Journal of Plant Biotechnology

  • 제34권1호
  • /
  • Pages.37-45
  • /
  • 2007
  • /
  • 1229-2818(pISSN)
  • /
  • 2384-1397(eISSN)
한국식물생명공학회 (The Korean Society of Plant Biotechnology)
권호 표지
DOI QR코드

DOI QR Code

캘러스 유도에 의한 수박 형질전환

Genetic Transformation of Watermelon (Citrullus vulgaris Schard.) by Callus Induction

  • 권정희 ((주)농우바이오 생명공학연구소) ;
  • 박상미 ((주)농우바이오 생명공학연구소) ;
  • 임미영 ((주)농우바이오 생명공학연구소) ;
  • 신윤섭 ((주)농우바이오 생명공학연구소) ;
  • 한지학 ((주)농우바이오 생명공학연구소)
  • 발행 : 2007.03.31
PDF 다운로드
⟨ 이전 논문 다음 논문 ⟩

초록

수박은 형질전환이 매우 어렵고 직접적인 신초 유도방법으로는 안정된 형질전환을 기대할 수 없어서, 다른 여러 작물에서 높은 효율을 보였던 캘러스 유래 신초 유도 방법을 도입하고자 하였다. 수박의 최적 캘러스 유도조건은 자엽 절편체의 경우 2.0 mg/L zeatin과 0.1 mg/L IAA이었으며 뿌리 절편체의 경우 2.0 mg/L BA와 0.1 mg/L 2,4-D이었다. NptII 유전자의 선발 항생제는 kanamycin보다는 paromomycin 빠르고 효과적이었으며, 수박의 절편체에 Agrobacterium을 접종 한 후 paromomycin을 125 mg/L 첨가한 배지에서 선발하였다. pmGFP5-ER vector로 형질전환한 후 캘러스 상태에서 형광현미경을 통해 GFP 유전자의 도입을 확인하였으며, 딱딱한 초록색의 캘러스에서 강한 GFP 발현을 관찰하였고, 자엽유래 캘러스의 경우 WM8에서 9.0%, 뿌리유래 캘러스의 경우 WM6에서 8.3%의 가장 높은 GFP 발현 효율을 보였다. GFP 유전자 도입과 같은 방법으로 WMV-CP 유전자가 있는 pWMV2300 vector로 형질전환한 후 캘러스 상태에서 PCR 및 Southern blot 분석을 한 결과, 두 점의 캘러스에서 WMV-CP 유전자가 도입되었음을 확인하였다. 본 연구를 통하여 확립된 수박의 캘러스 유도 시스템은 안정된 수박 형질전환 방법에 기초 자료로서 이용될 것이다.

The genetic transformation of watermelon by Agrobacterium has been known very difficult and a few successful cases have been reported by obtaining the direct shoot formation. However, since this direct shoot formation is not guaranteed the stable transformation, the stable transformation with reproducibility is required by a different approach such as a callus induced manner. The best conditions for inducing the callus from cotyledon and root explants of watermelon were 2 mg/L zeatin + 0.1 mg/L IAA and 2 mg/L BA + 0.1 mg/L 2,4-D, respectively. The GFP expression in the callus was identified and monitored through fluorescent microscopy after transformation with pmGFP5-ER vector. Paromomycin rather than kanamycin was used for selecting the nptll gene expression because it was more effective to select the watermelon explants. Four different callus types were observed and the solid green callus showed stronger GFP expression. The highest frequency of GFP expression in the callus developed from cotyledon was 9.0% (WM8 inbred line), while the highest frequency from root was 8.3% (WM6 inbred line). The WMV-CP was transformed using the method of GFP transformation and the genetic transformation of WMV-CP was confirmed by PCR and Southern blot analysis. Here we present a system for callus induction of watermelon explant and the callus induced method would facilitate the establishment of stable watermelon transformation.

