Development of Efficient Transformation Protocol for Soybean (Glycine max L.) and Characterization of Transgene Expression after Agrobacterium-mediated Gene Transfer = Development of Efficient Transformation Protocol for Soybean (Glycine max L.) and Characterization of Transgene Expression after Agrobacterium-mediated Gene Transfer

다국어 초록 (Multilingual Abstract)

Increasing vitamin E activity in economically important oil crops such as soybean will enhance the nutritional value of these crops. An improved soybean transformation system involving pre-culture of soybean seed on medium supplemented with 1 mg/L 6-benzylaminopurine under dark conditions was established. To improve the nutritional value of soybean by increasing the α-tocopherol content, soybeans were transformed with γ-tocopherol methyltransferase (γ-TMT) gene by Agrobacterium-mediated transformation. Frequency of soybean transformation was significantly increased from 0.5 to 4.3% by this modified system, and 13 lines of transgenic soybean plants containing γ-TMT were obtained. The molecular characterization using polymerase chain reaction (PCR), reverse transcriptase PCR, and Southern blot analysis confirmed insertion and inheritance of the transgene in the transgenic plants and their progeny. Morphologically normal and fertile transgenic plants were analyzed, and the majority of transgenic soybean transmitted herbicide resistance at 3:1 or 15:1 ratios to their progeny. Alpha-tocopherol content in transgenic soybean seeds was determined by high-performance liquid chromatography; over-expression of γ-TMT resulted in a 41-fold increase in α-tocopherol over wild-type soybean seeds.

참고문헌 (Reference)

1 김율호, "발아종자의 분열조직을 이용한 효율적인 콩 형질전환 방법" 한국육종학회 40 (40): 278-285, 2008

2 김경환, "들깨 γ-tocopherol methyltransferase cDNA 유전자의 분리 및 특성" 한국응용생명화학회 45 (45): 203-206, 2002

3 전은희, "국내콩 형질전환 기술개발" 한국식물생명공학회 36 (36): 344-351, 2009

4 Jiang Q, "γ-Tocopherol,but not α-tocopherol,decreases proinflammatory eicosanoids and inflammation damage in rats" 17 : 816-822, 2003

5 Traber MG, "Vitamin E in humans:Demand and delivery" 16 : 321-47, 1996

6 Bramley PM, "Vitamin E" 80 : 913-938, 2000

7 Finer JJ, "Transformation of soybean via particle bombardment of embryogenic suspension culture tissue" 27 : 175-182, 1991

8 Zhang Z, "The use of glufosinate as a selective agent in Agrobacteriummediated transformation of soybean" 56 : 37-46, 1999

9 Higgins TJV, "The sequence of a pea vicillin gene and its expression in transgenic tobacco" 11 : 683-695, 1988

10 Kamal-Eldin A, "The chemistry and antioxidant properties of tocopherols and tocotrienols" 31 : 671-701, 1996

11 McCabe, DE, "Stable transformation of soybean(Glycine max)by particle acceleration" 6 : 923-926, 1988

12 Saini R, "Stable genetic transformation of Vigna mungo L.Hepper via Agrobacterium tumefaciens" 21 : 851-859, 2003

13 Santarém ER, "Sonication-assisted Agrobacterium-mediated transformation of soybean immature cotyledons:optimization of transient expression" 17 : 752-759, 1998

14 Lee BK, "Seed-specific expression of perilla γ-tocopherol methyltransferase gene increases a-tocopherol content in transgenic perilla(Perilla frutescens)" 92 : 47-54, 2008

15 Malik KA, "Regeneration in Phaseolus vulgaris L.:High-frequency induction of direct shoot formation in intact seedlings by N6-benzylaminopurine and thidiazuron" 186 : 384-389, 1992

16 Zeng P, "Refined glufosinate selection in Agrobacterium-mediated transformation of soybean(Glycine max(L.)Merrill)" 22 : 478-482, 2003

