MATERIALS AND METHODS

This study conducted in Iranian Research Institute of Plant Protection, Tehran, Iran in May 2005-July 2006.

Plant material: One canola (Brassica napus L.) cultivar, Pionner was used. In order to establish a stock of plants, seeds that were obtained from Oilseed Research and development Company were sterilized in a commercial sodium hypochlorite solution (with 5% available chlorine) for 8 min and were rinsed 4 times with sterile water. Then sterile seeds were cultured in MS (Murashig and Skoog, 1962) medium without PGRs. Seven days later hypocotyls of (4-6 mm) seedlings were used as explant.

Culture medium, incubation conditions, evaluation procedure and statistical analysis: The basal medium that included the (MS formulation), sucrose 3% (w/v) and agar agar 1% supplemented with PGRs (2, 4-D, NAA, IBA, KN, BAP) and sodium chloride.

The media were adjusted to pH 5.8 and autoclaved at 121°C for 20 min. Cultures were maintained in 16 h photoperiod (for induction and growth of regenerated shoots) and dark (for induction and growth of calli and regenerated roots) at 25°C±1. After establishment, cultures were subcultured at 5-6 weeks intervals on fresh media. And 2 months later diameter of calli and the means number of regenerated shoots and roots per explant were evaluated. Experiment were set up in completely randomized design and repeated 4 times. Treatments has 4 replications. Data were subjected to SD.

RESULTS

Approximately two weeks after of establishment hypocotyl explants inducing of callusing, rooting and shooting were begun. The best medium for induction and growth of callus was MS medium with 6 mg L^-1 2, 4-D and 2 mg L^-1 BA. In medium with 1 mg L^-1 IBA and 1 mg L^-1 BA white calli produced regenerated shoots and earlier these results reported by Chamandoosti and Majd (2006). One milligram per liter NAA and 0.5 mg L^-1 KN was the suitable medium for production of regenerated roots on explants after a short period of callusing (very light yellow calli). After this primary selection, hypocotyl explants cultured in above media plus (0, 68.37, 102.56, 136.75, 170.94, 205.12, 239.31 and 273. 50 mM L^-1 ) sodium chloride.

Effect of sodium chloride on induction and growth of calli: After establishment, hypocotyl explants produced calli. Theses calli were compact, yellowish, nonorganogen and were similar to calli in medium with the same kind and concentration of PGRs but free from salt (sodium hypochlorite) (Fig. 1a). 0-68.37 mM L^-1 sodium chloride had a positive effect on growth of calli, so that in 68.37 mM L^-1 sodium chloride diameter of calli was the maximum (3.3125±0.4716) (Fig. 1b). The reducing effect of sodium chloride was in 102.56 mM L^-1 sodium chloride and higher. In 170.90-273.50 mM L^-1 sodium chloride diameter of calli was zero (Fig. 1c and Table 1).

Effect of sodium chloride on the means number of regenerated roots per explants: Approximately two weeks after establishment of explants in media containing 1 mg L^-1 NAA and 0.5 mg L^-1 KN plus different concentrations of sodium chloride very light yellow and compact calli on explants were appeared.

Fig. 1:	Effect of sodium chloride on callusing, rooting and shooting of Brassica naous L. Effect of sodium chloride on induction and growth of calli in 0 mML-1 (a), 68.37 mM L-1 (b), 273.50 mM L-1 (c), the mean numbers of regenerated roots in 0 mM L-1 (d), 102.56 mM L-1 (e), 273.50 mM L-1 (f), the mean number of regenerated shoots in 0 mM L-1 (g), 68.37 mM L-1 (h) and 273.50 mM L-1 (i)

Table 1:	Effect of NaCl on diameter of calli in Brassica napus L.
Data represent means of four replicates. There is no significant difference between treatments and control at p<0.01

Table 2:	Effect of NaCl on regenerated roots (Mean±SD) in Brassica napus L.
Data represent means of four replicates. There is significant difference between treatments and control at p<0.01

Theses calli were able to produce regenerated roots very well. Several kinds of roots were seen on explants. The difference of roots related to length and diameter of them. Also roots were always produced in one of the ends of explants.

Effect of sodium chloride on the means number of regenerated roots per explants was very clear. From 0-102.56 mM L^-1 effect of sodium chloride was increasing. (In 102.56 mM L^-1 sodium chloride the means number of regenerated roots was 7.2125±1.9049), (Fig. 1d and e). In concentrations 102.56 mM L^-1 the reducing effect of salt (sodium chloride) caused that number of regenerated roots were gradually reduced and in 273.50 mM L^-1 sodium chloride no rooting were seen (Fig. 1f and Table 2).

