• Current Research on Agriculture and Life Sciences
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• v.9
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• pp.21-28
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• 1991

The effect of subculture intervals and passages on plant regeneration from seed-derived callus was determined. Regeneration capacity of callus varied with rice cultivars and subculture intervars tested. The callus subcultured every 2 weeks produced more plants than that of 4 weeks. The calli from a Tongil-type rice cultivar, Milyang 23, lost easily their regeneration ability when the calli were subcultured every 2 weeks and 4 weeks. The callus induced from a japonica cultivar, "Yeongdeogbyeo", showed to maintain high frequency(>70%) of plant regeneration when it was subcultured every 2-week intervals. Casein hydrolysate supplemented in callus induction medium enhanced callus growth and its regeneration. High frequency of plant regeneration was obtained from the calli transferred on $N_6$ medium supplemented with kinetin(2mg/1) and NAA(1mg/1). The subcultured calli in the medium supplemented with casein hydrolysate(2 g/1), myo-inositol(200mg/1) and thiamine-HCl(2mg/1) increased the frequency of embryogenic callus formation and plant regeneration.

• Journal of Plant Biotechnology
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• v.32 no.2
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• pp.117-121
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• 2005

Immature embryos of 3 cultivars (Du Me Chal, Mi Baek Chal, Heug Jeom Chal) and 5 genotypes (HW1, KL103, HW3, HW4, KW7) were cultured on medium containing MS salts, Eriksson's vitamins, 1 mg/L 2,4-dichlorophenoxyacetic acid, 25 mM proline, 100 mg/L casamino acid, 3 mM MES, 1.7 mg/L $AgNO_3$ and 20 g/L sucrose (SIM). Frequency of somatic embryo formation on explant of immature embryos showed in HW1 (45.20%), KL103 (5.75%), HW3 (37.20%), HW4 (30.10%), KW70 (55.20%), Mi Baek Chal (18.74%), Heug Jeom Chal (22.41%), Du Me Chal (36.72%) and Hi II type (<10%), respectively. Yellowish friable embryogenic callus (YFEC) such as type II callus of Hi II genotype only produced from the HW3 and Heug Jeom Chal, whereas other cultivars and genotypes were directly formed somatic embryos with late-embryonic stages or expanded yellowish compact somatic embryo with morphological abnormality. The yellowish friable embryogenic callus (YFEC) could be proliferated on the same medium, which were maintained embryogenic capacity for 6 months over. Upon transfer to first regeneration and second regeneration medium, somatic embryos converted to plantlets at a frequency of approximately 100%. However, the expanded somatic embryos with abnormal morphology were slowly proliferated when subcultured on the same medium, and some of them were degenerated or converted to plantlets at a frequency of approximately 25%. Accordingly, The Heug Jeom Chal and HW3 genotype will be further used for development of high frequency transformation system in domestic maize germplasm.

• Journal of Plant Biotechnology
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• v.8 no.1
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• pp.21-25
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• 2006

Phragmites australis (reed) has received much attention as being one of the principle emergent aquatic plants for treating industrial and civil wastewater. Plant regeneration via plant tissue culture in p. australis was investigated. Three types of callus were identified from seeds on N6 medium plus 4.5 UM 2,4-dichlorophenoxyacetic acid (2,4-D). Yellow compact type showed the best redifferentiation, whereas white compact type and yellow friable were not competent to differentiate into plane. Solid medium culture was better than liquid suspension culture for enhancing callus growth when N6 medium supplemented with 4.5 ${\mu}M$ 2,4-D was used. Phytagel, as a gelling agent, was superior to agar in plant regeneration on N6 medium, supplemented with 9.4 ${\mu}M$ kinetin and 0.54 ${\mu}M$ $\alpha$-naphthaleneacetic acid (NAA). Transfer of the plantlets regenerated from kinetin and NAA-supplemented N6 medium to growth regulator-free MS medium enhanced the further development of the plantlets. Plantlets on subsequently grown to maturity when tansferred to potting soil. The regenerated plants exhibited morphologically normal. The system for plant regeneration of P. australis enables to propagate elite lines on a large scale for water purification in the ecosystem

• 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.

