2.1 Experimental design
Tomato plants were initially breed with seeds, which were collected from Baoshan, Yunnan Province. All seeds used in this study were surface sterilized by soaking in 70% ethanol for 1 min, 3% sodium hypochlorite for 10 min, and rinsed three times with sterile distilled water. The seeds were placed in a matrix with humus and perlite, when seeds grew to the stage with split symmetric leaves and central heartleaf (two leaves with one heart), picked out the similarity seedlings then according to the number of roots and averagely divided the roots into two parts, and transplanted to the hydroponics box (Fig. 1a), on the top of which all the seedlings were fixed with a groove foam board. The seedlings were cultivated with hydroponics (Hoagland’s nutrient solution), which was applied for tomato plant’s growth, and, was changed every 5 days to maintain salts balance, as showed in Fig. 1b. Tomato plants suffered three levels of salt stress with the application of NaCl concentration on both of left-right with uniformed (U) and non-uniformed distributed (N). Uniform (U) salt stress included: T1 (0, 0), T2 (0.2%, 0.2%), T3 (0.3%, 0.3%). Non-uniform (N) salt stress included: T4 (0.1%, 0.3%), T5 (0.1%, 0.5%), T6 (0.2%, 0.4%). Four groups of experiments were repeated for each treatment.
2.2 Measurement of plant parameters associated with salt tolerance
To effectively reflect plant physiological responses to salt stress, several indices were calculated. Those indices were relative decrease in plant biomass, the relative decrease in plant fresh weight, the relative decrease in plant height, the relative decrease in plant water content, plant K+ decrease rate, plant Na+ increase rate, the ratio of K+ to Na+ in plant, the salt injury index, and the death rate of plant. To comprehensively evaluate plant salt tolerance, the whole growth stage of plant was observed and analysis the indices that could effectively reflect plant physiological responses to salt stress. Details regarding measurements and calculation are followed below:
(1) Root measurement
took out the tomato plants from the hydroponic tank and clean them up. The side roots to be tested were laid flat in the glass dish to avoid overlapping. Use Hewlett-Packard scanner (ScanJet 3c/T) to scan the root system and specifies the scan color as black and white (the image is preserved, with the format of TIF., and the resolution is 300 dpi). Used root image analysis software DT-SCAN to determine the total root length.
roots were killed out at 105 oC first, then dry it at 80 oC to the constant weight, used an electronic scale (precision 0.001 g) weighed its dry mass.
(2) Plant leaves measurement
select three plants with similar growth conditions from each treatment, labeled the
biggest leaf from the top of the main stem before measuring the length and width it. Took the average and record the data. Using a photosynthetic meter to measure the index of leaf area (Li-6400) and calculated.
after 30 days of salt-stress treatment, measured the index with portable
photosynthesis system (Li-6400) from 11:00 am to 1:00 pm. Set up ventilation and red-blue light resource, the concentration of CO2 in the air was (394 ± 7) µmol·mol-1, the temperature was (30 ± 2) ℃, relative humidity was 37%, light intensity was 1400 µmol·m-2·s-1. Photosynthetic rate (Pn), stomatal conductance (Gs), and transpiration rate (Tr) were read directly.
Expanding index of leaf area
after the frutescence phase of the tomato started, three plants were selected from each treatment with the similar growing conditions. Newly expanded leaves are labeled and their leaf area was measured every 5 days with an average value. The tomato leaf area of the single leaf was calculated by the regression model of leaves product. The growth rate of leaves was calculated by the following formula
where LGR is the growth rate of leaf, cm/d, Q1 and Q2 is the leaf area of adjacent twice measured, cm², D1 and D2 is the time of leaf area of measuring adjacent twice, respectively day.
The formula of relatively extensional rate of leaf area is showed as below:
where RER is the relatively extensional rate of leaf area, cm2/(cm2·day), Q is the leaf area, cm².
(3) Fruit measurement and nutrient analysis
Start from the first day of tomato fruit setting, selected and labeled the target fruit from each plant that has been treated with salt stress with an interval of 10 days, respectively. Furthermore, selected three plants from each treatment, observed the transverse diameter and vertical diameter of fruit until their size and condition maintain. At the fruiting stage, the fresh weight of fruit and fruit yield were recorded.
The size of fruit, yield, quality (content of fruit soluble sugar, organic acid and vitamin C (Vc), the fruit ratio of sugar to acid, content of vitamin C, organic acid, content of starch in different parts of fruit) were measured. Mineral contents were calculated by multiplying mineral concentration by the dry weight of leaves. Na+ and K+ concentrations in leaves, roots, and fruits were determined by inductively coupled plasma-atomic emission spectrometry (ICP-AES) (Avio 200, PerkinElmer, USA). The soluble nitrate (NO3−) was measured by a flow autoanalyzer (San++System, SAKLAR, Netherlands). The chloride (Cl−) ions were measured by ion chromatography (ICS1100, Dionex, USA).