No abstract

Rice yield is severely affected by high-salt concentration in the vicinity of the plant. In an effort to engineer rice for improved salt tolerance Agrobacteriummediated transformation of rice cv. Binnatoa was accomplished with the Pennisetum glaucum vacuolar Na ? /H ? antiporter gene (PgNHX1) under the constitutive CaMV35S promoter. For the molecular analysis of putative transgenic plants, PCR and RT-PCR were performed. Transgenic rice plants expressing PgNHX1 showed better physiological status and completed their life cycle by setting flowers and seeds in salt stress, while wild-type plants exhibited rapid chlorosis and growth inhibition. Moreover, transgenic rice plants produced higher grain yields than wild-type plants under salt stress. Assessment of the salinity tolerance of the transgenic plants at seedling and reproductive stages demonstrated the potential of PgNHX1 for imparting enhanced salt tolerance capabilities and improved yield.

In an effort to improve salinity tolerance in rice, the Pennisetum glaucum vacuolar Na + /H + antiporter gene (PgNHX1) was transformed and expressed in Bangladeshi rice Binnatoa under the constitutive promoter CaMV35S. Transgenic status of the plant was confirmed by PCR and Southern blot hybridization. RT-PCR and Western dot-blot analyses were performed to validate the expression of the transgene at RNA and protein levels, respectively. At 160 mM (ECe of 16 dS/m) NaCl stress the transgenic seedlings showed enhanced tolerance compared to controls.

Salinity is one of the most critical environmental problems, which causes plant growth retardation by disturbing intracellular ion homeostasis. The Na + /H + antiporter plays an important role in resistance to salt stress by sequestering Na + in exchange for H + across the vacuolar membranes. In the current study, the coding regions of two Arabidopsis antiporters (AtNHX1 and AtNHX2) and one rice antiporter (OsNHX1) were amplified by target specific PCR. PCR amplicons were first cloned into pENTR/D-TOPO and later recombined with a destination vector (pK7WG2.0) by LR reaction. Positive clones were selected by PCR, restriction digestion (RD) and sequencing. They were then transformed into Agrobacterium tumefaciens (LBA4404 strain) for subsequent transformation of farmer popular tomato varieties.

The GATEWAY TM Binary Destination Vector pH7WG2 is available for easy insertion of genes for transformation into plants. The gene of interest integrates downstream of the Cauliflower Mosaic Virus Promoter CaMV 35S by recombination. The CaMV 35S promoter is however not suitable for transformation and expression of genes in monocots like rice. We isolated and cloned a ~1100 bp upstream region from two rice (Pokkali and IR64) Na/H antiporter genes into the GATEWAY TM promoter cloning vector pHGWFS7. The Pokkali promoter expressed the β-glucuronidase or GUS gene ~25-fold more efficiently than the CaMV 35S promoter in rice calli, while that of IR64 was 7-fold more. The IR64 promoter however showed efficient expression in transgenic rice leaves. The promoter from Pokkali Na/H antiporter was used to replace the CaMV 35S sequence in pH7WG2. The CaMV 35S region was cut out and the linear vector fragment blunted and T-tailed. After amplification of the promoter from Pokkali rice DNA, it was A-tailed and ligated to the modified T-vector. The resultant vector, named pH7WG3, following the nomenclature at the gateway site, www.plantgenetics.rug.ac.be/gatewayT, can now be used for recombination of any genes for efficient rice transformation.