Alleviation of the effects of NaCl salinity in spinach ( Spinacia oleracea L. var. All Green) using plant growth regulators

Salinity is one of the most devastating forms of land degradation which severely affects crop production worldwide especially in arid and semiarid regions (Shomeili et al., 2011). Attempts have been made to counteract the adverse effects of salinity on plants by pretreating the seeds with various plant growth regulators (Kasim and Dowidar, 2006). A report by Afzal et al. (2005) says that seed treatment with Kinetin can increase the ability of wheat to grow successfully under saline conditions. Mohammad (2007) has observed that gibberellic acid can be used to alleviate the adverse effects of salinity on mungbean seedlings. Similarly, it was reported by Bagdi and Afria (2008) that the adverse effects of salinity on growth and yield of wheat cultivar can be ameliorated by the use of benzyl adenine. Chauhan et al. (2009) suggested that plant growth regulators help in overcoming the harmful effects of salinity on growth by changing the endogenous growth regulators which affect plant water balance. Hence, in the present study an attempt has been made to study the ameliorative effect of plant growth regulators like gibberellic acid (GA3), 6-furfuryladenine (Kinetin) and benzyl adenine (BA) on Spinacia oleracea L. var. All Green, cultivated under saline conditions.

MATERIALS AND METHODS

Seeds of Spinacia oleracea L. were obtained from Indian Agricultural Research Institute, Pusa, New Delhi. After a pre-soaking treatment of six hours in 20 mg L^-1 solutions of gibberellic acid (GA 3 ), 6-furfuryladenine (Kinetin) and benzyl adenine (BA), seeds were sown in earthen pots (20 cm diameter, 29 cm height). The pots were irrigated using tap water till the phase of germination. A pot served as control where the seeds were irrigated with tap water. Remaining pots were irrigated with NaCl solutions. In order to acclimatize the plants to NaCl, the concentrations were raised after every three-four days in a stepwise manner starting from 5 mM till a final concentration of 60 mM NaCl. The plants were grown for forty-five days. Mature leaves of these plants were used to estimate organic constituents like chlorophyll a, chlorophyll b, total chlorophyll, reducing sugars, non-reducing sugars, proteins and proline using standard protocols of Arnon (1949), Miller (1959), Lowry et al . (1951) and Bates et al . (1973) respectively.

RESULTS AND DISCUSSION

From table 1, it is clear that chlorophyll a, chlorophyll b as well as total chlorophyll decreased with salinity as compared to control. A decrease in chlorophyll content has been observed in Spinacia oleracea by Kaya et al. (2001) under NaCl salinity. Similarly decreases in chlorophyll content have been reported in sweet sorghum (Almodares et al., 2008) and Vicia faba (El Sayed, 2011) under saline conditions. The reduction in chlorophyll content can be attributed to the inhibitory effect of accumulated ions such as sodium and chloride, on the biosynthesis of the pigments as suggested by Mohammed (2007). He suggested that the degradation of these pigments as well as the structure of the chloroplast can be affected by NaCl. According to Arulbalachandran et al. (2009), the reduction of photosynthetic pigment under salinity occurs due to degradation of chlorophyll by enzyme chlorophyllase and reactive oxygen species generated during photorespiration.

When the seeds were pretreated with plant growth regulators, ameliorative effects were observed as the chlorophyll content were observed to improve with such treatment. The present study showed that GA 3 , Kinetin and BA can successfully mitigate the adverse effect of NaCl salinity on pigment content (Table 1). Zeid (2011) also reported an alleviation of adverse effect of salinity on chlorophyll content in barley with GA 3 treatment. Ali et al . (2012) recorded restoration of altered pigments by application of GA 3 under saline condition in Hibiscus sabdariffa . Enhanced production of total chlorophyll due to grain priming with Kinetin in Sorghum bicolor has been observed by Alsokari (2009). Alleviation of salinity stress in wheat by the use of BA for presoaking has proved to be effective as total chlorophyll content has been reported to increase (Bagdi and Afria, 2008).

