• Journal of Plant Biotechnology
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• v.3 no.2
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• pp.89-94
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• 2001

In vitro plant regeneration of inbred breeding line of hot pepper (Capsicum annuum L.) was established using leaf and petiole segments as explants. About 28 days old plants were excised and cultured on MS medium supplemented with TDZ and NAA or in combination with Zeatin. In all of the media compositions tested, combination of TDZ 0.5 mg/L, Zeatin 0.5 mg/L, and NAA 0.1 mg/L was found to be the best medium for shoot bud initiation. Young petiole was the most appropriate explant type for the plant regeneration as well as genetic transformation in hot pepper. In this study, HpMADS1 gene isolated from hot pepper was introduced using Agrobacterium-mediated transformation system. Based on the analysis of Southern blot and RT-PCR, HpMADS1 gene was integrated in the hot pepper genome. It has been known that floral organ development is controlled by a group of regulatory factors containing the MADS domain. Morphological characteristics in these transgenic plants, especially flowering habit, however, were not significantly altered, indicating this MADS gene, HpMADS1 may be non-functional in this case.

• Plant Biotechnology Reports
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• v.5 no.2
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• pp.147-156
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• 2011

Efficient Agrobacterium-mediated genetic transformation of Scoparia dulcis L. was developed using Agrobacterium tumefaciens strain LBA4404 harboring the binary vector pCAMBIA1301 with ${\beta}$-glucuronidase (GUS) (uidA) and hygromycin phosphotransferase (hpt) genes. Two-day precultured leaf segments of in vitro shoot culture were found to be suitable for cocultivation with the Agrobacterium strain, and acetosyringone was able to promote the transformation process. After selection on shoot organogenesis medium with appropriate concentrations of hygromycin and carbenicillin, adventitious shoots were developed on elongation medium by twice subculturing under the same selection scheme. The elongated hygromycin-resistant shoots were subsequently rooted on the MS medium supplemented with $1mg\;l^{-1}$ indole-3-butyric acid and $15mg\;l^{-1}$ hygromycin. Successful transformation was confirmed by PCR analysis using uidA- and hpt-specific primers and monitored by histochemical assay for ${\beta}$-GUS activity during shoot organogenesis. Integration of hpt gene into the genome of transgenic plants was also verified by Southern blot analysis. High transformation efficiency at a rate of 54.6% with an average of $3.9{\pm}0.39$ transgenic plantlets per explant was achieved in the present transformation system. It took only 2-3 months from seed germination to positive transformants transplanted to soil. Therefore, an efficient and fast genetic transformation system was developed for S. dulcis using an Agrobacterium-mediated approach and plant regeneration via shoot organogenesis, which provides a useful platform for future genetic engineering studies in this medicinally important plant.

• Journal of Plant Biotechnology
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• v.48 no.4
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• pp.246-254
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• 2021

Environmental stresses are estimated to have reduced global crop yields of wheat by 5.5%. However, traditional approaches for the transfer of resistance to these stresses in wheat plants have yielded limited results. In this regard, genetic transformation has undoubtedly opened up new avenues to overcome crop losses due to various abiotic stresses. Particle bombardment has been successfully employed for obtaining transgenic wheat. However, most of these procedures employ immature embryos, which are not available throughout the year. Therefore, the present investigation utilized mature seeds as the starting material and used the calli raised from three Moroccan durum wheat varieties as the target tissue for genetic transformation by the biolistic approach. The pANIC-5E plasmid containing the SINA gene for drought and salinity tolerance was used for genetic transformation. To enhance the regeneration capacity and transformation efficiency of the tested genotypes, the study compared the effect of copper supplementation in the induction medium (up to 5 μM) with the standard MS medium. The results show that the genotypes displayed different sensitivities to CuSO₄, indicating that the transformation efficiency was highly genotype-dependent. The integration of transgenes in the T₀ transformants was demonstrated by polymerase chain reaction (PCR) analysis of the obtained resistant plantlets with primers specific to the SINA gene. Among the three genotypes studied, 'Isly' showed the highest efficiency of 9.75%, followed by 'Amria' with 1.25% and 'Chaoui' with 1%.

• Journal of Plant Biotechnology
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• v.49 no.1
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• pp.61-73
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• 2022

In this study, we developed a reliable and efficient Agrobacterium-mediated genetic transformation system by applying sonication and vacuum infiltration to six chickpea cultivars (ICCV2, ICCV10, ICCV92944, ICCV37, JAKI9218, and JG11) using embryo axis explants. Wounded explants were precultured for 3 days in shoot induction medium (SIM) before sonication and vacuum infiltration with an Agrobacterium suspension and co-cultivated for 3 days in co-cultivation medium containing 100 µM/l of acetosyringone and 200 mg/l of L-cysteine. Responsive explants with putatively transformed shoots were selected using a gradual increase in kanamycin from 25 mg/l to 100 mg/l in selection medium to eliminate escapes. Results showed optimal transformation efficiency at a bacterial density of 1.0, an optical density at 600 nm wavelength (OD₆₀₀), and an infection duration of 30 min. The presence and stable integration of the β-glucuronidase (gusA) gene into the chickpea genome were confirmed using GUS histochemical assay and polymerase chain reaction. A high transformation efficiency was achieved among the different factors tested using embryo axis explants of cv. JAKI 9218. Of the six chickpea cultivars tested, JAKI9218 showed the highest transformation efficiency of 8.6%, followed by JG11 (7.2%), ICCV92944 (6.8%), ICCV37 (5.4%), ICCV2 (4.8%), and ICCV10 (4.6%). These findings showed that the Agrobacterium-mediated genetic transformation system will help transfer novel candidate genes into chickpea.

