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

UPLC

Experiment: Identification of cytokinins by ultra-performance liquid chromatography (UPLC).

Introduction

We aim to make E. coli to produce cytokinins (especially iP: N6-(2-Isopentenyl)adenine and tZ : trans-zetain) by introducing AtIPT4 or AtIPT7 into E.coli. In order to confirm that E. coli synthesize iP and tZ, we will use ultra-performance liquid chromatography (UPLC). Before attempting the cytokinin biosynthesis, we determined the retention times of iP and tZ by using authentic samples.

Method

UPLC was carried out as described by Novák et al. (2008). Samples (5nM) were prepared by diluting each cytokinin DMSO solution with a mobile phase (initial conditions). 10 μL of each sample was injected onto a reversed phase column (BEH C18, 2.1 * 50 mm, 1.7 lm; Waters). The samples were eluted with an 8 min linear gradient of 90:10 = A:B to 50:50 = A:B (v/v) where A was 15 mM ammonium formate and B was methanol at a flow rate of 0.25 ml min-1. The column temperature was set to 40 ℃. At the end of the gradient, the column was washed with 100% B (1 min) and equilibrated to initial conditions for 3 min. Under these conditions, the retention times for the monitored compounds ranged from 2.5 to 7.5 min. The effluent was passed through an ultraviolet detector at 268 nm.

Result

Reference