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Journal of Integrative Agriculture  2020, Vol. 19 Issue (1): 23-32    DOI: 10.1016/S2095-3119(19)62601-8
Special Issue: 小麦遗传育种Wheat Genetics · Breeding · Germplasm Resources
Crop Science Advanced Online Publication | Current Issue | Archive | Adv Search |
Genetic and agronomic traits stability of marker-free transgenic wheat plants generated from Agrobacterium-mediated co-transformation in T2 and T3 generations
LIU Hui-yun1, 2, WANG Ke2, WANG Jing2, DU Li-pu2, PEI Xin-wu1, YE Xing-guo2  
1 Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R.China
2 Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R.China
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Abstract  
Genetically modified wheat has not been commercially utilized in agriculture largely due to regulatory hurdles associated with traditional transformation methods.  Development of marker-free transgenic wheat plants will help to facilitate biosafety evaluation and the eventual environmental release of transgenic wheat varieties.  In this study, the marker-free transgenic wheat plants previously obtained by Agrobacterium-mediated co-transformation of double T-DNAs vector were identified by fluorescence in situ hybridization (FISH) in the T1 generation, and their genetic stability and agronomic traits were analyzed in T2 and T3 generations.  FISH analysis indicated that the transgene often integrated into a position at the distal region of wheat chromosomes.  Furthermore, we show that the GUS transgene was stably inherited in the marker-free transgenic plants in T1 to T3 generations.  No significant differences in agronomic traits or grain characteristics were observed in T3 generation, with the exception of a small variation in spike length and grains per spike in a few lines.  The selection marker of bar gene was not found in the transgenic plants through T1 to T3 generations.  The results from this investigation lay a solid foundation for the potential application of the marker-free transgenic wheat plants achieved through the co-transformation of double T-DNAs vector by Agrobacterium in agriculture after biosafty evaluation.
Keywords:  wheat       marker-free transgenic plants       fluorescence in situ hybridization       genetic stability  
Received: 05 September 2018   Accepted:
Fund: The authors acknowledge the Ministry of Agriculture of China for the National Transgenic Research Program (2016ZX08010004) and the Chinese Academy of Agricultural Sciences for the Agricultural Science and Technology Innovation Program (ASTIP-2060302-2-19).
Corresponding Authors:  Correspondence PEI Xin-wu, E-mail: peixinwu@caas.cn; YE Xing-guo, E-mail: yexingguo@caas.cn   
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LIU Hui-yun, WANG Ke, WANG Jing, DU Li-pu, PEI Xin-wu, YE Xing-guo . 2020. Genetic and agronomic traits stability of marker-free transgenic wheat plants generated from Agrobacterium-mediated co-transformation in T2 and T3 generations. Journal of Integrative Agriculture, 19(1): 23-32.

Anand A, Trick H N, Gill B S, Muthukrishnan S. 2003. Stable transgene expression and random gene silencing in wheat. Plant Biotechnology Journal, 1, 241–251.
Bhalla P L. 2006. Genetic engineering of wheat-current challenges and opportunities. Trends in Biotechnology, 24, 305–311.
Cai Y P, Chen L, Liu X J, Guo C, Sun S, Wu C X, Jiang B J, Han T F, Hou W S. 2018. CRISPR/Cas9-mediated targeted mutagenesis of GmFT2a delays flowering time in soya bean. Plant Biotechnology Journal, 16, 176–185.
Chen J, Carlson A R, Wan J. 2003. Chromosomal location and expression of Green Fluorescent Protein (GFP) gene in microspore derived transgenic barely (Hordeum vulgare L.). Acta Genetica Sinica, 30, 697–705.
Cheng M, Fry J E, Pang S, Zhou H, Hironaka C M, Duncan D R, Conner T W, Wan Y. 1997. Genetic transformation of wheat mediated by Agrobacterium tumefaciens. Plant Physiology, 115, 971–980.
Cheng M, Hu T C, Layton J, Liu C N, Fry J E. 2003. Desiccation of plant tissues post-Agrobacterium infection enhances T-DNA delivery and increases stable transformation efficiency in wheat. In Vitro Cellular & Developmental Biology-Plant, 39, 595–604.
Cong L, Ran F A, Cox D, Lin S L, Barretto R, Habib N, Hsu P D, Wu X B, Jiang W Y, Marraffini L A, Zhang F. 2013. Multiplex genome engineering using CRISPR/Cas systems. Science, 339, 819–823.
