Improving Salt Tolerance in Tomato Lycopersicon esculentum Mill. by in Vitro Mutation Technique

This research was conducted to produce salt tolerant tomato plants by using mutation and tissue culture techniques. Seeds of two Tomato cultivars (Super Regina and Trescantos) were treated with 2.0 mM of Sodium Azide NaN3 for 4 h and sterilized with Sodium Hypochlorite (NaOCl) of (0.0, 1.0 or 3.0)%. The treated seeds were germinated on (MS) medium and shoot tips (1 cm) were used to induce calli on media supplemented with different hormones combinations. The produced calli were cultured on media of (6.5, 8.5, or 10.5) dSm -1 sodium chloride. Plants were then regenerated from the survival calli. The results showed that 3% NaOCl was the best sterilization treatment. Moreover the best growth regulator combinations were either 2 mg /L BA with 2 mg/L IAA or 0.5 mg of BA with 1 mg/L IAA. Since there was no significant difference between them, the last was recommended. Furthermore, salinity had negative effect on callus growth and (6.5 dSm -1 ) treatment showed significantly higher callus fresh and dry weights than the other treatments. No plants were produced from 10.5 dSm -1 treatment and Super Regina showed higher percentage of plant regeneration than Trescantos. Calli grown on 10.5 dSm -1 contained the highest Sodium and Chloride ions. Trescantos cultivar showed significantly higher calli sodium ions content compared with Super Regina. However, no significant differences between the two cultivars in the Chloride ion content were found. In conclusion it has been possible to induce and select salt tolerant tomato plants via in vitro mutation technique.


INTRODUCTION
Tomato (Lycopersicon esculentum Mill.) is one of the important vegetable crops of the Solanaceae family.Tomato is grown throughout the world due to its high contents of vitamins, minerals and antioxidants.Several breeding methods were used to improve its quantity and quality (Flowers, 2004;Foolad, 2004;Estan et al., 2005;Cuartero et al., 2006).However its yield is still severely affected by environmental stresses such as high temperature, drought, salinity, insects and various diseases.
High salinity stress is the most severe environmental stress, which affect crop production on at least 20% of irrigated land worldwide.Also salinity is the major problem in Iraq which has great impact on tomato production.Thus there is a need to improve the ability of tomato plants to tolerate soil salinity using any available breeding methods.
The fast method is through the selection from the genetic pool of this plant.However increasing the variations via chemical and physical mutation among the available cultivars will broaden the gene pool.Moreover tissue culture provides uniform environment for the selection under any stress.Combination of chemical mutation and in vitro selection has been used successfully by many researchers to select plants with desirable characteristics (Al-Hattab et al., 2002;Aslid et al., 2006;Shlahy and Al-Hattab, 2009a;Shlahy and Al-Hattab, 2009b).Therefore the current experiment was conducted to select tomato plants with good salt tolerance ability using in vitro chemical mutation and selection method.

MATERIALS AND METHODS
Two tomato cultivars (Super Regina and Trescantos) were used in this experiment.Mature dry seeds were treated with 2.0 mM Sodium Azide (NaN 3 ) in phosphoric buffer of pH 3 for 4 h.The seeds were sterilized for one mint with 70% ethanol and then with  Sodium Hypochlorite (0, 1, or 3 %) for 15 min and rinsed three times with distilled water 5 min each.The sterilized seeds were cultured on hormone free MS medium (Murashige and Skoog, 1962) supplemented with 30 g/L sucrose and 7 g/L agar to produce seedlings.Percentage of contamination was recorded after one week in culture.Shoot tips (1 cm) were excised from fourteen days old seedlings and used for callus induction.Shoot tips were cultured on MS media supplemented with different combinations of Benzyl adenine (BA) of (0.0, 0.5, 1.0, or 2.0 mg/L) and Indol Acetic Acid (IAA) of (0.0, 1.0, or 2.0 mg/L) with ten replications per treatment.Growth regulators effect on callus fresh weight was analyzed to select the best hormone combination.
Ten replications with (100 mg) /tube of the produced calli from all the above treatments were transferred to the selected medium with the addition of Sodium Chloride (NaCl) of (6.5, 8.5, or 10.5 dSm -1 ).After 6 weeks in culture, callus fresh and dry weights were recorded.The amounts of Sodium and Chloride ions in the dry calli of three replications were estimated according to Perez-Alfocea et al. (1994).Survival calli were transferred to regeneration medium supplemented with Sodium Chloride of the corresponding concentration.Numbers of regenerated plants from all the treatments were calculated.
The experiments were setup using (C.R.D) and the means were compared using Least Significant Differences (L.S.D) at p≤0.05 level.

