Momilactones A (MA) and B (MB) are the active phytoalexins and allelochemicals in rice. In this study, MA and MB were purified from rice husk of Oryza sativa cv. Koshihikari by column chromatography, and purification was confirmed by high-performance liquid chromatography, thin-layer chromatography, gas chromatography-mass spectrometry, liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS), and 1H and 13C nuclear magnetic resonance analyses. By in vitro assays, both MA and MB exerted potent inhibition on α-amylase and α-glucosidase activities. The inhibitory effect of MB on these two key enzymes was greater than that of MA. Both MA and MB exerted greater α-glucosidase suppression as compared to that of the commercial diabetic inhibitor acarbose. Quantities of MA and MB in rice grain were 2.07 ± 0.01 and 1.06 ± 0.01 µg/dry weight (DW), respectively. This study was the first to confirm the presence of MA and MB in refined rice grain and reported the α-amylase and α-glucosidase inhibitory activity of the two compounds. The improved protocol of LC-ESI-MS in this research was simple and effective to detect and isolate MA and MB in rice organs.

Abstract:In this study, phenotypic variation and genetic diversity, important factors to decide germplasm for rice breeding, were evaluated among 15 rice mutants attained from the MNU (N-Nitroso-N-methylurea) mutation. The correlation coefficient values among these phenotypic characteristics were calculated. The results showed that full grain number per plant was the most relevant factor contributing to grain yield per plant, and grain length to width ratio was the key parameter affected to amylose content. Furthermore, the genetic variation among mutants was estimated by Simple Sequence Repeat (SSR) markers related to amylose content trait. Fifty-six polymorphism markers covering across eleven rice chromosomes were recorded with an average of 3.02 alleles per locus. The average value of polymorphism information content was 0.47. By using the unweighted pair group method with arithmetic mean (UPGMA) clustering, four clusters were generated with the genetic similarities ranging from 0.52 to 0.91. The variation among groups was 34%, while the variation among individuals within groups was 66%. Findings of this study provided useful genetic background and phenotypic information of collected rice mutants to breed rice cultivars with improved quality.

This study was the first to detect the presence of the two compounds momilactone A (MA) and momilactone B (MB) in rice bran using liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). By in vitro assays, both MA and MB exhibited potent inhibitory activities on pancreatic α-amylase and α-glucosidase which were significantly higher than γ-oryzanol, a well-known diabetes inhibitor. Remarkably, MA and MB indicated an effective inhibition on trypsin with the IC 50 values of 921.55 and 884.03 µg/mL, respectively. By high-performance liquid chromatography (HPLC), quantities of MA (6.65 µg/g dry weight) and MB (6.24 µg/g dry weight) in rice bran were determined. Findings of this study revealed the α-amylase, α-glucosidase and trypsin inhibitors MA and MB contributed an active role to the diabetes inhibitory potential of rice bran.

Difficulties in breeding new rice cultivars that have a high yield, are acceptable quality, and are tolerant to environmental stresses have been the major constraint of rice production in many developing countries, as these traits are determined by multiple genes associated with complicated and uncontrollable gene segregations.Furthermore, the gene/QTL (quantitative trait locus) introduced to the cultivar is unstable due to the interaction among the active genes, which determine the phenotypic performance, not yet been well understood or controllable. In this study, the N-methyl-N-nitrosourea (MNU)-induced mutation was applied to the heterozygote of the F1 generation from the cross between TBR1 (female) and KD18 (male parent). The phenotype and genotype of the M2 and M3 generations were evaluated and showed that the mutant population phenotypes, including the plant height, semi-dwarfism, amylose content, protein content, gel consistency, grain yield, and spikelet fertility, varied. Interestingly, no segregation among the genotypes in the M2 and M3 generations was observed, while the genotypes of the control population were either paternally inherited or indeterminable when using 28 polymorphism simple sequence repeat (SSR) markers that were identified on parental lines from 200 markers. The MNU-induced mutation caused maternal inheritance in the segregating populations, as primarily important agronomic traits were maternally succeeded from the female line TBR1. The findings of this study indicated that, through the use of MNU, the breeding of rice cultivars with close genetic backgrounds (similarity coefficient = 0.52) could be shortened by the maternal control of important qualities, such as pest and disease resistance and high yield, thus contributing to sustainable rice production for rice farmers. Further examination of rice cultivars with a greater difference in the genetic background should be subsequently conducted.

Salinity stress is one of the most problematic constraints to significantly reduce rice productivity. The Saltol QTL (quantitative trait locus) has been known as one among many principal genes/QTLs responsible for salinity tolerance in rice. However, the introgression of the Saltol QTL from the donor (male) into the recipient (female) cultivars induces great recessions from the progeny generation, which results in heavy fieldwork and greater cost and time required for breeding. In this study, the F1 generation of the cross TBR1 (female cultivar, salinity tolerant) × KD18 (male cultivar, salinity susceptible) was preliminarily treated with N-methyl-N-nitrosourea (MNU) to induce the mutants M1. Results on physiological traits show that all the M2 (self-pollinated from M1) and M3 (self-pollinated from M2) individuals obtain salinity tolerant levels as the recurrent TBR1. Twelve SSR (simple sequence repeat) markers involved in the Saltol QTL (RM493, RM562, RM10694, RM10720, RM10793, RM10852, RM13197, RM201, RM149, RM508, RM587, and RM589) and other markers related to yield-contributing traits and disease resistance, as well as water and nitrogen use, have efficacy that is polymorphic. The phenotype and genotype analyses indicate that the salinity tolerant Saltol QTL, growth parameter, grain yield and quality, pest resistance, water and nitrogen use efficacy, and beneficial phytochemicals including antioxidants, momilactone A (MA) and momilactone B (MB) are uniparentally inherited from the recurrent (female) TBR1 cultivar and stabilized in the M2 and M3 generations. Further MNU applications should be examined to induce the uniparental inheritance of other salinity tolerant genes such as OsCPK17, OsRMC, OsNHX1, OsHKT1;5 to target rice cultivars. However, the mechanism of inducing this novel uniparental inheritance for salinity tolerance by MNU application needs elaboration.