Ding W J, Wei Y Q, Ye X G, Du L P, Xu H J. 2007. Genetic analysis of alien transferring genes in common wheat and their effects on agronomic characters. Acta Agronomica Sinica, 33, 955–960. (in Chinese)
Feng C, Su H D, Bai H, Wang R, Liu Y L, Guo X R, Liu C, Zhang J, Yuang J, Birchler J A, Han F P. 2018. High-efficiency genome editing using a dmc1 promoter-controlled CRISPR/Cas9 system in maize. Plant Biotechnology Journal, 16, 1848–1857.
Godfray H C, Beddington J R, Crute I R, Haddad L, Lawrence D, Muir J F, Pretty J, Robinson S, Thomas S M, Toulmin C. 2010. Food security: The challenge of feeding 9 billion people. Science, 327, 812–818.
Guo X, Su H, Shi Q, Fu S, Wang J, Zhang X, Hu Z, Han F. 2016. De novo centromere formation and centromeric sequence expansion in wheat and its wide hybrids. PLoS Genetics, 12, e1005997.
He Z H, Xia X C, Chen X M, Zhuang Q S. 2011. Progress of wheat breeding in China and the future perspective. Acta Agronomica Sinica, 37, 202–215. (in Chinese)
Hu T, Metz S, Chay C, Zhou H P, Biest N, Chen G, Cheng M, Feng X, Radionenko M, Lu F, Fry J. 2003. Agrobacterium-mediated large-scale transformation of wheat (Triticum aestivum L.) using glyphosate selection. Plant Cell Reports, 21, 1010–1019.
Ishida Y, Tsunashima M, Hiei Y, Komari Y. 2015. Wheat (Triticum aestivum L.) transformation using immature embryos. Methods in Molecular Biology, 1223, 189–198.
James C. 2018. Global Status of Commercialized Biotech/GM Crops in 2017: Biotech Crop Adoption Surges as Economic Benefits Accumulate in 22 years. International Service for the Acquisition of Agri-biotech Applications, USA. p. 53.
Jefferson R A, Kavanagh T A, Bevan M W. 1987. GUS fusions: Beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO Journal, 6, 3901–3907.
Jiang F G, Doudna J A. 2017. CRISPR-Cas9 structures and mechanisms. Annual Review of Biophysics, 46, 505–529.
Khanna H K, Daggard G E. 2003. Agrobacterium tumefaciens-mediated transformation of wheat using a super binary vector and a polyamine-supplemented regeneration medium. Plant Cell Reports, 21, 429–436.
Kharb P P, Dong J J, Islam-Faridi M N, Stelly D M, Hall T C. 2001. Fluorescence in situ hybridization of single copy transgenes in rice chromosomes. In Vitro Cellular & Developmental Biology-Plant, 37, 1–5.
Liang Z, Chen K L, Li T D, Zhang Y, Wang Y P, Zhao Q, Liu J X, Zhang H W, Liu C M, Ran Y D, Gao C X. 2017. Efficient DNA-free genome editing of bread wheat using CRISPR/Cas9 ribonucleoprotein complexes. Nature Communications, 8, 14261.
Li C X, Liu C L, Qi X T, Wu Y C, Fei X H, Mao L, Cheng B J, Li X H, Xie C X. 2017. RNA-guided Cas9 as an in vivo desired-target mutator in maize. Plant Biotechnology Journal, 15, 1566–1576.
Li J R, Ye X G, An B Y, Du L P, Xu H J. 2012. Genetic transformation of wheat: Current status and future prospects. Plant Biotechnology Reports, 6, 183–193.
Lu Y, Zhu J K. 2017. Precise editing of a target base in the rice genome using a modified CRISPR/Cas9 system. Molecular Plant, 10, 523–525.
Pedersen C, Zimny J, Becker D J, Jahne-Gartner A, Lorz H. 1997. Localization of introduced genes on the chromosomes of transgenic barley, wheat and triticale by fluorescence
in situ hybridization. Theoretical and Applied Genetics, 94, 749–757.
Qi L S, Larson M H, Gilbert L A, Doudna J A, Weissman J S, Arkin A P, Lim W A. 2013. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell, 152, 1173–1183.
Richardson T, Thistleton J, Higgins T J, Howitt C, Ayliffe M. 2014. Efficient Agrobacterium transformation of elite wheat germplasm without selection. Plant Cell Tissue and Organ Culture, 119, 647–659.
