유전자총을 이용한 형질전환 심비디움 식물체 생산체계 최적화
Optimization of particle gun-mediated transformation system in Cymbidium
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본 실험은 심비디움 원괴체 (PLB: protocorm-like bodies)를재료로 유전자총을 이용한 효율적인 형질전환 조건을 확립하고자 수행되었다. 이 PLB 조직에 제초제저항성 유전자인 bar 유전자와 reporter 유전자인 gus를 포함하고 있는pCAMBIA3301 벡터를 이용하여 유전자총으로 형질전환하였다. 형질전환 벡터에 포함되어 있는 제초제저항성유전자 (bar)를 이용하여 선발하게 되므로 선발배지에 첨가될 제초제로서 PPT (Phosphinotricin)의 적정 농도를 찾고자 실험한 결과, 5 mg/l에서 최적의 대부분의 PLB의 생육이 억제되고 신초형성이 이루어지지 않았다. 이를 기반으로 유전자총 실험에 맞는 최적 조건을 찾는 실험을수행하여 1.0 μm gold입자크기, 헬륨가스 압력은 1,100과1,350 psi사이에서는 차이가 없다는 전제 하에 물리적 피해가 덜 가는 1,100 psi를 조건으로 선택하였고, 유전자총과 목표물과의 거리는 6 cm 그리고 DNA 농도는 1회 유전자총 발사횟수당 1.0 μg 조건을 최적조건으로 하였다. 이 조건을 기반으로 100개의 PLB를 형질전환 하면 평균적으로 6 ~ 8개의 PLB가 제초제 저항성을 나타내는 개체로 성장하고 최종적으로 2개체 정도가 온실에서 순화과정을 거쳐 완전한 형질전환 식물체로 생산된다. 이외에도 유전자총 실험 전에 0.2 M sorbitol과 0.2 M mannitol을혼합처리하여 4시간 동안 배양시키면 2배 이상 효율을높일 수 있게 되어, 결론적으로 100개의 PLB를 형질전환수행하면 최종적으로 3.2 ~ 4.0개 정도의 형질전환 심비디움 식물체가 나오는 효율이라고 할 수 있다. 본 실험을 통해 생산된 형질전환 심비디움 개체들은PCR 분석을 통해 유전자 도입을 확인하였고, 형질전환개체 중 임의로 선발된 5계통들의 잎을 Basta 0.5% 용액에 침지한 결과, 3 계통은 제초제에 저항성을 가지는 것으로 확인되었고, 그중 1계통은 아주 강한 저항성을 보여주었다. 본 실험 결과들을 바탕으로 환경저항성 등의 유용유전자가 도입된 형질전환 심비디움 식물체 개발에 기여하리라 사료된다.
Abstract
This study is conducted to develop an efficient transformation system via particle bombardment with PLBs (Protocorm-like bodies) in Cymbidium. For this, pCAMBIA3301 vector which carries a herbicide-resistant bar gene and gus gene as a reporter gene was used for transformation with Cymbidium cultivars ‘Youngflower × masako’ line. To select transformants, proper concentration of herbicide, PPT (phosphinotricin), should be determined. As a result, 5 mg/l of PPT was selected as a proper concentration. Further,proper conditions for particle bombardment were determined to obtain a high frequency of transformation. Results showed that 1.0 μg of DNA concentration, 1,100 and 1,350 psi for helium gas pressure, 1.0 μm of gold particle and 6 cm of target distance showed the best result for the particle bombardment experiment. Also, pre-treatment with combination 0.2 M sorbitol and 0.2 M mannitol for 4 hrs prior to genetic transformation increased the transformation efficiency up to 2.5 times. Using transformation system developed in this study, 3.2 ~ 4.0 transgenic cymbidium plants can be produced from 100 bombarded PLBs on average. Putative transgenic plants produced in this system confirmed the presence of the bar gene by PCR analysis. Also, leaves from randomely selected five transgenic lines were applied for Basta solution (0.5% v/v) to check the resistance to the PPT herbicide. As a result, three of them showed resistance and one of them showed the strongest resistance with the maintenance of green color as non-transformed plants showed. Using this established transformation system, more genes of interests can be introduced into Cymbidium plants by genetic transformation in the future.
