6-benzyladenine (BA) and 0.1 mg/L of Naphthaleneacetic acid (NAA). Cotyledon explants produced the highest shoot regeneration frequency from fourday-old germinated seedlings in comparison with non-germinated seedlings. The vermicompost extracts have proved to be the best organic plant growth media to induce shoots from cotyledons compared to the MS media. Statistically significant difference (P = 0.008) for the root length, shoot length (P=0.000350) and the leaves (P=0.375) of the mustard plantlets were analyzed successfully. The survival rate was 98% in the mustard cotyledons on the Vermicompost extract media and 63% on MS media respectively. The coelomic fluid also is much suitable to induce shoots from cotyledons at lower concentrations. It was also shown that the vermicompost extract, which comprised of humic acids along with coelomic fluid, affected shoot regeneration from the cotyledons. An efficient and organic shoot regeneration study was standardized and it can be applicable in the improvement of the economically important crops.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.48 no.4
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• pp.292-296
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• 2003

The lateral root formation in soybean sprout culture declines its quality. This study was done to measure the effect of fluorescent light treatment during 24 hour imbibition and 6-day culture on seed germination and growth of soybean sprout. After 6 day culture, the sprouts were sorted as normal (＞4cm), abnormal (＜4cm) and non-germination by their hypocotyl lengths, and lateral roots, fresh and dry weights were measured. Lateral roots were less formed in the fluorescent light treatment lasted during the whole period of the imbibition than in the treatment for 50 minutes a day during the culture. The fluorescent light treatment during the imbibition mainly affected the germination and growth compared to the treatment done during the culture. Compared to the dark imbibition, the light treatment during the imbibition resulted in more normal sprouts, thicker diameters of hypocotyl and hook, and more fresh weights in cotyledon, hypocotyl, whole sprout, and economic yield. However, these results were reverse in lengths of hypocotyl and root, and fresh and dry weights of roots. It is concluded that the fluorescent lamp mainly irradiating red and blue lights can be used for the sprout production as an alternative light replacing blue and red lights treated during the imbibition because it blocked the lateral root appearance and stimulated growth of the sprout.

• Korean Journal of Agricultural Science
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• v.13 no.2
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• pp.157-167
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• 1986

The study was carried out to identify several factors affecting isolation and culture of cotyledone and leaf mesophyll protoplasts of cabbage (Brassica oleraceae), petsai (B.campestris subsp. pekinensis) and rape (B.napus). High viable protoplasts could be obtained when the cotyledon and the leaf mesophyll tissue of all species were treated with enzyme solution composed of 1% macerozyme 'R-10', 1.5% Onozuka 'R-10', 10% mannitol and 50,0 mM $CaCl_22H_2O$ for 4 hours. The protoplasts which obtained from the cotyledon of all species except the cabbage and the leaf mesophyll tissue of all species were divided on NN culture medium supplemented with 9.1% mannitol, 1% glucose, 1% sucrose, $1mg/{\ell}$ 2,4-D, $0.5mg/{\ell}$ NAA and $0.5mg/{\ell}$ BA. The division of the rape leaf mesophyll protoplasts were continued and led to colony.

• Korean Journal of Plant Resources
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• v.27 no.1
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• pp.51-59
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• 2014

In this study, we developed a high frequency watermelon regeneration system using three breeding lines ('B02', 'B05' and 'D04') of watermelon (Citrullus lanatus (thunb.) Matsum. & Nakai) which are differed in their fruits in shape, color of pericarp and flesh. The highest frequency of explants with callus was observed by using explants that consist of cotyledon proximal part end in all breeding lines, and the highest rate of callus induction was obtained on MS medium containing 1.0 mg/L BAP + 0.5 mg/L IAA for 'B02' (94%), 3.0 mg/L BAP for 'B05' (95%), 3.0 mg/L BAP + 0.1 mg/L IAA for 'D04' (90%). The highest shoot regeneration rates from derived callus were obtained on MS medium containing 1.0 mg/L BAP + 0.5 mg/L IAA for 'B05' (94%), and then a 'B02' (81%) with a same culture conditions, and the lowest regeneration was obtained on MS medium containing 1.0 mg/L BAP for 'D04' (56%). Regenerated plants showed the best rates of root formation on MS containing 0.1 mg/L IBA for 'B02' (67%), 0.1 mg/L NAA for 'B05' (87%), 0.5 mg/L IAA for 'D04' (88%). The regenerated plants showed a 100% survival rate in soil condition. The tissue culture and regeneration conditions obtained from this study will be useful for regenerating plants in breeding applications, and will be a useful tool for further genetic transformation studies on watermelons.