Sugar Beet Extract Acts as a Natural Bio-Stimulant for Physio-Biochemical analysis of Hordeum Vulgare L. under Induced Salinity Stress

Change in climate of the entire globe due to elevated temperature and minimum annual rainfall in barren zone frequently leads to salinity of soil. The current study was aimed to evaluate the importance of sugar beet extract (SBE) as a bio-stimulant to improve the adverse damages of induced salinity stress (40mM) on growth, oosmolytes and antioxidant defense system of barley (Hordeum vulgare L.). Pot experiment was carried in green house under different concentrations of SBE (10%, 20%, 30%, 40%, 50%) pre-soaked seeds of Hordeum vulgare for 5 hours SBE was analyzed for glycine betaine (100mmol/kg), betalains (1.3mg/l), phenolics (1.30g/100ml), flavonoids (0.59mg/ml), carotenoids (0.23ml/100ml), vitamin E (0.002%), vitamin C (8.04g/100ml), sugar (8g/100ml), protein (1.39mg/100ml), and oxalic acid (38mg/100ml) while Ca (13.72mg/l), Mg (7.121 mg/l) and K (11.45mg/l) contents were also determined. We found significant improvement in germination parameters of Hordeum vulgare L. via SB extract on coefficient of velocity of emergence (CVE), mean emergence time (MET), germination energy (GE), timson germination index (TGI), germination rate index (GRI) and time to 50% emergence (E50) under induced salinity stress. However, photosynthetic pigments, e.g., chlorophyll and carotenoids were enhanced using 40% SB extract, soluble sugar, protein, proline, POD, MDA with 50% SB extract while SOD and H2O2 in 20% SBE, respectively. Our findings suggested that SB extract promotes both agronomical and physiological attributes, is a positive way to enhance our economy by increasing crop yields in arid and semi-arid areas along with plant tolerance to under induced salinity stress. Highlights The recent study is of utmost importance owing to the following reasons: ➢ Changes in climatic condition results in salinity, droughts, floods, earthquakes, fluctuation in temperature and other environmental hazards. ➢ Salinity stress has myriad of damaging effects on crop growth and productivity and becoming a global issue of major concern especially in the Asian countries including Pakistan. Statistical analysis showed that 6.30 million of lands is affected with salinity. ➢ Barley (Hordeum Vulgare L.) being an important crop of economic and nutritional value has been facing salinity stress condition in Pakistan, resulting in productivity reduction and ultimately paving the way for food scarcity and crippling economy. ➢ The sole purpose of this research article is to assess and ameliorate the physio-biochemical responses via application of sugar beet extract as bio-stimulants in Barley (Hordeum Vulgare L.) grown under induced salinity stress; in order to cope with the dire consequences of salinity stress especially in the southern areas of Pakistan being badly affected by salt stress. ➢ Furthermore, there is a great need for adaptive measures suggested by the present research and other findings made by researchers in pursuit of tackling this alarming global menace.


Introduction
By the end of 2050, abiotic stresses such as salinity, drought and temperature will lead to huge losses in plants to alleviate salinity stress. Since sugar beet extract contains important nutrients like AsA, GB, vitamin E, sugars and amino acids, etc. Also, it can be used as a cheaper source of GB to improve and protect plants from the lethal effect of salt stress (Abbas et al. 2010; Ahanger et al. 2020). All the compounds in sugar beet, that is, play a vital role as a bio-stimulant in plant life during stressful conditions. Seed priming technique, best pregermination seed treatment has been revealed to boost seedling and yield germination (Nasri et al. 2013). The application of SBE as bio-stimulants is boosting speedily in agronomic crops for valuable effects under stressful conditions (Pinheiro et al. 2018;Mahdy et al. 2020). It was assumed priming of seeds with SBE could lower the damaging effects of salinity stress on seed seedling emergence, germination, and establishment. Therefore, the present experiment was performed to evaluate the capability of seed pre-sowing with SBE to regulate barley seed germination under 40 mM NaCl stress. In addition, to explore the degree of efficacy of SBE responses in terms of physiological and biochemical attributes were kept in focus to find out a viable strategy for blocking oxidative stress in barley.

Formation of Sugar beet extract (SBE)
The fresh roots of Beta vulgaris L. were obtained from local market, grind to obtain juice and stored at 10°C. Different concentrations of SBE i.e. (10%, 20%, 30%, 40% and 50% v/v) were then prepared and applied for the treatments.

