Objectives Here, we report the effect of overexpression of ginseng farnesyl diphosphate synthase on the transcription of three key regulatory enzymes involved in triterpene metabolism in hairy root of ginseng and Centella asiatica (L.) Urban. Materials and Methods A four-year-old root of Panax ginseng C.A. Meyer and Centella asiatica (L.) Urban whole plants were obtained from National Institute of Crop Science (Suwon, Korea) and Chonnam National University (Gwangju, Korea), respectively. Agrobacterium rhizogenes R1000 strain was kindly provided by Dr. In (Nongwoo Bio, Yeju, Korea). Results and Discussion The role of farnesyl diphosphate synthase (FPS) in triterpene biosynthesis (Fig. 1) was investigated. A pCAMBIA3101 vector was used to insert a exogenous gene into target plant genome (Fig. 2). After the transformation, we produced Panax ginseng and Centella asiatica hairy roots by introducing the coding region of the gene from Panax ginseng. In these hairy roots, integration of the transgenes into the C. asiatica nuclear genome was confirmed by PCR analysis using PgFPS (P. ginseng FPS) primers and by Southern hybridization using PgFPS-specific probe. FPS specific activity is increased 4-fold compared to controls. In RT-PCR analysis, overexpression of PgFPS in hairy roots was observed (Fig. 3) and two genes, cycloartenol and beta-amyrin synthase, related to triterpene biosynthesis were up-regulated. These results suggest that FPS overexpression might lead to an enhanced biosynthesis of triterpene saponins and phytosterols. However, we did not demonstrate whether or not the introduction of PgFPS gene in Centella asiatica genome directly enhances triterpene saponin production, although our results showed that gene expression related to triterpene saponin biosynthesis were obviously up-regulated. Therefore, additional experiments such as overexpression of FPS gene in triterpene saponin-deficient mutant plants will be required.

Objectives The objective of our research was to establish the gene transformation, expression and characterization system in transgenic Codonopsis lanceolata. Materials and methods Agrobacterium tumefaciens strain LBA 4404/ binary vector pYBI121Regeneration of transgenic shoots: MS medium supplemented with 0.1 mg/ℓ NAA and 1 mg/ℓ BAP, 3% sucrose and 0.8% agar at pH 5.8. Agrobacterium cell density OD 600 between 0.8 and 1.0, Infection: 5 minutes DNA isolation and Polymerase chain reaction: DNA was extracted from young leafs excised from kanamycin resistant shoots. Two primers used for PCR amplification of the 700 bp of the npt II gene were N 1 (5′ GAA GCT ATT CGG CGG CTA TGA CTG 3′) as a sense primer and N 2 (5′ ATC GGG AGC GGC GGC GAT ACC CTA 3′) as a anti sense primer. Result and Discussion Adventitious shoots regenerated 3 weeks after Agrobacterium infection on regeneration medium containing 0.1 mg/ℓ NAA and 1 mg/ℓ BAP, 100 mg/ℓ kanamycin 250 mg/ℓ cefatoxime. Numerous adventitious shoot inductions of putative transformants were observed from the cut surface of explants which initially resembled knob like structure and later developed into new plant. PCR analysis of showed the expected bands of npt II gene. PCR analysis was carried out to confirm the insertion of the npt II gene in the genome of transformed plant. The expected amplified npt II fragments of size 700 bp was found in the T0 transformed plants, indicating the integration of npt II gene.

본 연구는 Agrobacterium을 매개로 도라지에 PAT 유전자를 도입하여 ‘바스타’에 저항성을 가지는 형질전환 도라지를 개발하는 기술을 확립하기 위하여 수행되었다. 종자를 무균적으로 발아시킨 후 10일 된 미성숙 자엽과 성숙엽에 Agrobac-terium을 접종하고 1/10 MS 배지에서 48시간 동안 공동 배양하였다. 공동배양 후 부정아 유도를 위해 MS 선발배지 (0.2 mg/l NAA, 1.0 mg/l BA, 3% 설탕, pH 5.8; 3 g/l gelrite, 100 mg/l kanamycin, 500 mg/l carbenicillin)에 치상하여 배양한 결과 미성숙 자엽의 절편에서 형질전환체로 추정되는 부정아가 형성되었고, 선발배지에 2회 계대배양하여 형질전환 추정체를 선발하였다. 이러한 형질전환 추정체는 GUS, PCR 분석 및 RT-PCR 분석에 의하여 형질전환체로 확인되었다. 또한 10 mg/l 의 phosphinothricin이 함유된 배지에서 배양하여 형질전환 여부를 확인하였고, 순화재배 후 0.3% ‘바스타’를 살포한 결과 형질전환 도라지는 제초제에 저항성을 보였다. ‘바스타’에 저항성을 보인 도라지 식물체는 정상적인 생육을 계속하여 개화하였다.