Seedlings were raised in a greenhouse and transferred to 5 liter Mitscherlich pots filled with 2.0 kg of soil (RH loess serosiom) and 4.0 kg of sand. Four plants/pot with 8 replications of each variant were planted in spring 1986 and 1987. Five levels of N (NH4NO3) were used as follows. N0 = no nitrogen, N1 = 0.4 g N/pot, N2 = 0.8 g N/pot N3 = 1.2 g N/pot and N4 = 1.6 g N/pot. During planting, we applied 1.5 g/pot CaCO3, 5.0 g/pot Fe, 5.0 g/pot Mn, 2.5 g/pot Cu, 2.5 g/pot Zn, 0.5 g/pot B, and 0.05 g/pot Mo. The flowers were hand harvested at the medium stage of their development (Letchamo 1990) and dried at 38°C for 72 h. Circulatory steam distillation (2.0 g of the dry sample) was carried out in a Neoclevenger type apparatus (in 500 ml volume round bottomed flask with 300 ml deionized water) for 2 h using n-Pentan as a distillation receiver (Hölzl and Demuth 1979). The essential oil content was determined gravimetrically. Essential oil samples were diluted in 1 ml toluol and analyzed using Carlo Erba Instruments, Model GC-6000 Vega Series-2; equipped with a flame ionization detector (FID) interfaced with a Spectrophysics Integrator Sp. San Jose California for chromatography data acquisition, processing, and quantitation. A 30 m x 0.25 mm id fused silica capillary column packed with a stationary phase of 0.25 µm thickness was used for the separation of volatiles. Carrier gas flow rate was 30 ml N/min; sample size was 1 ul direct injection with Hamilton Microliter syringe. Injector system was split-splitless with 1:20 ratio. Injection port temperature was 220°C , detector oven temperature was adjusted to 240°C. Column temperature was programmed at 120°C for 0 min., 120° to 150°C at 30°C/min for 2 min., 150deg. to 175°C, 10 min., 175°C to 220°C at 30°C/min. n-Hexadecane was used as an internal standard. The data were analyzed using analysis of variance following established statistical procedures (Dospechov 1979; Köhler et al. 1984) using SPSS/pc+.
The number of the flower heads and the drug yield (g/pot) significantly increased due to additional levels of nitrogen application in all the genotypes (Fig. 3). The drug and straw yield of the diploid did not show much difference between N3 and N4. A difference of 1.9 and 6.1 g/pot between N3 and N4 was obtained for drug and straw, respectively. The maximum drug yield response for this genotype was reached at N3, as further additions of N did not result in further increase (Fig. 3). Similarly, the drug yield difference between N3 and N4 was only 0.8 g/pot of the tetraploid BK2-39, with a maximum being at N3. The weight of individual flower heads increased only up to N2 in the diploid and BK2-39 and up to N3 in R-43. The difference was 11.2 g/pot for BK2-39 and 11.8 g/pot for R-43.
Similarly, the lowest chamazulen content for all the genotypes was at N0 (Table 1) with a peak at intermediate N level depending on genotype. These results were in agreement with field experiments of Meawad et al. (1984). The overall contribution of N to Chamazulen increment was 17% in the diploid, 15% in BK2-39 and 28% in R-43.
The highest content of cis-trans-EID in the diploid was achieved at N2. The lowest value was recorded at N0. Further increment of N levels to N3 and N4 to the diploid brought about a decline in cis-trans-EID content (Table 1). However, the highest cis-trans-EID content in the tetraploids was obtained at N4, but a reasonably high concentration of cis-trans-EID was obtained at N0 in tetraploids (Table 1). The general contribution of N application for cis-EID was about 24% in the diploid, 12% in BK2-39 and 13% in R-43. This value for trans-EID was 16% for the diploid and 17% for BK2-39 and R-43.
We conclude that nitrogen application during growing conditions has a positive effect on the yield of camomile genotypes and favors the content of its active substances but, the response is affected by genotype. Establishment of the optimum level of N should be a compromise between drug yield, content of the essential oil, and the active substances in the flower heads.
|Genotype||NH4NO3 (g/pot)||Essential oil (%)||Bisabolol (mg/100 g)||Chamazulen (mg/100 g)||cis-EID (mg/100g)z||trans-EID (mg/100 g)z|
|Diploid (2n = 18)|
|Regression:||L||0.19*** (0.64)y||149*** (2.0)||2.4NS (0.06)||7.6* (0.40)||3.2NS (0.25)|
|Q||0.00NS (-0.01)||-71*** (-1.6)||-33.1** (-1.4)||-11.3* (-0.99)||-8.3* (-1.08)|
|Tetraploid BK2-39 (2n = 36)|
|Regression:||L||0.15*** (0.50)||32.2** (0.41)||61.9** (1.27)||9.5* (0.34)||1.5NS (0.16)|
|Q||-0.13NS (-0.69)||-20.5NS (-0.44)||-31.9* (-1.09)||11.2NS (0.68)||7.3** (1.31)|
|Tetraploid R-43 (2n = 36)|
|Regression:||L||0.14*** (0.52)||38.7* (0.36)||67.7** (1.47)||6.2NS (0.24)||4.9** (0.40)|
|Q||0.13NS (-0.79)||-42.0NS (0.65)||-31.9* (-1.09)||13.9NS (0.93)||1.9NS (0.25)|
Fig. 1. Diploid camomile plants under pot experiment, R. Holzhausen summer 1987.