BackgroundRice genome sequencing projects have generated remarkable amount of information about genes and genome architecture having tremendous potential to be utilized in both basic and applied research. Success in transgenics is paving the way for preparing a road map of functional genomics which is expected to correlate action of a gene to a trait in cellular and organismal context. However, the lack of a simple and efficient method for transformation and regeneration is a major constraint for such studies in this important cereal crop.ResultsIn the present study, we have developed an easy, rapid and highly efficient transformation and regeneration protocol using mature seeds as explants and found its successful applicability to a choice of elite indica rice genotypes. We have optimized various steps of transformation and standardized different components of the regeneration medium including growth hormones and the gelling agent. The modified regeneration medium triggers production of large number of shoots from smaller number of calli and promotes their faster growth, hence significantly advantageous over the existing protocols where the regeneration step requires maximum time. Using this protocol, significantly higher transformation efficiency (up to 46%) and regeneration frequency (up to 92% for the untransformed calli and 59% for the transformed calli) were achieved for the four tested cultivars. We have used this protocol to produce hundreds of independent transgenic lines of different indica rice genotypes. Upon maturity, these transgenic lines were fertile thereby indicating that faster regeneration during tissue culture did not affect their reproductive potential.ConclusionsThis speedy, yet less labor-intensive, protocol overcomes major limitations associated with genetic manipulation in rice. Moreover, our protocol uses mature seeds as the explant, which can easily be obtained in quantity throughout the year and kept viable for a long time. Such an easy, efficient and generalized protocol has the potential to be a major tool for crop improvement and gene-function studies on the model monocot plant rice.

Cytokinins play a significant role in determining grain yield in plants. Cytokinin oxidases catalyse irreversible degradation of cytokinins and hence modulate cellular cytokinin levels. Here, we studied the role of an inflorescence meristem-specific rice cytokinin oxidase - OsCKX2 - in reducing yield penalty under salinity stress conditions. We utilized an RNAi-based approach to study the function of OsCKX2 in maintaining grain yield under salinity stress condition. Ultra-performance liquid chromatography-based estimation revealed a significant increase in cytokinins in the inflorescence meristem of OsCKX2-knockdown plants. To determine if there exists a correlation between OsCKX2 levels and yield under salinity stress condition, we assessed the growth, physiology and grain yield of OsCKX2-knockdown plants vis-à-vis the wild type. OsCKX2-knockdown plants showed better vegetative growth, higher relative water content and photosynthetic efficiency and reduced electrolyte leakage as compared with the wild type under salinity stress. Importantly, we found a negative correlation between OsCKX2 expression and plant productivity as evident by assessment of agronomical parameters such as panicle branching, filled grains per plant and harvest index both under control and salinity stress conditions. These results suggest that OsCKX2, via controlling cytokinin levels, regulates floral primordial activity modulating rice grain yield under normal as well as abiotic stress conditions.

Crop plants face a multitude of diverse abiotic and biotic stresses in the farmers' fields. Although there now exists a considerable knowledge of the underlying mechanisms of response to individual stresses, the crosstalk between response pathways to various abiotic and biotic stresses remains enigmatic. Here, we investigated if the cytotoxic metabolite methylglyoxal (MG), excess of which is generated as a common consequence of many abiotic and biotic stresses, may serve as a key molecule linking responses to diverse stresses. For this, we generated transgenic rice plants overexpressing the entire two-step glyoxalase pathway for MG detoxification. Through assessment of various morphological, physiological and agronomic parameters, we found that glyoxalase-overexpression imparts tolerance towards abiotic stresses like salinity, drought and heat and also provides resistance towards damage caused by the sheath blight fungus (Rhizoctonia solani) toxin phenylacetic acid. We show that the mechanism of observed tolerance of the glyoxalase-overexpressing plants towards these diverse abiotic and biotic stresses involves improved MG detoxification and reduced oxidative damage leading to better protection of chloroplast and mitochondrial ultrastructure and maintained photosynthetic efficiency under stress conditions. Together, our findings indicate that MG may serve as a key link between abiotic and biotic stress response in plants.

Marker-free transgenic lines of rice are developed with enhanced trehalose accumulation that is associated with improved grain yield under salinity, sodicity, and drought stresses.

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Salinity, one of the most deleterious stresses, affects growth and overall yield of crop plants. To identify new "candidate genes" having potential role in salinity tolerance, we have carried out 'functional screening' of a cDNA library (made from a salt tolerant rice-Pokkali). Based on this screening, we identified a cDNA clone that was allowing yeast cells to grow in the presence of 1.2 M NaCl. Sequencing and BLAST search identified it as mannose-1-phosphate guanyl transferase (OsMPG1) gene from rice. Analysis of rice genome sequence database indicated the presence of 3 additional genes for MPG. Out of four, three MPG genes viz. OsMPG1, 3 and 4 were able to functionally complement yeast MPG mutant -YDL055C. We have carried out detailed transcript profiling of all members of MPG family by qRT-PCR using two contrasting rice genotypes (IR64 and Pokkali) under different abiotic stresses (salinity, drought, oxidative stress, heat stress, cold or UV light). These MPG genes showed differential expression under various abiotic stresses with two genes (OsMPG1 and 3) showing high induction in response to multiple stresses. Analysis of rice microarray data indicated higher expression levels for OsMPG1 in specific tissues such as roots, leaves, shoot apical meristem and different stages of panicle and seed development, thereby indicating its developmental regulation. Functional validation of OsMPG1 carried out by overexpression in the transgenic tobacco revealed its involvement in enhancing salinity stress tolerance.

Glyoxalase system consists of two enzymes glyoxalase I (Gly I) and glyoxalase II (Gly II). Gly I detoxifies methylglyoxal (MG), a cytotoxic byproduct of glycolysis, to S-lactoylglutathione (SLG) where it uses one molecule of reduced glutathione. Subsequently, SLG is converted to lactate by Gly II and one molecule of reduced glutathione is recycled back into the system. The level of MG, which is produced ubiquitously in all living organisms, is enhanced upon exposure to different abiotic stresses in plants. Overexpression of glyoxalase pathway genes in transgenic plants has been found to keep a check on the MG level under stress conditions, regulate glutathione homeostasis, and the transgenic plants are able to survive and grow under various abiotic stresses.

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