Buffalo (Bubalus bubalis) ES Cell–Like Cells are Capable of In Vitro Skeletal Myogenic Differentiation

When buffalo embryonic stem (ES) cell–like cells that expressed surface markers SSEA‐4, TRA‐1‐60, TRA‐1‐81, CD9 and CD90 and intracellular markers OCT4, SOX2 and FOXD3, as shown by immunofluorescence, and that expressed REX‐1 and NUCLEOSTEMIN as confirmed by RT‐PCR, were subjected to suspension culture in hanging drops in absence of LIF and buffalo foetal fibroblast feeder layer support, they differentiated to form three‐dimensional embryoid bodies (EBs). Of 231 EBs examined on Day 3 of suspension culture, 141 (61.3 ± 3.09%) were of compact type, whereas 90 (38.4 ± 3.12%) were of cystic type. The cells obtained from EBs were found to express NF‐68 and NESTIN (ectodermal lineage), BMP‐4 and α‐skeletal actin (mesodermal lineage), and α‐fetoprotein, GATA‐4 and HNF‐4 (endodermal lineage). When these EBs were cultured on gelatin‐coated dishes, they spontaneously differentiated to several cell types such as epithelial‐ and neuron‐like cells. When EBs were cultured in the presence of 1 or 2% DMSO or 10^?8 m or 10^?7 m retinoic acid for 25 days, ES cells could be directed to form muscle cell–like cells, the identity of which was confirmed by expression of α‐actinin by immunofluorescence and of MYF‐5, MYOD and MYOGENIN genes by RT‐PCR. MYOD was first detected on Day 10 in both treatment groups and on Day 15 in controls, whereas MYOGENIN was first detected on Day 10, Day 15 and Day 25 in the presence of retinoic acid, in the presence of DMSO and in controls, respectively. The present study demonstrates the ability of buffalo ES cell–like cells to undergo directed differentiation to cells of skeletal myogenic lineage.     

Assessment of DNA Damage during In Vitro Development of Buffalo (Bubalus bubalis) Embryos: Effect of Cysteamine

Comet assay was used in the present study to examine DNA damage to buffalo oocytes and embryos during in vitro culture. Embryos were produced in vitro from oocytes obtained from slaughterhouse ovaries in presence of cysteamine (IVM and IVC media supplemented with 50 and 100 μm , respectively) or in its absence (controls). Compared to controls, cysteamine supplementation increased (p < 0.01) cleavage rate and proportion of oocytes that developed to 8‐ to 16‐cell stage. The incidence of DNA damage was lower (p < 0.01) in cysteamine group than that in controls at 8‐ to 16‐ (19.3 ± 4.24 vs 72.0 ± 5.22%) but not in 2‐cell stage embryos (11.7 ± 5.63 vs 20.8 ± 5.49%) or in mature oocytes (5.3 ± 3.43 vs 10.3 ± 4.73%). The tail length, which indicates magnitude of DNA damage, was shorter (p < 0.01) in cysteamine group than in controls in mature oocytes (25.5 ± 0.5 vs 36.0 ± 0.71 pixels) and 8‐ to 16‐cell stage (49.2 ± 1.64 vs 152.7 ± 1.28 pixels) but not in 2‐cell stage embryos (36.3 ± 1.54 vs 36.4 ± 0.75 pixels). Also, exposure of oocytes/embryos to UV radiation or H₂O₂ caused extensive DNA damage. In conclusion, these results suggest that oocytes/embryos suffer from DNA damage during progress of in vitro culture, which can be partly ameliorated by cysteamine supplementation of culture media.     

N applications that increase plant N during panicle development are highly effective in increasing spikelet number in rice

Efficient use of nitrogen fertilizer is critical in improving yield stability in rice. The objective of this study was to determine the effect of nitrogen (N) top-dressing on the number of total spikelet (fertile plus sterile) production and evaluate the effect among rice cultivars. We analyzed 136 sets of experimental data on growth and spikelet production for three lowland cultivars, grown under various regimes of N over 10 seasons at Kyoto, Ibaraki and Kanagawa, Japan. In each season, one to three of the lowland cultivars, Nipponbare (japonica), Koshihikari (japonica) and Takanari (indica), were studied. In 1986, 1995 and 1999-2001, the N regimes included basal application only, light basal and heavy top-dressing from the panicle initiation stage onward, heavy basal and heavy top-dressing from the spikelet formation stage onward, and no applications. In 2002 and 2005-2008, we set up experimental plots with varied time of N top-dressing, with or without N basal application. Takanari had the largest spikelet number averaged over all plots and was considered better efficient in spikelet production per applied N than the other cultivars. Although the trend is not clear, the effect of time of top-dressing on spikelet number was generally the greatest when N was top-dressed from 35 to 30 days before heading. The variation of observed spikelet number was analyzed with a linear regression of plant N 14 days before heading and by a model that estimates spikelet production accounting for plant N 14 days before heading and crop growth rate (CGR) during the 14-day period preceding heading. For the variation of spikelet number within each cultivar, the linear function model expressed the observed spikelet number than the two function model with R² 0.43** versus 0.13*-0.28** for the former and later models, respectively. When the results of all cultivars were combined, the two function model was much better for estimation of spikelet number than the linear function model (R² = 0.36** vs. 0.20*). This indicates that yearly and varietal variation of spikelet number was caused mainly by plant N status at the late spikelet differentiation stage. The varietal variation in spikelet production efficiency is explained by CGR during this 14-day period. We concluded that N applications that increase plant N 14 days before heading is highly effective in maximizing spikelet production among cultivars.     

