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Agrobacterium-mediated genetic transformation of mint with E. coli glutathione synthetase gene

IR@IICB: CSIR-Indian Institute of Chemical Biology, Kolkata

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Title Agrobacterium-mediated genetic transformation of mint with E. coli glutathione synthetase gene
 
Creator Kumar, Akhilesh
Chakraborty, Amrita
Ghanta, Srijani
Chattopadhyay, Sharmila
 
Subject Drug Development/Diagnostics & Biotechnology
 
Description Morphologically identical transgenic mint (Mentha arvensis L.) with bacterial glutathione synthetase gene has been developed. Transformed plants were obtained by co-cultivation of leaf disks with Agrobacterium tumefaciens strain LBA 4404 harbouring a binary vector pCAMBIA-CpGS that carried E. coli glutathione synthetase (GS), b-glucuronidase as reporter gene and nptII as selective marker gene for kanamycin resistance. Using a constitutive double CaMV 35S promoter and an rbcS transit peptide, we successfully addressed CpGS to the chloroplasts through pJIT 117 vector. Preculture and the presence of AS in the co-cultivation medium played a significant role in enhancing transformation frequency. The highest transformation frequency was achieved with MS selection medium supplemented with 25% coconut water, 1.12 mg l-1 BAP, 0.2 mg l-1 NAA, 50 mg l-1 kanamycin and 125 mg l-1 cefotaxime. Robust rooting of regenerated shoots was obtained in half-strength liquid MS medium containing 0.2 mg l-1 NAA and 50 mg l-1 kanamycin. The presence and expression of transgenes in transgenics (T0) was evidenced by GUS histoenzymatic assay, PCR and RT-PCR analysis of nptII and the gene of interest, i.e., GS of putative transgenic leaves. Chromosomal integration of GS gene was confirmed by Southern blot analysis. Transgenic plants were successfully acclimatized in the greenhouse. An overall transformation frequency of 15% was achieved in approximately 3 months of time period. These results are discussed in relation to heavy metal trafficking pathways in higher plants and to the interest of using plastid expression of PCS for biotechnological applications.
 
Date 2009
 
Type Article
PeerReviewed
 
Format application/pdf
 
Identifier http://www.eprints.iicb.res.in/142/1/PLANT_CELL_TISSUE_AND_ORGAN_CULTURE%2C96(2)%2C117%2D126%2C__2009[104].pdf
Kumar, Akhilesh and Chakraborty, Amrita and Ghanta, Srijani and Chattopadhyay, Sharmila (2009) Agrobacterium-mediated genetic transformation of mint with E. coli glutathione synthetase gene. Plant Cell Tiss Organ Cult, 26 (2). pp. 117-126.
 
Relation http://dx.doi.org/10.1007/s11240-008-9467-9
http://www.eprints.iicb.res.in/142/