Skip to main content

Novel recombinant binary vectors harbouring Basta (bar) gene as a plant selectable marker for genetic transformation of plants

Abstract

Genetic transformation is one of the most widely used technique in crop improvement. However, most of the binary vectors used in this technique, especially cloning based, contain antibiotic genes as selection marker that raise serious consumer and environmental concerns; moreover, they could be transferred to non-target hosts with deleterious effects. Therefore, the goal of this study was reconstruction of the widely used pBI121 binary vector by substituting the harmful antibiotic selection marker gene with a less-harmful selection marker, Basta (herbicide resistance gene). The generated vectors were designated as pBI121NB and pBI121CB, in which Basta gene was expressed under the control of Nos or CaMV 35S promoter, respectively. The successful integration of the new inserts into both the vectors was confirmed by PCR, restriction digestion and sequencing. Both these vectors were used in transforming Arabidopsis, Egyptian wheat and barley varieties using LBA4404 and GV3101 Agrobacterium strains. The surfactant Tween-20 resulted in an efficient transformation and the number of Arabidopsis transformants was about 6–9 %. Soaked seeds of wheat and barley were transformed with Agrobacterium to introduce the bacteria to the growing shoot apices. The percentage of transgenic lines was around 16–17 and 14–15 % for wheat and barley, respectively. The quantitative studies presented in this work showed that both LBA4404 and GV3101 strains were suitable for transforming Egyptian wheat and barley.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Abbreviations

BA:

Benzyl adenine

CaMV 35S:

Cauliflower mosaic virus 35S promoter

DMSO:

Dimethyl sulfoxide

DMF:

N,N-Dimethyl formamide

LB medium:

Luria-Bertani medium

Nos:

Nopaline synthase

References

  1. Akbarzadeh A, Kordbacheh F, Jafari M, Salmanian A (2010) A binary vector for agrobacterium mediated plant transformation with new glyphosate tolerant gene as a selectable marker. Bih Biol 4:19–29

    Google Scholar 

  2. Assaad FF, Signer ER (1990) Cauliflower mosaic-virus p35S promoter activity in Escherichia-coli. Mol Gen Genet 223:517–520

    CAS  Article  PubMed  Google Scholar 

  3. Barampuram S, Allen G, Krasnyanski S (2014) Effect of various sterilization procedures on the in vitro germination of cotton seeds. Plant Cell Tiss Organ Cult. doi:10.1007/s11240-014-0472-x

    Google Scholar 

  4. Bechtold N, Ellis J, Pelletier G (1993) In planta Agrobacterium mediated gene transfer by infiltration of adult Arabidopsis thaliana plants. C R Acad Sci Paris Life Sci 316:1194–1199

    CAS  Google Scholar 

  5. Chen PY, Wang CK, Soong SC, To KY (2003) Complete sequence of the binary vector pBI121 and its application in cloning T-DNA insertion from transgenic plants. Mol Breed 11:287–293

    CAS  Article  Google Scholar 

  6. Clough SJ, Bent AF (1998) A simplified method for Agrobacterium mediated transformation of Arabidopsis thaliana. Plant J 16:735–743

    CAS  Article  PubMed  Google Scholar 

  7. Das P, Joshi NC (2011) Minor modifications in obtainable Arabidopsis floral dip method enhances transformation efficiency and production of homozygous transgenic lines harboring a single copy of transgene. Adv Biosci Biotech 2:59–67

    CAS  Article  Google Scholar 

  8. De Block M, De Brouwer D, Tenning P (1989) Transformation of Brassica napus and Brassica oleracea using Agrobacterium tumefaciens and the expression of the bar and neo genes in the transgenic plants. Plant Physiol 91:694–701

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  9. Dehestani A, Ahmadian G, Salmanian AH, Jelodar NB, Kazemitabar K (2010) Transformation efficiency enhancement of Arabidopsis vacuum infiltration by surfactant application and apical inflorescence removal. Trakia J Sci 8:19–26

    Google Scholar 

  10. Guilley H, Dudley RK, Jonard G, Balazs E, Richards KE (1982) Transcription of cauliflower mosaic virus DNA: detection of promoter sequences, and characterization of transcripts. Cell 30:763–773

    CAS  Article  PubMed  Google Scholar 

  11. Jones HD (2005) Wheat transformation: current technology and applications to grain development and composition. J Cereal Sci 41:137–147

    CAS  Article  Google Scholar 

  12. Joyce P, Kuwahata M, Turner N, Lakshmanan P (2010) Selection system and co-cultivation medium are important determinants of Agrobacterium-mediated transformation of sugarcane. Plant Cell Rep 29:173–183

    CAS  Article  PubMed  Google Scholar 

  13. Khuong TTH, Crete P, Robaglia C, Caffarri S (2013) Optimisation of tomato Micro-tom regeneration and selection on glufosinate/Basta and dependency of gene silencing on transgene copy number. Plant Cell Rep 32:1441–1445

    CAS  Article  PubMed  Google Scholar 

  14. Koehorst-van Putten HJJ, Sudarmonowati E, Herman M, Pereira-Bertram IJ, Wolters AMA, Meima H, de Vetten N, Raemakers CJJM, Visser RGF (2012) Field testing and exploitation of genetically modified cassava with low-amylose or amylose-free starch in Indonesia. Transgenic Res 21:39–45

    CAS  Article  PubMed  Google Scholar 

  15. Kopertekh L, Broer I, Schiemann J (2009) Developmentally regulated site-specific marker gene excision in transgenic B. napus plants. Plant Cell Rep 28:1075–1083

