Role of ZnO and TiO2 nanoparticles in improving seed germination, growth and biomass accumulation in Green gram (Cicer arietinum L.) cultivar

doi:10.21203/rs.3.rs-5153652/v1

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Role of ZnO and TiO₂ nanoparticles in improving seed germination, growth and biomass accumulation in Green gram (Cicer arietinum L.) cultivar

https://doi.org/10.21203/rs.3.rs-5153652/v1

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The world's food problem is partly caused by the enormous hurdles that the growing global population presents to agricultural output. Increased yield losses are caused by a number of stressors, including bacteria, fungus, salt, ozone, UV-B radiation, drought, and metal toxicity. Agricultural management uses a variety of techniques targeted at reducing yield loss and environmental impact to address these problems. In order to increase crop yields, nanoparticles are used in the creation of nano fertilizers, nano insecticides, and nano fungicides. Therefore, present study conducted to assess effectiveness of ZnO and TiO₂ nanoparticle on Green gram seed germination, root length, and shoot length and fresh weight and dry weight of plants. Results demonstrated that TiO₂ is more effective in seed germination than ZnO nanoparticles and showed 100% rate at 48-h observation. All nanomaterials stimulated root elongation and promoted shoot growth and biomass of plants at 50 ppm concentrations. The purpose of this study is to evaluate the mechanisms and efficacy of nanoparticles in promoting seed germination. The knowledge gathered from this research is essential for the creation of eco-friendly nanotechnologies, which will ultimately help to lessen the world's food crisis. According to the study, additional field research is necessary to have a more comprehensive understanding, especially in order to completely comprehend the impacts of NPs on the growth, improvement, and health of agricultural plants.

Agricultural Engineering

Food Science & Technology

Nanoscience

Agricultural Economics and Policy

Nanoparticles

Seed Germination

Growth and Biomass of Plants

The rising global population growth and simultaneous environmental challenges highlight the urgent need for innovative and sustainable solutions in agriculture to meet the growing demand for food worldwide (Ashraf et al., 2021). Agricultural seeds face susceptibility to environmental stressors, leading to compromised seed vigor, hindered crop growth, and diminished productivity. While conventional agrochemical-based seed treatments enhance seed germination, they often pose significant environmental risks (Tripathi et al., 2023). Consequently, there is a pressing need for sustainable technologies like nano-based agrochemicals to address these challenges. Nano agrochemicals offer the potential to mitigate the dose-dependent toxicity associated with seed treatments, thereby enhancing seed viability while ensuring the controlled release of active ingredients. However, the widespread adoption of Nano agrochemicals raises legitimate concerns regarding their safety, environmental exposure levels, and potential toxicological implications for both the environment and human health (Singh et al., 2021, Kale et al., 2024). Consequently, there is a critical need for comprehensive assessments and policy regulations to evaluate and manage these risks.

The agricultural industry plays a pivotal role in the economies of developing nations, serving as the primary provider of food for a rapidly expanding global population, which currently exceeds 7.5 billion people (Porter et al., 2014; Berners-Lee et al., 2018). Seeds constitute a fundamental input for sustainable agricultural productivity and production, with approximately 90% of food crops grown from seed. High-quality seeds are essential for generating healthier, more viable, and vigorous seedlings, thereby contributing to effective agricultural practices (Shelar et al., 2021). However, agriculture faces multifaceted challenges, including shifting environmental conditions such as salinity, drought, and heavy metal accumulation in soil, along with the impacts of climate change. These factors can detrimentally affect seed germination, seedling development, and ultimately, crop production. Moreover, seeds are susceptible to damage from seed-borne diseases and pests, leading to abnormal seed dormancy, reduced viability, and impaired water absorption, all of which negatively impact crop production and final yield (Nile et al., 2022).

