Abstract
BackgroundThe overall research objective was to develop single cell plant cultures as a model system to facilitate functional genomics of monocots, in particular wheat and barley. The essential first step towards achieving the stated objective was the development of a robust, viable single cell suspension culture from both species.ResultsWe established growth conditions to allow routine culturing of somatic cells in 24 well microtiter plate format. Evaluation of the wheat and barley cell suspension as model cell system is a multi step process. As an initial step in the evaluation procedure we chose to study the impact of selected abiotic stress elicitors at the physiological, biochemical and molecular level. We report the results of osmotic stress imposed by NaCl and PEG. As proline is an important osmoprotectant of the cereal cells, colorimetric assay for proline detection was developed for small volumes (200 μl). We performed RT-PCR experiments to study the change in the expression of the genes encoding Δ1-pyrroline-5-carboxylate synthetase (P5CS) and Δ1-pyrroline-5-carboxylate reductase (PC5R) in response to abiotic stress.ConclusionsWe found differences between the wheat and barley suspension cultures, barley being more tolerant to the applied osmotic stresses. We suggested a model to explain the obtained differences in stress tolerance between the two species. The suspension cell cultures have proven useful for determining changes in proline concentration and expression level of genes (P5CS, P5CR) under various treatments and we suggest that the cells can be used as a model host system to study gene expression and regulation in monocots.
Highlights
The overall research objective was to develop single cell plant cultures as a model system to facilitate functional genomics of monocots, in particular wheat and barley
It was found that using a pipette excluded micro callus, and after several cell passages the suspension began to loose viability
The essential first step towards achieving the stated objective was the development of a robust, viable single cell suspension culture that can be maintained in a multi-well format
Summary
The overall research objective was to develop single cell plant cultures as a model system to facilitate functional genomics of monocots, in particular wheat and barley. The greater proportion of work with plant cell suspensions relates to dicot ‘model’ plants i.e. Arabidopsis and tobacco, in the light of this we have chosen to develop single cell plant suspension cultures of wheat and barley as a model host system. It is being recognised that, under the appropriate conditions plant cell suspensions i.e. culturing cell in small multi-well plates could become an ideal platform to assist plant functional genomics by aligning with high-throughput (HTP) technologies, which have the capability to provide a global perspective on gene expression and gene product accumulation/interaction. Coupling HTP technologies with culturing cell in small multi-well format would serve two purposes; it would provide a vehicle whereby the effect of different elicitors on target gene expression can be assessed and the data acquired from somatic plant cells in culture under a range of environmental conditions can be compared with the expression profile in planta
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