    CAS  Article  PubMed  Google Scholar 

  16. Kuiper HA, Kleter GA, Noteborn HPJM, Kok EJ (2001) Assessment of the food safety issues related to genetically modified foods. Plant J 27:503–528

    CAS  Article  PubMed  Google Scholar 

  17. Kumari S, Vaishnav A, Jain S, Varma A, Choudhary DK (2016) Induced drought tolerance through wild and mutant bacterial strain Pseudomonas simiae in mung bean (Vigna radiata L.). World J Microbiol Biotech 32:4

    Article  Google Scholar 

  18. Leason M, Cunliffe D, Parkin D, Lea PJ, Miflin J (1982) Inhibition of pea leaf glutamine synthetase by sulphoximine, phosphinothricin and other glutamate analogues. Phytoch 21:855–857

    CAS  Article  Google Scholar 

  19. Li JF, Park E, Arnim AG, Nebenführ A (2009) The FAST technique: a simplified Agrobacterium-based transformation method for transient gene expression analysis in seedlings of Arabidopsis and other plant species. Plant Methods 5:6. doi:10.1186/1746-4811-5-6

    Article  PubMed  PubMed Central  Google Scholar 

  20. Logemann E, Birkenbihl RP, Ulker B, Somssich IE (2006) An improved method for preparing Agrobacterium cells that simplifies the Arabidopsis transformation protocol. Plant Methods 2:16

    Article  PubMed  PubMed Central  Google Scholar 

  21. Merkel U, Rathke GW, Schuster C, Warnstorff K, Diepenbrock W (2004) Use of glufosinate-ammonium to control cruciferous weed species in glufosinate-resistant winter oilseed rape. Field Crops Res 85:237–249

    Article  Google Scholar 

  22. Miyoshi K, Sato T (1997) Effects of ethanol on the germination of seeds of Japonica and Indica Rice (Oryza sativa L.) under anaerobic and aerobic conditions. Bot 79:391–395

    CAS  Article  Google Scholar 

  23. Piernas V, Guiraud JP (1997) Disinfection of rice seeds prior to sprouting. J Food Sci 62:611

    CAS  Article  Google Scholar 

  24. Pobjecky N, Rosenberg GH, Dintergottlieb G, Kaufer NF (1990) Expression of the beta-glucuronidase gene under the control of the CaMV-35S promoter in Schizosaccharomyces-pombe. Mol Gen Genet 220:314–316

    CAS  Article  PubMed  Google Scholar 

  25. Rohila JS, Chen M, Cerny R, Fromm ME (2004) Improved tandem affinity purification tag and methods for isolation of protein heterocomplexes from plants. Plant J 38:172–181

    CAS  Article  PubMed  Google Scholar 

  26. Shu QY, Liu GS, Xu SX, Li XF, Li HJ (2005) Genetic transformation of Leymus chinensis with the PAT gene through microprojectile bombardment to improve resistance to the herbicide Basta. Plant Cell Rep 24:36–44

    CAS  Article  PubMed  Google Scholar 

  27. Sretenović-Rajičić T, Ninković S, Vinterhalter B, Miljuš-Djukić J, Vinterhalter D (2004) Introduction of resistance to herbicide Basta® in Savoy cabbage. Biol Plant 48:431–436

    Article  Google Scholar 

  28. Subramanyam K, Arunachalam C, Thaneswari RM, Sulaiman AA, Manickavasagam M, Ganapathi A (2015) Highly efficient Agrobacterium-mediated in planta genetic transformation of snake gourd (Tricosanthes cucumerina L.). Plant Cell Tiss Organ Cult 123:133–142. doi:10.1007/s11240-015-0821-4

    CAS  Article  Google Scholar 

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

    CAS  Article  PubMed  Google Scholar 

  30. Szakasits D, Siddique S, Bohlmann H (2007) An improved pPZP vector for agrobacterium-mediated plant transformation. Plant Mol Biol Rep 25:115–120

    CAS  Article  Google Scholar 

  31. Tian X, Hao J, Fang B, Geng P, La H, Huang D, Wang H (2015) Transformation of upland rice with the bar gene and selection for resistance to the herbicide Basta. Euphytica 205(151):167

    Google Scholar 

  32. Wang JX, Zhao FY, Xu P (2006) Use of aro-A as a selectable marker for Brassica napus transformation. Crop Sci 46:706–711

    CAS  Article  Google Scholar 

Download references

Acknowledgments

I would like to thank Dr. Gaber M. Abogadallah (Associate Professor of Plant Genetics, Botany Department, Faculty of Science, Damietta University, New Damietta 34517, Egypt) for hosting this work in his laboratory. Agrobacterium GV3101 strain was kindly provided from Dr. Mohamed S. Tawfik (Agricultural Genetic Engineering Research Institute (AGERI), Agriculture Research Center (ARC), Giza, Egypt 12619).

Author information

Affiliations

Authors

Corresponding author

Correspondence to Reham M. Nada.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Nada, R.M. Novel recombinant binary vectors harbouring Basta (bar) gene as a plant selectable marker for genetic transformation of plants. Physiol Mol Biol Plants 22, 241–251 (2016). https://doi.org/10.1007/s12298-016-0360-4

Download citation

Keywords

  • pBI121CB
  • pBI121NB
  • Arabidopsis
  • Wheat
  • Barley
  • LBA4404
  • GV3101