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A quick, easy and cost-effective in planta method to develop direct transformants in wheat

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Abstract

Agrobacterium mediated in planta method was used to transform Indian elite wheat genotype HD2894 with herbicide-tolerant CP4-EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) gene. The apical meristems of germinated seeds were targeted for introgression of transgene. The obtained T1 plants were screened by spraying 1% glyphosate and only positive transformants survived. The presence of transgene was also confirmed by PCR and Southern hybridization. Using this method, 3.07% transformation rate was observed. To identify transgenic lines carrying stably integrated CP4-EPSPS gene, the transgenic populations were screened in T3 generation using 1% glyphosate and lines with 100% survival were considered as homozygous. No significant morpho-physiological variations were observed within the transgenic lines as compared to non-transgenic plants. The present study resulted in herbicide-tolerant transgenic wheat and provides a valuable tool for development of wheat genetic transformation.

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References

  • Anand A, Zhou T, Trick HN, Gill BS, Bockus WW, Muthukrishnan S (2003) Greenhouse and field testing of transgenic wheat plants stably expressing genes for thaumatin-like protein, chitinase and glucanase against Fusarium graminearum. J Exp Bot 54:1101–1111

    Article  CAS  Google Scholar 

  • Bohorova NE, Ginkel M, Rajaram S Hoisington DA (1995) Tissue culture response of CIMMYT elite bread wheat genotypes and evaluation of regenerated plants. Cereal Res Commun 23:243–249

    Google Scholar 

  • Cheng M, Fry JE, Pang H, Zhou CM, Hironaka CM, Duncan DR, Conner TW, Wan Y (1997) Genetic transformation of wheat mediated by Agrobacterium tumefaciens. Plant Physiol 115:971–980

    Article  CAS  Google Scholar 

  • Chhapekar S, Raghavendrarao S, Pavan G, Chopperla R, Singh VK, Phanindra MLV, Dhandapani G, Sreevathsa R, Kumar PA (2015) Transgenic rice expressing a codon-modified synthetic CP4-EPSPS confers tolerance to broad-spectrum herbicide, glyphosate. Plant Cell Rep 34:721–731

    Article  CAS  Google Scholar 

  • Chugh A, Vikrant S, Mahalakshmi A, Khurana A (2012) A novel approach for Agrobacterium-mediated germ line transformation of Indian bread wheat (Triticum aestivum) and pasta wheat (Triticum durum). J Phytol 4:22–29

    CAS  Google Scholar 

  • Cobb AH, Reade JPH (2010) The inhibition of amino acid biosynthesis. In: Cobb AH, Reade JPH (eds) Herbicides and plant physiology, 2nd edn. Wiley-Blackwell, Hoboken, pp 126–144

    Chapter  Google Scholar 

  • Duke SO (2011) Glyphosate degradation in glyphosate-resistant and-susceptible crops and weeds. J Agric Food Chem 59:5835–5841

    Article  CAS  Google Scholar 

  • Duke SO, Powles SB (2008) Glyphosate: a once-in-a-century herbicide. Pest Manag Sci 64:319–325

    Article  CAS  Google Scholar 

  • FAO (2018) https://www.fao.org/worldfoodsituation/csdb/en/

  • Friends of the Earth Europe (2013) https://www.foeeurope.org/sites/default/files/press_releases/foee_5_environmental_impacts_glyphosate.pdf

  • Funke T, Han H, Healy-Fried ML, Fischerm M, Schönbrunn E (2006) Molecular basis for the herbicide resistance of Roundup ready crops. Proc Natl Acad Sci USA 103:13010–13015

    Article  CAS  Google Scholar 

  • Geiger DR, Fuchs MA (2002) Inhibitors of aromatic amino acid biosynthesis (glyphosate). In: Boger P, Wakabayashi K, Hirai K (eds) Herbicide classes in development: mode of action, targets, genetic engineering, chemistry. Springer, New York, pp 59–85

    Chapter  Google Scholar 

  • Green JM (2012) The benefits of herbicide-resistant crops. Pest Manag Sci 68:1323–1331

    Article  CAS  Google Scholar 

  • Hamada H, Linghu Q, Nagira Y, Miki R, Taoka N, Imai R (2017) An in planta biolistic method for stable wheat transformation. Sci Rep 7:11443. https://doi.org/10.1038/s41958-017-11936-0

    Article  PubMed  PubMed Central  Google Scholar 

  • Han Y, Jin XL, Wu FB, Zhang GP (2011) Genotypic differences in callus induction and plant regeneration from mature embryos of barley (Hordeum vulgare L.). J Zhejiang Univ Sci B 12:399–407

