Project

Aim

Method

Plants were grown in growth chambers at Sheffield's Controlled Growth Facility. A number of phenotypic characteristics were studied whilst the plant was developing, e.g. flower stalk length, number of developing siliques and length of developing siliques.

Arabidopsis samples were collected as follows. Flower stalk length is measured on day of flowering. One flower and stalk should be harvested from each plant leaving one flower for later silique formation. Seven days after flowering count the number of developing siliques and measure the length of at least three. On the same day harvest two lots of rosette leaves and take two lots of phloem exudates. Fifteen days after flowering at least ten developing siliques are to be collected. All samples were placed in liquid nitrogen until they were stored in a -86°C freezer. Samples collected should not exceed 0.1g and if possible three samples of each type of tissue should be taken. All samples were harvested between 11am and 12am as amino acid concentrations are known to fluctuate throughout the day.

This is carried out to check the genotype of the plants and so allow differentiation between heterozygotes, homozygotes and mutant wild types.

The plant material was homogenised with an extraction buffer (50mM HEPES-KOH, pH 8.5) in a pestle and mortar on ice. The homogenate was centrifuged for two minutes at 14000 rpm at 4°C. The aqueous layer was then removed to a clean tube and stored at -86°C

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The amino acid content of the samples can be determined by HPLC with pre-column OPA derivatisation and fluorescence detection. Samples were prepared using a borate buffer and OPA and were then loaded in to the machine. Using the Perkin Elmer pump, detector and integrator the concentrations of the amino acids can be derived from a chromatogram using a data spreadsheet

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