Particle Bombardment에 의한 전처리 된 참나리(Lilium lancifolium Thunb.) 캘러스의 Agrobacterium tumefaciens을 통한 형질전환
Agrobacterium Mediated Transformation from Callus Pretreated with Particle Bombardment in Lilium lancifolium Thunb.
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초록
형질전환 효율을 높이기 위하여, NPT II와 GUS 유전자를 포함하고 있는 pIG121Hm을 미세 입자에 코팅하여 particle bombardment를 수행한 후 pIG121Hm가 도입된 Agrobacterium tumefaciens EHA101와 3 일간 공동배양하였다. 그후 캘러스를 1 mg/L 2,4-D, 0.1 mg/L BAP, 100 mg/L kanamycin, 그리고 200 mg/L carbenicillin이 포함된 MS배지로 옮겨 4 주 간 성장시킨 후 kanamycin 저항성 캘러스를 선발하여 GUS 발현을 관찰하였고, PCR 분석을 통하여 700 bp의 NPT II유전 자가 도입된 것을 확인하였다. Particle bombardment 후 Agrobacterium과 공동배양 한 처리구가 Agrobacterium과 공 동배양만 한 처리구보다 GUS 발현 분석에 의한 형질전환 효 율이 3 배 정도 더 높았다. 형질전환 된 재분화 식물체를 얻기 위해 다시 선발된 kanamycin 저항성 캘러스는 1 mg/L NAA 와 1 mg/L BAP가 포함된 재분화 배지에 옮겨져 4 주 후 형질 전환 된 소인경을 형성하였다.
Abstract
To improve transformation efficiency, the callus of Lilium lancifolium Thunb. were bombarded byparticles coated with pIG121Hm which include NPT IIand GUS genes, and then cocultivated with Agrobacteriumtumefaciens EHA101 which contain pIG121Hm binary vector, carrying neomycin phosphotransferase ( NPT II) andβ-Glucuronidase ( GUS) genes. Three days after cocultivation with Agrobacterium tumefaciens and particlebombardment, the callus clusters were transferred to MS medium containing 1 mg/L 2,4-D, 0.1 mg/L BAP, 100mg/L kanamycin and 200 mg/L carbenicillin. Four weeks after transfer to the selection medium, GUS expressionwas detected and PCR analysis revealed the presence of NPT IIfragment of the expected size (700 bp) in thetransformed callus. The GUS expression from Agrobacterium-mediated transformants after particle bombardmentincreased to over 3-folds compared with that of callus cocultivated with Agrobacterium tumefaciens without particlebombardment. The callus clusters containing kanamycin resistant gene were transferred to MS medium containing1 mg/L NAA and 1 mg/L BAP. Somatic embryos were developed in four weeks and microbulbs expressing GUSwere formed.
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