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Khan M.S.,University of Agriculture at Faisalabad | Kanwal B.,National Institute for Biotechnology and Genetic Engineering NIBGE | Nazir S.,Agricultural Biotechnology Research Institute
Frontiers in Plant Science | Year: 2015

Osmoprotectants stabilize proteins and membranes against the denaturing effect of high concentrations of salts and other harmful solutes. In yeast, arabitol dehydrogenase (ArDH) reduces D-ribulose to D-arabitol where D-ribulose is derived by dephosphorylating D-ribulose-5-PO4 in the oxidized pentose pathway. Osmotolerance in plants could be developed through metabolic engineering of chloroplast genome by introducing genes encoding polyols since chloroplasts offer high level transgene expression and containment. Here, we report that ArDH expression in tobacco chloroplasts confers tolerance to NaCl (up to 400 mM). Transgenic plants compared to wild type (WT) survived for only 4-5 weeks on 400 mM NaCl whereas plants remained green and grew normal on concentrations up to 350 mM NaCl. Further, a-week-old seedlings were also challenged with poly ethylene glycol (PEG, up to 6%) in the liquid medium, considering that membranes and proteins are protected under stress conditions due to accumulation of arabitol in chloroplasts. Seedlings were tolerant to 6% PEG, suggesting that ARDH enzyme maintains integrity of membranes in chloroplasts under drought conditions via metabolic engineering. Hence, the gene could be expressed in agronomic plants to withstand abiotic stresses. © 2015 Khan, Kanwal and Nazir. Source

Ullah I.,Agricultural Biotechnology Research Institute
Genetics and molecular research : GMR | Year: 2012

Aromatic basmati rice is vulnerable to bacterial blight disease. Genes conferring resistance to bacterial blight have been identified in coarse rice; however, their incorporation into basmati varieties compromises the prized basmati aroma. We identified bacterial blight resistance genes Xa4, xa5, Xa7, and xa13 in 52 basmati landraces and five basmati cultivars using PCR markers. The Xa7 gene was found to be the most prevalent among the cultivars and landraces. The cultivars Basmati-385 and Basmati-2000 also contained the Xa4 gene; however, xa5 and xa13 were confined to landraces only. Ten landraces were found to have multiple resistance genes. Landraces Basmati-106, Basmati-189 and Basmati-208 contained Xa4 and Xa7 genes. Whereas, landraces Basmati-122, Basmati-427, Basmati-433 were observed to have xa5 and Xa7 genes. Landraces Basmati-48, Basmati-51A, Basmati-334, and Basmati-370A possessed Xa7 and xa13 genes. The use of landraces containing recessive genes xa5 and xa13 as donor parents in hybridization with cultivars Basmati-385 and Basmati-2000, which contain the genes Xa4 and Xa7, will expedite efforts to develop bacterial blight-resistant basmati rice cultivars through marker assisted selection, based on a pyramiding approach, without compromising aroma and grain quality. Source

Ullah I.,Agricultural Biotechnology Research Institute
Genetics and molecular research : GMR | Year: 2012

The popularity of genetically modified insect resistant (Bt) cotton has promoted large scale monocultures, which is thought to worsen the problem of crop genetic homogeneity. Information on genetic diversity among Bt cotton varieties is lacking. We evaluated genetic divergence among 19 Bt cotton genotypes using simple sequence repeat (SSR) markers. Thirty-seven of 104 surveyed primers were found informative. Fifty-two primers selected on the basis of reported intra-hirsutum polymorphism in a cotton marker database showed a high degree of polymorphism, 56% compared to 13% for randomly selected primers. A total of 177 loci were amplified, with an average of 1.57 loci per primer, generating 38 markers. The amplicons ranged in size from 98 to 256 bp. The genetic similarities among the 19 genotypes ranged from 0.902 to 0.982, with an average of 0.947, revealing a lack of diversity. Similarities among genotypes from public sector organizations were higher than genotypes developed by private companies. Hybrids were found to be more distant compared to commercial cultivars and advanced breeding lines. Cluster analysis grouped the 19 Bt cotton genotypes into three major clusters and two independent entries. Cultivars IR-3701, Ali Akbar-802 and advanced breeding line VH-259 grouped in subcluster B2, with very narrow genetic distances despite dissimilar parentage. We found a very high level of similarity among Pakistani-bred Bt cotton varieties, which means that genetically diverse recurrent parents should be included to enhance genetic diversity. The intra-hirsutum polymorphic SSRs were found to be highly informative for molecular genetic diversity studies in these cotton varieties. Source

