Influence of seed priming with PbSO4 and FeSO4 on germination and seedling growth of cabbage under NaCl stress

The impact of salinity causes nutrients imbalance and accumulation of toxic elements in plants which reduce water in filtration and plant growth. In present study seeds were primed with Lead acetate (PbSO4) and Iron sulphate (FeSO4) at two dose levels(100 and 200 ppm) and then treated with 4 control groups in different experiments like seeds without priming grown in fresh water, primed seeds grown in distilled water, only sterilized seeds without priming grown in distilled water, treatment of seeds without priming with different Sodium chloride (NaCl) concentrations (60mM, 90mM, 120mM) and experimental group primed seeds grown in different concentrations of NaCl salinity (60mM, 90mM, 120mM). All growth factors like seedling growth percentage, rate of germination, root, shoot length, bio moisture contents of root and shoot, fresh and dry weight of seedlings were observed. It was estimated that seeds without priming exhibited reduced growth under NaCl stress. Priming of seeds with FeSO4 showed fairly better results whereas priming of seeds with PbSO4did not show any significant improvement in growth parameters


Introduction
Cabbage (Brassica oleracea L. var. capitata) is one of the most important vegetables grown worldwide and it belongs to the family Brassicaceae. The different cultivated types of cabbage show great variation in respect of size, shape and colour of leaves as well as the texture of the head [1]. The genus Brassica contains many species including (Brassica napus L.), mustard (Brassica juncea L.), cabbage (Brassica oleracea L.) and turnip (Brassica rapa L.) are commonly grown for vegetables, oils, condiments and fodder [2]. The genus comprises of approximately 30 wild species either belonging to wild taxa or have escaped cultivation [3]. It contains about 100 species, including the widely cultivated turnip, cabbage, cauliflower, broccoli, brussels sprouts, various mustards and weeds [4]. The species of this genus contain large amounts of fibers and vitamins especially the vitamin C [5]. Genus Brassicais pharmacologically important as some species contain nutrients that have potential for anti-cancer properties like 3, 3'di-indolylmethane, sulforaphane, and selenium [6,7]. Salinity is one of the major obstacles in crop growing areas throughout the world. Despite of extensive literature there is still controversy with regard to the mechanisms of salt tolerance in plants [8]. It is an issue that affects 6% of the World's land surface area or 12,780 million hectares (Mha) and secondary salinization from irrigation impacts 20% of irrigated land or 1474 Mha. According to the reports of United Nations, 20% of agricultural land and 50% of World cropland are salt affected. Salinity in soil or water is major stress and limits the crop production [9]. It starts at seed germination level, reduces nodule formation, retards plant growth and crops yield [10]. The plants that grow in saline soils have various ionic concentrations and compositions of dissolved salts. These concentrations alter due to changes in water source, evapo-transpiration, drainage, and solutes availability [11]. The establishment of seed/crop depends on the frequency and the amount of precipitation and the ability of the seed species to germinate [12].Salts inhibit seed germination and crop establishment [13]. Germination and seedling characteristics are the most viable criteria used for selecting salt tolerance in plants [14]. Poor crop establishment was considered as a major constraint for farmers. To control salinity or adverse environmental conditions, priming may be used for enhancing germination of seeded plants [15]. Priming is a controlled-hydration process followed by redrying that allows pre-germinative metabolic activities to carry on [16]. One of the most widely distributed heavy metals that is very toxic to plants is Lead (Pb) [17]. Pb affects mesophyll cells, pigment content and light and dark reactions of plants.
It also interferes with nutritional elements of seedlings and thus leading to deficiencies or adverse ion distribution within the plant [18] as well as growth inhibition [19,20]. In this context, an effort was made to investigate the effect of seed priming on germination and early seedling growth of cabbage and to find out a relationship between salt stress and growth.

