Cold treatments enhance responsiveness to gibberellin in stock (Matthiola incana (L.) R. Br.)

Summary

Endogenous GAs have been suggested as regulators of stem elongation and flowering of cold-requiring plants. Here, the relationship between temperature conditions and responsiveness to GA₄ on stem elongation and flowering of stock (Matthiola incana) was investigated. The optimum temperature for induction of flower bud initiation was 10°C, and the minimum duration was 20 d in the late flowering cv. Banrei; the type of cold treatment effect on flowering was classified as a “direct effect”. Stem elongation was markedly promoted by cold treatment regardless of flower bud initiation. The cold treatment amplified the stem elongation response to GA₄. The GA₄ level necessary for flower bud initiation was lower in the 10°C treatment than in the 15°C treatment, and it became lower at longer durations of cold treatment. These results indicate that the cold treatments enhance responsiveness to GA₄ not only in the stem elongation process but also in the flower bud initiation process and that the development of responsiveness to GA₄ may correlate with the temperature and duration of cold treatment.     

Design of hammerhead ribozymes that cleave murine Sry mRNA in vitro and in vivo

As the first step in investigating the possiblity of applying ribozyme technology to artificial control of the sex ratios at birth in farm animals, where the demand for females exceeds that for males, we designed a hammerhead ribozyme (HHRz) and 2 tRNA(val)-hammerhead ribozyme complexes (tRNARz3 and tRNARz4), and examined their effects upon murine Sry mRNA in vitro and in cells. We demonstrated that HHRz and tRNARz3 could effectively cleave the target Sry mRNA in vitro. For the purpose of experiments in vivo, HHRz was cloned into the highly efficient pUC-CAGGS mammalian expression vector (pCAG/HHRz), and the tRNA ribozyme complexes were cloned into the pol III promoter-driven pPUR-KE vector (pPUR/tRNARz3 and pPUR/tRNARz4); the ribozyme vectors were co-transfected with the target vector (pCAG/Sry). A suppressive action (up to approx. 60%) was confirmed for pCAG/HHRz and pPUR/tRNARz3 upon the transiently expressed exogenously introduced Sry in M15 cultured cells.     

Regulation of gibberellin biosynthesis and stem elongation by low temperature in Eustoma grandiflorum

Summary

Heat-induced rosetted Eustoma grandiflorum requires low temperature for induction of stem elongation and flowering. Although heat-induced rosetting is associated with a reduction of gibberellin A₁ (GA₁) content, how thermo-induction affects GA biosynthesis is unclear. Thus, we examined levels of GA, precursors including that of ent-kaurene which is the first committed step in GA biosynthesis. We used uniconazole, an ent-kaurene oxidase inhibitor to estimate the ent-kaurene biosynthesis activity. The accumulation level of ent-kaurene in stems of the cold-treated seedlings was approximately 1.8 times that of the non-cold-treated seedlings, whereas no difference was observed in the leaves. No change was observed in endogenous levels of GA₁ and GA₂₀ in stems of the heat-induced rosetted plants during the cold treatment, whereas their levels increased with stem elongation after transfer to warm conditions. In contrast to the levels of GA₁ and GA₂₀, endogenous levels of ent-kaurene, ent-kaurenoic acid, GA₅₃, GA₄₄ and GA₁₉ in the stems markedly increased at the end of cold treatment. These results indicate that ent-kaurene biosynthesis and its metabolism early in the GA biosynthetic pathway are stimulated by low temperature and, later, the stimulation leads to an increment of endogenous levels of GA₁ which is essential for stem elongation of the heat-induced rosetted E. grandiflorum.     

End-of-day far-red treatment enhances responsiveness to gibberellins and promotes stem extension in chrysanthemum

SUMMARY

A brief end-of-day (EOD) far-red (FR) exposure promotes extension growth in plants. This change suggests a role for gibberellins (GA). For chrysanthemum (Chrysanthemum morifolium Ramat.), we show that EOD-FR exposure enhances shoot extension and is mediated, at least in part, by increased responsiveness (sensitivity) to GA. In addition, our analysis showed how light, especially EOD-FR, influenced shoot extension. The effect of EOD-FR on promoting extension growth was maintained, not only during the dark period, but also in the subsequent light period. Furthermore, our observations suggest that dark reversion of certain phytochromes is involved in the determination of flowering time. We also discuss the applicability of EOD-FR treatment to reduce the overall duration of cultivation, while maintaining a sufficient stem height during cut chrysanthemum production.     

Variation in the effects of ethephon on flowering and extension growth in chrysanthemum as a function of temperature,season, and genetics

Summary

Ethephon (2-chloroethylphosphonic acid), an ethylene-releasing agent, is used in the production of Summer-to-Autumn-flowering chrysanthemums (Chrysanthemum morifolium Ramat.) to avoid early budding under long-day conditions. This study was carried out to show the variation in response to ethephon due to temperature, season, and cultivar. The seasonality of growth and flowering capacities, growing temperatures, and the genetic background of chrysanthemum make the effects of ethephon unstable and variable. The most important factor modifying the effect was the response to temperature, which showed seasonal variation. Ethephon briefly suppressed extension growth and flowering after chilling and at high growing temperatures. On the other hand, it induced dormancy and rosette formation, and suppressed flowering after Summer, and at lower temperatures. This suggests that ethylene is involved in the induction of dormancy.     

