Abstract
Objectives
In the plant transformation process, marker genes play a vital role in identifying transformed cells from non-transformed cells. However, once transgenic plants have been obtained, the presence of marker genes may provoke public concern about environmental or biosafety issues. In our previous study, a double T-DNA vector system has been developed to obtain marker-free transgenic plants, but the T-DNA left border (LB) and right border (RB) of the vector showed an RB–LB–RB–LB pattern and led to high linkage integration between the selectable marker gene (SMG) and the gene of interest (GOI). To improve this double T-DNA vector system, we inverted the first T-DNA direction such that a LB–RB–RB–LB pattern resulted to avoid transcriptional read-through at the LB and the subsequent linkage transfer of the SMG and GOI.
Results
We separately inserted the green fluorescent protein (GFP) gene as the GOI and the neomycin phosphotransferase II (NPTII) gene as the SMG in both optimized and original vectors and carried out Agrobacterium-mediated tobacco transformation. Statistical analysis revealed that the linkage frequency was 25.6% in T0 plants transformed with the optimized vector, which is a 42.1% decrease compared with that of the original vector (44.2%). The frequency of obtaining marker-free transgenic plants was 66.7% in T1 plants transformed with the optimized vector, showing a 33.4% increase compared with that of the original vector (50.0%).
Conclusion
Our results demonstrate that the optimized double T-DNA binary vector system is a more effective, economical and time-saving approach for obtaining marker-free transgenic plants.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Ahmad N, Mukhtar Z (2017) Genetic manipulations in crops: challenges and opportunities. Genomics 109:494–505. https://doi.org/10.1016/j.ygeno.2017.07.007
Cao X et al (2019) Development and characterization of marker-free and transgene insertion site-defined transgenic wheat with improved grain storability and fatty acid content. Plant Biotechnol J. https://doi.org/10.1111/pbi.13178
Chen S, Li X, Liu X, Xu H, Meng K, Xiao G, Wei X, Wang F, Zhu Z (2005) Green fluorescent protein as a vital elimination marker to easily screen marker-free transgenic progeny derived from plants co-transformed with a double T-DNA binary vector system. Plant Cell Rep 23:625–631. https://doi.org/10.1007/s00299-004-0853-4
Dale PJ, Clarke B, Fontes EM (2002) Potential for the environmental impact of transgenic crops. Nat Biotechnol 20:567–574. https://doi.org/10.1038/nbt0602-567
Darbani B, Eimanifar A, Stewart CN Jr, Camargo WN (2007) Methods to produce marker-free transgenic plants. Biotechnol J 2:83–90. https://doi.org/10.1002/biot.200600182
De Buck S, De Wilde C, Van Montagu M, Depicker A (2000) T-DNA vector backbone sequences are frequently integrated into the genome of transgenic plants obtained by Agrobacterium-mediated transformation. Mol Breed 6:459–468. https://doi.org/10.1023/A:1026575524345
De Neve M, De Buck S, Jacobs A, Van Montagu M, Depicker A (1997) T-DNA integration patterns in co-transformed plant cells suggest that T-DNA repeats originate from co-integration of separate T-DNAs. Plant J 11(1):15–29. https://doi.org/10.1046/j.1365-313X.1997.11010015.x
De BS, Jacobs A, Van MM, Depicker A (1999) The DNA sequences of T-DNA junctions suggest that complex T-DNA loci are formed by a recombination process resembling T-DNA integration. Plant J 20:295–304. https://doi.org/10.1046/j.1365-313X.1999.00602.x
Du D, Jin R, Guo J, Zhang F (2019) Construction of marker-free genetically modified maize using a heat-inducible auto-excision vector. Genes 10(5):374. https://doi.org/10.3390/genes10050374
Endo S, Sugita K, Sakai M, Tanaka H, Ebinuma H (2002) Single-step transformation for generating marker-free transgenic rice using the ipt-type MAT vector system. Plant J 30(1):115–122. https://doi.org/10.1046/j.1365-313X.2002.01272.x
Gilbert N (2013) Case studies: a hard look at GM crops. Nature 497:24–26. https://doi.org/10.1038/497024a
Hegemann JH, Heick SB (2011) Delete and repeat: a comprehensive toolkit for sequential gene knockout in the budding yeast Saccharomyces cerevisiae. Methods Mol Biol 765:189–206. https://doi.org/10.1007/978-1-61779-197-0_12
