2023.08.03.128

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Effect of Sodium Azide (NaN₃) on micropropagant and tolerance of Salt Stress of Strawberry cv. Albion

Raghad Abd Alhamza Juameer ¹, Ayad Assi Obaid²

¹University of Diyala ; [email protected]

² College of Agriculture \University of Diyala; [email protected].

* Correspondence: [email protected].

Available from. http://dx.doi.org/10.21931/RB/2023.08.03.128

ABSTRACT

Sodium azide (NaN₃) is widely used to induce in vitro plant systems mutagenesis.Current study aimed to develop mutants of Strawberry cv. Albion tolerant to salinity stress. Plant regeneration from runner explant cultured on multiplication Murashige and Skoog (MS) medium contain 0.5 mg l^-1 of 6-benzyladenine (BA) and 0.1 mg l^-1 of Naphthaleneacetic acid (NAA) and sodium azide (NaN3) as a chemical mutagens at concentrations (0.25.0.5) mMafter 4 weeks , the mutagens shoots were exposed to different concentrations of Sodium Chloride (NaCl) (6 , 10 , 14 , 22) ds m^-1. The results revealed that the superiority of 6 ds m^-1 of NaCl without mutation of sodium azide in giving the highest rate of the number of shoots reached 9.25 shoot explant^-1 and giving the highest average length of shoots,which was 3.04 cm and the highest rate of fresh weight reached 3.197 gm ,while the treatment sodium azide led to decrease in the number of shoots upon mutagenesis with NaN₃ at both concentrations and at all salt levels.A significant increasing of Sodium ion was observed in the shoots mutagenic with sodium azide at 0.5 mM and grown on a medium with 22 dS m^-1 of NaCl, as it reached 42.90 mg g^-1 and the highest calcium ion was 6.707 mg g^-1 when treated with 0.25 mM of sodium azide with 6 dS m^-1 NaCl ,It also gave the highest potassium concentration 31.50 mg g^-1.

Keywords: Strawberry cv. albion, microprpagation, Sodium azide, Mutagens, Salt tolerance

INTRODUCTION

Strawberry (Fragaria x ananassa Duch.) is a perennial herbaceous plant in the Rosaceae family. One of the fruits that grows in the southern hemisphere in temperate and semi-temperate regions ¹ , are plants from the Rosaceae. Their ploidy types range from diploid to decaploid ^2,3, while wild members of the genus distributed throughout the northern hemisphere and parts of western South America ⁴. and follow the fruit with small fruits and is the fourth most consumed fruit after apples, oranges and bananas ⁵. During the past few years, Iraq lands had witnessed an adverse environmental stress such as saline and drought conditions, which negatively affected crop yields ⁶.Generally, the foremost effect of several ecological stresses, producing salt stress, generates reactive oxygen/nitrogen species (ROS/RNS) and enhances oxidative stress load in plants⁷. Many studies have shown the role of chemical mutagens in stimulating genetic variation in plants because of their essential characteristics for the breeding program's success in plants that reproduce vegetatively and sexually ⁸. Sodium azide NaN3 is widely used to induce mutations in plant systems in vitro. However, these mutations are indirect, and Sodium azide is considered relatively safe to deal with chemical mutations, is inexpensive and does not cause cancer ⁹. The pH of the solution influences the mutagenic efficiency of sodium azide, and it has been shown that azide is most effective in inducing mutations at pH 3 ¹⁰. Despite the wide range of sodium azide concentrations, the mutagen generally induces point mutation in the genome, impairing metabolic activity, growth and development, inhibiting protein synthesis and DNA replication ¹¹. The present study was planned to assess the genetic variations through chemical mutation using sodium azide on strawberry cv. Albion at morphological level, as well as physiological level in order to evaluate the tolerance to salt stress.

MATERIALS AND METHODS

This study was conducted in 2021 at the Ministry of Science and Technology/ Directorate of Agricultural Research/ Genetic Engineering Department.

Plant material, in vitro methods

The uniform in vitro shoots of Strawberry cv. Albion from the multiplication stage (from the third sub-culture). These shoots were treated with mutagen during the fourth subculture of the multiplication stage.

Culture medium composition

Murashige and Skoog (MS) basal medium was used as a basal nutrient medium ¹² The basal medium was supplemented with 0.5 mg l^-1 6-Amino Benzyl Purine (BA) + 1.5 mg l^-1Kintin (Kin) ¹³ as imitation medium for culturing the sterilized shoot tip runners, After 4 weeks, the shoots are cultured on the medium of the multiplication supplemented with 0.5 mg l^-1 of BA and 0.1 mg l^-1 of NAA. pH of the medium was adjusted to 5.7. The cultures were incubated in a culture room under 16-h- and 8-h dark photoperiod at 23±1°C.

