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al., 1996 ; Sartain et al., 2004 ; Simonne and Hutchinson, 2005 ). Therefore, the objective of this publication is to describe and summarize laboratory, growth chamber, greenhouse, and field methods currently used to measure CRF-N release on

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established in 1994 by the Association of American Plant Food Control Officials to address issues regarding the effective regulation and analysis of SRF materials. To date, a soil incubation methodology and a short-term laboratory nutrient extraction method

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duration in DAH (0, 1, 2, 3, 4, 7, and 11) and drying condition (shade and sun) on the EO yield and on the concentrations of 47 major and minor constituents in the oil. Materials and Methods Field and laboratory experiments. The field and laboratory

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An efficient deoxyribonucleic acid (DNA) extraction procedure that yields large quantities of DNA would provide adequate DNA for a large number of different analytical procedures. This study was conducted to compare three DNA extraction procedures for cost, time efficiency, and DNA content while extracting DNA from Kentucky bluegrass (Poa pratensis L.). Three students at the Univ. of Illinois with varying levels of DNA extraction experience conducted DNA extractions using Plant DNeasy™ Mini Kits, Plant DNAzol® Reagent, and a PEX/CTAB buffer. Costs varied significantly with cost (US$) per DNA sample of $3.04 for the DNeasy™ method, $0.99 for the DNAzol® method, and $0.39 for the PEX/CTAB extraction. The DNAzol® method was the fastest; although extracting 2.8 ng less DNA than the DNeasy™ method, it did not require the use of hazardous organic solvents, and random amplified polymorphic DNA (RAPD) markers were satisfactory for DNA fingerprinting of Kentucky bluegrass cultivars. The PEX/CTAB method, which did not include a tissue homogenization step, did not have reproducible banding patterns due to miniscule and inconsistent quantities of DNA extracted, or possibly due to inadequate purification. The investigator with the least DNA extraction experience was the slowest, while extracting 75% more DNA. All three methods are easily adapted to laboratories having personnel with different levels of experience. The DNAzol® Reagent method should save time and money, with reproducible results when many individual plant samples need to be identified. Chemical names used: potassium ethyl xanthogenate (PEX); cetyltrimethyl ammonium bromide (CTAB)

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Abstract

The chlorophyll content of leaf tissues can be accurately determined in the laboratory by spectrophotometric measurement of the leaf extract obtained with ethanol (Knudson et al., 1977), acetone (Blessington and Rasberry, 1980), or N,N-dimethylformamide (Moran and Porath, 1980). However, extraction is laborious and destructive. A rapid, nondestructive method to estimate leaf chlorophyll using a portable chlorophyll meter (SPAD-501, Minolta Corp.) was recently reported (Marquard and Tipton, 1987; Yadava, 1986). However, the manufacture of this instrument has been discontinued.

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The rhizon soil solution sampler (RSSS) currently is being used for in situ extraction of the soil solution for nutrient analysis of mineral soils used to produce field-grown crops. In this study, laboratory and greenhouse experiments were conducted to test the effectiveness of the RSSS for in situ solution extraction from soilless container root media and to compare an RSSS extraction method for measuring root-medium pH, electrical conductivity (EC), and NO3-N and K concentrations with that measured with the saturated media extract (SME) method. A near 1:1 correlation was found between the pH, EC, and NO3-N and K concentrations measured in the extracted solution of the RSSS and SME method in media without plants and in media from ten species grown using three water-soluble fertilizer concentrations applied by subirrigation. More testing is needed with the RSSS, perhaps using composite samples form several pots for analysis. The RSSS shows promise for nutrient extraction in container-grown crops because it is fast, nondestructive, simple, economical, and has minimal effect on the nutritional status of the medium in the pot.