Hoa TT, Bong BB, Fau-Huq E, Huq E, Fau-Hodges TK, Hodges TK (2002) Cre/lox site-specific recombination controls the excision of a transgene from the rice genome. Theoretical and Applied Genetics 104:518–525. https://doi.org/10.1007/s001220100748
Horsch R, Fry J, Hoffmann N, Eichholtz D, Rogers SG, Fraley R (1985) A simple and general method for transferring genes into plants. Science 227:1229–1231. https://doi.org/10.1126/science.227.4691.1229
Jacob SS, Veluthambi K (2002) Generation of selection marker-free transgenic plants by cotransformation of a cointegrate vector T-DNA and a binary vector T-DNA in one Agrobacterium tumefaciens strain. Plant Sci 163:801–806. https://doi.org/10.1016/S0168-9452(02)00215-7
Kim SR, Lee J, Jun SH, Park S, Kang HG, Kwon S, An G (2003) Transgene structures in T-DNA-inserted rice plants. Plant Mol Biol 52(4):761–773. https://doi.org/10.1023/A:1025093101021
König A (2003) framework for designing transgenic crops–science, safety and citizen’s concerns. Nat Biotechnol 21(11):1274–1279
Koprek T, Rangel S, McElroy D, Louwerse JD, Williams-Carrier RE, Lemaux PG (2001) Transposon-mediated single-copy gene delivery leads to increased transgene expression stability in barley. Plant Physiol 125:1354–1362. https://doi.org/10.1104/pp.125.3.1354
Kuraya Y et al (2004) Suppression of transfer of non-T-DNA ‘vector backbone’ sequences by multiple left border repeats in vectors for transformation of higher plants mediated by Agrobacterium tumefaciens. Mol Breed 14:309–320. https://doi.org/10.1023/B:MOLB.0000047792.77219.bb
Lemaux PG (2008) Genetically engineered plants and foods: a scientist’s analysis of the issues (Part I). Annu Rev Plant Biol 59:771–812. https://doi.org/10.1146/annurev.arplant.58.032806.103840
Li X, Zhu Z, Xu J, Wu Q, Xu H (2001) Isolation of pea matrix attachment region and study on its function in transgenic tobaccos. Sci China, Ser C Life Sci 44:400–408. https://doi.org/10.1007/BF02879607
Li R et al (2011) Multiple inserts of gene of interest and selectable marker gene are co-integrated and stably transmitted as a single genetic locus in transgenic soybean plants. In Vitro Cell Dev Biol Plant 47:274–281. https://doi.org/10.1007/s11627-011-9359-2
Lu HJ et al (2001) Generation of selectable marker-free transgenic rice using double right-border (DRB) binary vectors. Aust J Plant Physiol 28:241–248. https://doi.org/10.1071/PP00129
Lyznik LA, Mitchell JC, Hirayama L, Hodges TK (1993) Activity of yeast FLP recombinase in maize and rice protoplasts. Nucleic Acids Res 4:969–975. https://doi.org/10.1093/nar/21.4.969
Maeser S, Kahmann R (1991) The Gin recombinase of phage Mu can catalyse site-specific recombination in plant protoplasts. Mol Gen Genet MGG 230:170–176. https://doi.org/10.1007/BF00290665
Manimaran P, Ramkumar G, Sakthivel K, Sundaram RM, Madhav MS, Balachandran SM (2011) Suitability of non-lethal marker and marker-free systems for development of transgenic crop plants: present status and future prospects. Biotechnol Adv 29:703–714. https://doi.org/10.1016/j.biotechadv.2011.05.019
Matthews PR, Wang MB, Waterhouse PM, Thornton S, Fieg SJ, Gubler F, Jacobsen JV (2001) Marker gene elimination from transgenic barley, using co-transformation with adjacent ‘twin T-DNAs’ on a standard Agrobacterium transformation vector. Mol Breeding 7:195–202. https://doi.org/10.1023/A:1011333321893
Miki B, McHugh S (2004) Selectable marker genes in transgenic plants: applications, alternatives and biosafety. J Biotechnol 107:193–232. https://doi.org/10.1016/j.jbiotec.2003.10.011
Miller M, Tagliani L, Wang N, Berka B, Bidney D, Zhao ZY (2002) High efficiency transgene segregation in co-transformed maize plants using an Agrobacterium tumefaciens 2 T-DNA binary system. Transgenic Res 11:381–396. https://doi.org/10.1023/A:1016390621482
Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x
Ooms G, Hooykaas PJ, Van Veen RJ, Van Beelen P, Regensburg-Tuink TJ, Schilperoort RA (1982) Octopine Ti-plasmid deletion mutants of Agrobacterium tumefaciens with emphasis on the right side of the T-region. Plasmid 7:15–29. https://doi.org/10.1016/0147-619X(82)90023-3
Ow DW (2002) Recombinase-directed plant transformation for the post-genomic era. Plant Mol Biol 48:183–200. https://doi.org/10.1023/A:1013718106742
Parisi C, Tillie P, Rodriguez-Cerezo E (2016) The global pipeline of GM crops out to 2020. Nat Biotechnol 34:31–36. https://doi.org/10.1038/nbt.3449
