Agrobacterium tumefaciens-mediated transformation protocol of Jatropha curcas L. using leaf and hypocotyl segments
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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.
KeywordsActin gene β-glucuronidase GUS histochemical assay Shoot organogenesis
Cauliflower mosaic virus
Hygromycin phosphotransferase gene
Neomycin phosphotransferase gene
Nopaline synthase promoter
Reverse transcription-polymerase chain reaction
Shoot induction medium
Transient transformation expression
The authors are thankful to the Director, National Botanical Research Institute (CSIR), for the facilities provided.
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