A simplified paraffin embedding method for small botanical samples

Abstract

We present a simplified paraffin embedding method suitable for unsuberized or unlignified small botanical samples (diameter < 0.3 cm). Only 2 h are required to yield plant tissues embedded in paraffin for anatomical observation and molecular analysis. Our method achieved morphological preservation of cell structures and conservation of nucleic acids that were equivalent to the traditional protocol. Fourier transform infrared spectrometry showed that the degree of degradation of the cytoplasmic components (e.g., protein) resulting from our simplified protocol was similar to that of the traditional protocol. The DNA samples embedded using the simplified method was extractable and could be used for PCR analysis. The DNA quality was equivalent to that embedded using the traditional method.     

A routine method for quantitative determination of soluble carbohydrates in small samples of plant material with gas-liquid chromatography

Aluminium in human tissue samples has been determined by atomic absorption spectrophotometry using the nitrous oxide-acetylene flame and the graphite furnace atomizer. The two methods are compared. Flameless atomic absorption was found superior when standard addition was applied. A routine method is thus described in detail in which aluminium levels down to 0.1 mg/kg of freeze-dried tissue and 0.01 mg/liter of serum can be determined.     

Ice-cap. A high-throughput method for capturing plant tissue samples for genotype analysis

High-throughput genotype screening is rapidly becoming a standard research tool in the post-genomic era. A major bottleneck currently exists, however, that limits the utility of this approach in the plant sciences. The rate-limiting step in current high-throughput pipelines is that tissue samples from living plants must be collected manually, one plant at a time. In this article I describe a novel method for harvesting tissue samples from living seedlings that eliminates this bottleneck. The method has been named Ice-Cap to reflect the fact that ice is used to capture the tissue samples. The planting of seeds, growth of seedlings, and harvesting of tissue are all performed in a 96-well format. I demonstrate the utility of this system by using tissue harvested by Ice-Cap to genotype a population of Arabidopsis seedlings that is segregating a previously characterized mutation. Because the harvesting of tissue is performed in a nondestructive manner, plants with the desired genotype can be transferred to soil and grown to maturity. I also show that Ice-Cap can be used to analyze genomic DNA from rice (Oryza sativa) seedlings. It is expected that this method will be applicable to high-throughput screening with many different plant species, making it a useful technology for performing marker assisted selection.     

A micro and rapid method for measuring P32 in plant samples

Summary A rapid method for measuring P³² in ground, undigested plant material is described.     

A method for transforming lymphocytes from very small blood volumes suitable for paediatric samples

Summary Permanent lymphoblastoid cell lines are important in the molecular analysis and characterization of human genetic disorders, when immortalized cells must be banked for future diagnostic or research purposes. However, routine methods for transformation using Epstein-Barr virus (EBV) require blood volumes that may be difficult to collect from clinically compromised neonates and small children. Here we report a modified transformation procedure utilizing blood samples of small volume (less than 1.0ml), which we have found to be particularly useful for the immortalization of lymphocytes destined for future molecular genetic studies.     

A new method for the authentication of plant samples by analyzing fingerprint chromatograms

Chemical analysis by high-performance liquid chromatography or capillary electrophoresis of plant pulverized samples, juices or extracts is an excellent method for the authentication of medicinal plant species and their products, particularly when morphological authentication is not possible. In the conventional procedure, chromatograms are integrated and the heights or areas of several peaks are used in a supervised pattern recognition method to confirm the authenticity of the product. We propose a new section approach in analysing chromatograms, where chromatograms are split into sections, which are described by four variables (number of peaks in the section, average retention time of peaks in the section, total area of peaks in the section and average area of peaks in the section), and these variables are then used in statistical analysis. The method is especially useful when the peaks on the chromatogram are not well separated and it is not easy to link individual peaks on one chromatogram with corresponding peaks on other chromatograms. In comparison with the standard procedure, our approach in analyzing chromatographic data of willow-herb (Epilobium and Chamaenerion spp.) extracts was more objective, gave better results and was also easier to perform.     

一种快速微量提取植物叶片DNA的方法

介绍了一种快速提取微量DNA的方法。该方法简单易行,无需任何特殊设备,所需样品量少。提取的DNA纯度高,D260nm/D280nm在1．9-2．1之间,可满足RAPD、SSR、转基因植株的PCR检测等以PCR扩增为基础的实验需要。     

Measurement of porosity in very small samples of plant tissue

The relative volume of internal gas spaces (i.e., porosity) of the shoot and roots of a plant largely determines its resistance to flooding, as oxygen may diffuse through these cavities from non-flooded parts of the plant into the submerged tissues. The current techniques to measure porosity either need relatively large amounts of plant tissue (200 mg per sample), or are time-consuming and not sufficiently accurate for specific types of plant material. These limitations were the reason to develop a new method of porosity measurement. Small segments of roots were taken from freshly harvested plants, placed in a two-piece hard gelatin capsule and weighed on a microbalance. The root segments were subsequently infiltrated with water under vacuum, blotted carefully and weighed again. Using the increase in weight and the specific weight of infiltrated tissue, derived from a larger sample of roots, it was possible to calculate the porosity of individual root segments as small as 3–5 mg with a length of 5 mm. The new method combines this use of small samples with a high accuracy, and proved useful for a variety of plant species. Porosity data obtained with this method will improve our knowledge of small-scale processes such as aerenchyma development in root tips.     

A small pressure chamber for use with plant leaves of small size

Summary A pressure chamber for use with small leaves can be constructed out of commercially available parts. re]19770203     

A method for sex assignment in mixed samples

A method for male sex assignment in mixed samples by amplifying a specific amelogenin Y sequence is described. The specificity and the high sensitivity make it suitable for basic research, forensic evaluations and clinical applications.     

A toolkit for analysing large-scale plant small RNA datasets

Recent developments in high-throughput sequencing technologies have generated considerable demand for tools to analyse large datasets of small RNA sequences. Here, we describe a suite of web-based tools for processing plant small RNA datasets. Our tools can be used to identify micro RNAs and their targets, compare expression levels in sRNA loci, and find putative trans-acting siRNA loci. AVAILABILITY: The tools are freely available for use at http://srna-tools.cmp.uea.ac.uk.