• Korean Journal of Plant Tissue Culture
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• v.25 no.5
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• pp.335-339
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• 1998

The factors affecting the isolation and culture of the protoplast of embryogenic callus, derived from immature ovule in Citrus junos, were examined. An incubation time in enzyme solution of 16 hrs was preferable for protoplast isolation. Efficient protoplast yields were obtained from the treatment of equal concentration of 0.7 M $\textrm{BH}_{3}$ to the enzyme solution containing 1.0% cellulase, 1.0% macerozyme and 0.2% pectolyase. Protoplast cultured in MT medium with 0.1 mg/L 2,4-D showed vigorous division and some of them formed callus. Induced callus was subcultured on solid MT medium but the callus showed very slow growth. The above results show the possibility to culture from protoplast fusion in Citrus genera.

• Korean Journal of Plant Tissue Culture
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• v.22 no.6
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• pp.323-327
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• 1995

This experiment was carried out to observe the origin and developmental pattern of somatic embryos of Oenanthe javanica ($B^{L}.$) DC. The experiment included observation of embryogenic cells and their development stages by light microscope, transmission electron microscope and scanning electron microscope. The embryogenic cells, which were smaller than non-embryogenic cells in size with expanded nucleus and dense cytoplasm. When stained with hematoxylin, the embryogenic cells were readily distinguished from the non-embryogenic cells of which cell walls were stained with safranin. It was observed at somatic embryos developed from single cells on the epidermis of developing embryos or in the surface or inside of embryogenic clumps by segmentation pattern. Observation with a transmission electron microscope revealed that the embryogenic cells had dense cytoplasm expanded nucleus, small vacuoles, large amyloplasts containing starch grains, and abundant organelles including lipid bodies. Under a scanning electron microscope, embryogenic callus was shown to consist of very smaller cells than non-embryogenic cells in an orderly arrangement and covered with a net-like structure, while the non-embryogenic callus consisted of large cells, irregular in size and arrangement, and covered with a gelatin-like material.

• Journal of Plant Biotechnology
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• v.5 no.4
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• pp.233-238
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• 2003

Sweet potato cells derived from Yulmi were isolated from embryogenic callus and irradiated with 50 Gy dose. Resistant cells were selected on a MS medium containing 1.0 mM S-aminoethyl-L-cysteine (AEC). This level of AEC approximately inhibits non-selected wild type cells. The callus resistant to this analog of lysine was subcultured for 30 days in absence of AEC to proliferate. The three resistant calli (AR-1, AR-2 and AR-3) with better growth were divvied into 0.5~1mm diameter and placed on MS medium with 0, 0.4, 0.6, 0.8 and 1.0 mM AEC. There are considerable growth difference between control callus and AEC resistant callus on the AEC-medium. The selected calli were placed on the hormone-free medium for regeneration. Three plantlets, five plantlets and six plantlets were recovered from AR-1, AR-2 and AR-3 calli, respectively. Each two regenerants in AR-1, AR-2 and AR3 were randomly selected for RAPD and SDS PAGE analysis. RAPD polymorph isms between Yulmi and AEC resistant plant from irradiated calli were detected in several Wako primers. Also, it was identified that two AEC resistant plants had higher protein than the original variety Yulmi.

• Journal of The Korean Society of Grassland and Forage Science
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• v.27 no.4
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• pp.235-240
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• 2007

Optimum tissue culture conditions for an efficient induction of embryogenic callus from mature seeds of perennial ryegrass (Lolium perenne L.) and regeneration of plants from callus tissues were investigated. MS medium containing 3 mg/L 2,4-D and 0.1 mg/L BA was optimal for embryogenic callus induction from mature seeds. The highest plant regeneration frequency (58.3%) was observed when the embryogenic callus tissues were cultured on N6 medium supplemented with 1 mg/L 2,4-D and 3 mg/L BA. Regenerated plants were grown normally when shoots transplanted to the soil. A short tissue culture period and high-frequency regeneration system would be helpful for molecular breeding of perennial ryegrass through Agrobacterium-mediated genetic transformation.

• Korean Journal of Plant Tissue Culture
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• v.24 no.3
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• pp.167-173
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• 1997

The embryogenic callus (EC), from which somatic embryos could be induced, was compared with nonembryogenic callus(NE) to study the origin and features of totipotent cell in water dropwort (Oenanthe stolonifera DC). To induce and maintain of EC and the NE, meristematic stem and immature floret were inoculated in MS media supplemented with 1 mg/L 2,4-D, and with 2.5 mg/L NAA and 5mg/L BA, respectively, The EC was not induced from the NE even after subculturing in MS medium supplemented with 1 mg/L 2,4-D. Plantlets were not regenerated from the NE in hormone-free medium. In histochemical comparison of the EC with the NE by light microscopy, the EC had smaller cells in size, dense cytoplasm, and more starch granules of cells compared to the NE cells. The cell from the EC, as observed by transmission electron microscopy, had smaller vaculoes, well developed ribosomes, mitochondria, and endoplasmic reticulum, whereas the cells from the NE had larger vacuoles and underdeveloped organelles. In protein pattern from NE, EC and Somatic embryo (SE), as analyzed by SDS polyacrylamide gel electrophoresis, different proteins specific for tissue were observed: 17 and 28 KD for NE, 50, 52, 57, 66, 68 KD for EC and 20 KD for SE. DNA polymorphism was also observed between EC and NE as analyzed by RAPD (randomly amplified polymorphic DNA) method. The origin of totipotent stem cell and the relationship between irreversible genomic change arose in differentiation and the loss of totipotency in plant were discussed.

