Development of a phenotyping protocol for combined drought and salinity stress at seedling stage in rice

Introduction: The case of combined drought and salinity stress is increasingly becoming a constraint to rice production, especially in coastal areas and river deltas where low rainfall not only reduces soil moisture levels but also reduces the flow of river water, resulting in intrusion of saline sea-water. A standardized screening method is needed in order to systematically evaluate rice cultivars under combined drought+salinity at the same time because sequential stress of salinity followed by drought or vice-versa is not similar to simultaneous stress effects. Therefore, we aimed to develop a screening protocol for combined drought+salinity stress applied to soil-grown plants at seedling stage. Methods: The study system used 30-L soil-filled boxes, which allowed a comparison of plant growth under control conditions, individual drought and salinity stress, as well as combined drought+salinity. A set of salinity tolerant and drought tolerant cultivars were tested, together with several popular but salinity and drought-susceptible varieties that are grown in regions prone to combined drought+salinity. A range of treatments were tested including different timings of the drought and salinity application, and different severities of stress, in order to determine the most effective that resulted in visible distinction among cultivars. The challenges related to determining a protocol with repeatable seedling stage stress treatment effects while achieving a uniform plant stand are described here. Results: The optimized protocol simultaneously applied both stresses by planting into saline soil at 75% of field capacity which was then allowed to undergo progressive drydown. Meanwhile, physiological characterization revealed that chlorophyll fluorescence at seedling stage correlated well with grain yield when drought stress was applied to vegetative stage only. Discussion: The drought+salinity protocol developed here can be used for screening rice breeding populations as part of a pipeline to develop new rice varieties with improved adaptation to combined stresses

Characterizing the Saltol quantitative trait locus for salinity tolerance in rice

This study characterized Pokkali-derived quantitative trait loci (QTLs) for seedling stage salinity tolerance in preparation for use in marker-assisted breeding. An analysis of 100 SSR markers on 140 IR29/Pokkali recombinant inbred lines (RILs) confirmed the location of the Saltol QTL on chromosome 1 and identified additional QTLs associated with tolerance. Analysis of a series of backcross lines and near-isogenic lines (NILs) developed to better characterize the effect of the Saltol locus revealed that Saltol mainly acted to control shoot Na +/K + homeostasis. Multiple QTLs were required to acquire a high level of tolerance. Unexpectedly, multiple Pokkali alleles at Saltol were detected within the RIL population and between backcross lines, and representative lines were compared with seven Pokkali accessions to better characterize this allelic variation. Thus, while the Saltol locus presents a complex scenario, it provides an opportunity for markerassisted backcrossing to improve salt tolerance of popular varieties followed by targeting multiple loci through QTL pyramiding for areas with higher salt stress

Stomatal responses in rainfed lowland rice to partial soil drying; evidence for root signals

The role of root signals in water deficit responses of rice (Oryza saliva L.) is important in the alternate flooding and drying conditions encountered in the rainfed lowlands, where the abundant roots in shallow soil layers may generate signals when droughted, with consequent reduction in stomatal conductance (g(s)) and growth, despite the likelihood of additional water in deeper soil layers. This study was conducted to confirm the presence of root signals, explore their nature and plant responses, consider the suitability of the methods, and discuss implications for adaptation under rainfed lowland drought. A split-root technique was used in greenhouse studies, whereby roots were divided into two sections: flooded and droughted. The decrease in g(s) and transpiration rate (Tr) due to drying of a portion of the roots, and their apparent recovery upon severing of this root portion, were consistent with the role for signals. The field study confirmed the evidence for root signals during progressive soil drying, whereby g(s) and Tr decreased before leaf water potential (Psi(L)) started to decline. The increase in leaf ABA concentration under field drought, and its strong association with soil moisture tension and g(s), suggested its involvement in mediating stornatal responses during early drought in rice. The recovery in Psi(L) after severing of droughted roots in the greenhouse could be attributed to increased hydraulic conductance. These responses imply a role for both chemical and hydraulic signals in rice, which have important implications for adaptation and crop performance in contrasting rice ecosystems

Stomatal responses in rainfed lowland rice to partial soil drying; comparison of two lines

Previously; we demonstrated that root Ups in drying soil communicate with shoots for stomatal closure. hi rainfed Wand rice, despite further water being available at depth. This study examines variation between two lines in root. signals. Rice lines CT9993 and IR62266 were grown in the held, and in die greenhouse with the split-root root-sever wax-layer system, to investigate their responses to mild and severe water deficit by monitoring stomatal conductance leaf water potential and leaf ABA concentration, In We greenhouse root systems were divided, withholding water from one portion, and in some cases, severing the droughted portion of roots to remove be signal. Wax layers differing in strength were placed at hardpan depth. Roots of CT9993 were better able to penetrate the wax layers. IR62266 exhibited stronger than CT9993 with W62266's stomatal conductance dropping sharply under water deficit, and recovering at slower rates but less completely, when roots subjected to drying soil were severed. The greater stomatal response in IR62266 was associated with a higher leaf, ABA concentration during Car, inner deficit which in turn was associated with its greater number or roots in drying soil In the Held, a second reduction hi g was observed under severe water deficit, with stronger signals in IR62266 associated with more conservative water toe as soil drying intensified. To better exploit subsoil water in mild or transient water deficit, selection for reduced root signals might he warranted

Newly Identified Wild Rice Accessions Conferring High Salt Tolerance Might Use a Tissue Tolerance Mechanism in Leaf

Cultivated rice (Oryza sativa\ua0L.) is very sensitive to salt stress. So far a few rice landraces have been identified as a source of salt tolerance and utilized in rice improvement. These tolerant lines primarily use Na+\ua0exclusion mechanism in root which removes Na+\ua0from the xylem stream by membrane Na+\ua0and K+\ua0transporters, and resulted in low Na+\ua0accumulation in shoot. Identification of a new donor source conferring high salt tolerance is imperative. Wild relatives of rice having wide genetic diversity are regarded as a potential source for crop improvement. However, they have been less exploited against salt stress. Here, we simultaneously evaluated all 22 wild\ua0Oryza\ua0species along with the cultivated tolerant lines including Pokkali, Nona Bokra, and FL478, and sensitive check varieties under high salinity (240 mM NaCl). Based on the visual salt injury score, three species (O.\ua0alta, O.\ua0latifolia, and\ua0O.\ua0coarctata) and four species (O.\ua0rhizomatis, O.\ua0eichingeri, O.\ua0minuta, and\ua0O.\ua0grandiglumis) showed higher and similar level of tolerance compared to the tolerant checks, respectively. All three CCDD genome species exhibited salt tolerance, suggesting that the CCDD genome might possess the common genetic factors for salt tolerance. Physiological and biochemical experiments were conducted using the newly isolated tolerant species together with checks under 180 mM NaCl. Interestingly, all wild species showed high Na+\ua0concentration in shoot and low concentration in root unlike the tolerant checks. In addition, the wild-tolerant accessions showed a tendency of a high tissue tolerance in leaf, low malondialdehyde level in shoot, and high retention of chlorophyll in the young leaves. These results suggest that the wild species employ tissue tolerance mechanism to manage salt stress. Gene expression analyses of the key salt tolerance-related genes suggested that high Na+\ua0in leaf of wild species might be affected by\ua0OsHKT1;4-mediated Na+\ua0exclusion in leaf and the following Na+\ua0sequestration in leaf might be occurring independent of tonoplast-localized OsNHX1. The newly isolated wild rice accessions will be valuable materials for both rice improvement to salinity stress and the study of salt tolerance mechanism in plants