Journal of Animal Science and Technology

Korean Society of Animal Sciences and Technology (한국축산학회)
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Factors Affecting Genetic Transformation of Italian Ryegrass

이탈리안 라이그래스의 형질전환에 미치는 몇 가지 요인의 영향

  • Lee, S.H. (Division of Applied Life Science Institute of Agriculture and Life Science, Gyeongsang National University) ;
  • Woo, H.S. (Division of Applied Life Science Institute of Agriculture and Life Science, Gyeongsang National University) ;
  • Lee, B.H. (Division of Applied Life Science Institute of Agriculture and Life Science, Gyeongsang National University)
  • 이상훈 (경상대학교 응용생명과학부 농업생명과학연구원) ;
  • 우현숙 (경상대학교 응용생명과학부 농업생명과학연구원) ;
  • 이병현 (경상대학교 응용생명과학부 농업생명과학연구원)
  • Published : 2004.04.30
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Abstract

A system for the production of transgenic plants has been developed for Italian ryegrass(Lolium mult리orum Lam.) via Agrobacterium-mediated transformation of embryogenic callus. Mature seed-derived calli were infected and co-cultured with Agrobacterium EHA101 carrying standard binary vector pIG121Hm encoding the hygromycin phosphotransferase(HPT), neomycin phosphotransferase II (NPTII) and intron-oontaining $\beta$g1ucuronidase( intron-GUS) genes in the T-DNA region. The effects of several factors on transformation and the expression of the GUS gene were investigated. Inclusion of 200${\mu}M$ acetosyringone(AS) in inoculation and co-cultivation media lead to a significant increase in stable transformation efficiency. Increasing Agrobacterium cell density up to 1.0 in $OD_{600}$ during infection increased transfonnation efficiency of embryogenic calli. The highest transfonnation efficiency was obtained when embryogenic calli were incoulated with Agrobacterium in the presence of 0.1% Tween20 and 200${\mu}M$ AS. Hygromycin resistant calli were developed into complete plants via somatic embryogenesis. GUS histochemical assay and PCR analysis of transgenic plants demonstrated that transgenes were integrated into the genome of Italian ryegrass.

유용유전자 도입을 통한 신품종 이탈리안 라이그래스를 개발할 목적으로 Agrobacterium을 이용한 효율적인 형질전환 체계를 확립하였다. 이탈리안 라이그래스 성숙종자 유래의 캘러스를 standard binary vector인 pIG121Hm을 가지는 Agrobacterium EHA101을 이용하여 감염시킨 후 공동배양하여 형질전환시켰다. Agrobacterium을 이용한 형질전환에 있어서 중요한 인자로 작용하는 몇 가지 요인에 대한 이탈리안 라이그래스 캘러스의 형질전환 효율을 GUS 유전자의 발현정도로 조사하였다. Agrobacterium 감염시에 접종배지와 공동배양배지에 200${\mu}M$의 acetosyringone(AS)을 첨가해 주었을 때 형질전환 효율이 현저히 증가되었다. 또한 Agrobacterium의 농도를 $OD_{600}$=10. 이상의 높은 농도로 1시간 감염시켜Tdfm 때 형질전환 효율이 증가되었다. 접종배지에 200${\mu}M$의 AS와 0.1%의 Tween20을 첨가해 주었을 때 가장 높은 형질전환 효율을 나타내었다. 50mg/L의 hygromycin이 첨가된 선발배지에서 살아남은 캘러스로부터 정상적인 식물체가 재분화되었으며, 이들 형질전환체의 잎으로부터 GUS 활성 염색과 PCR 분석을 통하여 발현벡터의 T-DNA 영역이 형질전환 식물체의 genome으로 도입되었음을 확인할 수 있었다. 본 연구를 통하여 확립된 효율적인 형질전환 시스템은 분자육종을 통한 신품종 이탈리안 라이그래스의 개발에 유용하게 이용될 수 있을 것이다.

