Production of 2n pollen of Asiatic hybrid lilies by nitrous oxide treatment

Tetraploid varieties of lilies have superior agronomic traits such as large flowers and resistance to physiological disorders. In the present study, we attempted to induce 2n pollen of Asiatic hybrid lilies by arresting the meiotic process with nitrous oxide (N₂O) gas. To determine which meiotic stage is optimal for induction of 2n pollen, plants with attached buds at different meiotic stages were treated with N₂O for 24 h in a pressure-tolerant cylinder. A few 2n pollen grains were induced using plants with anthers in prophase I, whereas mixed pollen grains of differing size were produced using plants undergoing meiotic metaphase predominantly in anthers. Although normal lily pollen grains are elliptical, nitrous oxide exposure induced giant pollen grains that appeared spherical. Flow cytometry analysis showed that the giant pollen grains were diploid. When mixed pollen that included normal and giant pollen was crossed to tetraploid cultivars, the resulting seedlings were tetraploid and aneuploid, indicating that the giant pollen grains were diploids that could generate tetraploid seedlings through fusion to diploid eggs supplied from a tetraploid female parent. Thus, treatment with N₂O is useful for the production of 2n lily pollen and may provide a new approach for tetraploid lily breeding.     

N2O induces mitotic polyploidization in anther somatic cells and restores fertility in sterile interspecific hybrid lilies

Fertile plants undergoing male gametogenesis can be treated with nitrous oxide (N₂O) gas to obtain 2n male gametes. N₂O treatment is also expected to restore the fertility of interspecific hybrids through meiotic restitution or mitotic amphidiploidization. However, this technique has few applications to date, and it is un-known how N₂O treatment restores fertility in sterile hybrids. To establish optimal N₂O treatment conditions and determine its cytological mechanism of action, we treated various sized floral buds with N₂O gas at different anther developmental stages from fertile and sterile hybrid lilies. N₂O treatment using the optimal 1–4 mm floral buds induced mitotic polyploidization of male archesporial cells to produce 2n pollen in fertile hybrid lilies. In sterile hybrid lilies, N₂O treatment doubled the chromosome number in male archesporial cells followed by homologous chromosome pairing and normal meiosis in pollen mother cells (PMC), resulting in restoration of pollen fertility. Backcrossing the resultant fertile pollen to Lilium × formolongi produced many triploid BC₁ plants. Thus N₂O treatment at the archesporial cell proliferating stage effectively overcame pollen sterility in hybrid lilies, resulting in fertile, 2n pollen grains that could produce progeny. The procedure presented here will promote interspecific or interploidy hybridization of lilies.     

Different effects of ionic and non-ionic compounds on the freeze denaturation of myofibrils and myosin subfragment-1

The effects of non-ionic (sorbitol, maltose, trehalose) and ionic compounds (Na-glutamate, Na-acetate, Na-sulfate, ammonium sulfate) on freeze denaturation of myosin subfragment-1 (S-1) and of myofibrils were compared. Sugars, Na-glutamate and Na-acetate well suppressed the freeze denaturation of myofibrils as well as S-1 in a concentration dependent manner. Although sulfate suppressed freeze denaturation of S-1 irregularly, it accelerated myofibril denaturation. It was concluded that sulfate salts were useless as cryoprotectant for myofibrils. Stabilization extent by F-actin in frozen storage was much less than that in heating.     

Effects of microbial growth on the preservation of scallop muscle in a vital state

ABSTRACT:   Adductor muscles dissected from live scallop Patinopecten yessoensis were stored in oxygenated artificial sea water. The initial muscle adenosine triphosphate (ATP) level, approximately 7.5 µmol/g, remained longer at 5°C than at either 0 or 10°C. The pH of sea water decreased continuously and the consumption of dissolved oxygen increased even after muscle ATP was almost exhausted. The number of viable microbes, measured as colony-forming units (c.f.u.) in the muscle, increased to reach a plateau at approximately 10⁷−10⁸ c.f.u./g, while muscle ATP remained at high levels. After this time, muscle ATP sharply decreased. Antibiotics or sorbate added into the oxygenated sea water effectively inhibited both the growth of microbes and the decrease in the pH of sea water. Under these conditions, the retention period of muscle ATP was greatly extended. Thus, it seems most likely that scallop adductor muscle cells are suffocated by the limitation of oxygen supply caused by aerobic microbes grown on the surface of muscle tissue.     

Seasonal changes and controlling factors of gross N transformation in an evergreen plantation forest in central Japan

We conducted a year-round measurement of gross N transformation rates using the ¹⁵N dilution method, and analyzed seasonal changes and the mechanisms regulating gross N transformation in the Kiryu Experimental Forest in central Japan. While soil microbial biomass C (SMB-C) decreased from the dormant to growing seasons at the organic (O) horizon, no significant trend was observed in SMB-N. This resulted in SMB-C/N being high in the dormant season and low in the growing season, and suggests that the microbial composition changed seasonally. No clear seasonal trend was found in gross NH₄ ⁺ production rates at either the O or surface mineral soil horizons. In contrast, the NH₄ ⁺ consumption rate varied seasonally, with high values in January and April during the dormant season and low values in July and October during the growing season. There was no clear trend in seasonal fluctuation of net NH₄ ⁺ production rates. Gross NH₄ ⁺ production and gross NH₄ ⁺ consumption rates were 10 times greater than the gross nitrification rate. Almost all of the produced NH₄ ⁺ was immobilized, indicating that N tightly cycles at this study site. Considered together with results of the gross N transformation rates, the dominance of high SMB-C/N microbes might stimulate immobilization in the dormant season. At this study site, the change in microbial composition likely influences gross N transformation through immobilization efficiency.     

