Environmental Engineering Reference
In-Depth Information
2.
M
ATERIALS AND
M
ETHODS
2.1. Plant Material and Treatment
The salt-sensitive
G. max
ZH13 cultivar (widely cultivated soybean in China) and the
salt-tolerant
G. soja
BB52 (collected from Yellow River Delta, Shandong Province, China)
were chosen for this study. Before germination, the seeds of ZH13 were fully soaked in
distilled water for 8h, while the seeds of BB52 were soaked in concentrated sulfuric acid for
10min to remove the hard shell over the seeds. Then, the treated seeds were sown into
germination dishes, lined with two layers of filter paper and moistened with 20ml Hoagland
nutrient solution and germinated at 25
℃
in the dark. After 5 days, the germinated seeds were
transferred to plastic pots filled with vermiculite and grown in artificial climatic chambers
(Huier, China). The vermiculite was kept wet by watering with Hoagland nutrient solution
every day, too. During the experiment, the photoperiod was 12 hours and day/night
temperature and humidity were respectively controlled at 25/18
℃
and 65% in the chambers.
After 20 days, seedlings with uniform growth pattern were selected for salt treatment. NaCl
was added to nutrient solution to provide final concentrations of 0 (control), 50, 100, 200 and
300mM/L. The higher NaCl concentrations (>50 mM/L) were imposed incrementally by
50mM/L step every day until final concentrations were reached. Seedlings of each group were
treated with NaCl nutrient solution of final concentration for 7 days. The newest fully
expanded leaves were used for measuring photosynthetic and fluorescent parameters. Leaves
and roots in each treatment group were harvested, weighed, frozen in liquid nitrogen and
stored at -80
℃
in a freezer for the subsequent experiments.
2.2. Growth Parameters
At the end of salt stress, plant height, root length, fresh weight, petiole number and stem
diameter were determined and leaf area at the same leaf location was measured by a leaf area
meter (portable area meter LI-3000A
，
LI-COR). All the indices were repeated five times.
2.3. Chlorophyll Content
Leaf samples (0.2g) were soaked in 20ml 95% (v/v) ethanol at 4
℃
in darkness until the
tissues became totally white. Extracts were used to measure the absorbance at 649nm and
665nm, the chlorophyll content was calculated according to Lichtenthaler and Wellburn [12].
2.4. Gas Exchange and Chlorophyll Fluorescence
Gas exchange and chlorophyll fluorescence were simultaneously detected by an open
photosynthetic system (LI-6400XT, Li-Cor, USA), which is equipped with a fluorescence leaf
chamber (6400-40 LCF, Li-Cor, USA). The leaves were dark-adapted for 30min before the
measurements. The minimal fluorescence level in the dark-adapted state (Fo) was measured
