Salinity is a major abiotic stress reducing the yield of a wide variety of
crops all over the world (Tester and Davenport 2003).
Salinity impairs seed germination, reduces nodule formation, retards plant development
and reduces crop yield. Generally, plants are sensitive to salinity during germination
and early seedling development (Maas, 1993), but they
might also be more or less sensitive to salinity at later growth stages. The
spotty pattern in crop stand at maturity, attributed to salinity, is actually
initiated at the time of germination and vegetative growth phases (Maas,
1993). The plants that grow in saline soils have diverse ionic compositions
and a range in concentrations of dissolved salts (Volkmar
et al., 1998).
Salinity inhibition of plant growth is the result of osmotic and ionic effects
and the different plant species have developed different mechanisms to cope
with these effects (Munns, 2002). The osmotic adjustment,
i.e., reduction of cellular osmotic potential by net solute accumulation, has
been considered an important mechanism to salt and drought tolerance in plants.
This reduction in osmotic potential in salt stressed plants can be a result
of inorganic ion (Na+, Cl¯ and K+) and compatible
organic solute (soluble carbohydrates, amino acids, proline, betaines, etc.)
accumulations (Hasegawa et al., 2000; Grattan
and Grieve, 1999). The osmotic adjustment in both roots and leaves contribute
to the maintenance of water uptake and cell turgor, allowing physiological processes,
such as stomatal opening, photosynthesis and cell expansion (Serraj
and Sinclair, 2002).
Salt resistance in plants is usually quantified in terms of survival rates
and/or growing abilities under stress conditions, but it is a complex phenomenon
that involves biochemical and physiological processes as well as morphological
and developmental changes (Yildiz and Kasap, 2007).
Although, the relationship between osmoregulation and salt tolerance is not
clear, there is evidence that the osmotic adjustment appears, at least partially,
to be involved in the salt tolerance of certain plant genotypes (Richardson
and McCree, 1985).
Canola (Brassicanapus L.) has some potential to cope with the toxicity
of salts (Francois, 1984) so it can be successfully
grown on salt affected soils. The present study was undertaken to assess the
effect of salt stress on different growth attributes of some genetically diverse
lines of canola at different growth stages.
Because the canola species used in this study are cultivated under various
conditions throughout the world, comparisons between varietals are questionable;
nonetheless, identification of varietal differences in response to salinity
between some Canola species was the aim of the present study. The differences
in nucleic acid content and nucleolytic enzyme activity were determined in order
to detect if they had similar responses to salinity stress during their growth.
It was also determined if the changes in nucleolytic enzyme activity, especially
ribonuclease II, could support the hypothesis that they could be used as a marker
for salt stress.Therefore, the objective of this investigation was to evaluate
the effects of salt stress on seed germination and seedling growth, proline
concentration and deoxyribonuclease (DNase I1) of canola under salinity conditions
to better understanding of the mechanisms of salt tolerance in these genotypes.
MATERIALS AND METHODS
An experiment was carried out in a growth room of the Department of agronomy and Plant Breeding, University of Zabol, Zabol, Iran during April-June 2009 to screen 5 canola (Brassica nupus L.) genotypes viz., Hyola308, Hyola401, Hyola60, Optlon50 and RGS003. These five genotypes are the most popular genotypes which can be cultivated in the north and south of Iran. The experiment was laid-out as a completely randomized factorial design with three replicates. Three hundred seeds of each canola geneotype were surface sterilized in 5% sodium hypochlorite solution for 5 min and then carefully rinsed with distilled water to remove the traces of sterilizing agent. There were four different regimes of salt stress i.e., S0 = 0, S1 = 100, S2 = 200 and S3 = 300 mM of NaCl.
The treatment solution in each Petri plate was changed every day by dripping out and adding fresh treatment solution. Germination started after two days of sowing and when the radicle reached up to 5 mm in length a seed was considered germinated. The data for germination was recorded daily up to the end of the experiment. Germination percentage was calculated using the following formula:
After fifteen days of the start of the experiment, plant seedlings were removed
carefully from the Petri plates and separated into shoots and roots and fresh
weights recorded. In this time biochemical components such as proline was determinated
in root and shoots. The extracts of shoot and root were used to determine proline
concentration in canola genotypes (Bates et al., 1973).
For free proline content, leaf samples were homogenized in 5 mL of sulpho salicylic acid (3%) using mortar and pestle. About 2 mL of extract was taken in test tube and to it 2 mL of glacial acetic acid and 2 mL of ninhydrin reagent were added. The reaction mixture was boiled in water bath at 100°C for 30 min. after cooling the reaction mixture, 6 mL of toluene was added and then transferred to a separating funnel. After thorough mixing, the chromophore containing toluene was separated and absorbance read at 520 nm in spectrophotometer against toluene blank.
Deoxyribonuclease (DNase II) assay: After fifteen days of the start
of the experiment, the plants were harvested, Fresh samples were immediately
used for the determination of nucleic acid content and enzyme activity. A known
weight of the fresh seedling was placed in a mortar and homogenised in 20 mL
of distilled water. The filtrate was separated from the residue and used in
the study. Estimation of DNase II was carried out by the method of Kunitz
(1950), in which 0.5 mL of sample was mixed with 2.5 mL of buffer substrate
(pH 5.0) and E/30 sec for 5 or 10 min at 260 nm was measured against a blank.
