In vivo gene transfer into skeletal muscle of neonatal chicks by electroporation

Chicks (Gallus gallus domesticus) show considerable growth of skeletal muscle during the neonatal period. The in vivo gene transfer method is useful for studying gene function and can be employed to elucidate the molecular mechanisms of skeletal muscle growth in chicks. We evaluated the following conditions for gene transfer to the skeletal muscle of neonatal chicks by electroporation: (i) voltage; (ii) age of the chick; (iii) plasmid DNA injected amount; and (iv) duration of gene expression. The results obtained from this study indicate that the most efficient gene transfer condition was as follows: 75 µg of plasmid DNA encoding β‐galactosidase was injected into the gastrocnemius muscle of chicks at 4 days of age electroporated at 50 V/cm. In addition, peak transferred gene expression was observed from 3 days to 5 days after electroporation. Our results provide optimal electroporation conditions for elucidating the gene function related to skeletal muscle growth and development in neonatal chicks.     

Rubisco small subunits of C4 plants,Napier grass and guinea grass confer C4-like catalytic properties on Rubisco in rice

ABSTRACT

Overexpression of Rubisco small subunit (RbcS) of C₄ plant, sorghum (sorghum bicolor) was shown to enhance the catalytic turnover rate (k _cat) of Rubisco in rice (Oryza sativa). In this study, the effects of other Rubisco small subunits of C₄ plants, Napier grass (Pennisetum purpureum) and guinea grass (Megathyrsus maximus) on kinetic properties of Rubisco in rice were studied. The expression levels of Napier grass RbcS (NgRbcS) and guinea grass RbcS (GgRbcS) proteins accounted for 41% and 45% of total RbcS, respectively in homozygous overexpression lines. The k _cat and K _m for CO₂ (Kc) of Rubisco were increased in all transgenic lines. Interestingly, the k _cat was markedly higher in NgRbcS homozygous line, whereas K _c was notably higher in GgRbcS homozygous line. Although its effects depend on species, these results suggest that the introduction of C₄ RbcS are effective approaches to alter the catalytic properties of Rubisco in rice.     

Semen Collection and Polymerase Chain Reaction‐Based Sex Determination of Black‐Headed and Straw‐Necked Ibis

This study aimed to develop a polymerase chain reaction (PCR)‐based sexing and effective semen collection methods for black‐headed and straw‐necked ibis species. However, most birds are not sexually dimorphic, that is, the sexes appear similar. Therefore, the gender should be determined before semen collection. DNA was extracted from the blood samples of 11 black‐headed and 4 straw‐necked ibis. The sex was determined after PCR amplification of the EE0.6 region of W‐chromosome. The PCR products were separated using gel electrophoresis. A single band indicated the presence of the EE0.6 region and that the individual was a female, while no band indicated that the individual was a male. Further, the single bands from seven specimens were amplified. Semen collection was performed by massage or a combination of massage with electro‐ejaculation and was attempted during all four seasons. The semen was successfully collected in March from male straw‐necked ibis using the massage method. Limited motility, viability and concentration of straw‐necked ibis sperm were observed. The sperm length was 180 μm and that of the nucleus was 30 μm with acrosome located at the tip of the nucleus. Thus, the PCR‐based sexing proved to be an accurate molecular sexing method for black‐headed and straw‐necked ibis. Furthermore, we successfully collected semen and observed the stained sperm nucleus and acrosome of the straw‐necked ibis sperm. We propose that the use of this PCR methodology can be applied as a routine method for sex determination and semen collection in ibis species for future ecological research. However, considering our limited success, further studies on semen collection method are required.     

Determination of antioxidant activity and characterization of antioxidant phenolics in the plum vinegar extract of cherry blossom (Prunus lannesiana)

Sakura-cha (salted cherry blossom tea) is a Japanese tea that is traditionally served at celebrations such as wedding ceremonies. The production of Sakura-cha includes the immersion of cherry blossom flowers in Japanese plum vinegar, and through this process, the byproduct (plum vinegar extract of cherry blossom) is obtained. In this study, the antioxidant activity of the plum vinegar extract of cherry blossom was examined. The plum vinegar extract of cherry blossom had a greater superoxide anion scavenging activity compared with red wine, which is a well-known strong antioxidant. Liquid chromatography-mass spectrometry analysis showed that cyanidin-3-glucoside, cyanidin-3-rutinoside, and caffeic acid were the major components in the phenolic extract prepared from plum vinegar extract of cherry blossom, and they possessed superoxide anion scavenging activity. Caffeic acid is mainly responsible for the scavenging activity of phenolic extract; the contributions of cyanidin-3-glucoside and cyanidin-3-rutinoside were minor.     