참고문헌 (28)
- 허연재, "Protocorm-like body를 이용한 호접란 형질전환 연구", 생명과학회지, 19(3), pp.378~383 (2009), 한국생명과학회
- Anzai H, "Transformation of Phaelaenopsis by particle bombardment", Plant Tiss Cult Lett, 13, pp.265~271 (1996)
- Azadi P, "Macro elements in inoculation and co-cultivation medium strongly affect the efficiency of Agrobacterium-mediated transformation in Liilum", Plant Cell Tiss Organ Cult, 101, pp.201~209 (2010)
- Chai ML, "Stable transformation of protocorm-like bodies in Phalaenopsis orchid mediated by Agrobacterium tumefaciens", Scientia hortic, 96, pp.213~224 (2002)
- Chin DP, "Agrobacterium-mediated transformation of protocorm-like bodies in Cymbidium", Plant Cell Rep, 26(6), pp.735~743 (2007)
- Coker J, "The genus Cymbidium, In Aistrailian orchid growing" (1993), Australian Orchid Council Inc, Australia
- Denchev PD, "Transgenic orchardgrass (Dactylis glomerata) plants by direct embryogenesis from microprojectile bombarded leaf cells", Plant Cell Rep, 16, pp.813~819 (1997)
- Deng XY, "Transgenic peanut plants obtained by particle bombardment via somatic embryogenesis regeneration system", Cell Res, 11, pp.156~160 (2001)
- Du Puy D, "The classification of Cymbidium", pp.50~194 (1988), Timber Press, Oregon, USA
- Gao C, "Comparative analysis of transgenic tall fescue (Festuca arundinacea Schreb.) plants obtained by Agrobacterium-mediated transformation and particle bombardment", Plant Cell Rep, 27, pp.1601~1609 (2008)
- Hong KA, "Optimization of Cymbdium transformation system by the particle gun techniques", Agri Chem Biotech, 39(4), pp.260~264 (1996)
- Jefferson RA, "GUS fusion: β -glucuronidase as a sensitive and versatile gene fusion marker in high plants", EMBO J, 6, pp.391~397 (1987)
- Kamo K, "Transforming embryogenic cell lines of Gladiolus with either a bar-uidA fusion gene or cobombardment", In Vitro Cell Dev Biol Plant, 36, pp.182~187 (2000)
- Kamo K, "Biolistic-mediated transformation of Lilium longifolium cv. Nellie white", Hortscience, 43(6), pp.1864~1869 (2008)
- Kim JB, "Efficient production of transgenic Alstroemeria plants by using Agrobacterium tumefaciens", ANNALS OF APPLIED BIOLOGY, 151, pp.401~412 (2007), BLACKWELL PUBLISHING
- 김미선, "생육이 강한 대형 백색계 심비디움 ‘White Princess’ 육성", 화훼연구, 16(4), pp.295~298 (2008), 한국화훼학회
- 김미선, "중형 백색계 다화성 심비디움 ‘Honey Girl’ 육성", 화훼연구, 16(4), pp.291~294 (2008), 한국화훼학회
- 김미선, "진한 자주색계 중형 심비디움 ‘Purple Princess’", 원예과학기술지, 28(4), pp.715~718 (2010), 한국원예학회
- Kuehnle AR, "Transformation of Dendrobium orchid using particle bomabrdment of protocorms", Plant Cell Rep, 11(9), pp.484~488 (1992)
- 이기종, "아그로박테리움을 이용한 제초제 저항성 콩 개발", Journal of Plant Biotechnology, 35(1), pp.69~74 (2008), 한국식물생명공학회
- 이유미, "심비디움 육종, 조직배양 및 형질전환 연구동향에 관한 고찰", Journal of Plant Biotechnology, 37(4), pp.357~369 (2010), 한국식물생명공학회
- Li SH, "Osmotic sucrose enhancement of single-cell embryogenesis and transformation efficiency in Oncidium", Plant Cell Tiss Organ Cult, 81, pp.183~192 (2005)
- Lin HS, "Genetic transformation of Alstroemeria using particle bombardment", Mol Breed, 6, pp.369~377 (2000)
- Men S, "Genetic transformation of two species of orchid by biolistic bombardment", Plant Cell Rep, 21, pp.592~598 (2003)
- Mishiba KI, "Agrobacterium-mediated transformation of Phalaenopsis by targeting protocorms at an early stage after germination", Plant Cell Rep, 24, pp.297~303 (2005)
- Popowich EA, "Agrobacterium-mediated transformation of Hyacinthus orientalis with thaumatin II gene to control fungal diseases", Plant Cell Tiss Organ Cult, 90, pp.237~244 (2007)
- Vain P, "Osmotic treatment enhances particle bombardment-mediated transient and stable transformation of maize", Plant Cell Rep, 12, pp.84~88 (1993)
- Yang J, "Genetic transformation of Cymbidium orchid by particle bombardment", Plant Cell Reports, 18(12), pp.978~984 (1999), Springer Verlag