• Journal of Plant Biotechnology
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• v.30 no.1
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• pp.109-113
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• 2003

Cryopreservation of embryogenic callus derived from apical meristem culture was attempted by slow prefreezing method (two-step method) with various cryoprotectants in sweetpotato cv. 'Yulmi' Precultured embryogenic calli on medium containing 10 mg/L ABA prior to slow prefreezing in liquid nitrogen indicated higher survival rate than 1.0 mg/L ABA preteatment. The cryoprotectant comprising 1.28 M DMSO in 0.4 M sucrose solution gave the best survival (over 46%) of sweetpotato cells exposed to liquid nitrogen as determined by TTC reduction and FDA staining method. Cryopreserved calli cultured on MS medium with 1.0 mg/L 2,4-D were grown for 4 weeks in the dark and induced embryos after another 4 weeks. They were subcultured on MS medium supplemented with 0.1 mg/L 2,4-D＋0.1 mg/L kinetin for 2 weeks and regenerated into normal plantlets in MS basal medium.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.54 no.4
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• pp.427-432
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• 2009

Seashore Paspalum (Paspalum vaginatum Swartz) is a warm season grass and indigenous to tropical and subtropical regions of coastal areas worldwide. The species is used as feed for cattle and horses and has been very successful for golf courses worldwide. One of the most outstanding characteristics of seashore paspalum is its tolerance to saline soils compared to other warm season turfgrasses. The development of new seashore paspalum cultivars with improved traits could be facilitated through the application of biotechnological strategies. The purpose of this study was to product for herbicide resistant seashore paspalum using Arobacterium-mediated transformation and this study is the first report on transformation and herbicideresistant transgenic plants in seashore paspalum. Embryogenic calli were induced from the seeded variety of pseashore paspalum. Embryogenic calli were transformed with Agrobacterium tumefaciens strain EHA105 carrying the binary vector pCAMBIA3301 with two genes encoding gusA and bar. Transformed calli and plants were selected on medium containing 3 mg/l PPT. PCR detected the presence of the gusA and bar gene, indicating both genes are integrated into the genome of seashore paspalum. A chlorophenol red assay was used to confirm that the bar gene was expressed. By application of herbicide BASTA, the herbicide resistance in the transgenic seashore paspalum plants was confirmed.

• Korean Journal of Plant Tissue Culture
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• v.22 no.1
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• pp.41-46
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• 1995

Leaf mesophyll protoplasts of Dianthus superbus were cultured in MSP1 liquid medium supplemented with 0.5 mg/L BAP, 2.0 mg/L NAA and 9% mannitol. Protoplast-derived colonies were formed after 3 to 4 weeks of culture in the dark at 27$^{\circ}C$. These colonies were kept under continuous illumination (21.5 $\mu$E. m-2 sec-1) for 2 weeks and finally most of the colonies became green microcalli, about 3 mm in diameter. When green microcalli were transferred to MS solidified medium with 2.0 mg/L 2,4-D, they formed embryogenic calli after 4 week of culture. These calli were then transferred onto $N_{6}$ medium containing 0.1mg/L 2,4-D, 0.1 mg/L NAA, 2.0 mg/L kinetin and 2.0 g/L casein hydrolysate and cultured under illumination. After 5 weeks of culture the calli gave rise to multiple shoots of 10 to 15 per callus. Upon transfer onto MS medium containing 2.0 mg/L NAA, they were noted. The regenerates were successfully transplanted into potting soil.

