Molecular Biology Reports

, Volume 46, Issue 2, pp 1845–1853 | Cite as

Optimization of Agrobacterium-mediated transformation in spring bread wheat using mature and immature embryos

  • Rakesh Kumar
  • Harohalli Masthigowda MamruthaEmail author
  • Amandeep Kaur
  • Karnam Venkatesh
  • Davinder Sharma
  • Gyanendra Pratap Singh
Original Article


Wheat is the most widely grown staple food crop in the world and accounts for dietary needs of more than 35% of the human population. Current status of transgenic wheat development is slow all over the world due to the lack of a suitable transformation system. In the present study, an efficient and reproducible Agrobacterium-mediated transformation system in bread wheat (Triticum aestivum L.) is established. The mature and immature embryos of six recently released high yielding spring bread wheat genotypes were used to standardize various parameters using Agrobacterium tumefaciens strain EHA105 harbouring binary vector pCAMBIA3301 having gus and bar as marker genes. The optimum duration for embryo pre-culture, inoculation time and co-cultivation were 2 days, 30 min and 48 h, respectively. The bacterial inoculum concentration of OD of 1 at 600 nm showed 67.25% transient GUS expression in the histochemical GUS assay. The filter paper based co-cultivation limits the Agrobacterium overgrowth and had 82.3% explants survival rate whereas medium based strategy had 22.7% explants survival only. The medium having picloram 4 mg/l along with antibiotics (cefotaxime 500 mg/l and timentin 300 mg/l) was found best suitable for initial week callus induction. The standardized procedure gave overall 14.9% transformation efficiency in immature embryos and 9.8% in mature embryos and confirmed by gene-specific and promoter-specific PCR and southern analysis. These results indicate that the developed Agrobacterium-mediated transformation system is suitable for diverse wheat genotypes. The major obstacle for the implication of the CRISPR-based genome editing techniques is the non-availability of a suitable transformation system. Thus, the present system can be exploited to deliver the T-DNA into the wheat genome for CRISPR-based target modifications and transgene insertions.


Wheat, Triticum aestivum, cereal, Agrobacterium bar gene Histochemical GUS assay Southern analysis Transformation 



We are highly thankful to Dr. Viswanathan Chinnusamy, Dr. Santosh Kumar, IARI, New Delhi and Dr. Monika Dalal, NRCPB, New Delhi for extending their guidance and facility for southern analysis.

Author Contributions

MHM conceived the project and designed the experiments with RK and AK. RK, AK and DS conducted experiments. RK, AK, and KV wrote the manuscript. GPS provides necessary infrastructure and facilities to conduct various experiments. All authors read and approved the final manuscript.


