인삼에 염류내성을 증진시키기 위해서 Arobidopsis에서 분리한 SAL1 (3‘(2’),5‘-bis-phosphate nucleotidase) 유전자를 Agrobacterium Tumefaciens을 이용하여 인삼자엽으로부터 형질 전환체를 유도하였다. Agrobacterium 과 공동배양 후 식물호르몬 무첨가 선발배지 (kanamycin 100 mg/l)에 치상한 결과 10%미만의 자엽에서 형질전환 인삼체세포배가 발생되었으나, Agrobacterium과 공동배양 후 1.0 mg/l 2.4-D와 0.5 mg/l kinetin의 식물호르몬을 첨가한 배지에 옮겨준 경우에는 74%의 형질전환율을 보였다. 발생한 체세포배는 초기에 250 mg/l 의 cefotaxime이 첨가된 MS배지에서 3주간 배양한 후 100 mg/l kanamycin과 250 mg/l cefotaxime이 첨가된 MS배지에 계대배양하여 선발하였다. 자엽단계로 발달한 체세포배들은 발아시키기 위해서 50 mg/l kanamycin과 10 mg/l 지베렐린이 첨가된 MS 배지로 옮겨 선발하였다. Kanamycin 첨가배지에서 선발된 체세포배들은 특이 프라이머로 PCR 증폭을 통하여 최종적으로 형질전환체를 확인하였으며, 줄기와 뿌리가 잘 발달된 형질전환체들은 성공적으로 토양에 순화시켰다.

The effects of sonication and vacuum infiltration on transformation efficiency was investigated by using immature embryos of Korean wheat as explants. Two Agrobacterium tumefaciens strains, KYRT1 and EHA105, carrying pCAMBIA 1305.1 were used. Transformation efficiency was demonstrated by the detection of β-glucu-ronidase (GUS) activity. GUS expression showed clear difference among Korean wheat cultivars. Geurumil showed higher GUS expression efficiency 79.1~% compared with other cultivars. The effects of the duration of vacuum infiltration and sonication treatment showed a tendency high GUS expression efficiency by their combination. In comparison with other Agrobacterium strains, KYRT1 showed high efficiency in most Korean cultivars.

우용유전자 도입을 통한 신품종 톨페스큐를 개발할 목적으로 Agrobacterium을 이용한 효율적인 형질전환 체계를 확립하였다 톨페스큐 성숙종자 유래의 캘러스를 standard binary vector인 pIG121Hm을 가지는 Agrobacterium EHA101을 이용하여 감염시킨 후 공동배양하여 형질전환시켰다. Agrobacterium을 이용한 형질전환에 있어서 중요한 인자로 작용하는 몇 가지 요인에 대한 톨페스큐 캘러스의 형질전환 효율을 GUS 유전자의 발현정도로 조사하였다. Agrobacterium 감염시에 접종배지와 공동배양배지에 200uM의 acetosyringone(AS)을 첨가해 주었을 때 형질전환 효율이 증가 되었으며, 공동배양기간을 5일까지 증가시켰을 때 형질전환효율이 증가되었다. 또한 Agrobacterium 감염시에 200uM의 AS와 0.l%의 Tween20을 동시에 첨가해 주었을 때 가장 높은 형질전환 효율을 나타내었다. 50 mg/L.의 hygromycin이 첨가된 선발배지에서 살아남은 캘러스로부터 정상적인 식물체가 재분화 되었으며 이들 형질전환체를 GUS 염색과 Southern blot 분석을 실시하여 본 결과 발현백터의 T-DNA 영역이 형질전환 식물체의 genome에 성공적으로 도입되었음을 확인할 수 있었다. 본 연구를 통하여 확립된 효율적인 형질전환 시스템은 분자육종을 통한 신품종 톨페스큐의 개발에 유용하게 이용될 수 있을 것이다

The objectives of this study were to establish the genetic transformation system of stilbene synthase in Rehmannia glutinosa. Resveratrol, which is both a phytoalexin with antifungal activity and a phytochemical associated with reduced cancer risk and reduced cardiovascular disease, is synthesized in a limited number of plant species including peanut. Resveratrol synthesis is catalyzed by the enzyme stilbene synthase including resveratrol synthase (RS). Stilbene synthase gene (RS3) obtained from peanut, Arachis hypogaea, Fabaceae has been transferred into chinese foxglove, Rehmannia glutinosa by using Agrobacterium mediated transformation. PCR analysis with RS3 primer confirmed that the targeted gene was introduced into the plant genome, 904 bp in size. Further analyses of identification of transformation using developed other molecular techniques and transgenic plants that RS t-DNA introduced to chinese foxglove (R. glutinosa L) and its reaction product, stilbene such as resveratrol will be isolate and characterize using NMR, MS, and HPLC.

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.

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~textrmmgL-1 kinetin, 0.5~textrmmgL-1 NAA, 2 % sucrose (or maltose), 3% sorbitol, and 500~textrmmgL-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.

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 200μM 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.