Bio assay

Experiment : Quantitative analysis of cytokinins using cucumber cotyledons

Introduction

We performed quantitative analysis of cytokinins using cotyledons of Cucumis sativus (cucumber). We have proposed to make E. coli produce cytokinins. We need to establish experimental system for quantitative analysis of cytokinins. The cucumber cotyledons bioassay is frequently used as a simple and rapid bioassay for cytokinins (1, 2). Previous works indicated that cytokinins enhance chlorophyll levels in plant cells. Using cytokinin samples, we attempted to acquire the technique of cucumber cotyledons bioassay.

Materials and Method

1. Cucumber (Cucumus sativus ) seeds were planted on absorbent cotton dampened with water and germinated in the dark at 27℃ for 5 days.

2. The cotyledons were excised in dim red light and placed in 3.5 cm plastic dishes containing 0.4 ml of cytokinin solutions.

3. The dishes were returned to the dark at 27℃ for 24 h and then moved into fluorescent light. After 24 h the weight of cotyledons was measured. The cotyledons were homogenized and the chlorophyll was extracted in 3 ml 80% cold acetone. The volume was brought up to 5 ml with acetone and then centrifuged (2000rpm, 5 min, 4℃). The absorbance of the supernatant were read at 663.6 and 646.6 nm. Calculation of concentration of the chlorophyll was carried out as described by Porra et al.,1989 (3).

Results

Two pictures of cytokinins treated cotyledons are shown in Figure1. We could see that cytokinins had effects of hypertrophy and greening on cotyledons. The rational concentrations of chlorophyll extracted from each dish are shown in Figure2. The concentrations of chlorophyll extracted from treated cotyledons were higher than those from non-treated cotyledons.

Refrences

(1) R. A. Fretcher and Dlanne McCullagh (1971) Planta, 101, 88-90

(2) R. A. Fretcher, V. Kallidumbil and P. Steele (1982) Plant Physiol, 69, 675-677

(3) R.J. Porra, W.A. Thompson and P.E. Kridemann (1989) Biochimica et Biophysica Acta, 975, 384-394

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