Please wait a minute...
Frontiers of Agriculture in China

ISSN 1673-7334

ISSN 1673-744X(Online)

CN 11-5729/S

Front. Agric. China    2008, Vol. 2 Issue (2) : 156-161     DOI: 10.1007/s11703-008-0024-4
Obtaining marker-free transgenic soybean plants with optimal frequency by constructing a three T-DNA binary vector
YE Xingguo, QIN Hua
Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, The National Key Facility for Crop Gene Resources and Genetic Improvement;
 Download: PDF(209 KB)   HTML
 Export: BibTeX | EndNote | Reference Manager | ProCite | RefWorks
Abstract Obtaining marker-free plants with high efficiency will benefit the environmental release of transgenic crops. To achieve this point, a binary vector with three T-DNAs was constructed using several mediate plasmids, in which one copy of BAR gene expression cassette and two copies of VIP1 gene expression cassette were included. EHA101 Agrobacterium strain harboring the final construct was applied to transform soybean cotyledon nodes. Through 2–3 months regeneration and selection with 3–5 mg·L-1 glufosinate, transgenic soybean plants were obtained at 0.83%–3.16%, and the co-transformation efficiency of both genes in the same individual reached up to 86.4%, based on the southern blot test. Using PCR analysis, southern blot and northern blot tests, as well as leaf painting of herbicide in T1 progenies, 41 plants were eliminated of BAR gene with the frequency of 7.6%. Among the T1 populations tested, the loss of the alien genes happened in 22.7% lines, the silence of the BAR gene took place in 27.3% lines, and VIP1 gene silence existed in 37.1% marker-free plants. The results also suggested that the plasmid with three T-DNAs might be an ideal vector to generate marker-free genetically modified organisms.
Issue Date: 05 June 2008
 Cite this article:   
YE Xingguo,QIN Hua. Obtaining marker-free transgenic soybean plants with optimal frequency by constructing a three T-DNA binary vector[J]. Front. Agric. China, 2008, 2(2): 156-161.
1 Clemente T La Vallee B Howe A Ward D Rozman R Hunter P Broyles D Kasten D Hinchee M 2000 Progeny analysis of glyphosate selected transgenic soybean derivedfrom Agrobacterium-mediated transformationCrop Sci40797803
2 Conner A J Glare T R Nap T P 2003 The release of genetically modified cropsinto the environmentPlant Journal331946.
3 Crawley M J Brown S L Hails R S Kohn D D Rees M 2001 Biotechnology-transgeniccrops in natural habitatsNature409682683.
4 Dale E Ow D 1991 Gene transferwith subsequent removal of the selection gene from the host genomeProc Natl Acad Sci USA881055810562.
5 Daley M Knauf V Summerfelt K Turner J 1998 Co-transformation with one Agrobacteriumtumefaciens strain containing two binary plasmids as amethod for producing marker-free transgenic plantsPlant Cell Rep17489496.
6 De Block M Debrouwer D 1991 Two T-DNAsco-transformed into Brassica napus by a double Agrobacterium infectionare mainly integrated at the same locusTheor Appl Genet82257263.
7 Dellaporta S Wood J Hicks J 1983 A plant DNA minipreparation: Version IIPlant Mol Biol Reptr11921.
8 Depicker G Herman L Jacobs A Schell J Van Montagu M 1985 Frequenciesof simultaneous transformation with different T-DNAs and their relevanceto Agrobacterium/plant cell interactionMol Gen Genet201477484.
9 Ebinuma H Sugita K Matsunaga E Yamakado M 1997 Selection of marker-free transgenic plants using the isopentenyltransferase gene. Proc Natl. Acad Sci USA9421172121.
10 Gleave A Mitra D Mudge S Morris B 1999 Selectable marker-free transgenic plants without sexual crossing:transient expression of cre recombinase and use of a conditional lethaldominant genePlant Mol Biol40223235.
11 Goldsbrough A Lastrella C Yoder J 1993 Transposition mediated repositioning andsubsequent elimination of marker genes from transgenic tomotoBio/Technology1112861292
12 Hinchee M Ward D Newell C McDonnell R Sato S Gasser C Fischhoff D Re D Fraley R Horsch R 1988 Production of transgenic soybean plants using Agrobacterium-mediated DNA transferBio/Technology6915922.
13 Hood E Helmer G Fraley R Chilton M 1986 The hypervirulence of Agrobacterium tumefaciens A281 is encoded in a region of pTiBo542 outside of T-DNAJ Bacteriol16812911301
14 James C 2002 Preview: Global status of commercialized transgenic cropsISAAA Briefs No. 27NYISAAA
15 John I Andrew Y Goldsbrough P 1994 Transformation system for generating marker-freetransgenic plantsBio/Technology12263267.
16 Keller B Carabias E H 2001 Transgenicplants for sustainable crop productionNösberger JGeiger H HStruik P CCrop Sciences:Progress and ProspectsNYCABI351367
17 Komari T Hiei Y Saito Y Murai N Kumashiro T 1996 Vectors carrying two separateT-DNAs for co-transformation of higher plants mediated by Agrobacterium tumefaciens and segregationof transformants free from selection markersPlant J10165174.
18 McKnight T Lillis M Simpson R 1987 Segregation of genes transferred to oneplant cell from two separate AgrobacteriumstrainsPlant Mol Biol8439445.
19 Sambrook J Fritch E F Maniatis T 1989 Molecular Cloning: Alaboratory Mannual.2nd ed.New YorkCold Spring Harbor Laboratory Press
20 Shirley S Xing A Ye X Schweiger B Kinney A Graef G Clemente T 2004 Production of γ-linolenic acid and stearidonic acid in seedsof marker-free transgenic soybeanCrop Sci44646652
21 Tzfira T Vaidya M Citovsky V 2001 VIP1, an Arabidopsis protein that interacts with Agrobacterium VirE2, is involved in VirE2 nuclear import and Agrobacterium infectivityEMBOJ2035963607.
22 Tzfira T Vaidya M Citovsky V 2002 Increasing plant susceptibility to Agrobacterium infection by overexpressionof the Arabidopsis nuclear proteinVIP1Proc Natl Acad Sci USA991043510440.
23 Xing A Zhang Z Shirley S Paul S Tom C 2000 The use of the two T-DNA binary systemto drive marker-free transgenic soybeansIn Vitro Cell Dev Biol Plant36456463.
24 Yohichi W Motoyasu O Koh I Takiko S 1998 Co-integration, co-expression and co-segregation of an unlinked selectablemarker gene and NtFAD3 gene intransgenic rice plants produced by particle bombardmentGenes Genet Syst73219226.
Full text