• Korean Journal of Microbiology
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• v.42 no.1
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• pp.59-61
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• 2006

Genetic transformation of a mushroom-forming fungus Coprinellus congregatus to antibiotic resistance gene had been successfully carried out by electroporation to oidia instead of protoplasts. Since there was no protoplast generation step which required not only cell wall degrading enzymes but many skillful procedures, commercial herbicide (basta) could be used without any difficulty with simple procedure. The transformation yield was 10-20 $transformants/{\mu}g$ DNA, and the transformants were very stable even after 10 consecutive transfers through the non-selective medium.

• Journal of Microbiology and Biotechnology
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• v.18 no.2
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• pp.234-241
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• 2008

Agrobacterum tumefaciens-mediated transformation (ATMT) is becoming an effective system as an insertional mutagenesis tool in filamentous fungi. We developed and optimized ATMT for two Colletotrichum species, C. falcatum and C. acutatum, which are the causal agents of sugarcane red rot and pepper anthracnose, respectively. A. tumefaciens strain SK1044, carrying a hygromycin phosphotransferase gene (hph) and a green fluorescent protein (GFP) gene, was used to transform the conidia of these two Colletotrichum species. Transformation efficiency was correlated with co-cultivation time and bacterial cell concentration and was higher in C. falcatum than in C. acutatum. Southern blot analysis indicated that about 65% of the transformants had a single copy of the T-DNA in both C. falcatum and C. acutatum and that T-DNA integrated randomly in both fungal genomes. T-DNA insertions were identified in transformants through thermal asymmetrical interlaced PCR (TAIL-PCR) followed by sequencing. Our results suggested that ATMT can be used as a molecular tool to identify and characterize pathogenicity-related genes in these two economically important Colletotrichum species.

• Journal of Plant Biotechnology
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• v.4 no.4
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• pp.151-154
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• 2002

Conditions for lily pollen growth in vitro and transformation were optimized. Active pollen tube development was achieved effectively in a medium containing 7% sucrose with pH adjusted to 5.7 at the temperature of 27$^{\circ}C$ for about 16-24 hours. Pollen growth was little impaired by the presence of kanamycin at concentration up to 100 mg/L. Pollen rains near the beginning of germination stage were more reliable for Agrobacterium-mediated GUS DNA transformation via vacuum infiltration lasted for 20-40 minutes. GUS DNA integration and its expression in fully developed pollen tubes could be confirmed by Southern blot hybridization, RT-PCR and histochemical staining.

• Journal of Plant Biology
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• v.39 no.2
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• pp.93-98
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• 1996

In order to enhance the system of potato transformation and further regeneration, potato was transformed using the Agrobacterium tumefaciens harboring $\beta$-glucuronidase (GUS) gene. We found that a series fo modified medium ttained 100% shoot regeneration within 5 weeks after the preincubated explants on stage I medium were infected with Agrobacterium. Callus appeared at the cut edges of stem segments on stage II medium, mainly at the basal parts. Some explants started to form shoots after two to three weeks on stage III medium containing kanamycin (50 mg/L). When transferred to MS medium containing 200 mg/L kanamycin, 81% of the transformed shoots formed roots at the cut edge of the plantlets. In contrast, untrasformed shoots never rooted and became yellowish after few weeks under the same conditions. Southern and northern analysis indicated in vitro shoot regeneration on the callus derived from the potato explants, which were incubated with Agrobacteria. The regeneration cycle was shortened after the transformatin and finally the transformation efficiency was highly enhanced.

• Journal of Plant Biotechnology
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• v.3 no.3
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• pp.135-140
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• 2001

An Agrobacterium tumefaciens LBA4404 (harboring antisense OMT gene)-mediated transformation method has been developed for poplar (P.nigra x maximowiczii) using prolonging co-cultivation time. Explants on LT (longterm) were induced transgenic calli one month earlier than those from ST (short-term) co-cultivation and remained healthier on LT than ST. With this approach, LT method reduced time to produce transgenic calli. Shoots were successfully regenerated from transgenic calli on SIM (Shoot Induction Medium) and rooted well on the basal medium spontaneously. The presence of antisense OMT gene was verified both by PCR and Southern analysis. Each transgenic poplar was phenotypically indishtinguishable when compared with controls for their growth pattern, leaf morphologies and xylem coloration.

• Journal of Microbiology and Biotechnology
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• v.2 no.2
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• pp.92-97
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• 1992

Microbial transformation of (-)kaur-l6-en-19-oic acid and (-)pimara-9(1l), 15-dien-19-oic acid from A. koreanum was investigated. Throughout the screening of the capability of metabolizing these bioactive diterpenoids, two microorganisms have chosen among various fungi and streptomycetes tested. Scale-up fermentation with Fusarium oxysporum KCTC 6051 produced two metabolites related to the precursor diterpenoids. The two metabolites were isolated by column chromatography and identified by chemical and spectroscopic methods as $2\beta$, $16\alpha$-dihydroxy kauran-19-oic acid and $16\alpha$-hydroxy kauran-19-oic acid. However any microorganisms capable to transform (-) pimara-9(11), 15-dien-19-oic acid was not screened in this condition.