In vitro Seed Germination Studies in Barley Exposed to Zinc Oxide Nanoparticles

Barley (commonly called “Jau”) is the main cereal grain in India. It is a very healthy grain that is rich in minerals and vitamins. It has a number of industrial applications and is also used as an animal feed. Due to increasing population in India, there is huge demand of such grains. To meet this increasing demand due to a growing population, it has become important to increase the production of crops. To meet these needs, researchers are trying to develop innovative eco-friendly techniques and methods for increasing the efficiency of crop production. Nanotechnology is one of the options nowadays to enhance the nutritional value of crops. Some engineered nanoparticles could be used as fertiliser and are being visualised as having a potential to revolutionise agriculture and food systems and thus improve the condition of the poor. Nanoparticles have unique physicochemical properties and have the potential to boost plant metabolism (Thakur et al., 2022). Reports in literature suggest that nanoparticle may also be used as an essential plant nutrient (Thakur et al., 2021; Rizwan et al., 2017; Moghaddasi et al., 2017) as they can interact with plants at various levels, thereby causing many morphological and physiological changes (Pathak et al., 2021; Khodakovskaya et al., 2012).
       
Zinc is an important micronutrient that regulates various physiological responses and metabolic processes in plants (Upadhyaya et al., 2017). At a zinc concentration of 25 mg/L, improvement in growth and physiology of cluster beans has been observed, whereas at 50 mg/L zinc, which is considered to be a higher concentration, reduced growth with an adverse effect on plant physiology has been observed (Manivasagaperumal et al., 2011). Zinc is known to play an important role in protecting and maintaining the stability of cell membranes. It is also used for protein synthesis, membrane function, cell elongation and tolerance to environmental stresses (Cabot et al., 2019). Plants emerging from seeds with low zinc have poor seedling vigour and field establishment in zinc-deficient soils. Germination also involves the movement of metal ions like zinc, so that it may be utilised efficiently. Due to the adverse potential and also having no negative effect on the ecosystem, it has been reported that zinc oxide nanoparticles are used worldwide in a number of applications (Rizwan et al., 2017). However, the majority of studies on nanoparticles to date concern toxicity. Comparatively few studies have been conducted on nanoparticles that show that they are also beneficial to plants.
       
Therefore, the present study was taken up to investigate the growth promotory or inhibitory effects of various concentrations of ZnO nanoparticles on the growth of barley seeds. The observations from the present study will prove useful in understanding the possible role of ZnO nanoparticles in barley during seed germination and will give an insight into the role of ZnO nanoparticles in understanding the physiology of barley seed germination.

Synthesis of ZnO nanoparticles
 
In order to synthesise ZnO nanoparticles, a wet chemical method using zinc nitrate and sodium hydroxide precursors was used (Talam et al., 2012). In this experiment, an alcoholic zinc nitrate solution (0.5 M) was prepared and a 0.9 M aqueous ethanol solution of sodium hydroxide (NaOH) was added drop-wise into it with high speed stirring and then it was allowed to settle overnight. The next day the supernatant was removed and the remaining solution was centrifuged at 6000 rpm for 5 minutes and precipitates were removed. These precipitates were washed with distilled water and ethanol solution and then dried at 60°C in an oven. After drying, ZnO nanoparticles were obtained (Talam et al., 2012). The characterization was done by taking the UV-Vis absorption spectrum of the zinc nitrate solution after the addition of NaOH. Also, the absorption spectrum of ZnO dispersed in water was obtained.
 
Pot experiments
 
Barley seeds were obtained from Punjab Agricultural University (PAU), Ludhiana, India. The experiments were performed in Biotechnology Department of Goswami Ganesh Dutta Santa Dharma College in the month of February and March in 2021. All the seeds were surface sterilised before germination by treating them with a 5% bavistin solution for 10 minutes and then washed several times with distilled water to remove the traces of bavistin from the seeds. The seeds were divided into five groups. There were 20 seeds in each treatment group. In the zinc treated group, the seeds were treated with a 0.1M zinc nitrate solution (Pathak et al., 2018). The nanoparticles were suspended in distilled water and dispersed by providing ultrasonic vibration.  Seeds of nanoparticle-treated groups were given 100, 200 and 500 ppm concentrations of ZnO nanoparticles. A similar experiment without nanoparticles was conducted as a control. The seeds belonging to all the groups were given respective treatments daily by watering them and all these treatments were continued for 3 weeks. During the period of three weeks, the number of seeds germinated each day was noted. After 3 weeks, seedlings were harvested and the shoots and roots of seedlings were separated. Seedling growth in terms of root length, shoot length, leaf length and leaf lamina was recorded and the results were compared with the controls to see the effect of nanoparticles on seed germination and early seedling growth.
       