Passricha N, Saifi S, Khatodia S, Tuteja N (2016) Assessing zygosity in progeny of transgenic plants: current methods and perspectives. J Biol Methods 3(3):46. https://doi.org/10.14440/jbm.2016.114
Podevin N, De Buck S, De Wilde C, Depicker A (2006) Insights into recognition of the T-DNA border repeats as termination sites for T-strand synthesis by Agrobacterium tumefaciens. Transgenic Res 15:557–571. https://doi.org/10.1007/s11248-006-9003-9
Ramana Rao MV, Parameswari C, Sripriya R, Veluthambi K (2011) Transgene stacking and marker elimination in transgenic rice by sequential Agrobacterium-mediated co-transformation with the same selectable marker gene. Plant Cell Rep 30:1241–1252. https://doi.org/10.1007/s00299-011-1033-y
Ramanathan V, Veluthambi K (1995) Transfer of non-T-DNA portions of the Agrobacterium tumefaciens Ti plasmid pTiA6 from the left terminus of TL-DNA. Plant Mol Biol 28:1149–1154. https://doi.org/10.1007/BF00032676
Rosellini D (2012) Selectable markers and reporter genes: a well furnished toolbox for plant science and genetic engineering. Crit Rev Plant Sci 31:401–453. https://doi.org/10.1080/07352689.2012.683373
Sallaud C et al (2003) Highly efficient production and characterization of T-DNA plants for rice (Oryza sativa L.) functional genomics. Theor Appl Genet 106:1396–1408. https://doi.org/10.1007/s00122-002-1184-x
Stewart CN Jr, Via LE (1993) A rapid CTAB DNA isolation technique useful for RAPD fingerprinting and other PCR applications. Biotechniques 14:748–750
Sun L, Zhou L, Lu M, Cai M, Jiang XW, Zhang QX (2009) Marker-free transgenic chrysanthemum obtained by Agrobacterium-mediated transformation with twin T-DNA binary vectors. Plant Mol Biol Rep 27:102–108. https://doi.org/10.1007/s11105-008-0062-3
Tuteja N, Verma S, Sahoo RK, Raveendar S, Reddy IN (2012) Recent advances in development of marker-free transgenic plants: regulation and biosafety concern. J Biosci 37:167–197. https://doi.org/10.1007/s12038-012-9187-5
Van DGE, Den DA, Hooykaas PJ (1996) Deviating T-DNA transfer from Agrobacterium tumefaciens to plants. Plant Mol Biol 31:677–681. https://doi.org/10.1007/BF00042239
Wang GP, Yu XD, Sun YW, Jones HD, Xia LQ (2016) Generation of marker- and/or backbone-free transgenic wheat plants via Agrobacterium-Mediated transformation. Front Plant Sci 7:1324. https://doi.org/10.3389/fpls.2016.01324
Woo HJ, Suh SC, Cho YG (2011) Strategies for developing marker-free transgenic plants. Biotechnol Bioprocess Eng 16:1053–1064. https://doi.org/10.1007/s12257-011-0519-3
Wu L, Nandi S, Chen L, Rodriguez RL, Huang N (2002) Expression and inheritance of nine transgenes in rice. Transgenic Res 11:533–541. https://doi.org/10.1023/A:1020331608590
Xing A, Zhang Z, Sato S, Staswick P, Clemente T (2000) The use of the two T-DNA binary system to derive marker-free transgenic soybeans. In Vitro Cell Dev Biol-Plant 36:456–463. https://doi.org/10.1007/s11627-000-0082-7
Zambryski P (1988) Basic processes underlying Agrobacterium-mediated DNA transfer to plant cells. Annu Rev Genet 22:1–30. https://doi.org/10.1146/annurev.ge.22.120188.000245
Acknowledgements
This work was supported by grants from National Science and Technology Major Project (2016ZX08010001, 2016ZX08001001 and 2016ZX08010002) and the State Key Laboratory of Plant Genomics of China (SKLPG2016B-21).
Supporting information
Supplementary Fig. 1—The full-length sequence of the vector pDTGFP-NPT. The shadowed parts represent different gene and element sequences of this vector, and the arrows indicate the direction of gene transcription. All primer sequences used for PCR amplification are underlined.
Supplementary Fig. 2—The full-length sequence of the vector pCDGFP-NPT. The shadowed parts represent different gene and element sequences of this vector, and the arrows indicate the direction of gene transcription. All primer sequences used for PCR amplification are underlined.
Author information
Authors and Affiliations
Corresponding authors
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Leng, C., Sun, B., Liu, Z. et al. An optimized double T-DNA binary vector system for improved production of marker-free transgenic tobacco plants. Biotechnol Lett 42, 641–655 (2020). https://doi.org/10.1007/s10529-020-02797-1
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10529-020-02797-1