    Article  CAS  Google Scholar 

  • Hensel G, Himmelbach A, Chen D, Douchkov K, Kumlehn J (2011) Transgene expression systems in the Triticaceae cereals. J Plant Physiol 168:30–44

    Article  CAS  Google Scholar 

  • Hu T, Metz S, Chay C, Zhou HP, 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 Rep 21:1010–1019

    Article  CAS  Google Scholar 

  • James C (2003) Global review of commercialized transgenic crops. Curr Sci 84(3):303–309

    Google Scholar 

  • James VA, Avart C, Worland B, Snape JW, Vain P (2002) The relationship between homozygous and hemizygous transgene expression levels over generations in populations of transgenic rice plants. Theor Appl Genet 104:553–561

    Article  CAS  Google Scholar 

  • Joshi AK, Ortiz-Ferrara G, Crossa J, Singh G, Sharma RC, Chand R, Parsad R (2007) Combining superior agronomic performance and terminal heat tolerance with resistance to spot blotch (Bipolaris sorokiniana) of wheat in the warm humid Gangetic Plains of South Asia. Field Crops Res 103:53–61

    Article  Google Scholar 

  • Kasirajan L, Kovilpillai B, Bansal KC (2013) Optimization of genetic transformation protocol mediated by biolistic method in some elite genotypes of wheat (Triticum aestivum L.). Afr J Biotechnol 6:531–538

    Google Scholar 

  • Kato K, Chowdhury SH, Harada S (1991) Effect of culture condition on plant regeneration capacity of mature embryo derived callus in wheat (Triticum aestivum L.). Wheat Inf Serv 72:95–97

    Google Scholar 

  • Li ZW, Hansen JL, Liu Y, Zemetra RS, Berger PH (2004) Using real-time PCR to determine transgene copy number in wheat. Plant Mol Biol Rep 22(2):179–188

    Article  CAS  Google Scholar 

  • Liao P, Wang H, Wang M, Hsiao A, Bach TJ, Chye M (2014) Transgenic tobacco overexpressing Brassica juncea HMG CoA synthase 1 shows increased plant growth, pod size and seed yield. PLoS One 9(5):e98264

    Article  Google Scholar 

  • Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCt method. Methods 25:402–408

    Article  CAS  Google Scholar 

  • Lu C, Kang J (2008) Generation of transgenic plants of a potential oilseed crop Camelina sativa by Agrobacterium-mediated transformation. Plant Cell Rep 27:273–278

    Article  CAS  Google Scholar 

  • Mason G, Provero P, Vaira A, Accotto GP (2002) Estimating the number of integrations in transformed plants by quantitative real-time PCR. BMC Biotechnol 2:20

    Article  Google Scholar 

  • Mukherjee S, Stasolla C, Brule-Babel A, Ayele BT (2015) Isolation and characterization of rubisco small subunit gene promoter from common wheat. Plant Signal Behav 10(2):e989033

    Article  Google Scholar 

  • Murashige T, Skoog FA (1962) Revised medium for rapid growth and bioassays with tobacco cultures. Plant Physiol 159:473–479

    Article  Google Scholar 

  • Padaria JC, Bhatt D, Biswas K, Singh G, Raipuria R (2013) In-silico prediction of an uncharacterized protein generated from heat responsive SSH library in wheat (Triticum aestivum L.). Plant Omics 6:150–156

    CAS  Google Scholar 

  • Padaria JC, Vishwakarma H, Biswas K, Jasrotia RS, Singh GP (2014) Cloning and in-silico characterization of heat responsive pAPX gene from heat tolerant Indian cv Raj3765. BMC Res Notes 7:149

    Article  Google Scholar 

  • Passricha N, Saifi S, Khatodia S, Tuteja N (2016) Assessing zygosity in progeny of transgenic plants: current methods and perspectives. J Biol Methods 3:e46

    Article  Google Scholar 

  • Razzaq A, Hafiz IA, Mahmood I, Hussain A (2011) Development of in planta transformation protocol for wheat. Afr J Biotechnol 10:740–750

    CAS  Google Scholar 

  • Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, New York

    Google Scholar 

  • Shinano T, Lei TT, Kawamukai T, Inoue MT, Koike T, Tadano T (1996) Dimethylsulfoxide method for the extraction of chlorophylls a and b from the leaves of wheat, field bean, dwarf bamboo and oak. Phytosynthetica 32(3):409–415

    CAS  Google Scholar 

  • Supartana P, Shimizu T, Shioiri H, Nogawa M, Nozue M, Kojima M (2005) Development of simple and efficient in planta transformation method for rice (Oryza sativa L.) using Agrobacterium tumefaciens. J Biosci Bioeng 100:391–397