Mutagen application

In the fourth subculture, mutant Sodium Azide (NaN3) was added after Cold sterilization of two mutagenic concentrations. Uniform multiplied shoot explants were longitudinally dissected and dipped into the mutagenic solution at (0.25, 0.5) mM for 30 min in addition to the control treatment, which was left without falling in any mutagens. Then, the explants were washed with sterile distilled water three times. Then, the explants were cultured in the nutrient medium for multiplication. Individual shoots were separated and transferred into the multiplying MS basal medium supplemented with 0.5 mg l^-1 of BA and 0.1 mg l^-1 of NAA.

Salinity treatment

The mutagenic and non-mutagenic shoots were grown on MS medium containing 0.5 mg. l^-1 of BA and 0.1 mg l^-1 NAA and supplemented with sodium chloride (NaCl) at concentrations of (6 , 10, 14 ,22) ds m^-1.

Data recorded

The culture was incubated at the same conditions previously mentioned above.4 weeks later, the mor phological parameters were recorded as follows:

1-shoots numbers.

2- shoots length (cm).

3-vegetative fresh and dry weight (g).

the physiological parameters were recorded as follows:

1-Sodium ion concentration (mg g^-1 dry weight).

2-calcium ion concentration (mg g^-1 dry weight).

3-Potassium ion concentration (mg g^-1 dry weight).

Ion Analysis

The vegetative shoots were washed with deionized distilled water several times, then the shoots were dried in the microwave and until the weight was stable (for 5 minutes), 150 mg of dry shoots were then taken and placed in a glass beaker of 50 ml capacity, and 9 ml of the digestion mixture was added to it. of nitric acid, perchloric acid and sulfuric acid in a ratio of 10: 4: 1, respectively, the pots were placed in a sand bath at a temperature of 80 ° C until the digestion was complete, and the digested material was dissolved for each treatment to 50 ml by adding deionized water. Calcium, potassium and sodium concentrations were estimated using a flame photometer model PFP7 of the British JENWAY company, while magnesium was determined using atomic absorption device AAS model NOVA 400 of the German company Analytikjena.

Experimental Layout and Statistical Analysis:

The experiment was designed as a factorial experiment based on a Completely Randomized Design (CRD), the factors included sodium azide and salt levels with 4 replications for each treatment. DATA analyzed by Genstat program version 12 Edition 12^ed and means were separated at 5% of probability using Duncan's test.

RESULTS

Effect of salinity levels and mutagenicity with sodium azide.

Average shoots number

The results in Table (1) show the superiority of 6 dS m^-1 Sodium chloride in giving the highest average number of shoots, reaching 5.92 while the lowest shoots number reached 1.33 projections in 22 dS m^-1NaCl. The treatments of 10 and 14 dS m^-1 of NaCl did not differ significantly between them, as for the effect of sodium azide ,It caused a significant decrease in the average number of shoots compared to the control treatment, and for interaction between salinity and NaN₃, it was noticed that there was a decrease in the number of nodes upon mutation with NaN₃ at both concentrations and at all levels of salinity, the control treatment superior in gave the highest number of shoots reached 9.25 shoots, while the lowest shoots number reached 0.75 nodes in 0.5 mM of sodium azide with 22 dS m^-1 of sodium chloride (Fig. 1)

According to Duncan's test, this means that the same column or their interactions followed by the same letters are not significantly different (P<0.05).

Table 1. Effect of salinity and NaN₃ levels on shoots number of strawberry cv. Albion after 4 weeks from cultured on MS medium.

Average length of shoots

The result shown in Table (2) shows that 6 dS m-1 NaCl was superior in giving the highest average length of shoots, reaching 2.47 cm. As for the effect of excess sodium azide on the average size of shoots, no significant differences between the treatments. For the interaction between salinity and NaN3 concentrations, 6 dS m-1 NaCl giving the highest average length of nodes reached 3.04 cm, while the interaction between the salt level of 14 ds m-1 with 0.5 mM of NaN3 gave the lowest shoot length reached 0.69 cm.

Means in the same column or their interactions followed by the same letters are not significantly different (P<0.05), according to Duncan's test.