• 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.

• Journal of Plant Biology
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• v.37 no.2
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• pp.237-244
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• 1994

Callus tissues derived from protoplast of Alnus hirsuta were shown to produce red pigments under the continuous light. The R4 cell line with high yield of the pigments was selected. One of the red pigments was identified as anthocyanin; glucose and galactose as sugar moiety and cyandin as aglycone, by spectroscopic analysis, cellulose TLC and GLC. Both callus growth and anthocyanin accumulation were remarkably stimulated by 2,4-D. The highest anthocyanin yield was observed when $1\;\mu\textrm{M}$ 2,4-D in combination with $0.1\;\mu\textrm{M}$ BAP was supplemented to the culture medium. In case of inorganic salts, anthocyanin yield was enhenced by reducing concentration of phosphate from 5.0 mM to 2.5 mM and by increasing the ratio of ${NO_3}^{-}$ type to 80% for total nitrogen source.source.

• Molecules and Cells
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• v.39 no.6
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• pp.484-494
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• 2016

Plant cells have a remarkable ability to induce pluripotent cell masses and regenerate whole plant organs under the appropriate culture conditions. Although the in vitro regeneration system is widely applied to manipulate agronomic traits, an understanding of the molecular mechanisms underlying callus formation is starting to emerge. Here, we performed genome-wide transcriptome profiling of wild-type leaves and leaf explant-derived calli for comparison and identified 10,405 differentially expressed genes (> two-fold change). In addition to the well-defined signaling pathways involved in callus formation, we uncovered additional biological processes that may contribute to robust cellular dedifferentiation. Particular emphasis is placed on molecular components involved in leaf development, circadian clock, stress and hormone signaling, carbohydrate metabolism, and chromatin organization. Genetic and pharmacological analyses further supported that homeostasis of clock activity and stress signaling is crucial for proper callus induction. In addition, gibberellic acid (GA) and brassinosteroid (BR) signaling also participates in intricate cellular reprogramming. Collectively, our findings indicate that multiple signaling pathways are intertwined to allow reversible transition of cellular differentiation and dedifferentiation.

• Plant Resources
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• v.7 no.1
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• pp.39-46
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• 2004

To demonstrate the importance of transformation efficiency in independent event, molecular and cytogenetic analysis were conducted with genomic DNA and chromosome of transgenic plants produced by Agrobacterium tumefeciens LBA4404 (pSBM-PPGN: gusA and bar). Selection ratios of putative transgenic calli were similar in independent experiments, however, transformation efficiencies were critically influenced by the type of regeneration media. MSRK5SS-Pr regeneration mediun, which contains 5 mgL$^{-1}$ kinetin, 2% (w/v) sucrose in combination with 3% (w/v) sorbitol, and 500 mgL$^{-1}$ proline, was efficient to produce transgenic plant of rice from putative transgenic callus in the presence of L-phosphinotricin (PPT). With MSRK5SS-Pr medium, transformation efficincies of Nagdongbyeo were significantly enhanced from 3.7% to 6.3% in independent callus lines arid from 7.3% to 19.7% in plants produced, respectively. Stable integration and expression of bar gene were confirmed by basta herbicide assay, PCR amplification and Southern blotting of bar gene, and fluorescence in situ hybridization (FISH) analysis using pSBM-PPGN as a probe. In Southern blot analysis, diverse band patterns were observed in total 44 transgenic plants regenerated from 20 independent PPT resistant calli showing from one to five copies of T-DNA segments, however, the transformants obtained from one callus line showed the same copy numbers with the same fractionized band patterns.

