The effects of calcium applications on the breaking forces in inflorescence stems of herbaceous peonies are shown in Table 1. Quantitative analysis showed that the force required to break the stems which were treated with calcium was significantly higher than that required for the control, it was at least 118.0%, 112.5% and 106.4% in the control stems for the top, middle and bottom segment, respectively. For the top segment, Treatment 3 (4% calcium three times) was the best, and significantly different from other schedules, Treatment 2 (4% calcium twice) was second best, and the effect of Treatment 4 (2% calcium three times) was the worst. For the middle segment and the bottom one, Treatment 3 was both the best, however, other schedules showed no clear difference. So we could reach the conclusion that preharvest sprays with 4% (w/v) calcium chloride three times after bud emergence were the best for the mechanical strength of “Da Fugui” peony stems. The effect on the breaking force was significantly reduced when increasing the concentration to 6%, which maybe in excess of the calcium absorption capacity of the plant. In addition, when decreasing the concentration to 2%, insufficient calcium supply may have occurred, regardless of the times of treatment.

The stems were sprayed three times with 4% (w/v) calcium chloride. Calcium concentrations together with cell wall materials (CWM) contents in the stems were analyzed to investigate the relationship between the breaking force and these factors.

Calcium sprays increased calcium concentrations in peony stems regardless of the segment (Table 2). The results were 111.1%, 106.9% and 105.9% in the control stem for the top, middle and bottom segment, respectively. There was significant difference between treatment and the control in the top segment, and none of the calcium sprays led to significant increase of calcium concentration in the middle or the bottom part.

The experiments were carried out with 3-year-old “Da Fugui”, one of the popular herbaceous peony cultivars in China, which were cultivated in the ornamental plants garden of Yangzhou university, Jiangsu Province, China (32°30′N, 119°25′E). The plants grew well in the natural environment with normal management.

Thirty-six plants of peonies were chosen for treatments. The chosen stems together with the leaves and buds on them were sprayed with calcium chloride (w/v) once a week after the bud emergence. The treatments were as follows: Control (water), Treatment 1 (4% calcium once), Treatment 2 (4% calcium twice), Treatment 3 (4% calcium three times), Treatment 4 (2% calcium three times), Treatment 5 (6% calcium three times). All the sprays including the control contained surfactant (0.01% Tween-20). About 1 week after all treatments were complete, we harvested the stems for experiments, the stage of peonies flower opening meanwhile was loose bud, the outer petals soft and loosening, firm inside [8,19].

The inflorescence stems were cut and separated into three segments, 5 cm length from the upper apex or distal of the stem as the top segment, the same length from the proximal and the midst as the bottom and the middle one, respectively. The breaking strength of each segment was measured by the method of Burk et al. [20] with a universal digital force testing device (Model NK-2; Huier, Hangzhou, China), and a force was applied manually until the stems were broken. Stems from 6 plants corresponding to each treatment were tested for the breaking force.

Calcium concentration in each part of the inflorescence stems was determined by the method as follows. Each part of the inflorescence stems was cleaned with deionized water and then dried to constant weight at 70 °C. The stems were then ground into powder and sieved. The weighed powder was digested with nitric acid and water (1:1, v/v) in the microwave digestion system (MARS 5, CEM, USA). Calcium concentration was determined by atomic absorption spectrophotometer (Solar S4 + Graphite Furnace System 97, Thermo Elemental, USA). The experiment was performed twice.

The cell wall materials (CWM) were fractioned following the method of Rose et al. [21] with some modifications. Briefly, the stems were ground into fine powder in liquid nitrogen and extracted with 95% alcohol, then washed twice with boiling alcohol and methyl alcohol: chloroform (1:1, v/v), respectively. Finally, the cell wall residues were dried overnight at 50 °C and used for analysis. The experiment was performed in triplicate.

Pectin content in different forms followed the methods of Manganaris et al. [22] for water soluble pectin and Selvendran & O’Neill [23] for water insoluble pectin fraction, and the uronic acid was determined by a colorimetric assay [24], using galacturonic acid as the standard. Total pectin content was the sum of pectin content in both fractions. Hemi-cellulose content was determined by the phenol colorimetric assay [25] and the cellulose content was measured by the anthrone colorimetric assay [26]. Lignin content was determined following the method of Müse et al. [27]. Measurements were done in triplicate.

Statistical analyses were conducted using SPSS 16.0. Differences in breaking strength and the content of cell wall fractions among treatments were analyzed by LSD test at a significance level of 0.05, one-way ANOVA.