In terms of agricultural innovation, nanotechnology is a promising new area that offers long-term solutions to the urgent problems affecting global food security. Through the utilisation of nanoparticles' distinctive characteristics, such as their heightened reactivity and surface area, nano-based agrochemicals and seed priming methods hold the potential to transform seed treatments, augment crop yields, and guarantee the enduring viability of farming methods. However, it is essential to proceed with caution and conduct comprehensive assessments to mitigate potential risks to the environment and human health, ensuring that nanotechnology is harnessed responsibly for the benefit of society as a whole. Nanotechnology emerged as a promising field in agriculture, offering innovative solutions to enhance crop productivity and stress tolerance (Chaudhary and Singh, 2020; Kale et al., 2024). In order to guarantee sufficient crop establishment and the effective use of production resources in commercial agriculture, rapid and uniform seed germination is crucial. Nano-enabled seed treatment has gained considerable attention due to its potential to improve seed germination, seedling vigor, and overall plant growth. Among the various nanoparticles utilized, Zinc oxide (ZnO) and Titanium dioxide (TiO₂) have shown remarkable effects on plant physiology.

The integration of nanotechnology into agriculture has ushered in a new era of innovation, contribution promising solutions to improve crop productivity and sustainability. Among the myriad applications of nanotechnology in agriculture, nano-enabled seed treatments have garnered significant attention for their potential to revolutionize conventional seed germination practices and seedling vigor indices. This dissertation delves into recent advancements in nano-enabled seed treatments and their profound impact on the germination process, seedling vigor, and growth physiology of Green gram (Cicer arietinum L.) cultivars, commonly known as chickpeas. As some staple crop rich in protein and essential nutrients, chickpeas play a vital role in global food security. Understanding the effects of nanoparticles, specifically Titanium dioxide (TiO₂) and Zinc oxide (ZnO), on seed germination and seedling growth is crucial for optimizing chickpea cultivation practices. This study examines the comparative effectiveness of TiO₂ and ZnO nanoparticles on seed germination rates and vigorous indices, elucidating their respective roles in promoting robust seedling growth and overall plant biomass. By elucidating the biochemical mechanisms underlying the observed effects, including enhanced antioxidant enzyme activity, chlorophyll content, and nutrient uptake, this research contributes valuable insights to the burgeoning field of nanotechnology-enabled agriculture. Through comprehensive investigation and analysis, this study aims to inform sustainable agricultural practices and pave the way for informed decision-making in crop management strategies.

In order to guarantee sufficient crop establishment and the effective use of production resources in profitable agriculture, fast and uniform seed germination is crucial. Many crop species have semi-permeable coatings in their seed coats that limit solute leakage while facilitating gas exchange and water absorption. The dense layer of aniline blue staining on the seed coats may have an impact on water permeability and, in turn, seed germination. With varied degrees of effectiveness, treatments like scarification, nicking, and removal of the seed coat have been studied to improve the permeability of the seed coat to water and oxygen, hence improving seed germination and vigor of the seedlings. Triploid seeds still have less seedling vigor than diploid seeds, nevertheless. Thus, new seed priming methods are required to increase seed germination and seedling vigor.

Understanding the mechanisms underlying the effects of ZnO and TiO₂ nanoparticles on seed germination and plant growth is crucial for harnessing their full potential in agriculture. Factors such as nanoparticle concentration, size, and shape play vital roles in determining their bio-effectiveness on plants. Therefore, this study conducted for betterment of seed germination by low cost treatment technology.

2.1. Experimental design:

The germination experiment was conducted using glass petri dish with a complete randomized design. Each petri dish contains 30 uniform seeds of Green gram (Cicer arietinum L.) treated with (T1) tap water, (T2) 25 ppm TiO₂, (T3) 50 ppm TiO₂, (T4) 25 ppm ZnO and (T5) 50 ppm ZnO, in room temperature. The 30 seeds were placed in dish which were lined with cotton and watered. These nanoparticles treated seed was also sowing in field experiment with same experimental design.

2.2 Selection of seed and nanoparticles

The local variety [Swetha (ICCV2)] of green gram (Cicer arietinum L.) was selected for seed treatment using nanoparticle ZnO & TiO₂.

2.3 Seed germination:

The germination experiment was conducted at room temperature. Experiment was continuously monitored till the radicle length touched half of seed length; the seed was measured to be germinated. The Germination characteristics were measures by following formulae:

Germination percentage (GP): GP = (number of germinated seeds ÷ total number of seeds sown) × 100 (Renal et al., 2009).

