Influence of seed priming with CuSO4 and ZnSO4 on germination and seedling growth of oat under NaCl stress

Salinity is a grave problem across the world which retards growth and productivity of plants. Seed priming is a technique which enhances growth and yield of crops by overcoming salt stress. Present study was conducted to examine the effect of grains priming of Avena sativa L. with different micronutrients (Cu & Zn) grown under NaCl stress. In this study grains of Avena were primed with two different concentration levels (100 & 200ppm) of CuSO4 & ZnSO4 solutions. After priming, these grains were treated with different levels of NaCl (60mM, 90mM & 120mM) and these results are compared with control groups. The effect of CuSO4, ZnSO4 and NaCl was observe on different physiological growth parameter including germination speed, germination percentage, root and shoot length, root and shoot fresh and dry biomass and biomoisture contents of root and shoot were observed. It was observed that grains without priming exhibited reduced growth under NaCl stress. Whereas priming of grains with CuSO4 and ZnSO4 showed improved growth in all growth traits as compared to non-primed seeds. It was evaluated that grains primed with ZnSO4 at 200ppm showed enhanced germination speed. Whereas grains treated with CuSO4 at 200ppm showed decreased germination speed. It was also demonstrated that priming of grains with both micronutrients (CuSO4 & ZnSO4) at 200ppm concentration exhibited higher shoot length as compared to Grains without priming grown in NaCl. It was concluded that seed priming technique can overcome the effects of salinity to some extent.

harmful effects of high levels of minerals like Na + and Cl + ions on plant is termed as Salt stress [6]. High concentrations of soluble salts are the characteristics of saline soils [7]. Among various environmental stresses one of the most important is the salt stress that influence the growth period of the whole plant and restrict the growth and productivity of crops by decreasing the osmotic potential and by impairing the absorption of certain nutrients. The absorption of essential ions such as potassium, calcium, ammonium and certain nitrate ions reduces as the level of sodium and chloride ions increases. Moreover increasing sodium and chloride ions blocks the structure of membrane and decreases the absorption of certain enzymes [8].
Increased salinity levels cause considerable decrease in different growth parameters including root and shoot dry weight, leaf length and leaf area [9]. Soil salinity is one of the major effective barriers that reduces the crop productivity [10]. In Pakistan, total irrigated area is 16.795 million ha. Slightly saline soil covers an area about 10%, 4% soil is moderately saline and 7% area is strongly saline, 6% is miscellaneous and 73% is termed as non-saline [11]. Salinity is the important abiotic stress, imposes negative effects on growth and productivity of crops [12]. The crop yield reduces upto 50% due to salinity in arid and semi-arid areas [13]. Stunted growth, decreased chlorophyll content and increased levels of reactive oxygen species are the most important symptoms of salt-stressed plants [14]. Around 9.5 billion hectares of the soil across the world are saline. This figure excludes large areas of secondarily salinized soil which is present in the currently cultivated land. Additionally, freshwater resources are getting scarce day by day. Under the present conditions it is essential to find plants which have economic value which can grow under saline conditions [15]. Seed priming is basically a pre-sowing approach for affecting the seedling development by balancing pre-germination metabolic processes before the development of radical and usually improves the rate of germination and plant performance [16]. Seed priming is one of the easiest and simplest technique to create tolerance in crops against salinity [17]. Before germination, seed priming lead to hydration in seeds but roots do not emerge Germination period was studied in 4 control groups with different experiments like seed without priming grown in fresh water, primed seed grown in distilled water, only sterilized seed without priming grown in distilled water, treatment of seeds without priming with different NaCl concentrations and experimental group in which seeds were primed with CuSO4 and ZnSO4 then grown in different concentrations of NaCl salinity (60mM, 90mM, 120mM). Repeating each experiment 3 times 64 petri plates were prepared. In each plate 7 seeds were placed which were lined with filter paper and moisten with 10-12 ml soln. All plates were kept at 10-13°C for germination being the optimum temperature. Germination started after 36 hrs (with the emergence of radical seeds were considered to be germinated).