Effects of Calcium Concentration in Potato Tuber Cells on the Formation of Cross-Links between Pectin Molecules by Ca<Superscript>2+</Superscript>

The formation of cross-links between pectin molecules via Ca²⁺ in the potato tuber cell wall is a determinant factor on processing properties of potato and the quality of its products such as French fries. Thus, in this study, potato tubers varying significantly in their calcium concentrations were analyzed to investigate whether an increased absorption of calcium by a potato tuber led to an increase in the calcium concentration in the cell wall and how the calcium concentration in the cell wall influenced on the formation of cross-links between pectin molecules via Ca²⁺. Correlation analysis revealed that calcium absorbed by a potato tuber was bound to the cell wall as a water insoluble form 99 days after planting or later. Furthermore, with an increase in the calcium concentration in the cell wall, the content of chelator soluble pectin increased throughout tuber bulking and maturation stages. However, the degree of methylation was not a limiting factor in the formation of cross-links between pectin chains via Ca²⁺. Atomic force microscopy images of parenchyma cell walls prepared from mature potato tubers indicated an increase in the amount of calcium cross-linked pectin molecules with an increase in the calcium concentration in the cell wall. The present study demonstrated that the calcium concentration of the cell wall of potato tubers significantly affected the formation of cross-linkages between pectin molecules and, consequently, contributed to an enhanced formation of pectin-calcium networks in the cell wall.     

Changes in freezing tolerance, plasma membrane H+-ATPase activity and fatty acid composition in Pinus resinosa needles during cold acclimation and de-acclimation

It has previously been suggested that plasma membrane ATPase (PM H+-ATPase, EC 3.6.1.3.) is a site of incipient freezing injury because activity increases following cold acclimation and there are published data indicating that activity of PM H+-ATPase is modulated by changes in lipids associated with the enzyme. To test and extend these findings in a tree species, we analyzed PM H+-ATPase activity and the fatty acid (FA) composition of glycerolipids in purified plasma membranes (PMs) prepared by the two-phase partition method from current-year needles of adult red pine (Pinus resinosa Ait.) trees. Freezing tolerance of the needles decreased from -56 degrees C in March to -9 degrees C in May, and increased from -15 degrees C in September to -148 degrees C in January. Specific activity of vanadate-sensitive PM H+-ATPase increased more than two-fold following cold acclimation, despite a concurrent increase in protein concentration. During de-acclimation, decreases in PM H+-ATPase activity and freezing tolerance were accompanied by decreases in the proportions of oleic (18:1) and linoleic (18:2) acids and increases in the proportions of palmitic (16:0) and linolenic (18:3) acids in total glycerolipids extracted from the plasma membrane fraction. This pattern of changes in PM H+-ATPase activity and the 18:1, 18:2 and 18:3 fatty acids was reversed during cold acclimation. In the PM fractions, changes in FA unsaturation, expressed as the double bond index (1 x 18:1 + 2 x 18:2 + 3 x 18:3), were closely correlated with changes in H+-ATPase specific activity (r2 = 0.995). Changes in freezing tolerance were well correlated with DBI (r2 = 0.877) and ATPase specific activity (r2 = 0.833) in the PM fraction. Total ATPase activity in microsomal fractions also closely followed changes in freezing tolerance (r2 = 0.969). We conclude that, as in herbaceous plants, simultaneous seasonal changes in PM H+-ATPase activity and fatty acid composition occur during cold acclimation and de-acclimation in an extremely winter hardy tree species under natural conditions, lending support to the hypothesis that FA-regulated PM H+-ATPase activity is involved in the cellular response underlying cold acclimation and de-acclimation.     

Monitoring of spatial variations of particulate matter (PM) pollution through bio-magnetic aspects of roadside plant leaves in an Indo-Burma hot spot region