Germination indices parameters like PI (Promptness index), GSI (Germination stress tolerance index), PHSI (Plant height stress tolerance index), RLSI (Root length stress tolerance index), seedling vigour index (SVI) and relative root elongation (RRE) were calculated by using different formulae (Dharanguttikar et al., 2015).
 
Statistical analysis
 
All the experimental values are expressed as means of 20 seeds. Comparisons between the control and treated groups were done by one-way ANOVA.

Nanotechnology has emerged as a new discipline and nanoparticles have become the centre of attraction for researchers because of their unique physico-chemical properties compared to bulk particles (Gatoo et al., 2014). Nanoparticle characterization is considered an important parameter for confirming whether nanoparticle formation has occurred or not. UV-visible absorption spectroscopy is widely being used to examine the optical properties of nanosized particles. The absorption spectrum of ZnO nanopowder is shown in Fig 1. It exhibits a strong absorption band at about 322 nm. This confirmed the proper synthesis of ZnO nanoparticles (AL-Asady et al., 2020; Shamhari et al., 2018).
 

       
Germination frequency was recorded after 8, 16 and 21 days. It was observed from Table 1 that the percent seed germination decreased in the zinc-treated group as compared to the control group. Also, the per cent germination was found to decrease with an increasing concentration of ZnO nanoparticles. Seed germination is an important phenomenon in modern agriculture because it is a thread of life for plants that guarantees their survival (Siddiqui et al., 2014). Results pertaining to seed germination and early seedling growth clearly indicate that ZnO nanoparticles at lower concentrations promoted seed germination, but at higher concentrations reduced seed germination and seedling growth. An increase in germination at low concentrations of nanoparticles may be due to the absorption and utilisation of ZnO nanoparticles by the seeds. Also, there could be the generation of superoxide and hydroxide anions by ZnO nanoparticles that might have encouraged the intake of water and oxygen needed for quick germination (Pathak and Bedi, 2015). ZnO nanoparticles have the potential to overcome seed germination issues in plant species that have low seed germination due to zinc alone.Therefore, a decrease in seed germination at a  higher nanoparticle concentration might be due to the physical or chemical toxicity exerted by ZnO nanoparticles on plants, depending on their chemicacomposition, size, surface energy and plant species (Talam et al., 2012).
 

       
After harvesting, root length, shoot length, leaf length and lamina of control as well as different treated groups were taken. The root length was found to decrease in the zinc-treated group as compared to the control group. The 100 ppm of nanoparticle treatment also caused a significant decrease in root length as compared to the control plants. However, the root length was found to increase at 200 ppm treatment and it again decreased significantly at 500 ppm nanoparticle treatment (Table 1). On the contrary, the shoot length was found to decrease after zinc treatment, further significantly decreased at 100 and 200 ppm nanoparticle treatments but was found to be increased at 500 ppm nanoparticle treatment. Zinc treatment also caused a decrease in leaf length in barley plants. However, the leaf length was found to increase with an increase in the concentration of ZnO nanoparticles.  Further, zinc treatment again caused a decrease in leaf lamina in barley plants. However, the leaf length was found to increase with an increase in the concentration of nanoparticles. Plant growth and development starts with the germination of seeds, followed by root elongation and shoot emergence. These are regarded as the earliest signs of growth and development. Therefore, it is important to understand the course of plant growth and development in relation to nanoparticles. The results obtained in the present study are in accordance with the results obtained by Lawre and Rasker (2014), who revealed that the higher dose of ZnO nanoparticles suspension reduced root and shoot growth of gram and mung seedlings, which may be due to toxicity levels of nanoparticles. ZnO nanoparticles were also reported to be one of the most toxic nanoparticles that could terminate root growth of radish, rape, ryegrass, lettuce, corn and cucumber (Tymoszuk et al., 2017; Lawre and Raskar, 2014). This can also be accepted because more ions are released from particles over time and accumulate in plantlets, making them more toxic. However, Prasad et al., (2012) found that lower concentrations of ZnO nanoparticles were beneficial to seed germination in peanut, Sedghi et al., (2013) in soybean, Ramesh et al., (2014) in wheat and Raskar and Laware (2014) in onion. The higher plant growth with nanoparticles might also be due to the mobilisation of nutrients in the soil as well as an increase in microbial population, especially in the rhizosphere (Raliya and Tarafdar, 2013). In the case of root/shoot ratio, an increasing trend was seen from lower (100 ppm) to higher concentrations (200 ppm) of ZnO nanoparticles (Table 1). However, the root/shoot ratio decreased concomitantly from 200 ppm to 500 ppm of ZnO nanoparticles. This indicates that at this higher concentration, the root length promotion is comparatively less when compared to shoot length. This indicates that roots were affected more by higher concentrations of ZnO nanoparticles as compared to percent seed germination and shoot growth.
       
