Korean Journal of Breeding Science (한국육종학회지)

The Korean Society of Breeding Science (한국육종학회)
권호 표지

Efficient Transformation Method of Soybean Using Meristematic Tissues of Germinating Seeds

발아종자의 분열조직을 이용한 효율적인 콩 형질전환 방법

  • Kim, Yul-Ho (National Institute of Crop Science, Rural Development Administration) ;
  • Park, Hyang-Mi (National Institute of Crop Science, Rural Development Administration) ;
  • Choi, Man-Soo (National Institute of Crop Science, Rural Development Administration) ;
  • Sohn, Soo-In (National Institute of Agricultural Biotechnology, Rural Development Administration) ;
  • Shin, Dong-Bum (National Institute of Crop Science, Rural Development Administration) ;
  • Lee, Jang-Yong (National Institute of Crop Science, Rural Development Administration)
  • 김율호 (농촌진흥청 작물과학원 환경생명공학과) ;
  • 박향미 (농촌진흥청 작물과학원 환경생명공학과) ;
  • 최만수 (농촌진흥청 작물과학원 환경생명공학과) ;
  • 손수인 (농촌진흥청 농업생명공학연구원 생물안전성과) ;
  • 신동범 (농촌진흥청 작물과학원 환경생명공학과) ;
  • 이장용 (농촌진흥청 작물과학원 환경생명공학과)
  • Received : 2008.09.08
  • Published : 2008.09.10
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Abstract

An efficient transformation method for soybean [Glycine max (L.) Merr.] using meristematic tissues of germinating seeds has been established. The embryonic axes were excised from germinating seeds of Korean soybean cultivar, Iksannamulkong and 0.5-2 cm long segment containing meristematic tissues were prepared by cutting hypocotyl region. The explants were inoculated with Agrobacterium tumefaciens strain LBA4404 harboring a binary vector with the bar gene as a selectable marker gene and a ${\beta}-glucuronidase$ (GUSINT) reporter gene, and then co-cultured for 7 days on co-cultivation medium (CCM). The meristematic tissues were cultured on shoot induction medium (SIMP6) supplemented with 0.4 mg/l $N_6-benzylaminopurine$ (BAP) and 0.1 mg/l indolebutyric acid (IBA) in the presence of 6 mg/l L-phosphinotricin (PPT) for 2 weeks and the surviving explants were transferred to shoot elongation medium (SEMP6). Transformation was confirmed by Southern blot analysis and the transformation efficiencies ranged from 1.48 to 2.07%. The new modified transformation method was successfully implemented for obtaining several transgenic lines with SMV-CP gene. It is expected that this method could efficiently be used for the transformation of recalcitrant soybean cultivars.

${\bullet}$ 국내 품종에 적합한 콩 형질전환 방법을 확립하기 위해 배축 절단 방법을 새롭게 개발하였으며 이 방법을 통해 목적 유전자가 콩 분열조직에 안정적으로 도입되고 선발 과정을 거쳐 형질전환 식물체를 작성할 수 있었다. ${\bullet}$ 콩 형질전환체의 도입유전자 copy 수는 1~2개이고 세대 진전에 따라 도입유전자가 안정적으로 유전되고 있음을 확인하였다. ${\bullet}$ 이러한 결과를 바탕으로 배축 절단 방법을 사용하여 우리나라 환경에 적합한 우수한 형질전환 콩 품종을 신속하게 개발할 수 있을 것으로 기대된다.