키워드

참고문헌

  1. Chaturvedi R, Bhatnagar SP (2001) High-frequency shoot regeneraton from cotyledon explants of watermelon cv. sugar baby. In Vitro Cell Dev Biol Plant 37: 255-258 https://doi.org/10.1007/s11627-001-0045-7
  2. Cho MA, Song YM, Park YO, Ko SM, Min SR, Liu JR, Lee JH, Choi PS (2005a) Production of transgenic melon from the cultures of cotyledonary-node explant using Agrobacterium-mediated transformation. Kor J Plant Biotechnol 32: 257-262 https://doi.org/10.5010/JPB.2005.32.4.257
  3. Cho MA, Song YM, Park YO, Ko SM, Min SR, Liu JR, Choi PS (2005b) The use of glufosinate as a selective marker for the transformation of cucumber (Cucumis setivus L.). Kor J Plant Biotechnol 32: 161-165 https://doi.org/10.5010/JPB.2005.32.3.161
  4. Choi PS, Soh WY, Kim YS, Yoo OJ, Liu JR (1994) Genetic transformation and plant regeneration of watermelon using Agrobacterium tumefaciens. Plant Cell Rep 13: 344-348
  5. Choi JY, Kim SH, Hyung NI (2003) Optimization of Agrobacterium-mediated transformation protocol in watermelon. J Kor Soc Hort Sci Technol Suppl 21: 59
  6. Compton ME (1999) Dark pretreatment improves adventious shoot organogenesis from cotyledons of diploid watermelon. Plant Cell Tiss Org Cult 58: 185-188 https://doi.org/10.1023/A:1006364013126
  7. Compton ME, Gray DJ (1993) Shoot organogenesis and plant regeneration from cotyledons of diploid, triploid and tetraploid watermelon. J Am Soc Hort Sci 118: 151-157
  8. Compton ME, Gray DJ (1999) Shoot organogenesis from cotyledon explants of watermelon. In: Trigiano RN.; Gray DJ (eds) Plant Tissue Culture Concepts and Laboratory Exercises. Ed 2, CRC Press, Boca Raton, FL, pp 149-158
  9. Compton ME, Gray DJ, Gaba VP (2004) Use of tissue culture and biotechnology for the genetic improvement of watermelon. Plant Cell Tiss Org Cult 77: 231-243 https://doi.org/10.1023/B:TICU.0000018428.43446.58
  10. Dabauza M, Bordas M, Salvador A, Roig LA, Moreno V (1997) Plant regeneration and Agrobacterium-mediated trasformation of cotyledon explant of Citrullus colocynthis (L.) Schrad. Plant Cell Rep 16: 888-892 https://doi.org/10.1007/s002990050340
  11. Dellaporta, SL, Wood J, Hicks J.B. (1983) A simple and rapid method for plant DNA preparation. Version II. Plant Mol Biol Rep 1: 19-21 https://doi.org/10.1007/BF02712670
  12. Dong JZ, Jia SR (1991) High efficiency plant regeneration from cotyledons of watermelon (Citrullus vulgaris Schrad.) Plant Cell Rep 9: 559-562
  13. Ellul P, Rios G, Atares A, Roig LA, Serrano R (2003) The expression of the Saccharomyces cerevisiae HAL1 gene increases salt tolerance in transgenic watermelon (Citrullus lanatus Thunb.) Matsum. & Nakai. Theor Appl Genet 107: 462-469 https://doi.org/10.1007/s00122-003-1267-3
  14. Franklin G, Lakshmi Sita G (2003) Agrobacterium tumefaciens-mediated transformation of eggplant (Solanum melongena L.) using root explants. Plant Cell Rep 21: 549-554
  15. Gaba V, Zelcer A, Gal-On A (2004) Cucurbit biotecholoqy-the importance of virus resistance. In Vitro Cell Dev Biol Plant 40: 346-358 https://doi.org/10.1079/IVP2004554
  16. 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
  17. Kim DH, Lee JM (1997) Shoot regeneration from watermelon explants as affected by cultivars, explant parts and cytokinins. J Kor Soc Hort Sci Suppl 15(1): 72-73
  18. Kusano M, Tohyama K, Bae CH, Riu KZ, Lee HY (2003) Plant regeneration and transformation of Kentuchy Bluegrass (Poa pratensis L.) via the plant tissue culture. Kor J Plant Biotechnol 30: 115-121 https://doi.org/10.5010/JPB.2003.30.2.115
  19. Lee YH, Kim HS, Kim JY, Jung M, Park YS, Lee JS, Choi SH, Her NH, Lee JH, Hyung NI, Lee CH, Yang SG and Ham CH (2004) A new selection method for pepper transformation: callus-mediated shoot formation. Plant Cell Rep 23: 50-58
  20. Lee HK, Paek KY, Seo YK, Rhee WY (1994) Genotypic effect of watermelon (Citrullus lanatus Thunb.) on organogenesis from shoot tip culture of seedlings. Kor J Plant Tiss Cult 21: 239-246
  21. Lee SH, Shon YG, Lee SI, Kim CY, Koo JC, Lim CO, Choi YJ, Han CD, Chung CH, Choe ZR, Cho MJ (1999) Cultivar variability in the Agrobacterium-rice cell interaction and plant regeneration. Physiol Plant 107: 338-340 https://doi.org/10.1034/j.1399-3054.1999.100311.x
  22. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tabacco tissue culture. Physiol Plant 15: 473-479 https://doi.org/10.1111/j.1399-3054.1962.tb08052.x
  23. Rashid H, Yokoi K, Toriyama K, Hinata K (1996) Transgenic Plant production mediated by Agrobacterium in Indica rice. Plant Cell Rep 15: 727-730 https://doi.org/10.1007/BF00232216
  24. Roa-Rodriuez and Nottenburg (1999) Nptll gene in combination with paromomycin as a selective agent. Patent EP927765A1
  25. Simmonds DH, Donaldson PA (2000) Genotype screening for proliferative embryogenesis and biolistic transformation of short-season soybean genotype. Plant Cell Rep 19: 485-490 https://doi.org/10.1007/s002990050760
  26. Southern, E (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98: 503-512 https://doi.org/10.1016/S0022-2836(75)80083-0
  27. Tricoli DM, Carney KJ, Russell PF, Quemada HD, McMaster RJ, Reynold JF & Deng RZ (2002) Transgenic plants expressing DNA constructs containing a plurality of genes to impart virus resistance. United States Patent No. 6,337,431