Recording of data
Germination of seeds were counted after every 24 hrs and after 8 days of germination, germination percentage and germination velocity were recorded but root and shoot length, fresh and dry biomass of seedlings were determined after 15 days of sowing. Root, shoot length were measured by using the simple ruler. Fresh weight was obtained using the electronics sartorius balance TE214S and after 24 hrs dry weight was determined for each case and percentage moisture content was obtained.

Statistical analysis
The data was statistically analysed using ttest.

Results and discussion Germination speed
Germination speed is badly affected by salinity. In the present studies, it was investigated that in control groups, the slowest germination speed was recorded at 120mM NaCl concentration as (804.25) in comparison to 60mM NaCl as (690.41) and 90mM NaCl as (775.22). And fastest germination speed was observed in seeds grown in fresh water as (508.87) and distilled water as (510.72) (Fig. 1).

Figure 1. Effects on germination speed of Avena sativa L. under different control groups
Seeds primed with CuSO4 100ppm showed higher germination speed as (602.57) in comparison to that of 200ppm as (635.73) which also showed that CuSO4 delayed germination in seeds of Avena sativa L. In case of priming with CuSO4, 100ppm had (0.02) and 200ppm had (0.04) P(T<=t) values. Both treatments of CuSO4 showed significant effects in this regard. In case of seeds priming with ZnSO4, 100ppm of ZnSO4 showed slow speed of germination as (540.96) than that of 200ppm as (531.66). Statistical analysis revealed that priming of ZnSO4, at 100ppm (0.009) and at 200ppm (0.018) P(T<=t) showed significant effect (Fig. 2).    (Fig 4).    6).    (Fig. 8).

Figure 8. Effects of seed priming with different concentrations of CuSO4 and ZnSO4 on %O.D weight of roots of Avena sativa L. under different levels of NaCl
Seedlings fresh biomass of root (g) Fig 5 clearly demonstrated that higher fresh biomass of seedlings root was found in seeds grown in distilled water (0.1105 g) as compared to fresh water (0.0853 g). While 60mM NaCl has fresh weight of root as (0.0765 g) and 90mM exhibited fresh weight of root as (0.0524 g) which was lower than 60mM NaCl treatment without priming. And 120mM NaCl showed great decline in fresh weight of root as (0.0398 g) (Fig. 9). The root fresh weight of germinated seeds or seedlings primed with CuSO4 at 100ppm was as (2.775 g) and was much more reduced as compared to that of 200ppm as (4.057 g) almost double in quantity. In case of priming with CuSO4, 100 and 200ppm concentrations showed (0.081) and (0.162) P(T<=t) values which had non-significant effects on this physiological parameter. A comparison between two concentrations of ZnSO4 used for Avena sativa L. seeds priming indicated that 100ppm exhibited much reduced root fresh weight as (0.0322 g) as compared to that of 200ppm as (0.0788 g). In case of ZnSO4, at 100 and 200ppm concentrations (0.110) and (0.220) P(T<=t) values were recorded as nonsignificant in this growth trait (Fig. 10).  priming with CuSO4, both 100 and 200ppm concentrations had (0.08) and (0.16) P(T<=t) values and both exhibited nonsignificant effects on this growth trait. 100ppm of ZnSO4 showed lower dry weight of root as (0.0285 g) than that of 200ppm as (0.0295 g). 100 and 200ppm of ZnSO4 exhibited (0.471) and (0.942) P(T<=t) values, show non-significant effects on root dry weight (Fig. 12).

