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
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Creator |
Kumar, Akhilesh
Chakraborty, Amrita Ghanta, Srijani Chattopadhyay, Sharmila |
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Subject |
Drug Development/Diagnostics & Biotechnology
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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.
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Date |
2009
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Type |
Article
PeerReviewed |
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Format |
application/pdf
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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. |
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Relation |
http://dx.doi.org/10.1007/s11240-008-9467-9
http://www.eprints.iicb.res.in/142/ |
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