    Article  CAS  Google Scholar 

  • Supartana P, Shimizu T, Nogawa M, Shioiri H, Nakajima T, Haramoto M, Nozue M, Kojima M (2006) Development of simple and efficient in planta method of transformation for wheat (Triticum aestivum L.), using Agrobacterium tumefaciens. J Biosci Bioeng 102:162–170

    Article  CAS  Google Scholar 

  • Vasil V, Castillo AM, Fromm ME, Vasil I (1992) Herbicide resistant fertile transgenic wheat plants obtained by microprojectile bombardment of regenerable embryogenic callus. Nat Biotechnol 10:667–674

    Article  CAS  Google Scholar 

  • Vasil V, Srivastava V, Castillo AM, Fromm ME, Vasil I (1993) Rapid production of transgenic wheat plants by direct bombardment of cultured immature embryos. Nat Biotechnol 11:1553–1558

    Article  Google Scholar 

  • Vickers CE, Xue GP, Gresshoff PM (2003) A synthetic xylanase as a novel reporter in plants. Plant Cell Rep 22:135–140

    Article  CAS  Google Scholar 

  • Weng H, Pan A, Yang P, Zhang C, Liu Z, Zhang D (2004) Estimating number of transgene copies in transgenic rapeseed by real-time PCR assay with HMG I/Y as an endogenous reference gene. Plant Mol Biol Rep 22(3):289–300

    Article  CAS  Google Scholar 

  • Xue G, Sadat S, Drenth J, Mclyntyre L (2014) The heat shock factor family from Triticum aestivum in response to heat and other major abiotic stresses and their role in regulation in heat shock protein genes. J Exp Bot 65(2):539–557

    Article  CAS  Google Scholar 

  • Yu T, Xu Z, Gou J, Wang Y, Abernathy B, Fu J, Chen X, Zhou Y, Chen M, Ye X, Ma Y (2016) Improved drought tolerance in wheat plants overexpressing a synthetic bacterial cold shock protein gene SeCspA. Sci Rep 7:44050. https://doi.org/10.1038/srep44050

    Article  Google Scholar 

  • Zhou H, Berg JD, Blank SE, Chay CA, Chen G, Eskelsen SR, Fry JE, Hoi S, Hu T, Isakson PJ, Lawton MB, Metz SG, Rempel CB, Ryerson DK, Sansone AP, Shook AL, Starke RJ, Tichota JM, Valenti SA (2003) Field efficacy assessment of transgenic roundup ready wheat. Crop Sci 43:1072–1075

    Article  CAS  Google Scholar 

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Acknowledgements

The authors are thankful to the Project Director NRCPB (National Research Centre on Plant Biotechnology), Pusa Campus, New Delhi, India, for providing facilities to carry out the research work. Funds provided by the ICAR (Indian Council of Agricultural Research) under NICRA (National Innovations in Climate Resilient Agriculture) project are duly acknowledged. Dr. P. A. Kumar, Ex-Project Director NRCPB, is duly thanked for providing the gene construct pCAMBIA 1301-Ubi-C4-EPSPS. The authors are thankful to the Director IARI, Pusa Campus, New Delhi, India, for providing the plant growth chambers at the National Phytotron Facilities.

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Authors and Affiliations

  1. National Research Centre on Plant Biotechnology, Pusa Campus, New Delhi, 110012, India

    Avijit Tarafdar, Harinder Vishwakarma, S. Gothandapani, Meenal Bhati, Koushik Biswas, Arul Prakash, Uttara Chaturvedi, Amolkumar U. Solanke & Jasdeep Chatrath Padaria

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  1. Avijit Tarafdar
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  2. Harinder Vishwakarma
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  3. S. Gothandapani
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  4. Meenal Bhati
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  5. Koushik Biswas
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  6. Arul Prakash
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  7. Uttara Chaturvedi
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  8. Amolkumar U. Solanke
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  9. Jasdeep Chatrath Padaria
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Contributions

Concept and design of experiment: JCP; performed the experiments: AT, HV, GS, MB, KB, AP, UC; analyzed the data: AT, HV, GS; manuscript writing and editing: AT, HV, GS, AUS, JCP.

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Correspondence to Jasdeep Chatrath Padaria.

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Tarafdar, A., Vishwakarma, H., Gothandapani, S. et al. A quick, easy and cost-effective in planta method to develop direct transformants in wheat. 3 Biotech 9, 180 (2019). https://doi.org/10.1007/s13205-019-1708-6

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