The results with respect to shoot and root length as well as root to shoot ratio are also consistent with previous studies that report nanoparticles had less of an effect on seed germination than on seedling growth (Lin et al., 2007; Ruffini et al., 2009). This may be explained by the protective effect of the seed coat (Adhikari et al., 2015). Since roots are the first target tissue to confront pollutants, toxic symptoms seem to appear more strongly in roots than in shoots (Garriga et al., 2014). The result clearly indicates that ZnO nanoparticles are effective in enhancing plant growth and yield. Studies on other types of nanoparticles have also shown that the engineered nanoparticles can boost seed germination, growth and development of plants (Siddiqui et al., 2014; Lahiani et al., 2013). As zinc is the structural component of phosphorous (P)-mobilizing phosphatase and phytase enzymes, it can be hypothesised that application of nanoZnO may help in more secretion of these enzymes, which are involved in the mobilisation of phosphorus for plant nutrition from unavailable organic sources. Excess zinc in the soil can often compete with the plant’s uptake of phosphorus, iron, or copper, resulting in deficiency in plants and, as a result, toxic ZnO nanoparticles at higher concentrations.The higher percentage of seed germination and significantly longer seedling length observed in barley seedlings obtained from ZnO nanoparticles treated plants can also be attributed to zinc movement from leaf tissues to seed during the seed development and maturation process.
       
Data with respect to the promptness index (PI) clearly showed that PI decreased with zinc treatment and also at all concentrations of nanoparticle treatment as compared to control, with a slight increase at 200 ppm (Table 2). A higher Germination stress tolerance index (GSI) (37.68) was observed in the 200 ppm ZnO nanoparticle treatment, while a lower GSI (34.78) was observed in the 100 and 500 ppm ZnO nanoparticle treatments. Zinc treatment alone also showed a decrease in GSI value. Plant height stress tolerance index (PHSI) values also showed a decrease at all concentrations of nanoparticle treatment compared to control.
 

       
A significant decrease in PHSI value, i.e., 68.55 at 100 ppm. However, a significant increase was noticed at 200 ppm and again at 500 ppm, a slight decrease was noted. A significant decrease in root length stress tolerance index (RLSI) values was observed at 100 ppm of ZnO nanoparticles treatment over control and a significant increase in RLSI values (77.59) was observed at 200 ppm, while again a decrease (35.23) was observed at 500 ppm concentrations of ZnO nanoparticles. Seedling vigour index (SVI) and relative root elongation (RRE) also showed a similar trend with decrease noticed at all concentrations of ZnO nanoparticles except at 200 ppm concentration, where a significant increase in SVI was noticed (Table 2).
       
The result regarding PI shows that ZnO at lower concentration increased seed germination, promptness index and seedling growth. This indicates the lower concentration is not harmful to the cell division and early seedling growth. GSI indicates the speed of seed germination in the seedlings. Thus, the results of PI, GSI, PHSI, RLSI, SVI and RRE demonstrated that nano particles have both positive and negative effects on crops, depending on solution concentration, particle size and chemical and physical properties of nano particles.

It can be concluded from the present study that the effect of Zn nanoparticles in the germination of growing seedlings of barley was significant at 200 ppm. As a consequence, the ZnO nanoparticles treated seeds unto 200 ppm showed better potential for germination. Overall, our results can help the fertilizer industries to decide the production of nanofertilizers especially ZnO nanoparticles for plant nutrition which will help to reduce the zinc deficiency in plants and finally humans. However, field studies under different nanostructure sizes, shapes, conditions and plants may further enhance the mechanistic understanding of the applicability of nanoparticles in this field. Also, more studies are needed to explore the mode of action of nanoparticles, their interaction with biomolecules and their impact on the regulation of gene expressions in plants.

We are thankful to the Principal, GGDSD College, Sector 32, Chandigarh, for providing the laboratory facilities in Department of Biotechnology.

None.