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Protocorm-like body를 이용한 호접란 형질전환 연구

Agrobacterium-Mediated Transformation of Phalaenopsis by Using Protocorm-Like Body

  • 발행 : 2009.03.31

초록

본 연구는 Agrobacterium을 이용한 효율적인 호접란 형질 전환 시스템의 확립을 위해 실시하였고, 호접란 PLB의 대량증식을 위한 배지 조성은 하이포넥스 기본 배지에 활성탄 1g/l, 티아민 0.1 mg/l 및 sucrose 30 g/l을 배지에 첨가한 경우 PLB가 안정적으로 증식되었다. 계대 배양시 PLB 절단 방법이 PLB의 생존과 증식에 큰 영향을 미치며, PLB 위쪽으로 부터 1/3 부위를 절단하여 계대 배양한 경우 90% 이상의 가장 높은 증식률을 나타났다. Agrobacterium 접종 시 균이 묻어 있는 침으로 PLB를 찔러 상처를 내는 dipping 방법을 이용하여 형질전환 효율을 높일 수 있었다. Agrobacterium 배양액의 흡광도 값이 0.8일 때 형질전환 효율이 가장 높았고, 형질전환된 PLB의 선발은 1 mg/l 정도의 저농도 hygromycin을 함유한 배지에 계대배양 하는 것이 효율적이었다. 호접란 형질전환체를 먼저 GUS assay를 통하여 선발하였고, 선발된 개체로부터 genomic DNA를 추출하여 GUS 특이적 primer와 probe로 PCR과 Southern blot 분석을 수행하고 유전자의 도입여부를 조사한 결과 GUS 염색된 형질전환체에서 정상적으로 GUS 유전자가 도입된 것을 확인할 수 있었다.

Agrobacterium tumefaciens-mediated transformation procedure for the phalaenopsis orchid, established by using Protocorm-like bodies (PLBs), was aimed at the introduction of target genes into individuals with divergent genetic backgrounds. PLBs obtained from the axillary bud of a peduncle were maintained on a hyponex medium supplemented with 1 g/l of activated charcoal, 30 g/l of sucrose and 0.1 mg/l thiamine. The multiplication rate of PLBs was about 90% in case of subculture PLBs to be cut transversely into 1/3 part from top position. The PLBs were inoculated with Agrobacterium strain EHA105 harboring both $\beta$-glucuronidase (GUS) and hygromycin-resistant genes for 20 minutes after dipping treatment. Transformation efficiency was the highest with a Agrobacterium culture medium and dipping treatment of O.D. 0.8. Newly induced PLBs were put on selection medium containing 1 mg/l hygromycin for 2 months. Hygromycin-resistant phalaenopsis plants that regenerated after the selection culture of PLBs showed histochemical blue staining due to GUS. Transgene integration of the hygromycin-resistant plants was confirmed by PCR and Southern blot using GUS specific primers and probe.

키워드

참고문헌

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피인용 문헌

  1. Recent trends in tissue culture and genetic transformation of Phalaenopsis vol.39, pp.4, 2012, https://doi.org/10.5010/JPB.2012.39.4.225
  2. Optimization of particle gun-mediated transformation system in Cymbidium vol.38, pp.4, 2011, https://doi.org/10.5010/JPB.2011.38.4.293