RESULTS AND DISCUSSION
The results showed (Table 1) that 3% Sodium Hypochlorite was the best sterilization treatment that reduced seed contamination to 0% in both cultivars Trescantos and Super Regina.Therefore 3% Sodium Hypochlorite was used throughout this experiment.Sterilization is the most important step to start successful culture.The disinfectant should not affect the viability of the explants and provide 100% sterilization.
The results (Table 2) showed that the growth regulators had significant effect on the fresh weight of the two cultivars.The medium with 2 mg/L BA showed significantly higher callus fresh weight in both cultivars compared with the other treatments.Moreover 1 and 2 mg/L of IAA gave significantly higher fresh weights than the control and there were no significant differences between them in both cultivars.Whereas the interaction analysis showed that the combination of 2 mg/L of both BA and IAA was the best for callus induction for Trescantos and Super Regina cultivars.However this treatment was not significantly different than the combination of 0.5 mg/L BA and 1 mg/L IAA.Therefore from this result it has been recommended to use the last treatment for callus induction.The results of the current study are in agreement with what was reported by other researchers that the cultivars have different callus induction response in the presence of different hormone combinations (El-Kaaby et al., 2012;Shlahi, 2003).
The results in Table 5 showed significant increase in Cl -concentration in the dry calli grown in medium supplemented with 10.5 dSm -1 salinity level compared with the control.Although Super Regina cultivar showed higher concentration of Cl -compared with Trescantos but it was not significant.For the interaction between the cultivars and the salinity level Super Regina dry calli showed significantly higher Cl - concentration on the 10.5 dSm -1 salinity level.At 8.5 dSm -1 salinity level, Trescantos dry calli contend higher Cl -concentration than Super Regina.
Calli from both cultivars grown on medium with 10.5 dSm -1 salinity level lost the ability to regenerate plants, whereas the control medium gave the highest percentage of plant regeneration (26%) (Table 6, Fig. 1A).Super Regina gave slightly significant higher percentage of plant regeneration than Trescantos (Fig. 1B).
Plants regenerated from salt tolerant calli might be salt tolerant too.The calli was induced from seeds treated with NaN 3 which has mutation affect, therefore it is possible some of the calli have genetic changes or due to the somaclonal variations that occur in culture.Al-Qurainy and Khan (2009) discussed the mutagenic effect of sodium azide and its application in crop improvement.In addition several studies showed that NaN 3 is effective in economical crop improvements in vitro such as tomato (Adamu and Aliyu, 2007), peas (Aslid et al., 2006) and sugarcane (Ikram et al., 2011).
Combination of tissue culture and mutation techniques were used to induce genetic changes to improve different crops (Irshad et al., 2010;Saif-Ur-Rashid et al., 2001).Selection of salt tolerant plants is difficult under field conditions, but plant tissue culture techniques are performed under aseptic and controlled environmental conditions.These advantages of plant tissue culture allow various opportunities for researcher to study the complex responses of plants against environmental stresses and select plants with desired traits (Lokhande et al., 2010;Lokhande et al., 2011;Safarnejad, 2004;Sakthivelu et al., 2008).
All the regenerated plants were transferred to the field and produced fruits.The seeds of the second generation will be used to test the salt tolerant ability of the regenerated plants under field conditions.

CONCLUSION
Sodium Hypochlorite at 3% concentration for 15 min is the best sterilization treatment for tomato seeds.Moreover, combination of 0.5 mg/L BA and 1 mg/L IAA is recommended for callus induction.Trescantos cultivar has the ability to accumulate Na + and Cl -ions in the cells with less effect on callus growth which is one of the mechanisms used by the plants to avoid the effect of high salinity levels in the growth environment.However, Super Regina gave higher percentage of plant regeneration than Trescantos.Tomato plants regenerated from salt tolerant calli were transferred to the field and grown to maturity.

Table 1 :
Effect of sodium hypochlorite concentration on contamination percentage of the seeds of two tomato cultivars

Table 2 :
Effect of growth regulators on callus fresh weight (mg) of two Tomato cultivars