Asian Journal of Turfgrass Science (아시안잔디학회지)

Turfgrass Society of Korea (한국잔디학회)
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Establishment of a transformation protocol combination particle bombardment with Agrobacterium tumefaciens in different zoysiagrass cultivars

유전자총과 아그로박테리움을 이용한 여러 가지 한국 잔디류의 형질전환체계 확립

  • Kim Jong-Bo (Turfgrass and Environment Research Institute, Samsung Everland Inc.) ;
  • Kim Kyong-Duck (Turfgrass and Environment Research Institute, Samsung Everland Inc.) ;
  • Park Dae-Sup (Turfgrass and Environment Research Institute, Samsung Everland Inc.)
  • 김종보 (삼성에버랜드 잔디환경연구소) ;
  • 김경덕 (삼성에버랜드 잔디환경연구소) ;
  • 박대섭 (삼성에버랜드 잔디환경연구소)
  • Published : 2004.09.01
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Abstract

In this report, several factors such as infection time and concentration of bacterial suspension, influencing on transient gene expression in Agrobacterium-mediated transformation were evaluated. An appropriate concentration (O.D 600nm = 1.0-1.2) of bateria and 30 min of infection time showed a higher level of GUS expression. To improve transformation efficiency (TE), friable embryogenic calli (FEC) were bombarded by tungsten particles without plasmid DNA, and then co-cultivated with A. tumefaciens LBA4404 which contains pTOK233 super binary vector, carrying neomycin phosphotransferase (NPTII), hygromycin phosphotransferase (hpt) and$\beta-glucuronidase$ (GUS) genes. Three days after co-cultivation with A. tumefaciens and particle bombardment, FEC cultures were transferred to the selection medium (SM: MS medium supplemented with BA 1mg/l, hygromycin 100mg/l, cefotaxime 250 mg/l and vancomycin 200mg/l). They were cultured for 2 weeks and then transferred to the second SM containing hygromycin 50mg/l, cefotaxime 200 mg/l and vancomycin 100mg/l. Later, stable GUS expression was detected 4 to 6 weeks after transfer to the SM. Further, TE from Agrobacterium-mediated transformation after particle bombardment increased to about 3-folds compared with Agrobacterium-mediated transformation without particle bombardment. In the present study, we established an efficient transformation protocol of zoysiagrass by using A. tumefaciens in the combination with particle bombardment for the first time.

다양한 Zoysiagrass 4가지 품종들을 식물재료로 사용하여 Agrobacterium만 이용한 방법 그리고 particle bombardment로 배발생캘러스에 상처를 낸 후, Agrobacterium으로 공동배양 시키는 2가지 다른 형질전환 방법을 비교하였다. 예비실험에서 일반적으로 형질전환에 널리 사용되는 kanamycin과 PPT(phospinitricin)의 적적선발농도에 대해서 실험하였는데, kanamycin의 경우 300mg/l 그리고 PPT의 경위 50mg/l의 농도에서 가장 효과적인 선발 효율을 나타내었다. Agrobacterium을 이용한 형질전환은 Agrobacterium을 2일간 배양시킨 다음, 박테리아 농도를 O.D 600nm=1.0-1.2로 맞추고, 배발생캘러스를 30분간 간염 시키는 방법이 효과적이었는데, particle bombardment를 이용하여 캘러스에 상처를 유발시킨 후, Agrobacterium으로 감염시키면 3배 이상 높은 형질전환 수율을 획득할 수 있었다. 이상의 결과는 한국잔디 형질전환에 있어서 particle bombardment과 Agrobacterium을 병행하여 실시한 최초의 보고이고, 이러한 시스템을 기반으로 하여 향후 한국잔디를 포함하여 다른 난지형 및 한지형 잔디의 품종개량에 널리 이용되리라 생각된다.

Keywords

References

  1. Chai, M. L., D. H. Kim. 2000. Agro-bacterium-mediated transformation of Korean Lawngrass (Zoysia japonica) J. of Korean Soc. Hort. Science 41:455-458
  2. Christou, P. 1997. Rice transformation: bombardment. Plant Molecular Biology 35:197-203 https://doi.org/10.1023/A:1005791230345
  3. Goldman, J. J., Hanna, W. W., Fleming, G. H, Ozias-Akins, P. 2004. Ploidy variation among herbicide-resistant ber-mudagrass plants of cv. TifEagle trans-formed with the bar gene. Plant Cell Rep. 22:553-560 https://doi.org/10.1007/s00299-003-0736-0
  4. Hiei, Y., Ohta, S., Komari, T., Kumashiro, T. 1994. Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J 6:271-282 https://doi.org/10.1046/j.1365-313X.1994.6020271.x
  5. Hiei, Y., Komari, T., Kubo, T. 1997. Transformation of rice mediated by Agrobacterium tumefaciens. Plant Mol BioI 35: 205-218 https://doi.org/10.1023/A:1005847615493
  6. Jefferson, R. A., Kavanaugh, T. A., Bevan, N. H. 1987. GUS fusions: $\beta$-glucuronidase as a sensitive and versatile gene fusion marker for higher plants. EMBO J 6: 3901-3907
  7. Lucas O, Kallerhoff J, Alibert G. 2000. Production of stable transgenic sun-flowers (Helianthus annuus L.) from wounded immature embryos by particle bombardment and co-cultivation with Agrobacterium tumefaciens. Molecular Breeding 6: 479-487 https://doi.org/10.1023/A:1026583931327
  8. Murashige, T., and F, Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15:473-497 https://doi.org/10.1111/j.1399-3054.1962.tb08052.x
  9. Zuker A, Ahroni A, Tzfira T, Ben-Meir H, Vainstein A. 1999. Wounding by bombardment yields highly efficient Agrobacterium-mediated transformation of carnation (Dianthus caryophyllus L). Molecular Breeding 5: 367-375 https://doi.org/10.1023/A:1009671131200
  10. 남상욱, 김혜영. 2004. Particle Bombard-ment 의해 전저리된 참나리 (Lilium Iancifoliutti Thunb.) 캘러스의 Agrobacterium tumefaciens를 통한 형질전환. Korean J. Plant Biotechnol. 31(1):13-17 https://doi.org/10.5010/JPB.2004.31.1.013
  11. 배창휴, 遠山統一, 이성춘 임용표, 김호일, 송필순, 이효연. 2001. 성숙종자 유래 캘러스를 이용한 들잔디 (Zoysia japonicaSteud.)의 효과적인 식물체 재분화. Korean J. of Plant Tissue Culture 28(2):61-67
  12. 임선형, 강병철, 남궁용, 신홍균. 2001. 유전자총을 이용한 잔디 형질전환 체계 확립. Kor. Turfgrass Sci., 15(1) 9-14
  13. 임용우, 김기용, 김맹중, 성병렬, 임영철, 정으이수, 신홍균, 김용선. 2003. 한국잔디 수집계통들 중에서 우수계통들의 생육특성비교. Kor. Turfgrass Sci. 17(2,3) 75-80