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Plants were grown for 6 weeks before leaf samples were taken for measurement of ��13C of leaf-respired CO2. The objective of this initial experiment was to obtain a time-course of ��13CRl values in the dark, so as to determine the sampling time at which ��13CRl is steady (see further discussion). A second experiment was conducted to assess the relationship between ��13CRl and A/gs with plants grown in the field. Barley cultivars Booma, Dash, Omaka and Sherwood were grown at Lincoln in the South Island of New Zealand in a randomized factorial combination with different irrigation and nitrogen treatments. Cultivars were chosen for the field trial based FKBP on previously observed differences in leaf-level transpiration efficiency (Barbour et?al. 2010a; Dash and Omaka), or results of cultivar trials conducted by the Foundation for Arable Research, NZ (R. Craigie pers. comm.; Booma and Sherwood). Seeds were sown in Templeton silt loam on 1st October 2009. Two levels of water availability were applied; an irrigation treatment where Pexidartinib irrigation was applied to keep the soil moisture deficit above 40?mm, and a control with no irrigation. Irrigation was applied weekly commencing on 11 November. Irrigation main-plots were split on 25 November into two nitrogen treatments, one receiving 150?g?N per ha and the other receiving none. There were four replicates of each treatment for each cultivar, resulting in 64 plots (4?��?4?m). To assess the appropriate time to measure the isotope composition in CO2, pre-illuminated leaves of cereal plants (Triticale and Barley cv. Dash) grown in the controlled environment chambers were sampled and the ��13C of evolved CO2 was followed over 4?h: leaves were immediately sealed into 2-L Tedlar bags fitted with taps and placed in the dark. The bags were flushed twice with CO2-free air, then re-sealed and placed in complete darkness until the CO2 concentration within the bag was >320??mol?mol?1. The bags were then connected to a pump via check details the tap, gas within the bag pumped to a tunable diode laser absorption spectrometer (TGA100A; Campbell Scientific) and analysed for ��13C (i.e. relative to the VPDB standard) as described previously (Barbour et?al. 2007, 2010a). Replicate samples were taken from three individual plants. After analysis, the bags were emptied, flushed with CO2-free air twice and re-sealed. This was repeated five to seven times for each sample, so that ��13CRl was measured over a 240?min period after sampling. The time taken for the air inside the bag to reach the required concentration varied between 6 and 20?min (for leaves that had been in the dark for more than 60?min). Leaves remained in complete darkness during incubation and analysis, and spent approximately 2?min at very low light (