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S L Singla-Pareek

Publications and source records attributed to S L Singla-Pareek.

2 recordsLinked to original sources

Genetic engineering of the glyoxalase pathway in tobacco leads to enhanced salinity tolerance.

The glyoxalase pathway involving glyoxalase I (gly I) and glyoxalase II (gly II) enzymes is required for glutathione-based detoxification of methylglyoxal. We had earlier indicated the potential of gly I as a probable candidate gene in conferring salinity tolerance. We report here that overexpression of gly I+II together confers improved salinity tolerance, thus offering another effective strategy for manipulating stress tolerance in crop plants. We have overexpressed the gly II gene either alone in untransformed plants or with gly I transgenic background. Both types of these transgenic plants stably expressed the foreign protein, and the enzyme activity was also higher. Compared with nontransformants, several independent gly II transgenic lines showed improved capability for tolerating exposure to high methylglyoxal and NaCl concentration and were able to grow, flower, and set normal viable seeds under continuous salinity stress conditions. Importantly, the double transgenic lines always showed a better response than either of the single gene-transformed lines and WT plants under salinity stress. Ionic measurements revealed higher accumulation of Na+ and K+ in old leaves and negligible accumulation of Na+ in seeds of transgenic lines as compared with the WT plants. Comparison of various growth parameters and seed production demonstrated that there is hardly any yield penalty in the double transgenics under nonstress conditions and that these plants suffered only 5% loss in total productivity when grown in 200 mM NaCl. These findings establish the potential of manipulation of the glyoxalase pathway for increased salinity tolerance without affecting yield in crop plants.

Blotting, Southern↗

Directional genome walking using PCR.

We describe here a PCR-based "directional genome walking" protocol. The basic procedure for the amplification consists of two rounds of PCR. A primary PCR was performed, on the genomic DNA using a biotinylated primer specific to a known sequence in the genome along with four universal walker primers that were designed with partial degeneracy. The biotinylated primary PCR products were immobilized on streptavidin-linked paramagnetic beads. This step removed all nonspecific amplification products, and the purified template was used for the second PCR using a nested primer and the walker primer-2 to increase specificity. This technique is potentially useful for cloning promoter regions and has been successfully used to isolate 5'-flanking genomic regions of many cDNA clones previously isolated by us.

Base Sequence↗