Using Agrobacterium-me야ated transformation method the auxin-regulated cotton GST (Gh-5) constructs were used to transform Rehmannia glutinosa L. The PCR analysis was conducted to verify transgenicity. Based on the PCR analysis, there was verified that the 988 bp DNA band had showed in transgenic pla...
Using Agrobacterium-me야ated transformation method the auxin-regulated cotton GST (Gh-5) constructs were used to transform Rehmannia glutinosa L. The PCR analysis was conducted to verify transgenicity. Based on the PCR analysis, there was verified that the 988 bp DNA band had showed in transgenic plant genomes in PCR anaJysis using Gh5-1 and Gh5-2 primers. The effects of cocultivation with Agrobacterium tumefaciens, regeneration and selection conditions on the transformation efficiency of Chinese foxglove (Rehmannia glutinosa L.) were investigated. Factors such as cocultivation period, use of acetosyringone, postcultivation in darkness, and different kanamycin concentrations for selection were assessed. In vitro regeneration, the number of leaves, shoot lengths and numbers on MS medium were superior to on B5 and WPM medium, and the shoot formation rate was highest level of 95% in cultured base part containing leaf stalk. Addition of acetosyringone at concentration of $200{\mu}M$ to cocultivation medium and 3-day of cocultivation improved transformation frequencies. Exposure of explants to darkness for 4 weeks on selection medium resulted in further increased the regeneration frequency of transgenic shoots. In PCR analysis, the amplified fragments of Gh5 gene were detected (988 bp), and GST-expressing transgenic R. glutinosa L. plants had approximately three-fold higher activity in leaf extracts compared with control plant.
Using Agrobacterium-me야ated transformation method the auxin-regulated cotton GST (Gh-5) constructs were used to transform Rehmannia glutinosa L. The PCR analysis was conducted to verify transgenicity. Based on the PCR analysis, there was verified that the 988 bp DNA band had showed in transgenic plant genomes in PCR anaJysis using Gh5-1 and Gh5-2 primers. The effects of cocultivation with Agrobacterium tumefaciens, regeneration and selection conditions on the transformation efficiency of Chinese foxglove (Rehmannia glutinosa L.) were investigated. Factors such as cocultivation period, use of acetosyringone, postcultivation in darkness, and different kanamycin concentrations for selection were assessed. In vitro regeneration, the number of leaves, shoot lengths and numbers on MS medium were superior to on B5 and WPM medium, and the shoot formation rate was highest level of 95% in cultured base part containing leaf stalk. Addition of acetosyringone at concentration of $200{\mu}M$ to cocultivation medium and 3-day of cocultivation improved transformation frequencies. Exposure of explants to darkness for 4 weeks on selection medium resulted in further increased the regeneration frequency of transgenic shoots. In PCR analysis, the amplified fragments of Gh5 gene were detected (988 bp), and GST-expressing transgenic R. glutinosa L. plants had approximately three-fold higher activity in leaf extracts compared with control plant.
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제안 방법
To assess factors affecting the transformation frequency, different treatments were performed. (1) The explants were cocultivated with Agrobacterium on solid cocultivation medium for 1, 2, 3, 4 or 5 days. (2) Cocultivation was done on cocultivation medium or cocultivation medium plus acetosyringone (0-400 μM).
glutinosa grown in vitro were used for this study. In vitro regeneration and growth of plant materials were done on MS, WPM and B5 basal medium containing different type and various concentrations of plant growth regulators and maintained with a 16-h photoperiod under illumination at 45 μmol/m2s1 and 60% relative humidity at 25 ℃ for 3 additional weeks. The BAP, TDZ, 2iP, kinetin, NAA and 2, 4-D were used at the concentration of 0.
Selection marker nptll gene was detected with N-l (5'-GAAGCTATTCG GCTATGACTG-3') and N-2 (5'-ATCGGGAGCGGCGATACCCTA-3'). PCR reactions were performed with TOUCHDOWNTM (HYBRID), and amplification conditions were: 45 cycles of pre-denature (at 94 ℃, 5 min.), denaturation (at 94℃, 1 min.), annealing (at 35℃, 1 min.), and extension (at 72℃, 2 min.)f 40 cycle of postelongation (at 72℃, 10 min.). To analyse the introduction of Gh5 cDNA fragment in the R.
To investigate the shoot formation and regeneration in different part of leaf, it divided to base part containing leaf stalk, intermediate, and tips containing leaf blade and cultured on a half of MS salt containing 0.5 mg/ℓ BAP, 0.5 mg/ℓ TDZ, and 0.1 mg/ℓ 2iP. In base part containing leaf stalk, shoot formation rate was highest level of 95% than other parts.
성능/효과
1 mg/ℓ of NAA for regeneration (data not shown). After 4 weeks culture on MS, B5 and WPM medium combined with growth regulators (1 mg/ℓof BAP and 0.1 mg/ℓ of NAA), leaf number, shoot length and number on MS medium were superior to on B5 and WPM medium. The shoot number was highest on MS medium and 4.
plants had approximately three-fold higher activity in leaf extracts compared with control plant (Table 2). The results from this assay confirmed that Gh5 transgenes encode an active GST in transgenic R. glutinosa L. plants.
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