Estimation of glycine betaine (GB) and Betalain content
SB extract was analyzed for glycine betaine (GB) by following the methodology of Grieve and Grattan (1983). Betalain content was investigated by the following method Nilsson (1970), the absorbance of betalains were measured at 538 and 480 nm using the method of Cai (2005)

Estimation of phenolic and flavonoid content
Sugar beet extract was examined phenolics in SBE following method of Waterhouse (2002) with optical density at 765 nm. Similarly, flavonoid content was calculated as per described by the methodology of Bushra et al. (2009) by reacting the dilute SBE with 0.5% of NaNO 2 and 10% AlCl 3 . After that, 0.2 ml of sodium hydroxide was added. optical density was recorded at 415 nm.

Estimation of carotenoid content
Carotenoid contents in sugar beet extract were determined by Mohdaly et al. (2010). Using spectrophotometer and measured at 450 nm by the following equation.

Estimation of alpha-tocopherol (Vitamin E) and ascorbic acid (Vitamin C)
According to Backer et al. (1980) method was used to study alpha-tocopherol content in sugar beet extract at 520nm whereas, following the standard protocol of Mukherjee and Choudhuri (1983) ascorbic acid was determined in sugar beet extract. The absorbance of vitamin C was measured at 530 nm.

Mean Germination Time (MGT)
Mean germination time for seed emergence investigated by Kader (2005). Greater will be the inhabitants germinated lower is mean germination time.
Hence "f" shows the numbers of seed germinated on X days.
Hence "N" refers to the final emerged seeds number, where nj and ni are inconsistent number of seeds germinated by the adjoining count at tj and ti, correspondingly, when ni<N/2>Nj.
Hence "G1" is the germinated seed percentage at day first and "G2" is germinated seed percentage at second day after sowing.

Coefficient of Velocity of Emergence (CVE)
Coefficient of velocity of emergence was determined by the method of Kader (2005).
Hence "n" is the number of seed germinated per/day while T represents the time.

Timson Germination Index (TGI), Germination Energy (GE)
Timson germination index and Germination energy was measured by the method of Al-Ansari and Siksi (2016).
Hence G is the germination percentage per day, while T is the entire germination period.
Germination energy was determined by the following formula Al-Ansari and Siksi (2016). Eq.6 Hence, X n = numbers of seed germination on last (nth) counting date and Y n represents the number of days from sowing to last (nth) counting date. nm were observed against 80% acetone blank through spectrophotometer.

Estimation of Osmolytes Content
Leaf soluble sugar content was evaluated by the methodology of Dubois et al. (1956). Foliar material of 0.5 g was grounded in 5 ml distilled water and centrifuged for 10 minutes. About 4 ml of 35% concentrated H 2 SO 4 was added to 1ml supernatant its optical density was found at 490 nm.
Total were grounded 3% (10 ml) sulfosalicylic acid and filtered. After filtration, 2 ml of the filtrate was dissolved in 2 ml acid ninhydrin (40 ml glacial acetic acid + 1.87 g ninhydrin + 30 ml of 6 M phosphoric acid) and glacial acetic acid in a test tube and warm for 1 h at 100°C. Solution extraction was completed with toluene (4 ml) and OD was measured at 520 nm.

Content (H 2 O 2 )
Soluble protein was found by the standard protocol of Rostami and Ehsanpaur (2009)

Determination of Antioxidant Enzymes
Leaf MDA content was estimated by Zhang and Huang (2013). 0.25 g of leaves were homogenized in 3 ml of 1.0% Trichloro acetic acid and centrifuge for 10 minutes. 1 ml of the supernatant was mixed with 0.5% (4 ml) 2-thiobarbituric acid and heated for one hour at 95 °C, then cooled with ice for 10 minutes and its optical density was determined at 532 nm.

Statistical Analysis
The statistical analysis was three factors (barley, SBE, salinity) which arranged in CRD. Data collected for various germination attributes, physiological and biochemical components by using IBM SPSS Statistics 22.
Mean separation and standard deviation were determined by Tukey's test.

Results
The

Germination attributes of Hordeum vulgare L.
In the present study, the signs of germination showed a significant role of SBE in terms of resistance to salinity stress and maintaining good germination. The results ( concentration of 30% and 40% SBE used for the priming was found to be more useful in terms of G% for the seeds exposed to salinity stress.