Materials and methods
Experiments were carried out to investigate the effects of seed priming of Brassica oleracea var. capitata L. with FeSO4 and PbSO4 under different levels of NaCl concentrations following the method with slight modification as described by [21]. Seeds priming and experimental design Seeds were surface sterilized with 70% ethanol for 30 seconds and then washed with distilled water. Seeds were then primed with two concentration levels of FeSO4 and PbSO4i.e, 100 and 200 ppm for 1hour and then dried. 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 FeSO4 and PbSO4 and 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 radicle 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 calculated 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 by using the electronics sartorius balance TE214S and after 24 hrs dry weight was determined in each case and percentage moisture content was calculated. The number of seeds germinated was counted on daily basis till the completion of germination % germination and speed of germination was calculated as described by [22].

Statistical analysis
The data was statistically analyzed using ttest and Pearson Correlation was found.

Results and discussion Speed of germination
Speed of germination is strongly influenced by salinity. Among control groups highest rate of germination was recorded in seeds grown in distilled water without priming (424.6) than in fresh water (435.3) and very slow germination speed were recorded in seeds without priming grown in different concentrations of NaCl i.e.,

Figure 3. Effects on germination percentage of Brassica oleracea under different control groups
When we compare germination percentage of PbSO4 at 100 and 200 ppm concentration, its 200 ppm showed lower germination percentage (60.02%) than that at 100 ppm (69.15%) but in FeSO4 primed seeds at 100 ppm showed less germination percentage (88.25%) as compared to that at 200 ppm (93.75%).When seeds were primed with PbSO4 its P(T<=t) value was (0.0002) for 100 ppm and (0.0005) was recorded for 200 ppm of PbSO4, and priming of seeds with FeSO4 for 100 ppm gave P(T<=t) value (0.0001) and for 200 ppm (0.0003).From the P(T<=t) value it was concluded that 100 and 200 ppm ofboth nutrients showed significant effects on germination percentage (Fig 4). Data also explained the strong positive correlation between the two concentrations i.e 100 & 200ppm of PbSO4 and FeSO4 in this growth parameter.   The results of %age O.D weight of root showed that roots grown without primingin fresh water had higher O.D weight (15.5%) than that of distilled water (6.8%). Similarly the %age O.D wt. of roots grown in 120mM NaCl without priming was higher (22%) than that of 90mM (12.4%), and 60mM (8.6%) (Fig 7).

Seedlings fresh biomass of root (g)
In control groups the highest rate of fresh biomass of root was observed in distilled water (0.0434 g), fresh water (0.0394 g) and then in NaCl concentrations 60mM(0.0325 g), 90mM (0.0307 g) and 120mM (0.0282 g) with the lowest value (Fig 9). Lower fresh weight was observed at 200 ppm (0.024 g) than that of 100 ppm(0.028 g) inseeds primed with PbSO4.In FeSO4 primed seeds, 100 ppm showed less fresh weight (0.043 g) as compared to (0.045 g) at 200 ppm. The P(T<=t) value recorded in 100 ppm of PbSO4 was (0.003) and (0.006) were recorded in 200 ppm of PbSO4, and priming of seeds with FeSO4 at 100 ppm gave P(T<=t) value (0.017) and at 200 ppm (0.035).From the P(T<=t) value it was concluded that 100 and 200 ppm of both nutrients showed significant effects on fresh weight of root (Fig 10). Data also explained the strong positive correlation between the two concentrations i.e 100 & 200ppm of PbSO4 and FeSO4 in this growth parameter.

Seedlings dry biomass of root (g)
The dry biomass of root was considerably reduced than that of the fresh biomass but the distilled water biomass (0.0406 g) was still greater than that of the fresh water biomass (0.0341 g). Both fresh and distilled water exhibited greater dry biomass of root seedlings as compared to tested three NaCl concentrations 60mM, 90mM and 120mM with 0.0299, 0.0274, and 0.0231g respectively (Fig 11). Distilled water > Fresh water > 60mM > 90mM > 120mM.