Seedlings fresh biomass of shoot (g)
The fig 7 clearly described that seeds grown in fresh water (1.0567 g) attained high fresh biomass of shoot. It can be seen that shoot fresh biomass of 60mM as (0.4285 g), 90mM as (0.3213 g) and 120mM as (0.2931 g) NaCl showed reduction in this growth trait as compared to fresh water (1.0567 g) and distilled water (0.8675 g) (Fig. 13). L. In case of priming with CuSO4, 100ppm had (0.29) and at 200ppm P(T<=t) value was as (0.58) and both treatments of CuSO4 showed non-significant effects for this growth parameter. Seeds primed with 100ppm of ZnSO4 showed reduced fresh weight of shoot (0.418 g) than that of 200ppm as (0.632 g). In ZnSO4 for 100ppm data the P(T<=t) value was (0.09) and for 200ppm it was (0.19), both being nonsignificant as far as this physiological parameter is concerned (Fig. 14).

Seedling dry biomass of shoot (g)
The dry biomass of shoot in fresh water (0.8827 g) was observed that was found to be higher as (0.8827 g) than in distilled water as (0.5971 g). Among various concentrations of NaCl it was observed that  CuSO4 at 100ppm exhibited reduction in dry shoot biomass of shoot as (0.275 g) when compared to that of 200ppm as (0.5008 g) and sufficiently increased this physiological parameter regarding dry biomass of shoot of Avena sativa L. In case of seeds priming with CuSO4, 100ppm showed non-significant value as (0.056) while 200ppm also exhibited nonsignificant values recorded as (0.113).
Results indicated that priming of seeds at 100ppm of ZnSO4 declined in dry biomass of shoot as (0.221 g) than that of 200ppm as (0.357 g). In case of ZnSO4 Avena seeds priming at 100 and 200ppm concentrations, (0.071) and (0.142) P(T<=t) values were recorded. Results showed that priming at 100ppm and 200ppm of ZnSO4 showed non-significant effect in this parameter (Fig. 16).

Seedlings root length (cm)
It was clear from the fig 9 that seeds grown in distilled water (6.276 cm) without priming have higher root lengths with respect to fresh water (5.855 cm). It was observed that root length in 60mM (4.123 cm) and 90mM (3.213 cm) levels of NaCl have very minute differences but root length in 120mM (2.143 cm) level of NaCl showed great decline in this growth trait (Fig. 17).   (Fig. 18).

Seedlings shoot length (cm)
Seeds grown in fresh water (12.767 cm) showed greater shoot lengths than in distilled water as (12.676 cm). Whereas treatments of 60mM and 90mM NaCl showed shoot lengths as (6.437 cm) and (5.725 cm) respectively. Results indicated that shoot length was much reduced in treatment of 120mM NaCl (4.105 cm) (Fig.  19).

Figure 19. Effects on shoot length of Avena sativa L. under different control groups
In case of priming of seeds with CuSO4, 100ppm reduced shoot length as (6.77 cm) in comparison to that of 200ppm as (7.99 cm) which indicated that CuSO4 showed enhancement in this parameter of Avena sativa L. seedlings. In case of priming of  Both these two treatments showed nonsignificant effects on this growth parameter (Fig. 20).