Environmental controls on photosynthetic production and ecosystem respiration in semi-arid grasslands of Mongolia

The Mongolian steppe zone comprises a major part of East Asian grasslands. The objective of this study was to separately evaluate the quantitative dependencies of gross primary production (GPP) and ecosystem respiration (R_eco) on the environmental variables of temperature, moisture, radiation, and plant biomass in a semi-arid grassland ecosystem. We determined GPP and R_eco using transparent and opaque closed chambers in a grassland dominated by Poaceae species in central Mongolia during five periods: July 2004, May 2005, July 2005, September 2005, and June 2006. Values of GPP were linearly related to live aboveground biomass (AGB) enclosed by the chamber. The amount of GPP per unit ground area differed among the study periods, whereas GPP normalized by the amount of AGB did not differ significantly among the periods, suggesting that plant production per unit green biomass did not depend on the phenological stage. GPP/AGB fit well a rectangular hyperbolic light–response curve for all the study periods. When the air and soil were dry, considerable reduction in GPP was observed. The GPP/AGB ratio was also expressed as individual functions of air temperature, vapor pressure deficit, and volumetric soil water content. R_eco was exponentially related to the soil temperature and the relationship was modified by soil moisture. The amount of R_eco and its temperature sensitivity (Q₁₀) declined with decreasing soil moisture. Sharp increases of R_eco after rainfall events were observed. The values of R_eco, even including the rain-induced pulses, were expressed well as a bivariate function of soil temperature and soil moisture near the soil surface.     

Seed abortion of ‘Tosa-Buntan’ pummelo pollinated with soft-X-irradiated pollens

Cross-pollination was performed with soft-X-irradiated hyuga-natsu pollens (1000 Gy) for ‘Tosa-Buntan’ pummelo (Citrus grandis (L.) Osbeck). This resulted in the transformation of large and complete seeds into small and empty ones (practically seedless). Although fruit set, fruit retention, total soluble solids content (TSS) and titratable acidity of the juice were not affected, decrement in the fruit size was observed. Two weeks after the pollination, endosperm cell division with free nuclei began in both the non-irradiated and irradiated pollen treatment conditions. Seven weeks after pollination, endosperm division with the cell wall occurred in the non-irradiated pollen treatment conditions; however, the endosperm development ceased in most ovules that underwent the irradiated pollen treatment, and the ovules remained in their free nuclear stage. The delayed degeneration of the ovules, following successful fertilization and commencement of endosperm cell division, allow these seedless fruits to be categorized as pseudo-parthenocarpic.     

Effect of high temperature stress on the reproductive growth of strawberry cvs. ‘Nyoho’ and ‘Toyonoka’

High temperatures are known to reduce fruit size and fruit weight in strawberry, but cultivar differences in the response to high temperature stress during the reproductive stage up to the second inflorescence have not been sufficiently reported. We examined the effect of two day/night temperature regimes on fruit set and fruit growth in two cultivars, ‘Nyoho’ and ‘Toyonoka’. A high day/night temperature of 30/25 °C reduced the number of inflorescences, flowers, and fruits in both cultivars compared with plants grown at 23/18 °C. The percentage of fruit set in ‘Nyoho’ was not significantly different between the two temperature treatments, while that in ‘Toyonoka’ was much lower at 30/25 °C than at 23/18 °C. Days to ripening was shorter at 30/25 °C than at 23/18 °C, and no cultivar differences were observed. Fresh weight of primary, secondary, and tertiary fruits was greater at 23/18 °C than at 30/25 °C in both cultivars, and no cultivar differences were observed, except in tertiary fruits. The diameter of fruits from all positions was also reduced at 30/25 °C in both cultivars. Relative growth rates of fruits showed two peaks in both cultivars and in both temperature treatments. Both peaks appeared earlier at 30/25 °C than at 23/18 °C. Percentage of fruit set at 30/25 °C in the second inflorescence was also significantly lower in ‘Toyonoka’ than in ‘Nyoho’. These results indicate that high temperature stress negatively affects the reproductive process in strawberry and that plant response to high temperature stress is cultivar-related in such responses.     

Isolation and structural characterization of rhamnogalacturonan II-borate complex fromPinus densiflora

A boron-containing pectic polysaccharide was isolated from Driselase digests of akamatsu (Pinus densiflora) cell wall. The boron-containing polysaccharide was purified by successive column chromatography of Cosmosil C₁₈ OPN, DEAE Sepharose FF, and Superdex 75. The complex had 0.12% (w/w) boron (B) and¹¹B-NMR spectroscopy showed the complex to be the tetrahedral diester form of boron. The sugar composition and glycosyl linkage analyses showed that the sugar portion of the complex was rhamnogalacturonan-II (RG-11). The boron-attached glycosyl residue was identified to be 2-O-Me-xylose-containing apiosyl side chain in RG-II. These results established that the location of borate in the cell wall is conserved among dicots, gramineous monocots, and coniferous gymnosperm.Part of this study was presented at the 8th Cell Wall Symposium, Norwich, September 3, 1998     

Differentiation in competitive ability and cold tolerance between diploid and tetraploid cultivars in Lolium perenne

In a diploid grass, perennial ryegrass (Lolium perenne L.), many autotetraploid cultivars have been released and widely used in many cool temperate countries. In this study, competitive ability and cold tolerance, which seem to be important factors in determining geographical distribution and persistency of this species, were compared between six diploid and five autotetraploid cultivars. The tetraploid cultivars had significantly higher competitive ability and lower cold tolerance than the diploid cultivars. Furthermore, there was no overlap in the two traits between them. These results suggest that chromosome doubling has a larger effect on competitive ability and cold tolerance than does the genetic difference between the populations within a single ploidy level. This revised version was published online in August 2006 with corrections to the Cover Date.