• Korean Journal of Medicinal Crop Science
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• v.15 no.5
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• pp.346-351
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• 2007

An efficient somatic embryogenesis and plant regeneration protocol was developed for Schisandra chinensis Baill, using embryogenic cell suspensions and optimized media conditions. Friable embryogenic callus was induced from cotyledonary leaf and hypocotyl explants of 7 days old seedlings on MS agar medium supplemented with 1.0 to $4.0\;mg\;l^{-1}$ of 2,4-dichlorophenoxyacetic acid (2,4-D). Fast growing and well dispersed embryogenic cell suspensions were developed within two months when embryogenic calli were transferred to MS liquid medium containing $1.0\;mg\;l^{-1}\;2,4-D$. One third strength of MS medium was the best for both overall growth and development of somatic embryos in liquid culture. Over 3400 viable somatic embryos were produced from each 150 ml flask with an initial cell density of 30 mg in 30 ml medium. Germinated somatic embryos developed in liquid medium converted into plantlets after transferred to half-strength MS semi-solid medium. Approximately 90% of the converted plantlets were successfully transplanted to soil and grew into fertile plants.

• Plant Resources
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• v.2 no.1
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• pp.1-9
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• 1999

The callus from the hypocotyl region of immature embryo of Citrus junos Sieb. was efficiently induced in the $\frac{1}{2}$ MS medium containing 45uM BA after 8 weeks culture. The callus was developed into the two callus type, embryogenic callus and nonembryogenic callus, which can be distinguished by visual examination depending on color and appearence. In vitro regeneration of callus established efficiently in the hormone-free MS medium from the embryogenic callus. In order to investigate the physiological changes depending on the developmental stage of embryo, the embryo was formed in the MS medium. The embryogenic and nonembryogenic callus, and the various stages of the somatic embryo were examimed the changes of esterase activity, and their isozyme patterns as well. The protein content and esterase activities was gradually increased on the developmental stages of embryo. Total protein pattern were different by the SDS-PAGE and were appeared strong band of 23 KD in the torpedo stage. The pattern of the esterase isozyme was exhibited a difference between embryogenic callus and nonembryogenic callus. It was appered pI 6.0, 8.0, 8.2 in the embryogenic callus. Also the new band of pI 4.75 was appeared in the cotyledon. These results suggest that the changes of esterase activities and isozyme patterns are importent factor in the differentiation and development of citrus.

• Korean Journal of Plant Tissue Culture
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• v.21 no.5
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• pp.309-314
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• 1994

To develop simple and efficient transformation methods of monocotyledonous plane, electroporation-mediated delivery of DNA into intact embryogenic cell clumps was investigated in zoysiagrass and rice. Calli of zoysiagrass, induced from 3-week-old immature embryos, were suspension-cultured in MS basic medium supplemented with 1.0 mg/t 2.4-D and used for elechuporation. Calli, derived from immature inflorescences of 20 mm lenth of rice, were also suspension-cultured on N6 basic medium supplemented with 1.0 mg/L 2.4-D. Suspension-cultured embryogenic cell clumps were electroporated in liqid MS medium added with a Plasmid DNA (30 $\mu\textrm{m}$/ml), pGA1074, encoding ${\beta}$-glucuronidiase (GUS). DNA delivery into the cells through cell walls and cell membrane was confirmed by the transient expression of the GUS gene. Cell clumps of zoysiagrass and rice, electroporated with 400 volt at 800 pF capacitance, expressed GUS gene activity at a mean frequency of 25 units (one unit = one clony of blue cells) per 200 ${\mu}\ell$ of packed cell volume. Untreated cells and healed non-embryogenic cells did not exhibit GUS activity These results indicate that electroporation-mediated transformation can use intact embryogenic cells (thus avoiding the use protoplasts) in zoysiagrass and rice.

