ABSTRACT
Stomata regulates conductance, transpiration and photosynthetic traits in plants. Increased stomatal density may contribute to enhanced water loss and thereby help improve the transpirational cooling process and mitigate the high temperature-induced yield losses. However, genetic manipulation of stomatal traits through conventional breeding still remains a challenge due to problems involved in phenotyping and the lack of suitable genetic materials. Recent advances in functional genomics in rice identified major effect genes determining stomatal traits, including its number and size. Widespread applications of CRISPR/Cas9 in creating targeted mutations paved the way for fine tuning the stomatal traits for enhancing climate resilience in crops. In the current study, attempts were made to create novel alleles of OsEPF1 (Epidermal Patterning Factor), a negative regulator of stomatal frequency/density in a popular rice variety, ASD 16, using the CRISPR/Cas9 approach. Evaluation of 17 T0 progenies identified varying mutations (seven multiallelic, seven biallelic and three monoallelic mutations). T0 mutant lines showed a 3.7-44.3% increase in the stomatal density, and all the mutations were successfully inherited into the T1 generation. Evaluation of T1 progenies through sequencing identified three homozygous mutants for one bp insertion. Overall, T1 plants showed 54-95% increased stomatal density. The homozygous T1 lines (# E1-1-4, # E1-1-9 and # E1-1-11) showed significant increase in the stomatal conductance (60-65%), photosynthetic rate (14-31%) and the transpiration rate (58-62%) compared to the nontransgenic ASD 16. Results demonstrated that the genetic alterations in OsEPF1 altered the stomatal density, stomatal conductance and photosynthetic efficiency in rice. Further experiments are needed to associate this technology with canopy cooling and high temperature tolerance.
ABSTRACT
Traditional rice landraces are treasures for novel genes to develop climate-resilient cultivars. Seed viability and germination determine rice productivity under moisture stress. The present study evaluated 100 rice genotypes, including 85 traditional landraces and 15 improved cultivars from various agro-ecological zones of Tamil Nadu, along with moisture-stress-susceptible (IR 64) and moisture-stress-tolerant (IR 64 Drt1) checks. The landraces were screened over a range of osmotic potentials, namely (-) 1.0 MPa, (-) 1.25 MPa and (-) 1.5 MPa, for a period of 5 days in PEG-induced moisture stress. Physio-morphological traits, such as rate of germination, root and shoot length, vigor index, R/S ratio and relative water content (RWC), were assessed during early moisture stress at the maximum OP of (-) 1.5 MPa. The seed macromolecules, phytohormones (giberellic acid, auxin (IAA), cytokinin and abscisic acid), osmolytes and enzymatic antioxidants (catalase and superoxide dismutase) varied significantly between moisture stress and control treatments. The genotype Kuliyadichan registered more IAA and giberellic acid (44% and 35%, respectively, over moisture-stress-tolerant check (IR 64 Drt1), whereas all the landraces showed an elevated catalase activity, thus indicating that the tolerant landraces effectively eliminate oxidative damages. High-performance liquid chromatography analysis showed a reduction in cytokinin and an increase in ABA level under induced moisture stress. Hence, the inherent moisture-stress tolerance of six traditional landraces, such as Kuliyadichan, Rajalakshmi, Sahbhagi Dhan, Nootripathu, Chandaikar and Mallikar, was associated with metabolic responses, such as activation of hydrolytic enzymes, hormonal crosstalk, ROS signaling and antioxidant enzymes (especially catalase), when compared to the susceptible check, IR 64. Hence, these traditional rice landraces can serve as potential donors for introgression or pyramiding moisture-stress-tolerance traits toward developing climate-resilient rice cultivars.
