Agrobacterium-mediated genetic transformation of selected tropical inbred and hybrid maize (Zea mays L.) lines

  • Omwoyo Ombori
  • John Vincent Omondi Muoma
  • Jesse Machuka
Original Paper

Abstract

The study was carried out to evaluate the amenability of tropical inbred and hybrid maize lines, using Agrobacterium mediated transformation technique. Agrobacterium tumefaciens strains EHA101 harbouring a pTF102 binary vector, EHA101, AGL1, and LBA4404 harbouring pBECK2000.4 plasmid, LBA4404, GV and EHA105 harbouring pCAMBIA2301 plasmid, and AGL1 harbouring the pSB223 plasmid were used. Delivery of transgenes into plant tissues was assessed using transient β-glucuronidase (gus) activity on the 3rd and 4th day of co-cultivation of the infected Immature Zygotic Embryos (IZEs) and embryogenic callus. Transient gus expression was influenced by the co-cultivation period, maize genotype and Agrobacterium strain. The expression was highest after the 3rd day of co-culture compared to the 4th day with intense blue staining was detected for IZEs which were infected with Agrobacterium strains EHA105 harbouring pCAMBIA2301 and EHA101 harbouring pTF102 vector. Putative transformants (To) were regenerated from bialaphos resistant callus. Differences were detected on the number of putative transformants regenerated among the maize lines. Polymerase chain reaction (PCR) amplification of Phosphinothricin acetyltransferase (bar) and gus gene confirmed the transfer of the transgenes into the maize cells. Southern blot hybridization confirmed stable integration of gus into PTL02 maize genome and segregation analysis confirmed the inheritance of the gus. A transformation efficiency of 1.4 % was achieved. This transformation system can be used to introduce genes of interest into tropical maize lines for genetic improvement.

Keywords

Agrobacterium tumefaciens Genetic transformation gus expression PCR Southern blot hybridization Zea mays 

Abbreviations

CaMV

Cauliflower mosaic virus

CTAB

Cetyltrimethylammonium bromide

2,4-D

2,4-Dichlorophenoxyacetic acid

bar

Phosphinothricin acetyltransferase gene

gus

β-Glucuronidase

IZEs

Immature zygotic embryos

MS

Murashige and Skoog

nptII

Neomycin phosphotransferase II gene

To

Primary transformants

YEP

Yeast peptones extract

Notes

Acknowledgments

We are grateful to Prof. K. Wang and Dr. J. Kumlehen for generously providing us with Agrobaterium strains. The authors are also thankful to students in the Plant Transformation Lab, Kenyatta University, Kenya for their technical assistance. This work was sponsored by Germany Exchange Service (DAAD) and Rockefeller Foundation.

Supplementary material

11240_2012_247_MOESM1_ESM.docx (15 kb)
Supplementary material 1 (DOCX 16 kb)

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Copyright information

© Springer Science+Business Media Dordrecht 2012

Authors and Affiliations

  • Omwoyo Ombori
    • 1
  • John Vincent Omondi Muoma
    • 2
  • Jesse Machuka
    • 3
  1. 1.Department of Plant and Microbial SciencesKenyatta UniversityNairobiKenya
  2. 2.Department of Biological SciencesMasinde Muliro UniversityKakamegaKenya
  3. 3.Department of Biochemistry and BiotechnologyKenyatta UniversityNairobiKenya

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