Symbiotic efficiency and compatibility of native rhizobia in northern Thailand with different soybean cultivars

Symbiotic efficiency and compatibility of 81 isolates of native bradyrhizobia from irrigated areas in northern Thailand with four soybean cultivars and one cowpea cultivar were evaluated under laboratory conditions. Effectiveness and / or compatibility of the tested isolates were compared with those of a standard strain (Bradyrhizobium japonicum CB 1809) by using plants grown on plastic seed bags. Effectiveness of the isolates was also estimated using uninoculated control plants grown in a nitrogen-free solution. Nodulation of a wide range of host plants by the majority of the tested isolates was observed, which agreed well with the results of our previous field experiment (Shutsrirung et al. 2002: Soil Sci. Plant Nutr., 48, 491–499). Up to 75% of the tested isolates induced a higher growth efficiency than that of the uninoculated control in association with one of the tested cultivars, Black soybean. Comparision with uninoculated control plants, enable to estimate the proportion of the tested isolates leading to effective growth promotion (E + e) of each cultivar, namely, Black soybean (local Thai cultivar), 75%; Cowpea, 82%; SJ5 (commercial Thai cultivar), 33%; Bragg (US cultivar), 33%; and Improved Pelican (US cultivar), 9%. These results indicated that although isolates with a high infectiveness with both “Asian-type” and “US-type” soybeans could be found, a high frequency of isolates leading to inefficient nodules was observed in the US cultivar, suggesting the presence of genetic differences in the soybean cultivars that express high-preference (efficient nodules) or low-preference (inefficient nodules) for a certain group of tested isolates. Based on the results of this laboratory experiment together with our previous field experiment, native rhizobial populations in the irrigated area of northern Thailand could be separated into three groups; Group 1: rhizobium strains showing a high effectiveness with only Asian cultivars, Group 2: strains showing a high effectiveness with only US origin cultivars, and Group 3: strains showing a high effectiveness with both Asian and US origin cultivars. The majority of the native rhizobial populations belonged to Group 1. The isolates in Group 3 may display a high potential for manipulating useful rhizobial inoculant.     

Aquaporin 1 expression in tissues of canines possessing inherited high K+ erythrocytes

We investigated the expression of aquaporin 1 (AQP1) in tissues from canines with an inherited anomaly that causes their erythrocytes to have high K⁺. Northern blot analysis revealed abundant AQP1 expression in lung and kidney, though little expression was found in spleen. Using anti-C-terminus for dog AQP1, abundant expression was shown in kidney, trachea, and eye, but little expression was shown in pancreas and cerebrum, indicating that AQP1 expression in canine tissues is similar to that noted in other mammals.     

Comparison of glycerol, lactamide, acetamide and dimethylsulfoxide as cryoprotectants of Japanese white rabbit spermatozoa

The rabbit is considered to be a valuable laboratory animal. We compared glycerol, lactamide, acetamide, and dimethylsulfoxide (DMSO) as cryoprotectants in egg-yolk diluent of ejaculated Japanese white rabbit spermatozoa for improvement of sperm cryopreservation methods. Rabbit semen was frozen with 1.0 M glycerol, lactamide, acetamide, or DMSO in plastic straws. Forward progressive motility and plasma membrane integrity of the post-thaw spermatozoa were examined. The rate of forward progressive motile spermatozoa in lactamide (37.8 +/- 3.0%) was significantly (P<0.05) higher than in glycerol (17.0 +/- 3.3%). In addition, the rates of sperm plasma membrane integrity in lactamide and acetamide (35.9 +/- 3.3% and 30.2 +/- 3.0%, respectively) were significantly (P<0.05) higher than in glycerol (17.0 +/- 2.6%). The results indicate that 1.0 M lactamide and acetamide have higher cryoprotective effects than 1.0 M glycerol for cryopreservation of Japanese white rabbit spermatozoa.     

Molecular detection and characterization of Haemobartonella felis in domestic cats in Japan employing sequence-specific polymerase chain reaction (SS-PCR)

A novel PCR assay was developed in order to examine the prevalence of Haemobartonella felis (H. felis) in Japanese domestic cats and which was able to differentiate of the Ohio strain and the California strain of H. felis. Blood samples from a total of 21 cats suspected of having haemobartonellosis were examined employing a novel PCR assay and demonstrated positive results in 18 cats which was confirmed by cytological examination of blood smears. Four out of 18 positive cats (22%) were infected with the California strain, whilst the other 12 cats (67%) were infected with the Ohio strain and two animals (11%) were infected with both strains. As most of the cats with moderate to severe anemia were infected with the Ohio strain, it is suggested that the most prevalent strain of H. felis in Japanese domestic cats might be the Ohio strain. In the present study, it was thought that molecular detection and characterization of H. felis may provide valuable information regarding the severity and prognosis of this illness.     

Knockout of targeted gene in porcine somatic cells using zinc‐finger nuclease

Targeted genome editing is a widely applicable approach for efficiently modifying any sequence of interest in animals. It is very difficult to generate knock‐out and knock‐in animals except for mice up to now. Very recently, a method of genome editing using zinc‐finger nucleases (ZFNs) has been developed to produce knockout rats. Since only injection of ZFNs into the pronuclear (PN) embryo is required, it seems to be useful for generating gene‐targeted animals, including domestic species. However, no one has reported the successful production of knockout pigs by direct injection of ZFNs into PN embryos. We examined whether ZFN works on editing the genome of porcine growth hormone receptor in two kinds of cell lines (ST and PT‐K75) derived from the pig as a preliminary study. Our data showed that pZFN1/2 vectors were efficiently transfected into both ST and PT‐K75 cells. In both cell lines, results from Cel‐I assay showed that modification of the targeted gene was confirmed. We injected ZFN1/2 mRNAs into the nucleus of PN stage embryos and then they were transferred to the recipients. However, pups were not delivered. Taken together, ZFN can be an available technology of genome editing even in the pig but further improvement will be required for generating genome‐modified pigs.