Table 2. Effect of salinity and NaN₃ levels on shoots length (cm)of strawberry cv. Albion after 4 weeks from cultured on MS medium

Figure 1. Effect of Sodium azide and salt stress, 8 weeks after the treatment on in vitro Strawberry shoot cv. Albion. A- 6 dS m^-1 NaCl B-0.25 mM NaN₃interaction with 10 dS m^-1 NaCl C- 0.5 mM NaN₃interaction with 10 Ds m^-1 D- 0.25 Mm NaN₃interaction with 22 dS m^-1.

Average of fresh weight (g)

The results (Table 3) revealed that 6 ds m^-1 NaCl gave the highest average fresh weight, reaching 1,990 g, significantly superior to the rest of the treatments, while 22 dS m^-1NaCl gave the lowest fresh weight, 0.344 g. Regarding sodium azide, 6 ds m^-1 NaCl gave the highest average of new weight, reaching 2.255 g, while the lowest fresh weight at 0.5 mM sodium azide went 0.685 g. For the interaction between NaN₃and NaCl, the results showed that 6 ds m^-1 NaCl, significantly superior to the other treatments, reached 3.197 gm fresh weight. In comparison, 22 dS m^-1 interaction with 0.25 mM sodium azide gave the lowest new weight, running 0.120 gm.

Means in the same column or their interactions followed by the same letters are not significantly different (P<0.05), according to Duncan's test.

Table 3.Effect of salinity and NaN3 levels on shoots fresh weight (gm) of strawberry cv. Albion after 4 weeks from cultured on MS medium

Average of dry weight (g)

The results (Table 4) showed the superiority of 6 ds m^-1 in giving the highest dry weight, reaching 0.180 g, while 22 dS m^-1NaCl gave the lowest dry weight, going 0.052 g. Regarding the sodium azide effect, the control treatment was superior in providing the highest dry weight, reaching 0.176 g, while 0.25 mM gave the lowest dry weight, calling 0.010 g. Concerning the interaction between NaCl and the mutagenic sodium azide, 10 dS m^-1 NaCl without mutagenic delivered the highest dry weight, reaching 0.273 g in both treatments, while 22 dS m^-1 of sodium chloride with 0.25 mM of sodium azide gave a minimum dry weight 0.033 g.

According to Duncan's test, the means in the same column or their interactions followed by the same letters are not significantly different (P<0.05).

Table 4. Effect of salinity and NaN₃ levels on shoots dry weight (gm) of strawberry cv. Albion after 4 weeks from cultured on MS medium

Sodium ion concentration (mg g^-1 dry weight)

The results showed in Table (5) that the superiority of 14 dS m^-1 sodium chloride in giving the highest ion concentration reached (32.04 mg sodium g^-1 dry weight of the shoots). In contrast, the control treatment gave the lowest attention reached (22.40 mg sodium gm^-1dry weight of projections) and showed that the concentration of sodium ion increased with the increase of the concentration of the chemical mutagen. The interactions between NaCl and NaN₃ had a significant effect on the sodium ion concentration, which reached the highest concentration (42.90 mg sodium g^-1 dry weight of the shoots) when treated with 0.5 mM sodium azide interaction with NaCl 10 g l^-1, while control treatment giving the lowest concentration of sodium ion reached (11.93 mg sodium g^-1 dry weight of the shoots).

According to Duncan's test, the means in the same column or their interactions followed by the same letters are not significantly different (P<0.05).

Table 5. Effect of salinity and NaN₃ levels on strawberry cv's Sodium ion concentration (mg g-1 dry weight). Albion after 4 weeks from cultured on MS medium

Calcium ion concentration (mg g^-1 dry weight)

The results shown in Table (6) revealed that (6 and 10) dS m^-1 NaCl gave the highest concentration of calcium reached (5.968 and 5.988 mg calcium g^-1dry weight of shoots) respectively, and the two treatments were significantly superior to the rest of the treatments while 22 dS m^-1 of NaCl gave the lowest concentration reached (4.910 mg of calcium gm^-1 of the dry weight of the shoots), while 0.25 mM of sodium azide gave the highest concentration of the ion called (6.019 mg calcium g^-1 dry weight of nodes), and the control treatment showed the lowest concentration of ion reached (5.191 mg calcium g^-1 dry weight of shoots) and did not differ significantly from the treatment of 0.5 mM sodium azide, which gave (5.333 mg calcium g^-1 dry weight of nodes) with regard to the interaction between NaCl and sodium azide, the concentration of 0.25 mM of sodium azide and 6 ds m^-1 was superior in giving the highest concentration of calcium ion reached (6.707 mg calcium g^-1 dry weight of the shoots), while 0.5 mM of sodium azide interaction with 22 ds m^-1 of sodium chloride had the lowest ion concentration reached (4.357 mg calcium gm^-1 dry weight of shoots).

