• Microbiology and Biotechnology Letters
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• v.22 no.2
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• pp.143-151
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• 1994

In the course of the study on strain inprovement by protoplast fusion, Lactobacillus acidophilus 88 protoplasts production and regeneration conditions were investigated. This strain produced a bacteriocin that revealed strong inhibitory activity against various indicator strains, especially L. helveticus CNRZ 1096. Protoplasts of L. acidophilus 88 strains were very efficiently obtained by treatment with 125 $\mu $g/ml lysozyme in a protoplast forming buffer containing 20 mM N-2 hydroxy-ethtl-piperazine-N'-2-ethane-sulfonic acid(HEPES, pH 7.0) and 1M sucrose at 37$\circ $C for 30 min. Hovever, treatment with mutanolysin was not effective for the production of L. acidophilus 88 protoplasts under the same conditions. High protoplast yield was obtained form the cells at the middle to late logarithmic growth phase in the de Man, Rogosa and Sharpe(MRS) medium. Regeneration was efficiently accomplished with the MRS medium containing 10% sucrose.

• Microbiology and Biotechnology Letters
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• v.20 no.1
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• pp.6-13
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• 1992

- Protoplast fusion between lincomycin resistant Lactobacillus casei KCTC 1121 and rifarnpicin resistant Lactobacillus delbrueckii JK-414 was attempted to obtain the improved strains. Protoplasts of L. casei and L. delbrueckii were produced by mutanolysin digestion at $42^{\circ}C$ for 15 min. L. casei cells were converted to protoplasts by treating with 5 $\mu g$ / m l of mutanolysin in 20 mM HEPES buffer (pH 7.0) containing 0.75 M sucrose at the middle logarithmic growth phase. In case of L. delbrueckii 1.0 M sucrose was used osmotic stabilizer. Regeneration of protoplast in both strains was efficiently accomplished on the regeneration medium containing 10% sucrose, 6 mM $MgC1_2, 6 mM CaC1_2$, and 2.5% gelatin. Protoplast fusion between L. casei and L. delbrueckii was carried out in the presence of 40% of PEG 4,000. The frequency of protoplast fusion was found to be about $3.2\times 10^4$. Acid production of L. casei was better than that of L. delbrueckii. Among fusants, F23 and F35 exhibited excellent lactic acid production. F23 and F24 exhibited the improved proteolysis compared to that of the parent strains and they had twice as much as DNA content of the parents.

• Journal of The Korean Society of Grassland and Forage Science
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• v.22 no.3
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• pp.209-212
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• 2002

We obtained regenerated Italian ryegrass (Lolium multiflorum Lam.) plants by anther culture. When Italian ryegrass anther was incubated for 20 days on callus induction medium, MS medium containing 30 g/$\ell$ of sucrose, 2 mg/$\ell$ of NAA and 1 mg/$\ell$ of kinetin, its callus was induced. The ratio of callus induction was 9.2 %, the mean of callus weight was 8.6 mg/callus/anther. When Italian ryegrass callus was incubated for 50 days on plant regeneration medium, MS medium containing 30 g/$\ell$ of sucrose, 1 mg/$\ell$of NAA and 2 mg/$\ell$of kinetin, Italian ryegrass plant was regenerated. The ratio of plant regeneration was 26%.

• Journal of The Korean Society of Grassland and Forage Science
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• v.24 no.3
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• pp.265-270
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• 2004

Plant regeneration from seed-derived callus of Kentucky bluegrass(Poa pratensis L. cv. Kenblue) was investigated. Callus induced on the medium supplemented with 2 mg/L 2,4-D and 0.2 mg/L BAP showed highest frequency of plant regeneration on the regeneration medium supplemented with 1 mg/L NAA and 5 mg/L kinetin. Callus induced in the dark condition showed higher regenerability than that induced in the dim light. MS medium was better than N6 and B5 medium in enhancing plant regeneration. Maltose was superior to sucrose in plant regeneration as carbon source in the medium.