Sergei K S, David A S. 2002. Characterization of transgene loci in plants using FISH: A picture is worth a thousand words. Plant Cell Tissue and Organ Culture, 69, 205–214.
Shan Q W, Wang Y P, Chen K L, Liang Z, Li J, Zhang Y, Zhang K, Liu J X, Voytas D F, Zheng X L, Zhang Y, Gao C X. 2013. Rapid and efficient gene modification in rice and Brachypodium using TALENs. Molecular Plant, 6, 1365–1368.
Shewry P R. 2009. Wheat. Journal of Experimental Botany, 60, 1537–1553.
Susana S, Javier G H, Carmen V O, Mar?a J G, Carolina S, Daniel F V, Francisco B. 2018. Low-gluten, nontransgenic wheat engineered with CRISPR/Cas9. Plant Biotechnology Journal, 16, 902–910.
Teng W, He X, Tong Y P. 2017. Transgenic approaches for improving use efficiency of nitrogen, phosphorus and potassium in crops. Journal of Integrative Agriculture, 16, 2657–2673.
Tester M, Langridge P. 2010. Breeding technologies to increase crop production in a changing world. Science, 327, 818–822.
Tuteja N, Verma S, Sahoo R K, Raveendar S, Reddy I N. 2012. Recent advances in development of marker-free transgenic plants: Regulation and biosafety concern. Journal of Bioscience and Bioengineering, 37, 167–197.
Vishnudasan D, Tripathi M N, Rao U, Khurana P. 2005. Assessment of nematode resistance in wheat transgenic plants expressing potato proteinase inhibitor (PIN2) gene. Transgenic Research, 14, 665–675.
Wang K, Liu H Y, Du L P, Ye X G. 2017. Generation of marker-free transgenic hexaploid wheat via an Agrobacterium-mediated co-transformation strategy in commercial Chinese wheat varieties. Plant Biotechnology Journal, 15, 614–623.
Wang K, Riaz B, Ye X G. 2018. Wheat genome editing expedited by efficient transformation techniques: Progress and perspectives. The Crop Journal, 6, 22–31.
Wang Y, Cheng X, Shan Q, Zhang Y, Liu J, Gao C, Qiu J L. 2014. Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew. Nature Biotechnology, 32, 947–951.
Wu H, Sparks C, Amoah B, Jones H D. 2003. Factors influencing successful Agrobacterium-mediated genetic transformation of wheat. Plant Cell Reports, 21, 659–668.
Wu X, Doherty A, Jones H D. 2008. Efficient and rapid Agrobacterium-mediated genetic transformation of durum wheat (Triticum turgidum L. var. durum) using additional virulence genes. Transgenic Research, 17, 425–436.
Ye X G, Cheng H M, Xu H J, Du L P, Lu W Z, Huang Y H. 2005. Development of transgenic wheat plants with chitinase and β-1,3-glucosanase genes and their resistance to fusarium head blight. Acta Agronomica Sinica, 31, 583–586. (in Chinese)
Ye X G, Cheng M, Du L P, Xu H J. 2011. Description and evaluation of transformation approaches used in wheat. Hereditas, 33, 422–430. (in Chinese)
Ye X G, Xu H J, Du L P, He G Y, Wang K, Lin Z S. 2014. Establishment and application of large-scale transformation systems in wheat. Scientia Agricultura Sinica, 47, 4155–4171. (in Chinese)
Yuan J, Guo X, Hu J, Lv Z L, Han F P. 2014. Characterization of two CENH3 genes and their roles in wheat evolution. New Phytologist, 206, 839–851.
Zhang Y, Liang Z, Zong Y, Wang Y P, Liu J X, Chen K L, Qiu J L, Gao C X. 2016. Efficient and transgene-free genome editing in wheat through transient expression of CRISPR/Cas9 DNA or RNA. Nature Communication, 7, 12617.
Zhou H, Berg J D, Blank S E, Chay C A, Chen G, Eskelsen S R, Fry J E, Hoi S, Hu T, Isakson P J, Lawton M B, Metz S G, Rempel C B, Ryerson D K, Sansone A P, Shook A L, Starke R J, Tichota J M, Valenti S A. 2003. Field efficacy assessment of transgenic roundup ready wheat. Crop Science, 43, 1072–1075.
 
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