3.4 Plant analysis:

Plants were taken for growth analysis at 10 and 20 DAG (days after germination). Measured root length, shoot length, total length, fresh plant weight, dry plant weight.

2.3 Growth and biomass analysis:

The lengths of the roots and shoots were measured in centimeters plant^− 1 using a meter scale. The leaf area was measured graphically. For this purpose, plant leaves were laid out on graph paper, and the area covered by each leaf was measured and reported as cm² plant^− 1. Following meticulous cleaning and drying, the plant sections were divided, weighed, and maintained in a hot air oven at 80^°C until a consistent weight was reached for the purpose of measuring biomass.

2.4. Biochemical analysis:

2.4. 1 Total chlorophyll and carotenoids:

A 0.1 g leaf sample was chopped and mixed with 10 mL of an 80% acetone (v/v) solution. In a test tube, the solution was kept overnight at 4 C. The solution’s OD (optical density) was measured at 663 nm and 645 nm. Total chlorophyll and carotenoids were calculated following the formula described by Machlachlan and Zalik (1963) and Duxbury and Yentsh (1956):

2.4.2 Estimation of ascorbic acid:

Using the Keller and Schwager (1977) approach, the ascorbic acid concentration was extracted and determined based on the reduction of 2, 6-dichlorophenolindophenol (DCPIP). After homogenizing a 5 g fresh leaf sample in 20 ml of extraction solution (which contained 5 g of oxalic acid and 0.075 g of EDTA in 100 ml of distilled water), the sample was centrifuged for 15 minutes at 12,000x. The solution turned pink when 1 milliliter of the obtained supernatant and 5 milliliters of 2, 6-dichlorophenolindophenol were added. The absorbance of the pink color solution (Es) was determined at 520 nm. After adding a drop of ascorbic acid solution to the mixture, the absorbance (Et) at the same wavelength was measured in order to lower DCPIP. Both absorbances were measured in comparison to the extraction medium blank (Eo), which included 1 ml of extraction medium plus 5 ml of DCPIP.

Using the following formula, a standard curve made from an aqueous solution of ascorbic acid was used to compute the total amount of ascorbic acid:

Ascorbic acid (mg g^− 1 fresh leaf) = [{Eo− (Es − Et)} × V] / (v × W × 1000).

3.1 Effectiveness of Nanoparticles on Seed Germination and Vigorous Index:

TiO₂ and ZnO nanoparticles both positively impacted seed germination. The effectiveness ranking based on vigorous index is as follows: 50 ppm TiO₂ > 50 ppm ZnO > 25 ppm TiO₂ > 25 ppm ZnO > control (Fig. 1). ZnO nanoparticles showed improved germination rates and increased seedling vigour. TiO₂ nanoparticles stimulated seed germination and promoted robust seedling growth. TiO₂ particularly excelled in root and shoot growth, contributing to overall plant biomass. TiO₂ demonstrated a higher effectiveness in seed germination compared to ZnO nanoparticles, reaching a 100% germination rate at 48 hours. Nanoparticle treatment, specifically with ZnO and TiO₂, positively impacted seed germination in Gram cultivars compared to untreated seeds. Germination rate, defined as the percentage of seeds that successfully sprouted, increased in nanoparticle-treated seeds (Chandrika et.al. 2024; Kale et al., 2024). Nanoparticle-treated seeds exhibited faster and more uniform germination compared to untreated seeds. The improvement in seed germination indicates that nanoparticle treatment enhanced the physiological processes necessary for seedling emergence and establishment (Rhaman et al., 2022).

The vigorous index, which reflects the overall health and vigor of seedlings, was higher in nanoparticle-treated Gram cultivars compared to untreat ones (Fig. 1). Nanoparticle-treated seedlings displayed stronger and healthier growth characteristics, including robust shoot and root development. Vigorous index calculations may involve parameters such as seedling height, root length, shoot length, leaf area, and biomass accumulation. The increase in the vigorous index suggests that nanoparticle treatment promoted enhanced seedling vigor and physiological performance in Gram cultivars. Nanoparticles ZnO and TiO₂, resulted in improved seed germination and vigorous index of Gram (Cicer arietinum L.) cultivars. Nanoparticle-treated seeds exhibited higher germination rates and more vigorous seedling growth compared to untreated seeds, indicating the potential for enhanced crop establishment and productivity.