Reducing as well as non-reducing sugars were found to increase with increasing salinity in the present study (Table 2). While photosynthesis is the main source of carbohydrate accumulation, Munns (1993) reported that the concentrations of sugars (and reserve polysaccharides) always rise after plants are exposed to salinity. Similarly, Siringam et al. (2011) reported an increase in sugars in the roots of Oryza sativa under salt stress. They related such accumulation of soluble sugars with increased sodium content. Sugars in such cases may function as an osmoticum and help in maintaining water use efficiency in the root cells.

The present study showed further rise in sugars in pretreated seeds. Pretreating seed with Kinetin has improved the sugar content at a maximum level (Table 2). Similarly, Niazi et al . (2005) observed that Kinetin pretreatment has enhanced the sugar content in fodder beet grown under NaCl salinity. An increase in sugar content in wheat seedlings with GA 3 pretreatment has been observed by Jat and Sharma (2006) when grown under saline conditions.

In the present study, the protein content was observed to decrease with increase in salt concentration (Table 2). There are several reports indicating a decrease in protein content under saline conditions as in Dioscorea rotundata (Jaleel et al ., 2008), rice (Amirjani, 2010) and tomato (Doganlar et al ., 2010). Decreased protein content under the influence of NaCl salinity has also been reported in Triticum aestivum roots by Tammam et al . (2008).

The pretreatment with plant growth regulators showed an improvement in protein content, Kinetin pretreatment was found to be most effective. Similarly Kinetin was recorded beneficial when used for pretreating the seeds of Hordeum vulgare thereby increasing the protein content under saline irrigation by Sarwat and El-Sherif (2007). Shomeili et al . (2011) recorded a positive effect of GA 3

treatment on protein content in sugarcane under NaCl salinity. Bagdi and Afria (2008) have also observed that presoaking the seeds of wheat with BA helped by improving protein content over control.

In the present study, an increase in the proline contents was observed with increasing salinity (Table 2) Accumulation of osmolytes such as proline are beneficial under stress conditions as they make the water potential low inside the cell and prevent the intracellular loss as suggested by Mahajan and Tuteja (2005). There are several reports where an increase in proline content have been reported under saline conditions as in alfalfa (Mezni et al., 2010), Jatropha curcas (Patel et al ., 2010), Brassica genotypes (Siddiqui et al ., 2010) and Capsicum annuum (Chookhampaeng, 2011).

Presoaking treatment with all the three PGRs has resulted in lowering the proline content under saline conditions, most effective plant growth regulator being GA 3 (Table 2). Similarly, Kasim and Dowidar (2006) observed lower proline content in GA 3 primed radish seedlings under NaCl treatment. They suggested that, this decrease in the amino acid in GA 3 primed seedlings may be due to their incorporation in some new proteins or their utilization in the synthesis of certain existing proteins.

CONCLUSION

On the basis of above findings it is clear that all the three plant growth regulators used in the present study are found to be effective in mitigating the adverse effect of NaCl on Spinacia oleracea L. var. All Green.

Table 1 Effect of seed priming with PGRs on chlorophyll content from the mature leaves of 45 day old Spinacia oleracea L. var. All Green grown under saline (60 mM NaCl) environment

Results are the mean of three determinants.

*One-way ANOVA was carried out and F ratio was significant at 5% level of significance.

# Significant at p < 0.05 (t-test was carried out to test whether there is significant difference between control and individual salt concentration).

Table 2 Effect of seed priming with PGRs on reducing sugars, non-reducing sugars, protein and proline content from the mature leaves of 45 day old Spinacia oleracea L. var. All Green grown under saline (60 mM NaCl) environment

Results are the mean of three determinants.

• One-way ANOVA was carried out and no significant difference was observed in the group, so data is not analyzed further for pair-wise comparisons among the treatments.