According to Duncan's test, the means in the same column or their interactions followed by the same letters are not significantly different (P<0.05).

Table 6. Effect of salinity and NaN₃ levels on strawberry cv's calcium ion concentration (mg g-1 dry weight). Albion after 4 weeks from cultured on MS medium

NaCl Level (dS m^-1)

Potassium ion concentration (mg g^-1 dry weight

The results in Table (7) that the superiority (6 and 10) dS m^-1 NaCl in giving the highest concentration of ion (26.31 and 26.88 mg potassium g^-1 dry weight of shoots) respectively, while the lowest concentration of Potassium ion at 22 dS m^-1 NaCl (14.62 mg potassium gm^-1 dry weight of shoots), and as for the chemical mutagenic effects of sodium azide, it was significantly superior to 0.25 mM sodium azide and gave the highest potassium ion concentration reached (29.46 mg potassium g^-1 dry weight of shoots ) and the treament was very special to the rest of the treatments and the control treatment gave the lowest concentration of the ion reached (13.82 mg potassium g^-1 dry weight of the shoots), and regarding the interaction between NaCl and NaN₃ ,sodium azide was superior at the concentration 0.25 mM interaction with 6 ds m^-1 NaCl reached (33.06 mg potassium g^-1 dry weight of shoots), while 0.5 mM sodium azide interation with 22 ds m^-1 NaCl had the lowest concentration (8.29 mg potassium g^-1 dry weight of the sprouts).

According to Duncan's test, the means in the same column or their interactions followed by the same letters are not significantly different (P<0.05).

Table 7. Effect of salinity and NaN₃ levels on potassium ion concentration (mg g^-1 dry weight) of strawberry cv. Albion after 4 weeks from cultured on MS medium

DISCUSSION

Salinity stress is excessive amounts of soluble salt that negatively affect the functions needed for average plant growth and development. Salt stress comprises two harmful effects: osmotic stress, leading to reduced water uptake, and ionic stress, caused by the toxicity of specific ions (mainly Na⁺ and Cl^– ). Ionic stress leads to unrestrained overproduction of ROS (reactive oxygen species), such as superoxide radicals (O2·), hydrogen peroxide (H2O2), and hydroxyl radicals (OH·^– ). These reactive molecules accumulate to toxic levels and trigger oxidative damage in the cells and organelles by destroying membranes, proteins, enzymes, and nucleic acids (^14, ^15, ^16, ¹⁷).

Chemical mutagens are helpful tools in crop improvement and have been used to produce abiotic stress tolerance and disease resistance in various plant crops, improving their yield ¹⁸In the present study, while in vitro exposure to sodium azide decreased the number of shoots, fresh and dry weight due to Phenotypically, the rapid effects of sodium azide on the meristematic cells in the cultivated explant is to inhibit cell multiplication due to its obstruction of the multiplication process in the S phase (S-phased) during the cell cycle in addition to some biochemical reasons, as it causes inhibition of the respiratory chain and transport chain Electron ¹⁹. The mutagenic ability of the sodium azide mutagen depends on its production of an organic metabolic compound called B-acid alanine, which stimulates chromosomal aberrations at a lower rate than other mutagens ²⁰, which cause disruptions in growth, metabolic activity, protein inhibition, and DNA replication ¹¹ The mutagen changes the balance between growth stimulators and inhibitors ²¹. The results of this study are consistent with the findings of ²² of a decrease in vegetative growth when plants were subjected to chemical mutagenicity.

CONCLUSIONS

The results showed that salinity caused an increase in sodium ions. In contrast, it caused a decrease in the concentration of calcium and potassium ions, and this may be because the increased absorption of sodium and chlorine as a result of adding them to the food medium has reduced the absorption of other nutrients ²³. After all, it interferes and competes with absorption by the plant²⁴ found that using different mutants, such as sodium azide, reverses and reduces these adverse effects and increases potassium and calcium ion absorption in plant tissues.

Funding: "This research received no external funding.

Conflicts of Interest: "The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results."

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Received: 25 June 2023/ Accepted: 26 August 2023 / Published:15 September 2023

Citation: Juameer, R ., Obaid, A. Effect of Sodium Azide (NaN3) on micropropagant and tolerance of Salt Stress of Strawberry cv. Albion. Revis Bionatura 2023;8 (3) 128 http://dx.doi.org/10.21931/RB/2023.08.03.128