• Journal of Microbiology and Biotechnology
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• v.15 no.4
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• pp.683-688
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• 2005

A transformation system mediated by Agrobacterium tumefaciens is routinely used for the genetic engineering of plants. Here, we report an efficient and stable method for transformation of heterogeneous genes into an industrial Cephalosporium acremonium by using a similar transformation system established in plants. Both the phleomycin-resistant gene and vgb gene were used as screening markers to confirm the success of transformation by either Southern hybridization or PCR amplification. It was found that acetosyringone (AS) was necessary only for protoplast transformation and the heterogeneous genes transferred were integrated into the genome of C. acremonium. The transformation efficiency obtained with this system was much higher than the conventional techniques used for transformation of C. acremonium.

• Korean Journal of Microbiology
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• v.52 no.1
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• pp.110-115
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• 2016

A small plasmid (pDK4) from the Antarctic marine organism Pseudoalteromonas sp. PAMC 21150, was purified, sequenced and analyzed. pDK4 was determined to be 3,480 bp in length with a G+C content of 41.64% and contains three open reading frames encoding a replication initiation protein (RepA), a conjugative mobilization protein (Mob) and a hypothetical protein. PCR-amplified pDK4 was cloned in high-copy pUC19 to yield the fusion vector pDOC153. The chloramphenicol resistance gene was inserted into pDOC153 to give an ampicillin and chloramphenicol-resistant, Pseudoalteromonas - Escherichia coli shuttle vector (7,216 bp; pDOC155). The TonB-dependent receptor (chi22718_IV ) and exochitinase (chi22718_III ) genes from Arctic marine P. issachenkonii PAMC 22718 were cloned into pDOC155 to produce pDOC158 and pDOC165, respectively. Both vector derivatives were transferred into plasmid-free Pseudoalteromonas sp. PAMC 22137 by the triparental mating method. PCR experiments showed that the genes were stably maintained both in Pseudoalteromonas sp. PAMC 22137 and E. coli $DH5{\alpha}$ cells, indicating the potential use of pDOC155 as a new gene transfer system into marine Pseudoalteromonas spp.

• Korean Journal of Plant Resources
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• v.17 no.2
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• pp.161-168
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• 2004

Optimal regeneration conditions of ginseng transformants were studied. It has been known that Ferritin Light Heavy Chain (FLHC) gene remove the several heavy metal by combination, store and transport. To obtain the ginseng tolerant to heavy metal, binary vector was introduced in Agrobacterium by tri-parental mating and then Agrobacterium tumefaciens MP90/FLHC was selected on the AB media and MS media containing kanamycin. Explants were co-cultured with Agrobacterium tumefaciens MP90/FLHC, which contained NPT II as a selectable marker, tadpole ferritin heavy chain (FLHC) gene and human ferritin light chain gene and then a number of embryos were induced. The induced embryo transferred to shooting media consisting of MS medium supplemented with GA 10 mg/L. As a result of examination that induced the normal growth of transfomants, transformants showed the equivalent growth in both root and shoot on the media containing the 1/3 MS.

• Journal of Plant Biotechnology
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• v.1 no.1
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• pp.46-55
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• 1999

The expressed sequence tags(ESTs) from immature seed of rice, Oryza sativa cv Milyang 23, were partially sequenced and analyzed by homology. As of 1998, the partial sequences of about 6,600 cDNA clones were analyzed from normal and normalized immature seed cDNA libraries. About 2,200 ESTs were putatively identified by BLASTX deduced amino acid sequence homology analysis. About 20% of them were putatively identified as storage proteins. Also the clones were highly homologous to genes involved particularly in starch biosynthesis, glycolysis, signal transduction and defenses. Compared to 35% of redundancy in the ESTs of normal cDNA library, that from the substracted library was 15%. The Korea Rice Genome Network is maintained to provide the updated information of sequences, their homologies and sequence alignments of ESTs. For the stable expression of transgene in rice, diverse vectors were developed for overexpression, targeting and gene dosage effect with transit peptides (Tp) and matrix attachment region (MAR) sequence from chicken lysozyme locus. The rice calli were transformed via Agrobacterium tumefaciens LBA4404(pSB1) with the triparental mating technique and selected by herbicide resistance. The green fluorescent protein(GFP) gene in expression vector under the control of rbcS promoter-Tp was overexpressed upto 10 % of the total soluble protein. In addition, the Tp-sGFP fusion protein was properly processed during translocation into chloroplast. The expression of sGFP in the presence of MAR sequences was analyzed with Northern and immunoblot analysis. All the lines in which sGFP transgene with MAR sequence, showed position independent and copy number-dependent expression, while the lines without MAR showed the varied level of expression with the integration site. Thus the MAR sequence significantly reduced the variation in transgene expression between independent transformants.

