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Plant Cell Reports

, Volume 34, Issue 10, pp 1835–1848 | Cite as

Agrobacterium-mediated in planta genetic transformation of sugarcane setts

  • Subramanian Mayavan
  • Kondeti Subramanyam
  • Balusamy Jaganath
  • Dorairaj Sathish
  • Markandan Manickavasagam
  • Andy GanapathiEmail author
Original Article

Abstract

Key message

An efficient, reproducible, and genotype-independent in planta transformation has been developed for sugarcane using setts as explant.

Abstract

Traditional Agrobacterium-mediated genetic transformation and in vitro regeneration of sugarcane is a complex and time-consuming process. Development of an efficient Agrobacterium-mediated transformation protocol, which can produce a large number of transgenic plants in short duration is advantageous. Hence, in the present investigation, we developed a tissue culture-independent in planta genetic transformation system for sugarcane using setts collected from 6-month-old sugarcane plants. The sugarcane setts (nodal cuttings) were infected with three Agrobacterium tumefaciens strains harbouring pCAMBIA 1301–bar plasmid, and the transformants were selected against BASTA®. Several parameters influencing the in planta transformation such as A. tumefaciens strains, acetosyringone, sonication and exposure to vacuum pressure, have been evaluated. The putatively transformed sugarcane plants were screened by GUS histochemical assay. Sugarcane setts were pricked and sonicated for 6 min and vacuum infiltered for 2 min at 500 mmHg in A. tumefaciens C58C1 suspension containing 100 µM acetosyringone, 0.1 % Silwett L-77 showed the highest transformation efficiency of 29.6 % (with var. Co 62175). The three-stage selection process completely eliminated the chimeric transgenic sugarcane plants. Among the five sugarcane varieties evaluated using the standardized protocol, var. Co 6907 showed the maximum transformation efficiency (32.6 %). The in planta transformation protocol described here is applicable to transfer the economically important genes into different varieties of sugarcane in relatively short time.

Keywords

Agrobacterium tumefaciens BASTA® In planta transformation Sonication Southern blot hybridization Sugarcane setts Vacuum infiltration 

Abbreviations

MS

Murashige and Skoog medium

hpt II

Hygromycin phosphotransferase

gus A

β-Glucuronidase gene

CaMV 35S

Cauliflower mosaic virus 35S promoter

35 S poly A

35S Poly A terminator

Nos ter

Nopaline synthase terminator

Notes

Acknowledgments

The authors are grateful to University Grants Commission (UGC), Government of India, for the financial support [No.F.31-239/2005 (SR)] to carry out the present work. The corresponding author is thankful to University Grants Commission (UGC), Govt. of. India for providing Fellowship under UGC–BSR scheme. All the authors are thankful to Prof. A.S Rao, Department of Biotechnology & Genetic Engineering, Bharathidasan University, Tiruchirappalli for his valuable suggestions in improving the manuscript.

Conflict of interest

The authors declare that they have no conflict of interest.

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Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Subramanian Mayavan
    • 1
    • 2
  • Kondeti Subramanyam
    • 1
    • 3
  • Balusamy Jaganath
    • 1
  • Dorairaj Sathish
    • 1
  • Markandan Manickavasagam
    • 1
  • Andy Ganapathi
    • 1
    Email author
  1. 1.Department of Biotechnology and Genetic Engineering, School of BiotechnologyBharathidasan UniversityTiruchirappalliIndia
  2. 2.Center for Bioenergy, Cooperative ResearchLincoln University of MissouriJefferson CityUSA
  3. 3.Laboratory of Biochemistry and Glycobiology, Department of Molecular BiotechnologyGhent UniversityGhentBelgium

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