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Methods for extracting growing substrate root-zone solution include the saturated media extract (SME) and the 2 water: 1 substrate (v/v) suspension, neither of which are particularly suited to bedding plant plug systems. We have developed the press extraction method (PEM) as a simple and quick alternative to these methods. The grower simply collects a representative sample of plug trays and presses the top of the plug, collecting the expelled solution. Solution pH and EC can be measured immediately and the sample then sent to an analytical laboratory for nutrient analysis. Initial experiments demonstrated that differing manual pressures did not affect solution chemical properties. The PEM then was compared to the SME and 2:1 methods over a range of fertilizer levels and with peat- and coir-based substrates. Within substrates, pH, EC, and macronutrients were similar between the PEM and the SME. The level of dilution inherent in the 2:1 method resulted in much lower EC and nutrient levels when compared to the other two methods. Further experiments compared the PEM to the SME and 2:1 on plug flats collected from several commercial greenhouses and also those grown in the research greenhouse. The wide range of bedding plant species and fertility levels tested introduced variation needed to develop regression equations and correlations to create quantitative interpretation ranges for the PEM based on previously published sufficiency ranges for the SME and 2:1.

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Several probes have been been recently developed that can be inserted directly into the growing medium of container-grown crops to get electrical conductivity (EC) or pH measurements. However, for many floriculture and greenhouse crops, EC interpretation ranges are based on substrate solution extraction methods such as the 1:2 v/v dilution, saturated media extract (SME), and more recently, the pour-through. We tested the sensitivity and accuracy of four in situ EC probes at a range of substrate moisture content and fertilizer concentrations. We also compared results from in situ probes with currently used methods of EC measurement. Concerning the effects of substrate volumetric water content (VWC) on the in situ probes, our results indicate little differences exist among probes when VWC exceeds 0.50, though drier substrates yielded differences depending on the measurement method. The SigmaProbe and W.E.T Probe measure the EC of the pore water specifically and show a decrease in EC with increasing water content, as the fertilizer ions in the pore water becomes more diluted as VWC increases. Results with the Hanna and FieldScout probes increased with increasing water content as the added water helps conduct the current of these meters. The EC measured with the various in situ probes differed slightly among the probes, but was highly and positively correlated with all three of the solution extraction methods over the range of fertilizer concentrations. It would be possible to convert substrate EC guidelines that have been established for any of the laboratory methods for use with the in situ probes, though our results indicate the substrate VMC must be above 0.35 for the interpretation to be valid.

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Anther culture has been one of the most successful techniques for generating haploid plants over a wide range of species. It is a reasonably simple procedure that can be accomplished successfully without sophisticated laboratory facilities; yet, the plants generated through anther culture can be used to demonstrate the application of many modern methods that have direct applicability to plant breeding. Anthers of diploid potato clones that have been selected for competence in anther culture can be cultured in a simple medium to yield androgenic embryos after 5 weeks. Plant regeneration requires an additional 3 to 4 weeks. Regenerated plants should be large enough 2 weeks after transfer to basal medium for ploidy determination by any of three methods depending on available facilities: chromosome counts in root tips; chloroplast counts in stomatal guard cells; or flow cytometry of nuclei released from in vitro plantlets. DNA can be extracted from anther-derived plantlets using a rapid extraction procedure to demonstrate segregation of PCR (polymerase chain reaction)-based markers such as RAPD (randomly amplified polymorphic DNA), RAMPs (randomly amplified microsatellite polymorphisms), or microsatellites. Microsatellite markers that were heterozygous in the anther donor can be used to verify haploidy in anther-derived plants. If an anther culture laboratory is scheduled early in a semester, such molecular analysis can be planned for late in the same semester.

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Most commercial and university substrate testing laboratories' recommended floriculture nutritional values are based on the saturated media extract (SME) method. With the recent gain in popularity of pour-through nutritional monitoring, alternative recommended values are needed for nutrient analyses based on pour-through extracts. Pour-through nutritional values were compared to the SME values to develop calibration curves and recommended nutritional values. Euphorbia pulcherrima `Freedom Red' Willd. ex Klotzch. were grown for two consecutive growing seasons in 16.5 cm plastic pots with Fafard 4 P root substrate and fertigated with 200, 300, or 400 mg·L-1 N from a 13N-0.88P-10.8K fertilizer. Linear relationships existed and inverse calibration curves for pour-through and SME comparisons were developed for (r 2): EC (0.98), NO3 - (0.98), P (0.97 to 0.99), K (0.99), Ca (0.94 to 0.97), and Mg (0.91). In addition, recommended pour-through substrate value ranges were developed for comparison with SME values. The established calibration curves and pour-through substrate value ranges will allow substrate-testing laboratories to make nutritional recommendations based on pour-through extractions.

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