Figure 20. Effects of seed priming with different concentrations of CuSO4 and ZnSO4 on shoot length of Avena sativa L. under different levels of NaCl
The goal of this research work was to evaluate the effects of seeds priming of Avena sativa L. under salt stress. Results revealed that oat seedlings demonstrated decreased growth under salt stressed conditions. The results indicated that the germination of oat seedlings was negatively influenced by NaCl stress under different concentrations (60mM, 90mM and 120mM). Seeds germination percentage and seedlings emergence were delayed by higher salinity levels as described by [24] and our results are in agreement with these findings. The same results were reported in mustard by [25]. These results are also in conformity with our findings regarding salt stress.
The inhibition of germination induced by salts could be associated with specific ion toxicity or osmotic stress as reported by [26]. Increased salinity level of the medium caused great reduction in germination percentage as reported by [27,28] and these results are similar with our findings.
From our results it was also demonstrated that high salinity levels caused severe reduction in seedlings shoots and roots length and fresh and dry weights. Shoot and root lengths are the important growth parameters that are readily affected by salt stress due to the reason that roots take in water due to direct contact with soil and then shoots empower its supply in whole plant. Due to this reason shoot and root length administer critical indications of a plant's response to salt stress as reported by [29]. Our results showed reduction in fresh and dry biomass as salt stress increases which was strongly supported by [30,31] in which they described the reason that salinity influences the metabolic processes, by decreasing water potential. Reduction in seedling growth under salinity is due to increase in sodium chloride toxicity and aggregation of sodium ions in the photosynthetic tissues as demonstrated by [32] and our results are in agreement with these findings. Present results showed that seeds primed with 200ppm of ZnSO4 showed considerable increase in seedlings shoot length of Avena sativa L. as compared to their respective controls. Same results were demonstrated on Cumin plant by [33] and our results are in accordance with these findings. Osmo-priming and halo-priming caused in increased stem length of hot pepper seedlings as demonstrated by [34] and our results are in agreement with these findings. Priming in maize with 1% ZnSO4 (for 16h) extensively increased the crop growth, grain yield and Zn content as reported by [35]. Similar results were obtained in rice where seed priming with Zn caused enhanced growth and grain yield which was found more suitable than any other soil application [36]. Our results are also in conformity with these findings. Seeds priming with Zn considerably increased the yield and related traits in common bean (Phaseolus vulgaris L.) as reported in findings by [37]. Similar results were also reported in barley (Hordeum vulgare L.) where the improved germination and seedlings development was observed due to seeds priming with Zn as reported by [38]. Our findings are in conformity with these findings. Explained that In another research it was demonstrated that the improved growth after nutrient primings of seeds may be due to the reason that in newly developed radicals and coleoptiles during seed germination the Zn content was found to be much higher (upto 200 mg kg -1 ) thus proving the involvement of Zn in Physiological processes during early seed germination as reported by [39]. Copper and Zinc plays an important role in cellular metabolism because both of them are being involved in many proteins [40]. So all these findings support our findings for improved growth after seed priming with different micronutrients. CuSO4 treatments on Avena sativa L. seeds had no significant effects on germination speed in our present studies. Our results clearly demonstrated that seeds primed in 100ppm CuSO4 showed reduction in seedlings shoot length as compared to CuSO4 at 200ppm and their respective controls. Results revealed that CuSO4 had no significant effects on germination speed. Priming of wheat seeds with CuEDTA (0.04 to 0.16 kg Cu ha -1 ) considerably improved grain yields but had no significant effects on seedlings emergence as reported by [41]. These results were in conformity with another experiments on oats in which seeds treated with Cu (0.001% solution of CuSO4) had no considerable effects on seedling germination although caused increase in yield of 16.5% with respect to controls as described by [42]. A comparison was made between 100 and 200ppm of CuSO4 and controls which indicated that 200ppm promoted more growth. Similar results were obtained in pea plant as reported by [43]. These results were in accordance with our's results and thus support our findings.

Conclusion and recommendations
From the research work it was concluded that salinity caused reduction in seedlings germination and emergence and also affected the other growth physiological parameters of Avena sativa L. Our results revealed that seeds priming was proved to be helpful in overcoming the effects of salinity on seeds germination and growth. The negative effects of salinity were reduced by using the micronutrients (Cu and Zn) through seeds priming with CuSO4 and ZnSO4 at two concentration levels (100 & 200ppm). It was concluded that by the application of these micronutrients, all the growth parameters including seed germination, root and shoot length and fresh and dry biomass showed improvement in saline as well as in non-saline conditions. It is suggested that to produce tolerance in plants against NaCl stress, seeds should be primed with micronutrients before sowing. Moreover, priming techniques can enhance the germination rate, growth and ultimately yield of some important crops. Furthermore, it is environment friendly technique that's why it may be used in future for beneficial results.
analysis: I Dilshad, K Moatter & T Ahmed, Proof read the article: SA Gilani.