• Journal of Life Science
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• v.13 no.2
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• pp.143-149
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• 2003

This experiment was undertaken to investigate proper conditions for protoplast isolation and regeneration from the mycelia of Lyophyllum ulmnrium. Protoplast isolation and regeneration are influenced by a variety of factors such as enzyme, osmotic stabilizer, reaction time and age of mycelia. A combination of Novozyme 234 (10mg/ml) and cellulase Onozuka R-10 (10 mg/ml) with 0.6 M $MgSO_4$ was most effective for isolation of the protoplasts. The optimum reaction time of the mycelia with the lytic enzymes was 3.5~4 hours at $28^{\circ}C$ in shaking condition at 120 strokes per min. High yield of the protoplasts were obtained from its 4~5 days old mycelia on complete agar media. Its protoplasts were regenerated to normal hyphae. Regeneration media with 0.6 M sucrose were proper for regeneration of the protoplasts. Their regeneration frequency on complete agar media was 2.3~2.7%.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.58 no.4
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• pp.331-335
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• 2013

Buckwheat sprout is used as vegetable, and also flour for making noodles, and so on. Currently, information about tissue culture in buckwheat is limited and restricted to micro-propagation. We carried out somatic embryogenesis and plant regeneration using hypocotyl segments as explant of the cultivated buckwheat species, Fagopyrum esculentum which differs from existing studies in the growth regulator combinations used. Maximum callus regeneration was induced on MS medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) $2.0mg{\cdot}L^{-1}$, benzyladenine (BA) $1.0mg{\cdot}L^{-1}$ and 3% sucrose. Friable callus was transferred to solidified MS media containing BA ($1.0mg{\cdot}L^{-1}$) with various concentrations of 2,4-dichlorophenoxyacetic acid for the induction of embryogenesis. The optimum concentrations of growth regulators (for regeneration of plantlet) were indole-3-acetic acid ($2.0mg{\cdot}L^{-1}$), Kinetin ($1.0mg{\cdot}L^{-1}$), BA ($1.0mg{\cdot}L^{-1}$). Only 2,4-D did not show any significant effect on callus induction or embryogenesis. Regeneration of embryonic callus varied from 5% to 20%. Whole plants were obtained at high frequencies when the embryogenic calli with somatic embryos and organized shoot primordia were transferred to MS media with 3% sucrose. The main objective of this research was to develop an efficient protocol for plant regeneration for common buckwheat, and to apply in future for genetic transformation.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.49 no.1
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• pp.61-68
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• 2004

A reproducible transformation system via optimized regeneration media for Korean rice cultivars was established using Agrobacterium tumefeciens LBA4404 (pSBM-PPGN; gusA and bar). Although japonica rice genotypes were easier to produce transgenic plants compared to Tongil type cultivars, transformation efficiencies were not always correlated with regeneration efficiencies of non-transgenic callus on the control medium. Regeneration efficiencies of Donganbyeo, Ilmibyeo, and Manchubyeo were over 50% in non-transgenic control, however, transformation efficiencies were significantly low when only sucrose was added to the media as a carbon source. However, the medium, MSRK5SS-Pr (or MSRK5SM-Pr), that contains $5\textrm{mgL}^{-1}$ kinetin, $0.5\textrm{mgL}^{-1}$ NAA, 2 % sucrose (or maltose), 3% sorbitol, and $500\textrm{mgL}^{-1}$ proline, was the most efficient not only for regeneration of non-transgenic callus but also for regeneration of transgenic callus in the presence of L-phosphinotricin (PPT). Average transformation efficiencies of 16 Korean rice cultivars were significantly enhanced by using the optimized medium from 1.5% to 5.8% in independent callus lines and from 2.9% to 19.4% in tromsgenic plants obained. Approximately 98.9% (876 out of 885) transgenic plants obtained on optimized media showed basta resistance. Stable integration, inheritance and expression of gusA and bar genes were continued by GUS assay and PCR and Southern analysis of the bar gene. With Pst1 digestion of genomic DNA of transgenic plants, one to five copies of T-DNA segment were observed; however, 76% (19 out of 25 transgenic plants) has low copy number of T-DNA. The transformants obtained from one callus line showed the same copy numbers with the same fractionized band patterns.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2019.04a
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• pp.80-80
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• 2019