Particulate matter (PM) is an important air pollutant because of its adverse impacts towards human health. The existing and conventional methods of PMs monitoring are found to be inadequate in feasibility, which paved the way of magnetic biomonitoring approach. The magnetic measurement carried through the plant leaves is useful means in assessing the PM pollution. Plant species are found to be an effective biomonitors and may act as natural filters by trapping and retaining the PM on their leaf surfaces. Therefore, the aim of this communication is to demonstrate the magnetic properties [magnetic susceptibility (χ), anhysteretic remanent magnetization (ARM) and saturation isothermal remanent magnetization (SIRM)] of two roadside plant leaves (Hibiscus rosa-sinensis and Mangifera indica) at four spatially distant sites. The study measures and compares the capabilities of these plants to accumulate and retain the PMs. The study also assesses the PM pollution at selected sites and establishes the relationship between magnetic properties and PM in the city of an Indo-Burma hot spot region. The results indicated a significant correlation between the concentration of ambient PM and magnetic measurement (χ, ARM and SIRM) of both the roadside plant leaves. Similarly, reasonably good correlations are obtained between magnetic parameters (χ, ARM and SIRM) and Fe content in PMs. Present study is, perhaps, a novel contribution in the area of bio-magnetic monitoring studied with several magnetic parameters viz., χ, ARM and SIRM. Results indicated that the bio-magnetic monitoring is applied for environmental geomagnetism which act as proxy for ambient PM pollution and further employed as an eco-sustainable tool for environmental management in urban and peri-urban regions.     

Activation of Zona‐Free Buffalo (Bubalus bubalis) Oocytes by Chemical or Electrical stimulation,and Subsequent Parthenogenetic Embryo Development

Parthenogenetic activation using zona‐free oocytes offers an alternative model that could be applied to develop protocols for the activation of reconstructed embryos for cloning. The aim of this study was to compare the efficacy of different methods for the activation of zona‐free buffalo oocytes in terms of their effects on the developmental competence of parthenogenetic embryos. The effects of zona removal on parthenogenetic activation and in vitro developmental competence of metaphase II oocytes were also examined. All activation methods were followed by incubation of 2 mm 6‐dimethylaminopurine (6‐DMAP) for 4 h. Out of three different pulse strengths (1.2, 2.1 or 3.3 kV/cm) used, 2.1 kV/cm resulted in the highest blastocyst rate (25.3%). On comparing different chemical agents and electric pulse, highest blastocyst rate was observed for calcium ionophore (CaI) (28.6%) followed by ethanol (25.0%), electric pulse (22.5%) and combined CaI and ethanol treatment (16.7%) although differences among them were not significant. Furthermore, a significantly reduced developmental potential was observed in zona‐free oocytes when compared to zona‐intact ones up to the blastocyst stage (44.3% vs 27.1%). In conclusion, zona‐free buffalo oocytes can be successfully activated for parthenogenetic development using chemical or electrical stimulation. Out of different agents examined, CaI followed by 6‐DMAP resulted in the highest blastocyst rate.     

Use of stomatal index as a marker to screen backcross populations of two wild potato species segregating for freezing tolerance

The purpose of this study was to investigate differences in stomatal index among backcross progeny ofSolanum commersonii (freezing tolerant) andSolanum cardiophyllum (freezing sensitive) to assess the feasibility of using this trait as a marker for screeningSolanum populations for freezing tolerance. Measurements were taken on three terminal fully-expanded leaflets per genotype by completing microscopic examination of epidermal impressions made in partially dissolved cellophane tape. Freezing tolerance was estimated in parallel studies on the same plant material. Values of SI were significantly greater (Fisher T-test, 0.05) for theS. cmm. group (parent + backcross progeny) compared with theS. cph. group. Stomatal index of the F₁ was significantly greater than SI ofS. cph. parents and similar toS. cmm. parents. Values of SI for both backcross progenies were greater than parental values. Non-acclimated relative freezing tolerance values were in the following order:S. cmm. parents >S. cmm. backcrosses > F₁ >S. cph. back-crosses >S. cph. parents. Stomatal index values followed a similar pattern with the exceptionS. cmm. backcross >S. cmm. parents. These data suggest: a) increased SI is inherited as a dominant trait, b) SI may be a useful screening marker in breeding programs interested in improving freezing tolerance.     

Utilization of potatoes for life support systems II. The effects of temperature under 24-H and 12-H photoperiods

The growth and tuberization of Norland potatoes were studied under five different temperatures and two photoperiods. Treatment levels included 12, 16, 20, 24, and 28 C with either a 24-h (continuous light) or a 12-h photoperiod at 400 μmol m^-2 s^-1 PPF. Plants were grown in 6-liter containers and harvested at 56-days-age. Stem length increased with increasing temperature under both photoperiods. The highest tuber yield occurred at 16 C under the 24-h photoperiod (755 g/plant) and at 20 C under the 12-h photoperiod (460 g/plant). Little or no tuber formation occurred at 28 C under either photoperiod or at 24 C under continuous light. As with tuber yield, the highest total plant dry weights also occurred at 16 C under the 24-h photoperiod and at 20 C under the 12-h photoperiod. Harvest index (tuber dry weight to total dry weight ratio) decreased with increasing temperatures and with continuous light. Results indicate that good growth and tuberization can occur under continuous light, and that increasing the photoperiod from 12 to 24 h effectively decreased the optimal temperature for tuber formation from near 20 C to 16 C. Alternatively, the results imply that at cooler temperatures, the potato becomes less obligate for dark period stimulation of tuberization.     

Evidence for the existence of functional roots on potato tubers and stolons: Significance in water transport to the tuber

American Journal of Potato Research - Field observations of potato plants showed various types of roots. There were roots originating at the base of the main stem (basal roots); roots arising from...