Keywords

References

  1. Amoah, B. K, Wu, H, Sparks, C. and Jones, H D. 2001. Factors influencing Agrobaeterium-mediated expression of uidA in wheat inflorescence tissue. J. Exp. Bot. 52:1135-1142.
  2. An, G., Ebert, P. R., Mirata, A. and Ha, S. B. 1988. Binary vector. In: Gelvin, S. B., Schilperoot, R. A, Verma, D. P. S. (Eds) Plant Molecular biology mannual. Kluwer, Dordrecht, p. 1.
  3. Arencibia, A D., Carmona, E. R, Tellez, P., Chan, M -T., Yu, S. -M, Trujillo, L. E. and Oramas, P. 1998. An efficient protocol for sugarcane (Saccharum spp. L.) transformation mediated by Agrobacterium tumefaciens. Transgenic Res. 7:213-222.
  4. Bettany, A. J. E., Dalton, S. J., Timms, E., Manderyck, B., Dhanoa, M. S. and Morris, P. 2002. Agrobacterium tumefciens-mediated transformation of Festuca arundinacea and Lolium multiflorum. Plant Cell Rep. 21:437-444.
  5. Cheng, M., Fry, J. E., Pang, S., Zhou, H., Hironaka, C. M., Duncan, D. R, Conner, T. W. and Wan, Y. 1997. Genetic transformation of wheat mediated by Agrobacterium tumefaciens. Plant Physiol. 115:971-980.
  6. Henderson, J. L. and Davies, R. O. 1955. The yield and composition of mixed cereal-legume crops at different stages of growth. Empire J. Exp. Agr. 23:131-144.
  7. Dalton, S. J., Bettany, A J. E., Timms, E. and Morris, P. 1998. Transgenic plants of Lolium multiflorum, Lolium perenne, Festuca arundinacea and Agrostis stolonifera by silicon carbide fibre-mediated transfomation of cell suspension cultures. Plant Sci. 132:31-43.
  8. Dalton, S. J., Betanny, A. J. E., Timms, E. and Morris, P. 1999. Co-transfomed, diploid Lolium perenne, Lolium multiflorum and Lolium temulentum plants produced by microprojectile bombardment. Plant Cell Rep. 18:721-726.
  9. Hides, D. H., Kute, C. A. and Marshall, A. H. 1993. Seed development and seed yield potential of Italian ryegrass(Lolium multiflorum Lam.) populations. Grass and Forage Sci. 48:181-188.
  10. Hiei, Y., Ohta, S., Komari, T. and Kumasiro, T. 1994. Efficient transformation of rice mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J. 6:271-282.
  11. Hood, E. E., Helmer, G. L., Fraley, R T. and Chilton, M. D. 1986. The hypervirulence of Agrobacterium tumefaciens A281 is encoded in a region of pTiBo542 outside of T-DNA. J. Bacteriol. 168:1291-1301. https://doi.org/10.1128/jb.168.3.1291-1301.1986
  12. Ishida, Y., Saito, H, Ohta, S., Hiei, Y., Komari, T. and Kumashiro, T. 1996. High efficiency of transformation of maize(Zea amys L.) mediated by Agrobacterium tumefaciens. Nature Biotechnol. 14: 745-750.
  13. Isselstein, J. 1993. Influence of slight shading, sward density and nitrogen fertilization on yield and nutritive value of Lolium multiflorum Lam. J. Agro. and Crop Sci. 170:341-347.
  14. Jefferson, R A. 1987. Assay chimeric genes in plant: the GUS gene fusion system. Plant Mol. Biol. Rep. 5:387-405.
  15. Komari, T. 1990. Transformation of callus cells of Chenopodium quinoa by binary vectors that carry a fragment of DNA from the virulence region of pTiBo542. Plant Cell Rep. 9:303-306.
  16. McKersie, B. D. 1997. Improving forage production systems using biotechnolgy. In: McKersie, B. D. and Brown, D. C. W.(Eds), Biotechnology in Agriculture Series, No. 17, CAB International, Wallingford, p. 3.
  17. Murashige, T. and Skoog, F. 1962. A revise medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15:473-497.
  18. Murray, M. G. and Tompson, P. F. 1980. Rapid isolation of high molecular weight plant DNA. Nucleic Acid Res. 8:4321-4325. https://doi.org/10.1093/nar/8.19.4321
  19. Nadolska-Qrczyk, A., Orczyk, w. and Przetakiewicz, A. 2000. Agrobacterium-mediated transformation of cereals from technique development to application. Acta Physiol. Plant. 22:77-88.
  20. Park, B. H., Park, B. S. and Kang, J. H 1987. A comparison between diploid and tetraploid cultivars of Lolium multiflorum Lam. J. Kor. Grassland Sci. 7:135-139.
  21. Spangenberg, G., Wang, Z.-Y., Wu, X., Nagel, J. and Potrykus, I. 1995. Transgenic perennial rye-grass(Lolium perenne) plants from microprojectile bombardment of embryogenic suspension cells. Plant Sci. 108:209-217.
  22. Spangenberg, G., Wang, Z. Y. and Potrykus, I. 1998 Biotechnology in forage and turf grass improvement. In: Frankel et al(Eds), Monographs on theoretical and applied genetics, Vol. 23, Springer Verlag, Heidelberg, p. 192.
  23. Tingay, S., McElroy, D., Kalla, R., Fieg, S., Wang, M., Thronton, S. and Brettell, R 1997. Agrobacterium tumefaciens-mediated barley transformation. Plant J. 11:1369-1376.
  24. Trifonova, A., Madsen, S. and Olesen, A. 2001. Agrobacterium-mediated transgene delivery and integration into barley under a range of in vitro culture conditions. Plant Sci. 162:871-880.
  25. Usami, S., Morikawa, S., Takabe, L and Machida, T. 1987. Absence in monocotyledonous plants of the diffusible plant factocs inducing T-DNA circularization and vir gene expression in Agrobacterium. Mol. Gen. Genet. 209:221-226.
  26. Van Wijk, A. J. P., Boonman, J. G. and Rumball, W. 1993. Achievements and prospectives in the breeding of forage grasses and legumes. In: Baker, M. J. (Eds), Grasslands for our world, SIR, Wellington, p. 116.
  27. Wu, H., Sparks, C., Amoah, B. and Jones, H. D. 2003. Factors influencing successful Agrobacterium-mediated genetic transformation of wheat. Plant Cell Rep. 21:659-668.
  28. Ye, X., Wang, Z. Y., Wu, X., Potrykus, I. and Spangenberg, G. 1997. Transgenic Italian ryegrass (Lolium multiflorum) plants from microprojectile bombardment of embryogenic suspension cells. Plant Cell Rep. 16:379-384.
  29. Zhao, Z. -Y., Cai, T., Tagliani, L., Miller, M., Wang, N., Pang, H, Rudert, M., Schroeder, S., Hondred, D., Seltzer, J. and Pierce, D. 2000. Agrobacterium-mediated sorghum transformation. Plant Mol. Biol. 44:789-798.
  30. Zhong, H., Bolyard, M. G., Srinivasan, C. and Stickelen, M. 1993. Transgenic plants of turfgrass (Agrostis pa/ustris Huds.) from microprojectile bombardment of embryogenic callus. Plant Cell Rep. 13:1-6.