Abstract
For understanding the crucial Na+ uptake mechanism in rice plants imparting salinity tolerance, the experiments were conducted during 2014-18 on assessing salt tolerance performance of the genotypes based on Nat: K+ ion estimation, gene expression analysis, salt tolerant gene isolation and finally, by cloning and transforming salt tolerant gene (s). Sixty rice genotypes were screened against the increasing levels of salinity up to 15dSm-1(-150mM) and evaluated with the SES scores at 14 and 28 days after sowing. In the first instance, 13 genotypes were found tolerant, 7 susceptible and the rest (40) moderately tolerant. With exposure of the genotypes to stress longer, the tolerance level of the genotypes decreased further. The indica rice cultivar Pokkali appeared as the most tolerant and BRRI dhan29, the most susceptible. Further study evidenced that Pokkali and FL-478 (both salt tolerant) had higher root and shoot dry weights, in turn, higher total dry matter at all salinity levels than in BRRI dhan29. The uptake of Na+ and K+ through root and their transport in shoot and leaf, as estimated by the fluorescence microscopy and atomic absorption spectrophotometer, were lower in the salt tolerant rice cultivars Pokkali, BRRI dhan47, Binadhan-8 and Binadhan-10 compared to salt sensitive BRRI dhan29. Salinity stress induced higher K+/Na+ ratio in the salt tolerant genotypes having the highest value of cytosolic Na+/K+ homeostasis in Pokkali with increased K+ and decreased Na+ uptake through the induction of genes and possibly as by increasing the compartmentalization of cytosolic Na+ into the vacuoles as traced through the Na+/H+ aniporter activities of OsNHX1 and OsNHX2 genes. The expressions of the genes [OsNHX1, OsNHX2, OsSOS1 and OsHKT8 (OsHKT1;5)] conferring salinity tolerance showed variable expressions at different time points of salt stress in both Pokkali and BRRI dhan29. Up-regulations of OsNHX1, OsNHX2, OSSOS1 and OsHKT8 (OsHKT1;5) were found in Pokkali but not in BRRI dhan29. The high abundance of OSSOS1 transcript level of Pokkali was accompanied by a lower Na+ accumulation in comparison with BRRI dhan29. The high affinity K+ transporters (HKT) prevented shoot Na+ over-accumulation by mediating possibly by Na+ exclusion as evidenced by the over-expression of OsHKT1;5. The salinity induced cDNA was prepared from Pokkali, the coding regions of OsNHX1, OsNHX2, OSSOS1 and OsHKT8 (OsHKT1;5) genes were amplified and cloned into the entry vector PDONOR221 using BP Clonase through Gateway Cloning Technology. The entry clones were transformed into E. coli (DH5a) by heat-shock method and transformed colonies were confirmed by the colony PCR. The gene constructs were cloned into plant expression/destination vector pB2WG7 containing cauliflower mosaic virus. 35S promoter by using LR Clonase and mobilized into Agrobacterium tumefaciens strain GV3101. Several putative transgenic rice lines were obtained by Agrobacterium harbouring OsNHX2 gene from the rice genotype IR64, of which three transgenic lines viz., OE1, OE2 and OE3 were confirmed by PCR.