Liu S.,University of California at Riverside | Dunwell T.L.,Beckman Research Institute | Pfeifer G.P.,Beckman Research Institute | Dunwell J.M.,University of Reading | And 3 more authors.
PLoS ONE | Year: 2013

Epigenetic regulations play important roles in plant development and adaptation to environmental stress. Recent studies from mammalian systems have demonstrated the involvement of ten-eleven translocation (Tet) family of dioxygenases in the generation of a series of oxidized derivatives of 5-methylcytosine (5-mC) in mammalian DNA. In addition, these oxidized 5-mC nucleobases have important roles in epigenetic remodeling and aberrant levels of 5-hydroxymethyl-2′-deoxycytidine (5-HmdC) were found to be associated with different types of human cancers. However, there is a lack of evidence supporting the presence of these modified bases in plant DNA. Here we reported the use of a reversed-phase HPLC coupled with tandem mass spectrometry method and stable isotope-labeled standards for assessing the levels of the oxidized 5-mC nucleosides along with two other oxidatively induced DNA modifications in genomic DNA of Arabidopsis. These included 5-HmdC, 5-formyl-2′- deoxycytidine (5-FodC), 5-carboxyl-2′-deoxycytidine (5-CadC), 5-hydroxymethyl-2′-deoxyuridine (5-HmdU), and the (5′S) diastereomer of 8,5′-cyclo-2′-deoxyguanosine (S-cdG). We found that, in Arabidopsis DNA, the levels of 5-HmdC, 5-FodC, and 5-CadC are approximately 0.8 modifications per 106 nucleosides, with the frequency of 5-HmdC (per 5-mdC) being comparable to that of 5-HmdU (per thymidine). The relatively low levels of the 5-mdC oxidation products suggest that they arise likely from reactive oxygen species present in cells, which is in line with the lack of homologous Tet-family dioxygenase enzymes in Arabidopsis. © 2013 Liu et al. Source

Nawaz M.,Government College University at Faisalabad | Iqbal N.,Government College University at Faisalabad | Idrees S.,Government College University at Faisalabad | Ullah I.,Agricultural Biotechnology Research Institute
Turkish Journal of Botany | Year: 2014

The family of AP2/ERF plant specific transcription factors, including dehydration-responsive element binding (DREB) proteins, has been reported to activate and bind with the dehydration responsive element/C repeat (DRE/CRT) of stress-inducible gene promoters. We amplified DREB1A from Oryza sativa var. IR6 (807 bp) and subjected it to protein homology modelling and phylogenetic analysis. BLAST sequence analysis indicated high similarity (99%) with the O. sativa japonica group. Phylogenetic analysis was carried out with other 12 DREB sequences from cereals/grasses using Clustal Omega. Protein secondary structure and 3D models were determined using SOPMA and MODELLER v9.10, respectively. PROSA was used to determine the excellence and consistency of models. The results indicated that the Z-score value was –2.94 and 79.5% of the residues were found in the favoured region. A psi/phi Ramachandran plot was determined using PROCHECK. The molecular docking of the GCC-box binding domain (GBD) of the protein under investigation was done using atomic coordinates of Arabidopsis thaliana GBD- and GCC-box containing DNA, respectively. The tertiary structure of the modelled protein and template were found to be very similar as predicted by a root mean square deviation of 0.968 Å. It seemed that both the proteins interacted with the major groove of DNA using β-sheets. We attempted to expose the molecular basis of O. sativa var. IR6 DREB1A protein interaction with the target promoter sequence. The results highlighted that this gene could be a good candidate for production of abiotic stress-tolerant crop(s) in the future. © TÜBİTAK. Source

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