Figure 11. Effects on dry biomass root of Brassica oleracea under different control groups
When we compare the dry biomass of root seedlings with PbSO4 priming at 100 and 200 ppm concentrations, then 200 ppm showed lower dry weight (0.018 g) than that of (0.020 g) at 100 ppm. FeSO4 primed seeds at 100 ppm produced less dry weight (0.

Seedlings fresh biomass of Shoot (g)
The fresh biomass of shoot of seedlings of seeds grown in distilled water (0.1662 g) was higher than that of fresh water (0.1433 g). Among non-primed seeds grown at different levels of NaCl(60mM, 90mM and 120mM), 60Mm exhibited higher fresh biomass of shoot (0.1149 g) followed by 90mM (0.1006 g) and 120mM (0.0882 g), Fig. 13 showing decreasing trend as Distilled water > Fresh water > 60mM > 90mM >120mM.

Figure 13. Effects on fresh biomass shoot of Brassica oleracea under different control groups
The fresh weight of shoot of seeds primed with PbSO4 at 100 and 200 ppm concentrations, 200 ppm showed lower fresh weight (0.087 g) than that of 100 ppm exhibiting fresh biomass (0.103 g) considerably higher. In case of FeSO4 primed seeds 100 ppm concentration showed less fresh weight (0.172 g) as compared to that of (0.175 g) at 200 ppm. The P(T<=t) value recorded for PbSO4 at 100 ppm (0.004) and (0.009) were recorded at 200 ppm, and whereas priming of seeds with FeSO4 at 100 ppm gave P(T<=t) value (0.048) and at 200 ppm (0.09).From the P(T<=t) value it was concluded thatexcept200 ppm of FeSO4all nutrients showed significant effects on fresh weight of shoot (Fig 14). Data also explained the positive correlation between the two concentrations i.e 100 & 200ppm of PbSO4 and FeSO4 in this growth parameter.

Seedlings dry biomass of shoot (g)
The dry biomass of shoot was considerably reduced than that of the fresh weight. Results revealed that the dry biomass of seeds grown in distilled water (0.0982 g) was greater than that of the fresh water (0.0874 g) and it was subsequently reduced as (0.0831g), (0.0715 g) and (0.0688 g) at 60mM, 90mM and 120mM levels of NaCl respectively (Fig 15). The following decreasing trend was observed: distilled water > fresh water > 60mM > 90mM > 120mM.

Figure 15. Effects on dry biomass shoot of Brassica oleracea under different control groups
The dry weight of shoot of seeds primed with PbSO4 at two concentrations 100 and 200 ppm, its 200 ppm showed lower dry biomass (0.053 g) as compared to 100 ppm having dry biomass (0.059 g). But in case of FeSO4 primed seeds at 100 ppm dry shoot weight was found as (0.093 g) little lower as compared to that of 200 ppm (0.094 g). Seeds were primed with PbSO4, its P(T<=t) value was (0.0001) at 100 ppm and (0.0002) recorded at 200 ppm while priming of seeds with FeSO4 at 100 ppm gave P(T<=t) value (0.027) and 200 ppm (0.055) . From the P(T<=t) value it was concluded that both nutrients showed significant effects on dry weight of shoot (Fig. 16). Data also explained the strong positive correlation between the two concentrations i.e 100 & 200ppm of PbSO4 and FeSO4 in this growth parameter.

Seedlings root length (cm)
A great change was observed in root length of seeds grown in fresh and distilled water. Root length of seeds grown in fresh water (4.713 cm) was greater than that in distilled water (3.683 cm). Similarly root length of non-primed seeds treated at 60mM NaCl (3.104 cm) was greater than that in 90mM (2.596 cm) and so on in 120mM (2.015 cm) levels of NaCl. Fig. 17 showing the decreasing trend: fresh water > distilled water > 60mM > 90mM >120mM.