• Korean Journal of Agricultural Science
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• v.20 no.2
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• pp.133-144
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• 1993

Calli were induced on microtuber disks of potato(S.tuberosum) infected with three binary vectors transconjugated with C58, A281 and LBA 4404 of Agrobacterium tumefaciens and pBI121. The frequency inducing callus was the highest by infection of C121 carrying pC58 and pBI121, and shoots were differentiated on the calli without any hormonal application. Transformed calli were selected by their resistance to kanamycin and identified by GUS activity. The frequency of callus formation by infection of binary vector strain was affected according to the hormonal application.

• Korean Journal of Plant Tissue Culture
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• v.25 no.3
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• pp.207-217
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• 1998

Total of 78 strains were characterized based on the morphological characteristics of colonies isolated on Schroth, and New & Kerr's media for selection of hypervirulent wild-type Agrobacterium spp from galls, hairy root-like process and soil of Populus, Malus, Salix and Diopyros in Korea. Among them, 48 strains were able to induce tumors in carrot disc. Hypervirulent A. tumefaciens SP101 and SM042 were identified as biotype 1 and biotype 2, respectively, These strains formed fast growing, larger tumors as compared to those induced by other strains. The binary vector pGA643 with kanamycin resistant gene was mobilized from E. coli MC100 into A. tumefaciens strain SM042 isolated from soil, and/or disarmed vector PC2760 using a triparental mating method with E. coli HB101/pRK2013, and transconjugants, A. tumefaciens SM643 and PC643 were obtained in minimal media containing kanamycin and tetracycline. Tobacco tissues were cocultivated with conjugant Agrobacterium and then transferred to selective medium with 2,4-D and kanamycin to induce the transformants. Calli were formed more efficiently in cocultivation with A. tumefaciens SM643 than that with A. tumefaciens PC643. Most of calli transformed with A. tumefaciens PC643 were friable and regenerated into normal plantlets, while the calli transformed with A. tumefaciens SM643 were compact, hard, and mixed with friable calli. The friable calli formed normal shoots, while compact calli did not form shoots but only grew to typical compact tumor calli. When the shoots formed directly from tobacco stems without callus induction after transformation by A. tumefaciens SM643 with wild-type Ti-plasmid, normal transformed plants can be induced without using disarmed Ti-plasmid.

• The Journal of Natural Sciences
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• v.4
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• pp.103-142
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• 1991

These studies were carried out to select somatic hybrid using selectable marker genes of Nicotiana glauca transformed by NPTII gene and Solanum tuberosum transformed by T- DNA, and to study characteristics of transformant. The results are summarized as follows. 1. Crown gall tumors and hairy roots were formed on potato tuber disc infected by A. tumefaciens Ach5 and A. rhizogenes ATCC15834. These tumors and roots could be grown on the phytohormone free media. 2. Callus formation from hairy root was prompted on the medium containing 2, 4 D 2mg/I with casein hydrolysate lg/l. 3. The survival ratio of crown gall tumor callus derived from potato increased on the medium containing the activated charcoal 0. 5-2. 0mg/I because of the preventions on the other hand, hairy roots were necrosis on the same medium. 4. Callus derived from hairy root were excellently grown for a short time by suspension culture on liquid medium containing 2, 4-D 2mg/I and casein hydrolysate lg/l. 5. The binary vector pGA643 was mobilized from E. coli MC1000 into wild type Agrobacteriurn tumefaciens Ach5, A. tumefaciens $A_4T$ and disarmed A. tuniefaciens LBA4404 using a triparental mating method with E. ccli HB1O1/pRK2013. Transconjugants were obtained on the minimal media containing tetracycline and kanamycin. pGA643 vectors were confirmed by electrophoresis on 0.7% agarose gel. 6. Kanamycin resistant calli were selected on the media supplemented with 2, 4-D 0.5mg/1 and kanamycin $100\mug$/ml after co- cultivating with tobacco stem explants and A. tumefaciens LBA4404/pGA643, and selected calli propagated on the same medium. 7. The multiple shoots were regenerated from kanamycin resistant calli on the MS medium containing BA 2mg/l. 8. Leaf segments of transformed shoot were able to grow vigorusly on the medium supplemented with high concentration of kanamycin $1000\mug$/ml. 9. Kanamycin resistant shoots were rooting and elongated on medium containing kanamycin $100\mug$/ml, but normal shoot were not. 10. For the production of protoplast from potato calli transformed by T-DNA and mesophyll tissue transformed by NPTII gene, the former was isolated in the enzyme mixture of 2.0% celluase Onozuka R-10, 1.0% dricelase, 1.0% macerozyme. and 0.5M mannitol, the latter was isolated in the enzyme mixture 1.0% Celluase Onozuka R-10, 0.3% macerozyme, and 0.7M mannitol. 11. The optimal concentrationn of mannitol in the enzyme mixture for high protoplast yield was 0.8M at both transformed tobacco mesophyll and potato callus. The viabilities of protoplast were shown above 90%, respectively. 12. Both tobacco mesophyll and potato callus protoplasts were fused by using PEG solution. Cell walls were regenerated on hormone free media supplemented with kanamycin after 5 days, and colonies were observed after 4 weeks culture.