2.1 The objectives of this chapter are to
2.2.1 Materials and methods
The experiment was conducted according to Abel et al. (2006), with minor modifications. Thirteen genotypes: Alesi, H30, Mansholts, Samourai, Sollux, Gaoyou, Sansibar, Oase, Express, R53, Digger, DH14 and L16 were tested in the greenhouse. Six seeds of each genotype were sown in 7x7-cm pots filled with a soil mix of 50% composite and 50% sand. The soil and sand were sieved through a 5-mM mesh and dried for 24 h at 105°C. The pots were randomized on tables in a complete block design, where each genotype was represented by two pots in each replication. Each pot was watered by 100 ml tap water. After seedling emergence the soil was kept humid by overhead spraying for one week.
At day 12, plants were thinned into two plants per pot. The plants were watered every 2nd or 3rd day by placing them for 1 h into a bowl filled with tap water, ca 3-4 cm high. Plants were grown without saline solution till they had four leaves, around two weeks after sowing. The control plants continued to be watered with tap water until harvest. The salt-stressed plants were treated with two different salt concentrations. For acclimation, the saline concentrations were elevated gradually from the 1st day to the 4th day, to the final concentrations of 200 mM and 300 mM, respectively. The saline treatment continued for two weeks by applying saline solution via dipping into the bowl as described above. At day 28, the plants were harvested, whereby the shoot system was divided into different parts; 1st + 2nd leaves, 3rd and 4th leaves, hypocotyl and sprouts. The FW for each part was measured immediately, and the leaf area was measured. For DW estimation samples were dried for 72 h at 60 °C.
2.2.2 Results
A significant reduction in FW and DW was observed for all parts under both the 200 mM NaCl and 300 mM NaCl treatment compared to the controls. However, six parental lines, Mansholts, Samourai, Sollux, Gaoyou, Alesi and H30, showed higher biomasses compared to the other genotypes (Figures 1 and 2). Leaf chlorolysis, leaves dropping and plant loss were observed, especially under 300 mM NaCl.
2.2.3 Conclusion
The flooding method was suitable for phenotyping plants under salt stress in greenhouse experiments. The six parental lines of Mansholts, Samourai, Sollux, Gaoyou, Alesi and H30 were included in the following experiment. We also concluded that fertilization would be necessary to avoid the above-mentioned problems of leaf chlorolysis, leaf-drop and plant loss. The 200 mM NaCl treatment was more relevant than the 300 mM NaCl condition. Leaf area results were valuable, but this parameter made it impossible to harvest the whole plants at the same time. Moreover, the measurement process took too long, thus, this parameter was not considered in the next experiments.
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• No microenvironment differences because all genotypes were irrigated together, not independently.
• No logging or water deficiency because all pots were immersed in the tap water or in the saline solution. This method allows the plants to take up the optimum amount of water.
The disadvantages of the flooding method
• After watering the discharge of the rest of the water is laborious.
• The water and saline solution should be enriched with a fertilizer.
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Chapter IIــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ
** Significant at P = 0.01; * significant at P = 0.05, + significant at P = 0.1 and ns = non-significant. The significance test was done by simple ANOVA using the software Plabstat (Utz 2003).
0 2 4 6 8 10 12 14
Mansholts Samourai Sollux Gayou Alesi H30 Express DH14 R53 L16 Sansibar Oase Digger
Fresh weight (g)
Genotypes
Control 200 mM 300 mM
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Figure II-1: Total plant fresh weight (g) mean values and significance levels of the 13 parental lines under control and salt stress
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** Significant at P = 0.01; * significant at P = 0.05, + significant at P = 0.1 and ns = non-significant.
0 0.2 0.4 0.6 0.8 1 1.2 1.4
Mansholts Samourai Sollux Gaoyou Alesi H30 Express DH14 R53 L16 Sansibar Oase Digger
Dry weight (g)
Genotypes
Control 200 mM 300 mM
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Figure II-2: Total plant dry weight (g) and significance levels of the 13 parental lines under control and salt stress
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Chapter IIـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ 2.3 Experiment 2: Salinity effect on the selected six genotypes; Mansholts, Samourai, Sollux, Gaoyou, Alesi and H30.
2.3.1 Materials and methods
The six selected genotypes: Mansholts, Samourai, Sollux, Gaoyou, Alesi and H30 were tested under both control and the salt treatment of 200 mM NaCl. The soil contents, pot size, pots and randomization were the same a mentioned above.
Experiment one (2.2): In experiment one, some leaves became chlorotic earlier;
therefore, to avoid nutrient deficiency the control treatment and saline solution were both supplied with Hakaphos blue (COMPO, Netherlands).
At day 15, the salt treatment was started with 100 mM NaCl supplemented with 0.5 g/l Hakaphos in the saline solution. At day 17, the saline solution concentration was increased to 150 mM NaCl enriched with 1 g/l Hakaphos. Finally, at day 19 the final concentration of 200 mM NaCl was reached. From day 19 until the end of the experiment the stressed plants were watered with the final concentration on alternative days; one day with water only and the second day with the saline solution.
The control plants were always watered with tap water enriched with 1 g/l Hakaphos.
The chlorophyll content was measured with a SPAD-meter Minolta 502 (Osaka, Japan). The measurement was made one week from the beginning of salt treatment.
The measurements were scored for two different plant parts: the 1st and the 2nd leaves, and the 3rd and the 4th leaves. The plants were harvested two weeks from the beginning of the salt treatment. The shoot system was separated into 1st and 2nd leaves, 3rd and 4th leaves, stem and rest. Fresh weight was determined immediately after harvesting. For DW estimation, the plant parts were dried for 72 h at 60°C.