Seedlings shoot length (cm)
Like root length, shoot length of seeds grown in fresh water also exhibited the highest value (6.446 cm) than that of the seeds grown in distilled water (5.968 cm). But shoot length of seeds grown in distilled water was greater than 60mM NaCl (5.432 cm), then 90mM (5.026 cm) and then 120mM (4.835 cm) of NaCl concentration. Fig 19, showing the trend in decrease of shoot length as: fresh water > distilled water > 60mM > 90mM > 120mM. lower shoot length as (6.42 cm) as compared with that at 200 ppm as (7.78 cm). P(T<=t) value of seeds primed with PbSO4 was (0.018) and (0.037) were recorded at 100 ppm and 200 ppm respectively, and priming of seeds with FeSO4 at 100 ppm gave P(T<=t) value (0.002) and at 200 ppm as (0.005). From the P(T<=t) value it was concluded that both nutrients showed significant effects on root length (Fig. 20). Data also explained the strong positive correlation between the two concentrations i.e 100 & 200ppm of PbSO4 and FeSO4 in this growth parameter.

Figure 20. Effects of seeds priming with different concentrations of PbSO4 and FeSO4 on shoot length of Brassica oleracea seedlings under different levels of NaCl
The results of the present studies indicate that non-primed seeds grown under different salinity levels (60mM, 90mM and 120mM of NaCl) inhibit the germination speed and percentage. Similar decrease in germination speed and percentage due to salinity was also observed by [23]. It was also assumed that higher salts concentration hinders the absorption of water in germinating seeds and cause delay in germination [24]. In our study the highest germination percentage were obtained in control groups when compared with that of the results obtained from saline treatments and our results are in agreement with that of the findings of [25]. In another study germination percentage was highest in control groups as compared to that of saline conditions where it was reported low by [26]. In present findings seeds treated with PbSO4 have shorten the roots and shoots length. It may be due to absorption of Pb in roots that would decrease Ca in root tips and thus cause decrease in cell division or cell elongation [35].Pb also inhibits the growth of rice shoot due to its harmful effects on photosynthesis, membrane structure and permeability, mineral nutrition, water balance, and hormonal status as reported by [36].Pb increased rates resulted in reduced growth of root and shoot when a comparison was made between 100 and 200ppm of PbSO4 and control, the results indicated that 200 ppm retarded the growth [37]. In our findings shoot height was also shortened in seedlings of PbSO4primed seeds (4.74cm, 3.85cm) seeds at the two concentration levels. Same phenomena in wheat shoot height was also reported with increasing Pb application by [38]. Iron (Fe) has a great role in photosynthesis and plant growth regulation [39].In present study germination speed and percentage was high in FeSO4 primed seed. These results were supported by the findings of [40] according to which the application of spray of FeSO4 on corn leaf showed considerable enhancement in chlorophyll concentration. The germination speed and percentage is high in FeSO4 primed seeds because the application of priming increases growth rate, possibly due to increase in photosynthetic activities and various plant metabolites that are responsible for cell division and elongation [41]. The deficiency of iron might be limiting factor for vegetative growth [42].
In present study the highest mean dry and fresh weight of stems and roots were obtained in FeSO4 primed seeds. Same results of highest mean dry and fresh weight of stems, roots and leaves of Ocimumbasilicumwere obtained in the nano-iron chelated treatment as described by [43]. The present studies reveal that FeSO4 primed seeds show best results. Other researchers are in agreement to [44] who reported that application of FeSO4 increased seed protein and phosphorus concentration.

Conclusion and recommendations
It was concluded that salinity affects all the processes of seedlings growth from emergence of seedlings to seedlings rootshoot length and weight. To overcome adverse effects of salinity, nutrients priming of seeds through Fe showed best results on germination and seedling growth whereas priming of seeds with (Pb) did not show any improvement in these parameters.The present studies suggest that priming of seeds of Cabbage through micro-nutrients may be beneficial as it can enhance the speed rate or percentage of germination, increase salt tolerance, improve the micronutrient value of plants that may lead to increased micronutrient content of the seeds resulting in better nutrition of the crop progeny and also it is basis for future research in this field.