• Korean Journal of Weed Science
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• v.6 no.2
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• pp.174-190
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• 1986

A series of laboratory and greenhouse experiments were conducted to find out the possibility of tissue culture and cell culture methods as a tool to study herbicidal behaviour and herbicide tolerance screening from 1985 to 1986 at the Yeongnam Crop Experiment Station. For dehulled-rice culture, pure agar medium was the most appropriate in rice growth campared to other media used for plant tissue culture method. All the media but the pure agar medium resulted in growth retardance by approximately 50% and this effect was more pronounced to root growth than shoot growth. Herbicidal phytotoxicity was enhanced under light condition for butachlor, 2.4-D, and propanil while this effect was reversed for DPX F-5384 and CGA 142464, respectively. And also, herbicides of butachlor, chlornitrofen, oxadiazon, and BAS-514 resulted in more phytotoxic effect when shoot and root of rice were exposed to herbicide than root exposure only while other used herbicides exhibited no significant difference between two exposure regimes. Similar response was obtained from Echinochloa crusgalli even though the degree of growth retardance was much greater. Particularly, butachlor, 2.4-D, chlornitrofen, oxadiaxon, pyrazolate and BAS-514 totally inhibited chlorophyll biosynthesis even at the single contact of root. Apparent cultivar differences to herbicide were observed at the young seedling culture method and dehulled rice cultivars were more tolerant in DPX F-5384, NC-311, pyrazolate and pyrazoxyfen, respectively. For derant than other types or rice cultivar in butachlor, pretilachlor, perfluidone and oxadiazon while Tongil-type rice cultivars were more tolerant in DPXF-5384, NC-311, Pyrazolate and Pyrazoxyfen, respectively. For dehulled rice culture, on the other hand, Japonica-type rice cultivar was less tolerant to herbicides of butachlor, propanil, chlornitrofen and oxadiazon that was reversed trend to young seedling culture test. Cultivar differences were also exhibited within same cultivar type. In general, relatively higher tolerant cultivars were Milyang 42, Cheongcheongbyeo, Samgangbyeo, Chilseoungbyeo for Tongil-type, Somjinbyeo for Japonica-type and IR50 for Indica-type, respectively. The response of callus growth showed similar to dehulled rice culture method in all herbicides regardless of property variables. However, concentration response was much sensitive in callus response. The concentration ranges of $10^{-9}M-10^(-8)M$ were appropriate to distinguish the difference between herbicides for E. crusgalli callus growth. Among used herbicides, BAS-514 was the most effective to E. crusgalli callus growth. Based on the above results, tissue culture method could be successfully used as a tool for studying herbicidal behaviour and tolerance screening to herbicide.

• Korean Journal of Plant Resources
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• v.8 no.3
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• pp.237-246
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• 1995

In this study, the callus was induced and regenerated from the immature embryo and ultrastructural characteristics of developmental stages in Citrus junos SIEB, were investigated. The yellowish callus was induced by 5 to 6 week of culture of citrus. In proliferation callus after 6 weeks of culture, large vacuole was formed by fusion between adjacent small ones. In the non-embryogenic callus cultured for 12weeks, re-differentiated cells of callus showed the large nucleus with globular nucleus and amyloplast with large size of starches. In the embryogenic callus cltured for 14-16 weeks, the active exocytosis occurred in cells, secretory vesicles appeared on cell membrane and small particles from cytoplasm were released to intercelluar space. In the embryogenic callus cultured for 24 weeks, a sperical type of chloroplast bounded on cytoplasm by double membrane and typical grana was dispersed equally among matrix. In the normal plantlet after 26 weeks of culture, a lot of vessels and companion cells apperaed in the leaf cell of plantlet. In the normal plantlet after 30 weeks of culture, the immature leaf showed many small companion cells, sieve tubes and central vacuole. Also, the secondary vacuole protruded into the central vacuole and elongated chloroplasts near plasma membrane. In the matured plant habituated on the soil, palisada tissue composed of orderly arranged cells contained the nucleus in the center of the cell and large vacuoles on either side of the nucleus.