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Journal of Plant Biochemistry and Biotechnology

, Volume 21, Issue 1, pp 128–133 | Cite as

Agrobacterium tumefaciens-mediated transformation protocol of Jatropha curcas L. using leaf and hypocotyl segments

  • Pratibha Misra
  • Dibya Dolridge Toppo
  • Manoj Kumar Mishra
  • Syed Saema
  • Gaurav Singh
Short Communication

Abstract

In the present study, Agrobacterium tumefaciens-mediated transformation protocol has been optimized for Jatropha curcas—an important biofuel plant using leaf segments (LS) and hypocotyl segments (HS) from in vitro-seedlings. Among different accessions used, ‘GJ-Ran-C2’ was selected as the best for transformation. A. tumefaciens strain EHA101, containing the binary vector pIG121Hm was used for transformation, along with the GUS reporter gene containing intron and CaMV 35S as promoter. Various parameters were optimized to get the highest transient transformation expression (TTE). Augmentin® was used in the medium to control A. tumefaciens. Selection of transgenic shoots was done in the presence of kanamycin. Integration of GUS gene was confirmed by PCR analysis, whereas the expression of GUS transgene was confirmed by RT-PCR, using actin gene as internal control. GUS histochemical analysis of explants just after cocultivation showed blue color in approximately all the LS and HS but stable transformation efficiency was 5% in case of LS, whereas 4% in HS.

Keywords

Actin gene β-glucuronidase GUS histochemical assay Shoot organogenesis 

Abbreviations

CaMV

Cauliflower mosaic virus

CCM

Cocultivation medium

GUS

β-glucuronidase gene

hptII

Hygromycin phosphotransferase gene

nptII

Neomycin phosphotransferase gene

Pnos

Nopaline synthase promoter

RT-PCR

Reverse transcription-polymerase chain reaction

SIM

Shoot induction medium

TTE

Transient transformation expression

X-Gluc

5-bromo-4-chloro-3-indolyl-β-d-glucuronide

Notes

Acknowledgements

The authors are thankful to the Director, National Botanical Research Institute (CSIR), for the facilities provided.

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Copyright information

© Society for Plant Biochemistry and Biotechnology 2011

Authors and Affiliations

  • Pratibha Misra
    • 1
  • Dibya Dolridge Toppo
    • 1
  • Manoj Kumar Mishra
    • 1
  • Syed Saema
    • 1
  • Gaurav Singh
    • 1
  1. 1.National Botanical Research Institute, Council of Scientific and Industrial Research (CSIR)LucknowIndia

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