Repeated reaching of the harmful algal bloom of Karenia spp. around the Pacific shoreline of Kushiro,eastern Hokkaido,Japan, during autumn 2021

Unprecedented large-scale algal blooms were observed during autumn 2021 around the Katsurakoi fishing port, Kushiro, eastern Hokkaido, Japan. Monitoring of shoreline water showed that chlorophyll a (Chl a) concentrations and the cell density of Karenia spp., dominated by Karenia selliformis, repeatedly increased synchronously between September and November 2021. These increases were associated with a southerly wind-driven current, which transported offshore water on the shelf towards the shoreline at the sea surface. The blooms were prolonged as a result of algal accumulation in the semi-closed fishing port. The maximum Chl a concentration and cell density exceeded 50 µg Chl a/L and 10⁴ cells/mL, respectively. During the autumn bloom of Karenia spp., the nitrate?+?nitrite and phosphate concentrations in the water were lower than those in 2019 and 2020, and the silicate concentration was comparable. The ammonium concentration during the bloom was notably higher than before the bloom period, reaching 15 µM. Mass mortality of several fish species and echinoderms that were cultured using rearing water intake from the same shoreline occurred synchronously with the increase in Karenia spp.

     

The Colletotrichum higginsianum secreted effector protein ChEC91 induces plant cell death

ChEC91, a novel cell death-inducing effector protein from the fungal pathogen Colletotrichum higginsianum, causal agent of crucifer anthracnose disease, is described. Both transient expression of ChEC91 and infiltration of purified recombinant protein induced necrotic lesions in Nicotiana benthamiana leaves. The recombinant protein also induced electrolyte leakage and callose deposition in Arabidopsis thaliana leaf tissue and the expression of defence marker genes. Moreover, fungal mutants constitutively over-expressing ChEC91 in C. higginsianum were impaired in appressorial penetration on Brassica rapa cotyledons. These results suggest that inappropriate expression of ChEC91 might negatively affect the early stage of C. higginsianum infection by inducing plant defence responses. Protein domain deletion analysis showed that the C-terminal region of ChEC91 was necessary, but not sufficient, for activity in N. benthamiana. Homologous effector proteins cloned from C. gloeosporioides, Fusarium graminearum, and Pyricularia oryzae differed in their cell death-inducing activity, which appeared related to sequence variations in the C-terminal region of these proteins. Moreover, this region contained amino acid residues that were well conserved within Colletotrichum species. These results suggest that the amino acid residues in the C-terminal region may be important for inducing cell death in plants.

     

Development of a SYBR green real-time polymerase chain reaction assay for quantitative detection of Babesia gibsoni (Asian genotype) DNA.

A real-time fluorogenic polymerase chain reaction (PCR) assay based on SYBR green that allows for sensitive, reproducible, and accurate quantification of Babesia gibsoni (Asian genotype). DNA from peripheral blood of infected dogs was developed. Standard curves were created by plotting the input amount of a standard template, constructed with plasmid DNA containing 182 base pairs (bp) of the p18 gene, against threshold cycle numbers. The curves showed a wide dynamic range (1,000,000-fold input) and high correlation values (>0.99). The PCR amplification efficacy of the standard template was similar to that of intact genomic DNA obtained from peripheral blood with B. gibsoni infection. The detection limit of the assay was 9 parasites/microl of blood with B. gibsoni infection. The intra-assay and interassay coefficients of variation of the threshold cycles ranged from 0.70% to 1.89% and from 1.18% to 1.92%, respectively. This assay system was found to be reproducible and accurate for the quantification of parasite DNA in experimentally infected dogs and far more sensitive than traditional microscopic examination.     

Metagenomic profiles of the rumen microbiota during the transition period in low‐yield and high‐yield dairy cows

We investigated potential relationships between rumen microbiota and milk production in dairy cows during the transition period. Twelve dairy cows were divided into a low‐yield (LY) or high‐yield (HY) group based on their milk yield. Rumen samples were taken from dairy cows at 3 weeks before parturition, and at 4, 8, and 12 weeks after parturition. 16S rDNA‐based metagenomic analysis showed that diversities of rumen microbiota in both groups were similar and the number of operational taxonomic units (OTUs) was lower in the postpartum than prepartum period in both groups. The abundance of Bacteroidetes and ratio of Bacteroidetes:Firmicutes was higher in the HY than the LY group. OTUs assigned to Prevotella bryantii, Fibrobacter succinogenes, Ruminococcus albus, Butyrivibrio fibrisolvens, and Succinivibrio sp. were abundant in the HY group. These OTUs were significantly related to the propionate molar proportion of rumen fluids in the HY group. OTUs assigned to Lachnospiraceae, Bifidobacterium sp. and Saccharofermentans were dominant in the LY group. Predictive functional profiling revealed that abundance of gene families involved in amino acid and vitamin metabolism was higher in the HY than the LY group. These results suggest that the community structure and fermentation products of rumen microbiota could be associated with milk production of dairy cows.