Effect of sodium chloride on the means number of regenerated shoots per explants: In media suitable for producing regenerated shoots (1 mg L^-1 IBA and 1 mg L^-1 BA), explants produced calli at first, similar earlier cases. These calli were white to light yellow and able to regenerating shoots (Fig. 1g). The means number of regenerated shoots related to the concentration of sodium chloride. 68.37 mM L^-1 sodium chloride and 273.50 mM L^-1 of this salt had the high level of increasing effect (Table 3) and reducing effect on the means number of regenerated shoots per explants, respectively (Fig. 1h and i).

Table 3:	Effect of NaCl on regenerated shoots (Mean±SD) in Brassica napus L.
Data represent means of four replicates. There is significant increase in 100 mm L-1 and significant reduction in 200 and 250 mm L-1 at p<0.01

Growth of this regenerated shoots was letter than to regenerated shoots in media with the same concentrations of PGRs but free from salt (sodium chloride) but similar to these shoots, rooted in MS medium with 1 mg L^-1 IBA and potted successfully.

DISCUSSION

There are many reports about effect of salt stress on growth and constituent of plants in tissue culture systems. For example Omar et al. (1993 ) conducted to determine the effects of sodium chloride cellular content of sunflower callus culture as the first step towards using such technique for production of salinity tolerant sunflower. These researcher resulted that sodium chloride caused reduction in callus fresh weight. They stressed that reduction in callus fresh weight might be a result of reduced water availability in the culture medium due to increase sodium chloride concentration. Mercado et al. (2000) used of growth of apical stem sections and adventitious organogenesis to evaluate salinity tolerance in cultivated tomato (Lycopersicon esculentum L.) and resulted that this approach may not be a reliable tool to evaluate salt tolerance in tomato because the lack of agreement between the results obtained with the in vitro tests, adventitious shoot formation and growth of apical stem section. However, Rus et al. (2000) studied salt responses induced by long-term callus culture in leaf callus tissue of the cultivated tomato species and its wild salt-tolerant relative (L. pennellii Correl Darcy). They concluded that the salt responses varied according to the precedence of calli (from control to saline medium). Selection a proper scale for evaluating salinity by tissue culture is very important, because breeding for water stress tolerance by the traditional methods is time consuming procedure (Droffling et al., 1993). In this research we used diameter of calli and the means number of adventitious shoots and roots in saline media and resulted that this approach is a suitable tool to evaluate effect salt in canola. Present results demonstrated that hypocotyl explants of canola not only can live in saline media but also in some concentrations of salt (68.37-102.56 mM L^-1), diameter of calli, also the means number of adventitious roots and shoots per explant were higher. In fact salt stress caused that to induce a variation in these reactions. As we know variation observed in plants regenerated from tissue culture termed somaclonal variation (Larkin and Scowcroft, 1981). Induction of resistance or tolerance in canola for salt stress (75 mM L^-1) need to gene transfer (Srivastava et al., 2004). These researchers for the first time reported that the constitutive expression of a pea PR10 gene in Brassica napus enhances their germination and growth in the presence of 75 mM L^-1 sodium chloride. PR10 proteins are encoded by a gene family and have been characterized from various plant species (Liu et al., 2003). In this research resistance in canola in 68.37-102.56 mM L^-1 sodium chloride has been induced by somaclonal variation. It seems that there is a positive correlation between our results and results of transgenic plant production. We can find that variation induced in regenerated roots and shoots, also in prolonged cells (callus) were lead to increasing the means number of adventitious root and shoots and diameter of calli. In this experiment regeneration of roots and shoots were by passing phase callus culture and regenerated organs (roots and shoots) originated from calli. So far somaclonal variation could induced new characteristic such as enhancement salinity tolerance, on the other hand new organs (roots and shoots) were completely regenerated. It is interesting that according to our findings effect of sodium chloride on rooting are similar to effect of this salt on somatic embryogenesis. Effect of sodium chloride on somatic embryogenesis reported earlier (Majd et al., 2006). The similarity between somatic embryogenesis with induction of adventitious roots has been earlier reported (Neumann, 1995).

Present findings suggest that somaclonal variation can yield stable, salt-tolerant plants. We decides that produce whole regenerated plants in the next researches.