In Vitro Gamma Mutagenesis Techniques in Rice (Oryza sativa L. var. Lazarroz FL).

Gamma radiation (60Co)-induced mutagenesis offers an alternative to develop rice lines by accelerating the spontaneous mutation process and increasing the pool of allelic variants available for breeding. Ionizing radiation works by direct or indirect damage to DNA and subsequent mutations. The technique can take advantage of in vitro protocols to optimize resources and accelerate the development of traits. This is achieved by exposing mutants to a selection agent of interest in controlled conditions and evaluating large numbers of plants in reduced areas. This chapter describes the protocol for establishing gamma radiation dosimetry and in vitro protocols for optimization at the laboratory level using seeds as the starting material, followed by embryogenic cell cultures, somatic embryogenesis, and regeneration. The final product of the protocol is a genetically homogeneous population of Oryza sativa that can be evaluated for breeding against abiotic and biotic stresses.

A Temporary Immersion System Improves Regeneration of In Vitro Irradiated Recalcitrant Indica Rice (Oryza sativa L.) Embryogenic Calli.

The development of gamma ray-mutated rice lines is a solution for introducing genetic variability in indica rice varieties already being used by farmers. In vitro gamma ray (60Co) mutagenesis reduces chimeras and allows for a faster selection of desirable traits but requires the optimization of the laboratory procedure. The objectives of the present work were sequencing of matK and rbcL, the in vitro establishment of recalcitrant rice embryogenic calli, the determination of their sensitivity to gamma radiation, and optimization of the generation procedure. All sequenced genes matched perfectly with previously reported matK and rbcL O. sativa genes. Embryogenic calli induction improved using MS medium containing 2 mg L-1 2,4-D, and regeneration was achieved with MS medium with 3 mg L-1 BA and 0.5 mg L-1 NAA. The optimized radiation condition was 60 Gy, (LD20 = 64 Gy) with 83% regeneration. An immersion system (RITA®, Saint-Mathieu-de-Tréviers, France) of either 60 or 120 s every 8 h allowed systematic and homogeneous total regeneration of the recalcitrant line. Other well-known recalcitrant cultivars, CR1821 and CR1113, also had improved regeneration in the immersion system. To our knowledge, this is the first study reporting the use of an immersion system to allow for the regeneration of gamma-ray mutants from recalcitrant indica rice materials.

In vitro attenuation of a virulent swine isolate of Brachyspira hampsonii.

Brachyspira hampsonii causes dysentery-like disease in infected pigs. Serial passage of a virulent swine isolate (P13) one-hundred times in laboratory culture medium was conducted to produce an attenuated strain, and to identify genomic determinants of virulence through comparison of genome sequences of the original and passaged strains. The resulting strain, P113, did not differ from P13 in terms of diagnostic biochemical characteristics but had an enhanced growth rate in culture, indicating laboratory adaptation. Whole genome sequencing of P113 revealed several single-nucleotide changes including a T to C transition that results in an R to G amino acid change in a putative mannose-1-phosphate guanylytransferase that is implicated in production of lipo-oligosaccharide. P113 was partially attenuated in a mouse model of infection, indicated by significantly fewer observations of abnormal feces in mice infected with P113 relative to P13. No differences were detected in bacterial shedding in feces, demonstrating that the ability of the organism to colonize mice was not affected. Passage through a mouse did not further alter the virulence of P113. Results of this study provide insight into genomic determinants of virulence in B. hampsonii and a live attenuated vaccine candidate.

An optimized swine dysentery murine model to characterize shedding and clinical disease associated with "Brachyspira hampsonii" infection.

BACKGROUND: The development of a mouse model as an in vivo pathogenicity screening tool for Brachyspira spp. has advanced the study of these economically important pathogens in recent years. However, none of the murine models published to date have been used to characterize the clinical signs of disease in mice, instead focusing on pathology following oral inoculation with various Brachyspira spp. The experiments described herein explore modifications of published models to characterize faecal consistency, faecal shedding and pathology in mice challenged with "Brachyspira hampsonii" clade II (Bhamp). METHODS AND RESULTS: In Experiment 1, 24 CF-1 mice were randomly allocated to one of three inoculation groups: sham (Ctrl), Bhamp, or B. hyodysenteriae (Bhyo; positive control). Half of each group was fed normal mouse chow (RMH) while the other received a low-zinc diet (TD85420). In Experiment 2, eight CF-1 mice and nine C3H/HeN mice were divided into Ctrl or Bhamp inoculation groups, and all fed TD85420. In Experiment 1, mice fed TD85420 demonstrated more severe mucoid faeces (P = 0.001; Kruskal Wallis) and faecal shedding for a significantly greater number of days (P = 0.005; Kruskal Wallis). Mean faecal scores of Bhamp inoculated mice trended higher than Ctrl (P = 0.06; Wilcoxon rank-sum) as did those of Bhyo mice (P = 0.0; Wilcoxon rank-sum). In Experiment 2, mean faecal scores of inoculated CF-1 mice were significantly greater than in C3H mice (P = 0.049; Kruskal Wallis) but no group differences in faecal shedding were observed. In both experiments, mice clustered based on the severity of colonic and caecal histopathology but high lesion scores were not always concurrent with high fecal scores. CONCLUSION: In our laboratory, CF-1 mice and the lower-zinc TD85420 diet provide a superior murine challenge model of "Brachyspira hampsonii" clade II.