3.2 Effectiveness of Nanoparticles on growth and developments

3.2.1 Root Length:

Nanoparticle-treated plants showed an increase in root length compared to untreated plants. Both ZnO and TiO₂ nanoparticles likely stimulated root growth, resulting in longer and more extensive root systems. Longer root length is beneficial for nutrient uptake, water absorption, and anchorage, ultimately contributing to improved plant health and productivity. 50 ppm TiO₂ has shown higher growth followed by 50 ppm ZnO then 25 ppm TiO₂ and then 25 ppm ZnO and then control shown less growth effect on root length (Fig. 2).

Shoot length, which refers to the length of the above-ground portion of the plant, also increased in nanoparticle-treated Gram cultivars. Enhanced shoot length indicates increased vegetative growth and biomass accumulation. The promotion of shoot growth by nanoparticle treatment suggests improved photosynthetic capacity and overall plant vigor. 50 ppm TiO2 has shown higher growth followed by 50 ppm ZnO then 25 ppm TiO2 and then 25 ppm ZnO and then control shown less growth effect on plant shoot height (Fig. 2).

Treatment with nanoparticles, specifically ZnO and TiO₂, led to an increase in the total height of Gram (Cicer arietinum L.) cultivars compared to untreated plants. Nanoparticle-treated plants exhibited taller stature, indicating enhanced vertical growth. The increase in total height suggests that nanoparticle treatment promoted overall plant growth and development. 50 ppm TiO2 has shown higher growth followed by 50 ppm ZnO then 25 ppm TiO2 and then 25 ppm ZnO and then control shown less growth effect on height. The application of nanoparticles, specifically ZnO and TiO2, positively influenced the growth and development of Gram (Cicer arietinum L.) cultivars. Nanoparticle-treated plants exhibited increased total height, root length, and shoot length compared to untreated plants. These improvements in growth parameters indicate enhanced overall plant vigor.

Both TiO₂ and ZnO nanoparticles had a positive impact on the growth of Gram cultivars. TiO₂ nanoparticles notably promoted both root and shoot growth. ZnO nanoparticles also contributed to enhanced growth, although the effect might have been more pronounced in terms of seedling vigor rather than direct growth promotion. 50 ppm TiO₂ has shown higher growth followed by 50 ppm ZnO then 25 ppm TiO₂ and then 25 ppm ZnO and then control shown less growth effect (Fig. 2).

3.3 Effectiveness of Nanoparticles on Biomass

TiO₂ nanoparticles showed significant efficacy in promoting biomass accumulation in Gram cultivars. The promotion of root and shoot growth by TiO₂ likely led to increased biomass production. ZnO nanoparticles also had a positive effect on biomass, albeit perhaps to a lesser extent compared to TiO2. 50 ppm TiO₂ has shown higher biomass followed by 25 ppm TiO₂ then 50 ppm ZnO and then 25 ppm ZnO and lastly control. Nanoparticle-treated Gram (Cicer arietinum L.) cultivars exhibited higher fresh weight compared to untreated plants. The increase in fresh weight indicates greater water content and overall biomass accumulation in nanoparticle-treated plants. Enhanced fresh weight suggests improved growth and physiological activity in response to nanoparticle treatment.

Nanoparticle-treated Gram cultivars also showed an increase in dry weight compared to untreated plants. Dry weight represents the mass of the plant’s tissues after removing water content, providing a measure of the plant’s structural biomass. The increase in dry weight indicates enhanced accumulation of structural components such as cellulose, lignin, and proteins in nanoparticle-treated plants (Helal et.al. 2022). Higher dry weight reflects improved biomass production and potential for increased yield in nanoparticle-treated Gram cultivars. The application of nanoparticles led to increased fresh and dry weight in Gram (Cicer arietinum L.) cultivars. These changes indicate enhanced biomass accumulation, growth, and physiological activity in nanoparticle-treated plants compared to untreated plants. The improvements in plant weight suggest that nanoparticle treatment positively influenced the growth and biomass production of Gram cultivars, potentially leading to increased agricultural productivity (Fig. 3). The application of nanoparticles, particularly TiO₂, resulted in improved growth and biomass production in Gram cultivars (Kumar et.al. 2023). Enhanced root and shoot growth contributed to increased biomass, indicating the potential for improved plant productivity. While both TiO₂ and ZnO nanoparticles showed positive effects on growth, TiO₂ appeared to be more effective in promoting biomass accumulation, suggesting its potential for use as a seed treatment to enhance crop productivity. The application of nanoparticles, especially TiO₂, holds promise for improving the growth and biomass production of Gram cultivars, which could have significant implications for agricultural productivity and sustainability.