• Journal of Plant Biotechnology
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• v.37 no.4
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• pp.511-516
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• 2010

The process to acquire intron-GUS gene-expressed transformants from somatic embryos (including embryogenic calli) of Rosa hybrida cv. 'Sweet Yellow' using Agrobacterium-meditated transformation method was reported in this study. Somatic embryos including embryogenic calluses were infected with Agrobacterium tumefaciens AGL1 strain (O.D = 0.7~1.6) including intron-GUS gene for 30 min, and were co-cultured for 3 days. After co-cultivation, they were cultured on embryo germination medium (EGM) supplemented with $250\;mg{\cdot}L^{-1}$ cefotaxim at $4^{\circ}C$ for 7 days. Then, transient GUS gene expression was observed. Shoots were regenerated from the shoot primodia induced from the intron-GUS gene-transferred either somatic embryos or embryogenic calli cultured on EGM supplemented with both cefotaxim $250\;mg{\cdot}L^{-1}$ and ppt $2\;mg{\cdot}L^{-1}$. Before induction of rooting from shoots cultured on shoot growing medium supplemented with both cefotaxim $250\;mg{\cdot}L^{-1}$ and ppt $2\;mg{\cdot}L^{-1}$, the shoots were cultured on multi-shoot induction medium supplemented with both cefotaxim $250\;mg{\cdot}L^{-1}$ and ppt $2\;mg{\cdot}L^{-1}$ to induce multi-shoots. When expression of the gene from a part of the multi-shoots was identified by GUS transient assay, the putative transgenic multishoots were transferred to rooting medium supplemented with cefotaxim $250\;mg{\cdot}L^{-1}$. After the formation of healthy roots, transgenic plantlets were transferred to the greenhouse after acclimatization. The expression rate of the intron-GUS gene in the multi-shoots was 100%.

• Journal of Plant Biotechnology
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• v.6 no.2
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• pp.87-90
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• 2004

An efficient plant regeneration system in alfalfa (Medicago sativa L.) through somatic embryogenesis was established. Embryogenic callus was obtained by culture of hypocotyl segments on MS medium with 0.02mg $L^{-1}$ IAA and 1.0mg $L^{-1}$ zeatin after 45 days of culture. Embryogenic calli were converted to the somatic embryos when transferred to either MS medium without plant growth regulators (PGRs) or MS medium containing various cytokinin (BA, kinetin and zeatin). Most of the somatic embryos were developed into plantlets on MS medium supplemented with 0.1 mg $L^{-1}$ kinetin. Also, secondary embryos appeared on the surface of primary embryo but they showed abnormal growth. Regenerated plantlets were transplanted to pots containing vermiculite and perlite for further analysis.

• Journal of Plant Biotechnology
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• v.33 no.4
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• pp.237-242
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• 2006

Commercial cultivars and elite germplasms of barely (Hordeum vulgare L) are still recalcitrant to genetic transformation because of the lack of an efficient regeneration system. In this study, we established an efficient plant regeneration procedure from embryogenic calli derived from mature embryos. Callus induction from germinated mature embryos was best as over 95% in CIM medium (CI medium containing $2.5mg/{\ell}$ dicamba) under dark incubation. Development of embryogenic callus was highest as over 50% in CI3D medium (EC medium supplemented with $3mg/{\ell}$ 2,4-D). The highest regeneration of plants from embryogenic callus (40%) was obtained with CIS medium ($SI+1mg/{\ell}IAA\;and\;2mg/{\ell}\;BA$). These plant regeneration conditions could be useful in improving barley transformation efficiency.

• Plant Resources
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• v.8 no.2
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• pp.81-86
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• 2005

An efficient plant regeneration system of Gentiana scabra through somatic embryogenesis was established. Leaves and roots of seedlings of Gentiana scabra excised after germination were cultured on MS basal medium with 2,4-D, NAA or BA. Embryogenic callus was obtained on MS medium with 0.5 mg/L 2,4-D alone or 0.1 mg/L 2,4-D combimation with 1.0 mg/L BA after 45 days of culture. These embryogenic calli gave rise to somatic embryos, which subsequently developed into plantlets on MS medium without PGRs. Also, shoots were effectively differentiated from embryogenic callus when root segments were cultured on MS medium supplement with 0.1 mg/L 2,4-D and 1.0 mg/L BA. Shoots were effectively rooted on MS medium without PGRs. In vitro flowers were formed from plantlets cultured on MS medium with $5\%$ sucrose after 60 days of culture.