Keywords

Acknowledgement

Grant : 밭작물 콩 병해 저항성 신품종 개발

Supported by : 농촌진흥청

References

  1. Buhr T, Sato S, Ebrahim F, Xing AQ, Zhou Y, Mathiesen M 2002. Nuclear localization of RNA transcripts downregulate seed fatty acid genes in transgenic soybean. Plant J. 30:155-163 https://doi.org/10.1046/j.1365-313X.2002.01283.x
  2. Cheng T-Y, Saka H, Voqui-Dinh TH. 1980. Plant regeneration from soybean. cotyledonary node segments in culture. Plant Sci. Lett. 19:91-99 https://doi.org/10.1016/0304-4211(80)90084-X
  3. De block M 1993. The cell biology of plant transformation: Current state, problems, prospects and the implications for the plant breeding. Euphytica 71:1-14 https://doi.org/10.1007/BF00023461
  4. Finer JJ, McMullen MD 1991. Transformation of soybean via particle bombardment of embryogenic suspension culture tissue. In Vitro Cell and Develop Biol-Plant 27P:175-182
  5. Franklin G, Carpenter L, Davis E, Reddy CS, Al-Abed D, Alaiwi WA, Parani M, Smith B, Goldman SL, Sairam RV 2004. Factors influencing regeneration of soybean from mature and immature cotyledons. Plant Growth Regul. 43: 73-79 https://doi.org/10.1023/B:GROW.0000038359.86756.18
  6. Gamborg OL, Miller RA, Ojima K 1968. Nutrient requirement of suspensions cultures of soybean root cells. Exp. Cell Res. 50:151-157 https://doi.org/10.1016/0014-4827(68)90403-5
  7. Hari B, Krishna. 2005. Engineering soybean for enhanced sulfur amino acid content. Crop Sci. 45:454-461 https://doi.org/10.2135/cropsci2005.0454
  8. Hinchee MAW, Conner-Ward DV, Newell CA, McDonnell RE, Sato SJ, Gasser CS, Fischhoff DA, Re DB, Fraley RT, Horsch RB. 1988. Production of transgenic soybean plants using Agrobacterium-mediated DNA transfer. Bio/Technology 6:915-922 https://doi.org/10.1038/nbt0888-915
  9. Kimball SL, Bingham ET 1973. Adventitious bud development of soybean. hypocotyl sections in culture. Crop Sci. 13:758-760 https://doi.org/10.2135/cropsci1973.0011183X001300060053x
  10. Ki Jong Lee, Hong Jae Park, Bu Young Yi, Kyeong Ryeol Lee, Myung Sik Kim, Hee Jong Woo, Yong Moon Jin, Soon Jong Kweon. 2008. Development of herbicide tolerant soybean using Agrobacterium tumefaciens. Plant Biotechnology Vol.35, No.1, 69-74 https://doi.org/10.5010/JPB.2008.35.1.069
  11. Liu, HK, Yang C, Wei ZM 2004. Efficient Agrobacterium tumefaciens-mediated transformation of soybeans using an embryonic tip regeneration system. Planta 219:1042-1049 https://doi.org/10.1007/s00425-004-1310-x
  12. Macrae TC, Baur ME, Boethel DJ, Fitzpatrick BJ, Gao AG, Gamundi JC, Harrison LA, Kabuye VT, Mcpherson RM, Miklos JA, Paradise AS, Toedebusch AS, and Viegas A 2005. Laboratory and field evaluations of transgenic soybean exhibiting high-dose expression of a synthetic Bacillus thuringiensis cry1A gene for control of Lepidoptera. J. Econ. Entomol. 98(2):577-587 https://doi.org/10.1603/0022-0493-98.2.577
  13. Mante, S, Scorza R, Cordts J 1989. A simple, rapid protocol for adventitious shoot development from mature cotyledons of Glycine max cv Bragg. In Vitro Cell. Dev. Biol. 25:385-388 https://doi.org/10.1007/BF02624603
  14. McCabe, DE, Swain, WF, Martinell BJ Christou, P 1988. Stable transformation of soybean (Glycine max) by particle acceleration. Bio/Tech. 6:923-926 https://doi.org/10.1038/nbt0888-923
  15. Micheal D, Owen K 2000. Current use of transgenic herbicide-resistant soybean and corn in the USA. Crop Protection 19:765-771 https://doi.org/10.1016/S0261-2194(00)00102-2
  16. Murashige T, Skoog F 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant 15:473-479 https://doi.org/10.1111/j.1399-3054.1962.tb08052.x
  17. Olhoft PM, Lin K, Galbraith J, Nielsen NC, Somers DA 2001. The role of thiol compounds in increasing Agrobacterium-mediated transformation of soybean cotyledonarynode cells. Plant Cell Rep. 20:731-737 https://doi.org/10.1007/s002990100388
  18. Paz MM, Martinez JC, Kalvig AB, Fonger TM, Wang K 2006. Improved cotyledonary node method using an alternative explant derived from mature seed for efficient Agrobacterium-mediated soybean transformation. Plant Cell Rep. 25:206-213 https://doi.org/10.1007/s00299-005-0048-7
  19. Rogers S, Benich A 1994. Extraction of total DNA from plants, algae and fungi In: Plant Molecular Biology Manual. S. A. Gelvin SB. Dordrecht, The Netherlands, Kluwer Academic Publisher. 1-8
  20. Sairam RV, Franklin G, Hassel R, Smith B, Meeker K, Kashikar N, Parani M, Al. Abed D, Ismail S, Berry K, Goldman SL 2003. A study on the effect of genotypes, plant growth regulators and sugars in promoting plant regeneration via organogenesis from soybean cotyledonary nodal callus. Plant Cell Tiss. Organ Cult. 75:79-85 https://doi.org/10.1023/A:1024649122748
  21. Saka H, Voqui-Dinh TH, Cheng T-Y 1980. Stimulation of multiple shoot formation on soybean stem nodes in culture. Plant Sci. Lett. 19:193-201 https://doi.org/10.1016/0304-4211(80)90072-3
  22. Shan Z, Raemakers K, Tzitzikas EN, Ma Z, Visser RGH 2005. Development of a highly efficient, repetitive system of organogenesis in soybean (Glycine max (L.) Merr). Plant Cell Rep. 24:507-512 https://doi.org/10.1007/s00299-005-0971-7
  23. Sohn SI, Kim YH, Cho JH, Kim JK, Lee JY 2006. An efficient selection scheme for Agrobacterium-mediated cotransformation of rice using two selectable marker genes hpt and bar. Korean J. Breed. 38(3):173-179
  24. Trick HN, JJ Finer. 1997. SAAT:Sonication Assisted Agrobacterium-mediated Transformation. Transgenic Research 6:329-336 https://doi.org/10.1023/A:1018470930944
  25. Wang G, Yinong X. 2008. Hypocotyl-based Agrobacteriummediated transformation of soybean (Glycine max) and application for RNA interference. Plant Cell Rep. 27:1177-1184 https://doi.org/10.1007/s00299-008-0535-8
  26. Wright, MS 1987. Initiation and propagation of Glycine max L. Merr.:Plants from tissue-cultured epicotyls. Plant Cell Tissue and Organ Cult. 8:83-90 https://doi.org/10.1007/BF00040735