This work is financially supported by the Indian Council of Agricultural Research, New Delhi, India, under the project entitled “ICAR Network Project on Functional Genomics and Genetic Modification in Crops (NPFGGM)” (Project No. 1006474).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. 1.
    IWGSC Infographic. Facts about Wheat (2016) Accessed 16 Jan 2017
  2. 2.
    Cheng M, Fry JE, Pang SZ, Zhou H, Hironaka CM, Duncan DR, Conner TW, Wan Y (1997) Genetic transformation of wheat mediated by Agrobacterium tumefaciens. Plant Physiol 115:971–980CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Wu H, Sparks C, Amoah B, Jones HD (2003) Factors influencing successful Agrobacterium-mediated genetic transformation of wheat. Plant Cell Rep 21:659–668PubMedGoogle Scholar
  4. 4.
    Yu Y, Wang J, Zhu ML, Wei ZM (2008) Optimization of mature embryo-based high frequency callus induction and plant regeneration from elite wheat cultivars grown in China. Plant Breed 127(3):249–255CrossRefGoogle Scholar
  5. 5.
    Parmar SS, Sainger M, Chaudhary D, Jaiwal PK (2012) Plant regeneration from mature embryo of commercial Indian bread wheat (Triticum aestivum L.) cultivars. Physiol Mol Biol Plants 18(2):177–183CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Kumar R, Mamrutha HM, Kaur A, Venkatesh K, Grewal A, Kumar R, Tiwari V (2017a) Development of an efficient and reproducible regeneration system in wheat (Triticum aestivum L.). Physiol Mol Biol Plants 23:945–954CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Kumar N, Gulati A, Bhattacharya A (2013) L-glutamine and L-glutamic acid facilitate successful Agrobacterium infection of recalcitrant tea cultivars. Appl Biochem Biotechnol 170:1649–1664CrossRefPubMedGoogle Scholar
  8. 8.
    Kumar R, Mamrutha HM, Kaur A, Grewal A (2017b) Synergistic effect of cefotaxime and timentin to suppress the Agrobacterium overgrowth in wheat (Triticum aestivum L.) transformation. Asian J Microbiol Biotechnol Environmental Sci 19(4):961–967Google Scholar
  9. 9.
    Mamrutha HM, Kumar R, Venkatesh K, Sharma P, Kumar R, Tiwari V, Sharma I (2014) Genetic transformation of wheat - present status and future potential. J Wheat Res 6:107–119Google Scholar
  10. 10.
    Kumar R, Mamrutha HM, Venkatesh K, Tiwari KN, Kumar S (2018) Application and achievements of Recombinant DNA in crop improvement. In: Bharadwaj DN (ed) Advanced molecular plant breeding. Apple Academic Press, Inc., New Jersey, USA, p 299–328Google Scholar
  11. 11.
    Ding L, Li S, Gao J, Wang Y, Yang G, He G (2009) Optimization of Agrobacterium-mediated transformation conditions in mature embryos of elite wheat. Mol Biol Rep 36:29–36CrossRefPubMedGoogle Scholar
  12. 12.
    Ishida Y, Tsunashima M, Hiei Y, Komari T (2015) Wheat (Triticum aestivum L.) transformation using immature embryos. In: Kan Wang (ed) Agrobacterium Protocols: Volume 1, Methods in Molecular Biology, vol 1223, Springer Science+Business Media New York, p 189–198CrossRefGoogle Scholar
  13. 13.
    Medvecka E, Harwood WA (2015) Wheat (Triticum aestivum L.) transformation using mature embryos. In: Kan Wang (ed) Agrobacterium Protocols: Volume 1, Methods in Molecular Biology, vol 1223, Springer Science+Business Media New York, p 189–198CrossRefGoogle Scholar
  14. 14.
    Patnaik D, Vishnudasan D, Khurana P (2006) Agrobacterium-mediated transformation of mature embryos of Triticum aestivum and Triticum durum. Curr Sci 91:307–317Google Scholar
  15. 15.
    Tao LL, Yin GX, Du LP, Shi ZY, She MY, Xu HJ, Ye XG (2011) Improvement of plant regeneration from immature embryos of wheat infected by Agrobacterium tumefaciens. Agric Sci China 10:317–326CrossRefGoogle Scholar
  16. 16.
    Amoah BK, Wu H, Sparks C, Jones HD (2001) Factors influencing Agrobacterium-mediated transient expression of uidA in wheat inflorescence tissue. J Exp Bot 52:1135–1142CrossRefPubMedGoogle Scholar
  17. 17.
    Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Saghai-Maroof MA, Soliman KM, Jorgensen RA, Allard RW (1984) Ribosomal DNA spacer-length polymorphisms in barley: Mendelian inheritance, chromosomal location, and population dynamics. Proc Natl Acad Sci USA 81:8014–8018CrossRefPubMedGoogle Scholar
  19. 19.
    Binka A, Orczyk W, Nadolska-Orczyk A (2012) The Agrobacterium-mediated transformation of common wheat (Triticum aestivum L.) and triticale (x Triticosecale Wittmack): Role of the binary vector system and selection cassettes. J Appl Genet 53:1–8CrossRefPubMedGoogle Scholar
  20. 20.
    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 21:1010–1019CrossRefPubMedGoogle Scholar
  21. 21.
    Jones HD, Doherty A, Wu H (2005) Review of methodologies and a protocol for the Agrobacterium-mediated transformation of wheat. Plant Methods 1:5CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Cheng M, Hu TC, Layton J, Liu CN, Fry JE (2003) Desiccation of plant tissues post-Agrobacterium infection enhances T-DNA delivery and increases stable transformation efficiency in wheat. In Vitr Cell Dev Biol – Plant 39:595–604CrossRefGoogle Scholar
  23. 23.
    He Y, Jones HD, Chen S, Chen XM, Wang DW, Li KX, Wang DS, Xia LQ (2010) Agrobacterium-mediated transformation of durum wheat (Triticum turgidum L. var. durum cv Stewart) with improved efficiency. J Exp Bot 61:1567–1581CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Han SN, Oh PR, Kim HS, Heo HY, Moon JC, Lee SK, Kim KH, Seo YW, Lee BM (2007) Effect of antibiotics on suppression of Agrobacterium tumefaciens and plant regeneration from wheat embryo. J Crop Sci Biotechnol 10(2):92–97Google Scholar
  25. 25.
    Yu Y, Wei Z (2008) Increased oriental armyworm and aphid resistance in transgenic wheat stably expressing Bacillus thuringiensis (Bt) endotoxin and Pinellia ternate agglutinin (PTA). Plant Cell Tiss Organ Cult 94:33–44CrossRefGoogle Scholar
  26. 26.
    Gelvin SB (2003) Agrobacterium-mediated plant transformation: the biology behind the “Gene-Jockeying” tool. Microbiol Mol Biol Rev 67(1):16–37CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.ICAR-Indian Institute of Wheat and Barley Research (IIWBR)KarnalIndia
  2. 2.ICAR-National Bureau of Animal Genetic Resources (NBAGR)KarnalIndia

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