17 Hinchee MAW, "Production of transgenic soybean plants using Agrobacterium-mediated DNA transfer" 6 : 915-922, 1988

18 Barwale UB, "Plant regeneration from callus cultures of several soybean genotypes via embryogenesis and organogenesis" 167 : 473-481, 1986

19 Birch RG, "Plant Transformation:Problems and strategies for practical application" 48 : 297-326, 1997

20 Koeun Lee, "Overexpression of Arabidopsis Homogentisate Phytyltransferase or Tocopherol Cyclase Elevates Vitamin E Content by Increasing γ-tocopherol Level in Lettuce (Lactuca sativa L.)" 한국분자세포생물학회 24 (24): 301-306, 2007

21 Gamborg OL, "Nutrient requirements of suspension cultures of soybean root cells" 50 : 151-158, 1968

22 Barker RF, "Nucleotide sequence of the T-DNA region from the Agrobacterium tumefaciens octopine Ti plasmid pTi15955" 2 : 335-350, 1983

23 Kamal-Eldin A, "Normal-phase high-performance liquid chromatography of tocopherols and tocotrienols" 881 : 217-227, 2000

24 Hood EE, "New Agrobacterium helper plasmids for gene transfer to plants" 2 : 208-218, 1993

25 Lippmann B, "Induction of somatic embryos in cotyledonary tissue of soybean,Glycine max L" 3 : 215-218, 1984

26 Tavva VS, "Increased α-tocopherol content in soybean seed overexpressing the Perilla frutescens γ-tocopherol methyl transferase gene" 26 : 61-70, 2007

27 Verma DPS, "In Soybean: Genetics, Molecular Biology and Biotechnology" CAB International 1996

28 Grusak M, "Improving the nutrient composition of plants to enhance human nutrition and health" 50 : 133-161, 1999

29 Paz MM, "Improved cotyledonary node method using an alternative explant derived from mature seed for efficient Agrobacterium-mediated soybean transformation" 25 : 206-213, 2005

30 Wang G, "Hypocotyl-based Agrobacteriummediated transformation of soybean(Glycine max)and application for RNA interference" 27 : 1177-1184, 2008

31 James C, "Global status of commercialized biotech/GM crops: 2009" ISAAA 2009

32 Jefferson RA, "GUS fusions:β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants" 6 : 3901-3907, 1987

33 Franklin G, "Factors influencing regeneration of soybean from mature and immature cotyledons" 43 : 73-79, 2004

34 Rogers SO, "Extraction of DNA from plant tissues" A6 : 1-10, 1988

35 Van Eenennaam AL, "Engineering vitamin E content:From Arabidopsis mutant to soy oil" 15 : 3007-3019, 2003

36 Shintani DK, "Elevating the vitamin E content of plants through metabolic engineering" 282 : 2098-2100, 1998

37 Olhoft PM, "Efficient soybean transformation using Hygromycin B selection in the cotyledonary-node method" 5 : 723-735, 2003

38 Liu HK, "Efficient Agrobacterium tumefaciens-mediated transformation of soybeans using an embryonic tip regeneration system" 219 : 1042-1049, 2004

39 Padgette, SR, "Development,Identification,and Characterization of a Glyphosate-Tolerant Soybean Line" 35 : 1451-1461, 1995

40 Shan Z, "Development of a highly efficient,repetitive system of organogenesis in soybean(Glycine max(L.)Merr)" 24 : 507-512, 2005

41 Sairam RV, "A study on the effect of genotypes,plant growth regulators and sugars in promoting plant regeneration via organogenesis from soybean cotyledonary nodal callus" 75 : 79-85, 2003

42 Murashige T, "A revised medium for rapid growth and bioassays with tobacco tissue cultures" 15 : 473-497, 1962

43 Jiang Q, "A combination of aspirin and γ-tocopherol is superior to that of aspirin and α-tocopherol in anti-inflammatory action and attenuation of aspirin-induced adverse effects" 20 : 894-900, 2009