This study describes an efficient and widely applicable droplet-vitrification following cryopreservation for shoot tips of (Fragaria x ananassa DUCH. cvs. 'Derunoka' and 'Jumbo pure berry'. The shoot tips of strawberry were precultured in Murashige and Skoog (MS) liquid medium supplemented with sucrose (0.3-0.7M). Precultured explants were treated with loading solution (LS, C4) containing glycerol 17.5% and sucrose 17.5% for 40 min and exposed to dehydration solution (B1) containing 50% of glycerol and 50% of sucrose for 60 min at $25^{\circ}C$, and then transferred onto droplets containing $2.5{\mu}l$ PVS3 on sterilized aluminum foils ($4cm{\times}0.5cm$) prior to direct immersion in liquid nitrogen (LN) for 1 h. The highest regeneration rate (%) was obtained when shoot tips were precultured with treatment-2 (exposing of shoot tips to MS + 0.3M Sucrose for 30 h and then treated with MS+0.5 M sucrose for 16 h) at $25^{\circ}C$ in both the cultivars. The viability of cooled samples, followed by culturing on MS medium for 4 weeks was 77.8% and 60.0% for 'Derunoka' and 'Jumbo pure berry', respectively. This result shows droplet-vitrification would be a promising method for cryobanking strawberry germplasm.

• Korean Journal of Plant Resources
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• v.31 no.6
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• pp.675-683
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• 2018

This study describes an efficient and widely applicable droplet-vitrification following cryopreservation for shoot tips of Chrysanthemum morifolium (Ramat.) cvs. 'Borami' and 'Yes morning'. The shoot tips of Chrysanthemum were precultured in Murashige and Skoog (MS) liquid medium supplemented with sucrose (0.3-0.7 M). Precultured explants were treated with loading solution (LS, C6) containing glycerol 20% and sucrose 20% for 30 min and exposed to dehydration solution (B5) containing 40% of glycerol and 40% of sucrose for 60 min at $25^{\circ}C$, and then transferred onto droplets containing $2.5{\mu}l$ PVS3 on sterilized aluminum foils ($4cm{\times}0.5cm$) prior to direct immersion in liquid nitrogen (LN) for 1 h. The highest regeneration rate (%) was obtained when shoot tips were precultured with treatment-2 (exposing of shoot tips to MS + 0.3M sucrose for 30 h and then treated with MS+0.5 M sucrose for 16 h) at $25^{\circ}C$ in both the cultivars. The viability of cooled samples, followed by culturing on $NH_4NO_3$-free MS medium for first 5 days was increased to two-fold (80.7%) regrowth rate over those cultured on normal MS medium or MS medium containing plant growth regulators. This result shows droplet-vitrification would be a promising method for cryobanking chrysanthemum germplasm.

• Journal of Plant Biotechnology
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• v.3 no.2
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• pp.101-106
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• 2001

In order to establish efficient plant regeneration from embryogenic suspension cultures of soybean, Glycine max L, we examined the effects of auxin type and concentration, cytokinin type and concentration, and amino acid type and concentration on the growth of embryogenic clumps from induced callus, and the effect of desiccation of mature somatic embryos obtained from these clumps on the frequency of somatic embryo germination. Embryogenic callus was induced from the edge of the cotyledons cultured on MS medium containing 6% sucrose, 40 mg/L 2,4-D, 0.2% gelrite and pH 5.7. The growth of embryogenic clumps was best in early staged, embryogenic callus that was placed in suspension culture of MS medium containing 5 mg/L 2,4-D and 0.5 mg/L asparagine. Single somatic embryos were isolated from the clumps and plated on the same medium for maturation. When the mature single somatic embryos were desiccated for 96 h, somatic embryo germination came up to approximately 90%. The plantlets germinated after embryos desiccation for 2 weeks were transfered to MS medium containing 3% sucrose,0.2% gelrite and pH 5.7.