Physiological and biochemical attributes
It is suggested that the degradation of plant chlorophyll content and increase in cellular respiration rate under stress condition is related with the accumulation of reactive oxygen species (Camejo et al. 2006). Plant foliar material was analyzed for photosynthetic pigments such as chlorophyll a/b ratio, total Chlorophyll and carotenoid content were significantly enhanced by increasing concentration from 20-40% SBE under salinity stress. According to ( Fig. 1-3) results revealed that maximum values of chlorophyll a/b ratio, total chlorophyll content and carotenoid contents were recorded in T5, T10, and T6 while minimum results have been obtained by applying the extract without salinity indicated that SBE is a natural bio-stimulant as well as creating plant ability to stress tolerance by activating different physiological processes respectively. Furthermore, the results showed that there is a big difference among all the primed and non-primed treatments under induced salinity stress (40 mM NaCl).
Plants perceive a change in their physiological events in response to several abiotic stress and therefore respond shortly by accumulating different protective osmolytes mostly comprising sugar, proline and glycine betaine to provide favorable environment for metabolic activities and also protecting the plant by reducing the harmful damages caused by oxidative stress (Hayat et al. 2012). Similarly, plant osmolyte such as soluble sugar content has been recorded maximum in T11 followed by T9 under induced salinity stress in (Fig. 4). Results revealed that primed seeds have a great influenced on the soluble sugar content up to a significant level. Therefore, it is investigated that increasing the SBE concentration, sugar content will also be enhanced under salinity.
Consequently, soluble protein content (Fig. 5) was measured maximum in T11 (50% SBE) up to significant level (p<0.05) under induced salinity stress revealed that the soluble protein content has been promoted by increasing the concentrations of SBE. Nonetheless, total proline content (Fig. 6)  Regarding SOD activity, 20% SBE concentrations are significantly more effective at improving SOD activity under salinity stress than other SBE levels (Fig. 7). However, in terms of POD activity, this improvement was greater in plants grown with 30% SBE primed under induced stress conditions (Fig. 8). Similarly, hydrogen peroxide activity studied in ' Figure 9' has been reported maximum in 30% sugar beet extract followed by 50% SBE. MDA is marker of lipid peroxidation/membrane loss under stress and indicates plant resistance to stress. In the current study, the MDA concentration augmented significantly enhanced under salinity in T11 stress as compared to plants grown under control and without salinity (Fig. 10).

Regression and correlation analysis of the measured traits
Statistical analysis showed that sugar beet extract affected on hydrogen peroxide activity in crop plants.
Analysis of variance of measured traits with significant differences between treatments and within treatments was carried out between the priming factors under Salinity Stress (Table 4).
According to the results of (Table 5, 6), regression and correlation analysis showed a significant positive correlation (p ≤0.05) between total chlorophyll and carotenoids content. Similarly, a positive correlation was also found between SSC, SOD, POD and MDA content in the leaves at p<0.05.

Table 5. Regression and correlation analysis between physiological and biochemical parameter in Hordeum vulgare L. under induced salinity stress.
Chlo a/b ratio=chlorophyll a/b ratio, TCC=total chlorophyll content, CC=caroteniod content, SCC=soluble sugar content, SPC=soluble protein content, TPC=total proline content, POD=peroxidase content, SOD=superoxide dismutase content, H 2 O 2 =hydrogen peroxide, MDA=malendyaldehyde, * is significant up to p=0.01,** is significant to p=0.01,*** is significant up to p=0.001.

Principle component analysis of biological component
Principle component analysis were studied on 10 characters (Table 7) enclosed over all 55.009% of the whole variation. The PC1 explained 23.562% of complete variance which were significantly correlate with protein, proline, soluble sugar content and carotenoid content particularly related with osmolytes. while PC2 investigated 17.694% of complete variance which was particularly correlate with total chlorophyll content, chlorophyll "a/b" ratio, SOD and POD corresponded to plant antioxidant enzymes and growth components.

Discussion
Irrespective of hundreds of factors causing impediments in plant growth and development, water shortage is reckoned as a main abiotic factor that affects the crop growth and limits the global food production (Noman et al. 2018 Differences in photosynthetic pigments are a key indicator for determining the photosynthetic pigments of plants under salinity stress. Our results ( Fig. 1-3) showed a noticeable increase in chlorophyll "a/b ratio, total chlorophyll and carotenoids by pre-sowing seeds with SBE both with and without salinity stress. Our findings are correlated with the study of Du-Jardin (2015), Ali and Ashraf (2011) who suggested that SBE act as a natural biostimulant against salinity stress. However, carotenoids content increased under salinity due to SBE seed priming might have a defensive role by reducing the damages to photosystem II proteins due to reduced light harvesting.
The Similarly increase in soluble protein under salinity stress via seed priming with 50% SBE may be due to broadspectrum of stress resistance that has been unlocked by osmolytes to survive under several biotic and abiotic stresses.

Conclusion
The present study investigated that seed priming with SBE is a reliable and cost-effective technique for the agricultural and seed quality. It can be evaluated that seed priming treatment with sugar beet extract is of significant