• Journal of Plant Biotechnology
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• v.31 no.1
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• pp.67-72
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• 2004

An efficient protocol has been developed for rapid mass propagation of sweetpotato from shoot-tips derived embryogenic callus. Optimal embryogenic callus was induced from shoot apical meristem explants on Murashige and Skoog (MS) medium supplemented with 1mg/L 2,4-D. The addition of casein hydrolysate in the media increased the embryogenesis efficiency of sweetpotato. Somatic embryos were easily induced from the embryogenic callus on MS basal medium containing 300-500mg/L casein hydrolysate without phytohormon. Treatment of casein hydrolysate (100∼300mg/L) with 1mg/L 2,4-D also improved the secondary embryonic efficiency from somatic embryos below 2mm in length. Plant regeneration was achieved via somatic embryogenesis and direct organogenesis. Regenerated planlets with well developed shoots and roots on MS basal medium were successfully transferred to soil.

• Korean Journal of Plant Tissue Culture
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• v.26 no.1
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• pp.15-19
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• 1999

For the induction of somatic embryogenic callus, the penduncle explants of Corydalis remota for peatinata were cultured on MS basal media supplemented with 2,4-D, kinetin and zeatin. The highest embryogenic callus formation was observed on the media containing 2.0 mg/L of 2,4-D and 2.0 mg/L of zeatin. The somatic embryogenesis on the media with 0.5 mg/L of cytokinin (zeatin or kinetin) were excellent under light condition, however somatic embryos abnormally developed into plantlets. Normal dicotyledonary plantlets were found on MS medium supplemented with 1.0 mg/L of zeatin. When MS medium with 2,4-D plus cytokinin and with BAP were used, the secondary somatic embryogenesis took place in root explants of the regenerants derived from in vitro somatic embryogenic callus.

• Korean Journal of Plant Tissue Culture
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• v.26 no.2
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• pp.121-126
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• 1999

Calli were induced from the petiole explants of Pimpinella brachycarpa on MS medium supplemented with 0.5 mg/L 2,4-D and 0.1 mg/L BA after four weeks of culture. Compact clusters of small and dense cells among these calli were selected and suspension-cultured as the source of embryogenic calli. When transferred to MS medium with 0.1 mg/L NAA, the suspension-cultured cells grew to embryogenic callus. Somatic embryos derived from these embryogenic calli developed into plantlets. The cDNA library was constructed in the embryogenic callus and in order to screen the cDNA library, these cDNAs were plated at a density 1.5 $\times$ 10^5 plaques per 15 cm petridish. Among 19 clones showing preferential hybridization with petiole HD-Zip gene, five clones were obtained after second screening. Four clones among them, were highly homologous to P. brachycarpa shoot-tip Phz4 gene, but one clone, Phc5 was about 1.5 kb which has an extra 163 bp to 5' upstream of Phz4. The Phc5 was 1,531 bp containing poly A tails of 18 bases. ATG start codon for Phc5, was located at position 284 with an open reading frame of 906 by which encodes a polypeptide of 302 amino acids. The Phc5 protein revealed that the polypeptides between 135 and 195 contain a homeodomain as the `leucine zipper' motif.

• Journal of Plant Biotechnology
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• v.30 no.3
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• pp.233-239
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• 2003

We have developed a reliable and high-frequency genetic transformation and regeneration system via somatic embryogensis of Eleutherococcus sessiliflorus. Embryogenic callus obtained from seed were co- cultivated with Agrobacterium tumefaciens strain EHA101/pIG121Hm harboring genes for intron-$\beta$-glucoronidase(GUS), kanamycin and hygromycin resistance. Following co-cultivation, two types of samples(fine embrogenic calli and early globular embryo clusters) were cultivated on Murashige and Skoog(MS) medium containing 1 mg/L2.4-D for 3day in dark. Transient expression of GUS gene was found to be higher in the early globular embryo clusters than in the embryogenic calli. Also, co-cultivated period affected expression of GUS gene; the best result was obtained when globular embryo clusters were co-cultivated with Agrobacterium for 3 days. Subsequently, this callus transferred to selective MS medium containing 1mg/L2.4-D, 50mg/L kanamycin or/and 30mg/L hygromycin and 300mg/L cefortaxime. These embryogenic calls were subcultured to the same selection medium at every 2 weeks intervals. Approximately 24.5% of the early globular embryos co-cultivated with Agrobacterium for 3days produced kanamycin or/and hygromycin-resistant calli. Transgenic somatic embryos were converted into plantlets in half strength MS medium supplemented with 3mg/L GA$_3$ kanamycin and were confirmed by GUS histochemical assay and polymerase chain reaction analysis. Genomic Southem blot hybridization confirmed the incorporation of NPT II gene into the host genome.

• Journal of The Korean Society of Grassland and Forage Science
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• v.20 no.1
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• pp.13-18
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• 2000

An efficient method for Agrobacterium-mediated transformation of forage crop alfalfa (Medicago sativa L.) was established using secondary somatic embryogenic calli. Agrobacterium tumefaciens strain EHAlOl and a binary vector pIG121-Hm which has selection markers for kanamycin and hygromycin have been shown to be an efticient materials for alfalfa transformation. The secondary somatic embryogenic calli originated from hypocotyl explants of alfalfa were efficient infection materials for Agrobacterium EHAlOl and normally germinated into plantlets. The introduced gene (GUS) was constitutively expressed in all tissues of transgenic alfalfa with different expression levels. These results indicate that the use of pIG121-Hm vector, Agrobacterium EHAlOl and improved culture system of callus facilitate the transformation of alfalfa. (Key words : Agrobacterium, Alfalfa, Gene transfer, Transformation)