The volume activity was equal to (3.0x1000)/0.5xΔE (units mL-1
Statistical analyses: Data quantification and statistical analysis were performed using MS Excel software (Microsoft Excel 2003) and then all data were analyzed with SAS Institute Inc 6.12. Data were first analyzed by ANOVA to determine significant (p = 0.05) treatment effects. Significant differences between individual means were determined using Fishers protected Least Significant Difference (LSD) test. Data points in the figures represent the Mean±SE of three independent experiments at least three replications per cultivar per treatment combination each.
Germination percentage and seedling growth: The results revealed that the germination of canola, was strongly affected by all salt treatments. Increased salt concentration caused a decrease in germination. Strong reduction was observed mainly at the higher level of salt concentration compared to control. Lowest germination was observed in case of Hyola308 at high salinity treatments (Fig. 1).
The studies were laid to investigate the influence of salinity on seedling
growth of germinating seeds of canola genotypes. The results indicated that
emergence of root and shoot delayed as the salt stress increases compared to
controls. The continuous increase in length of root and shoot was observed in
frequent hours of germination in four canola in control as well as salt treatments.
The data on the average length (Fig. 2 and 3)
of root and shoot shows that RGS003 genotype had the highest length of root
and shoot in control treatment. The results presented in Fig.
2 and 3 indicated that RGS003 had the greatest reduction
of shoot length and Hyola60 had the highest root length in 300 mM NaCl treatment.
Decrease in length of root was more pronounced as compared to shoot in all NaCl
||Effects of salt stress on root length
||Effects of salt stress on shoot length
Proline concentration and nucleic acid: With a NaCl concentration of 300 mM, the proline concentration in root and shoot in canola genotypes increased to two-fold values, compared to the control treatment (Fig. 4 and 5) and salinity stress had significantly effect on it. Among the genotypes, Hyola401 and Hyola60 had the maximum concentration of proline in root and shoot in S3 treatment. In this study root proline content was substantially higher than in shoot.
Salinity disturbed nucleic acid metabolism and caused growth inhibition. Along
with increased salinity stress in all of the studied plants, salinity significantly
affects on nucleic acid metabolism and increased the level of the activity of
DNase II (Fig. 6).
||Effects of salt stress on proline content in root
||Effects of salt stress on proline content in shoot
||Effects of salt stress on DNase II activity
At the highest concentration NaCl treatment, enzyme activity was significantly
enhanced. Among the genotypes, RGS003 had the highest the activity of DNase
II in all of salinity treatments and at the S3 level.
Seeds of canola genotypes germinated rapidly in non saline (control) treatment
and reached final germination percentage in less than 10 days. Change in salinity
significantly affected the final germination of canola seeds. In general, increased
NaCl level, led to the reductions in germination percentage (Fig.
1), germination rate and seedling fresh weight. This can be attributed to
prevent of water uptake created by salinity condition. This can be also due
to the toxic effects of Na and Cl ions on the germination process. NaCl may
be inhibitory to the activities of some enzymes that may play critical roles
in seed germination (Khajeh-Hosseini et al., 2003).
Seedling growth was recorded in terms of Shoot/Root length at different levels of NaCl salinity. The increase in NaCl concentrations decreased the shoot and root length of all the canola genotypes. All genotypes responded in same manner to salinity stress. However, the intensity of stress varied with the genotypes. The reduction in root length was greater than shoot length (Fig. 2 and 3). Maximum decrease in root and shoot length at 300 mM NaCl were recorded in Hyola60 and RGS003 genotypes.
Among the genotypes tested RGS003 appeared to be more sensitive at 300 mM NaCl
in germination stage then others. Although RGS003 genotype had comparatively
low germination at higher salinity levels but performed quiet satisfactorily
at seedling stage for activity of DNase II. Among the genotypes, RGS003 had
the highest the activity of DNase II in all of salinity treatments and at the
S3 level (Fig. 6). Abo-Kassem
(2007) studied effects of salinity: calcium interaction on growth and nucleic
acid metabolism in five species of Chenopodiaceae, he found that, the activity
of DNase II increased along with increased salinity stress in all of the studied
plants. At the highest concentration NaCl treatment, enzyme activity was significantly
enhanced (4 times that of the control).
All of the studied plants, showed a progressive increase in DNase II activity
with increased salinity. This showed that for plant cells protecting the DNA
when under salt stress was a priority, which is confirmed by the data reported
by Hasegawa et al. (2000), who observed a salinity
stress-induced reduction in cell elongation, but no reduction in cell division.
Addition of CaSO4 to NaCl increased DNase I activity in most of the
examined plants, whereas NaCl stress reduced DNase- and RNase-specific activity
in alfalfa and lentil (Yupsanis et al., 2001).
In this study, in root and shoot of canola genotypes, proline accumulated but
in root tissue, accumulation of proline was 2 times higher than that in shoot
tissue. Among the genotypes, Hyola401 had the maximum concentration of proline
in root and shoot in S3 treatment than control. In addition, accumulation
of solute such as water soluble carbohydrate, polyos and amino acids in tissues
is an important adaptation mechanism for plants in response to osmotic stress
like water deficit and high salinity levels (Yang et
The results obtained showed that of all genotypes studied, Hyola60 was the most salt- tolerant. It also showed the highest germination percentage and seedling growth (length of root and shoot) in 300 mM NaCl. Solute accumulation such as proline in the root and shoot as a result of salt stress appeared to play an important role in the acclimation of this genotypes to salt stress, suggesting that they could be used as physiological markers during the screening for salt tolerance.