3.4 Biochemical changes

3.4.1 Chlorophyll a, b & total chlorophyll

Both ZnO and TiO₂ nanoparticles resulted in an increase in chlorophyll a, chlorophyll b, and total chlorophyll content in Gram cultivars. ZnO nanoparticles might have contributed to enhanced chlorophyll synthesis, leading to increased chlorophyll a and b content (Rai-Kalal and Jajoo, 2021). TiO₂ nanoparticles might have stimulated chlorophyll biosynthesis pathways, resulting in higher levels of chlorophyll a and b. The total chlorophyll content, representing the sum of chlorophyll a and b, showed a significant increase following the application of both types of nanoparticles. Higher chlorophyll content suggests improved photosynthetic capacity and light absorption, which can positively influence plant growth and biomass accumulation.

These are pigments crucial for photosynthesis. Chlorophyll a absorbs primarily blue and red light, while chlorophyll b absorbs blue light. Together, they capture light energy for photosynthesis. Total Chlorophyll: It represents the overall chlorophyll content in the plant, indicating its photosynthetic capacity. Higher levels of chlorophyll suggest enhanced photosynthesis and better growth potential.

In this study conducted for assessment of effectiveness of nanoparticles. The higher chlorophyll a b and total content was noted in treatment 50ppm TiO₂ followed by ZnO showed least increment of chlorophyll content as compared to control. Age wise chlorophyll a, b and total content was increased and enhance level of chlorophyll a, b and total content was seen at 20 DAS than 10 DAS in all treatments. 50 ppm TiO₂ has shown higher chlorophyll a, b and total content followed by 50 ppm ZnO then 25 ppm TiO₂ and then 25 ppm ZnO as compared to control (Fig. 4). Resultant all chlorophyll pigment positively impacted due to application selected nanoparticles. Result of the study also showed that higher concentration of selected nanoparticle improved chlorophyll contents of selected cultivar.

3.4.2. Carotenoids:

Both ZnO and TiO₂ nanoparticle treatments led to elevated levels of carotenoids in Gram cultivars. Carotenoids play a crucial role in photoprotection and light harvesting during photosynthesis (Chaudhary and Rathore, 2021a; Simkin et al., 2022; Patankar et al., 2024). Increased carotenoid content indicates enhanced photoprotection against excess light and oxidative stress, which can contribute to improved plant resilience and productivity. Auxiliary pigments known as carotenoids help chlorophyll absorb light energy and offer photoprotection by releasing excess energy as heat. Additionally, they aid in the scavenging of reactive oxygen species (ROS), shielding the plant from oxidative damage brought on by stressors like temperature swings and intense light. In this study conducted for assessment of effectiveness of nanoparticles. The higher carotenoid content was noted in treatment 50ppm Ti₂O followed by ZnO showed least increment of ascorbic acid content as compared to control. Age wise carotenoid content was increased and enhance level of carotenoid content was seen at 20 DAS than 10 DAS in all treatments (Fig. 4).

3.4.3. Ascorbic acid:

Ascorbic acid is a water-soluble antioxidant that scavenges reactive oxygen species (ROS) and protects cells from oxidative damage (Chaudhary and Rathore, 2021b, 2022). Elevated levels of ascorbic acid in nanoparticle-treated Gram cultivars indicate enhanced antioxidant defense mechanisms. Ascorbic acid helps to neutralize ROS generated during stress conditions, such as high light intensity or drought, thereby protecting cellular structures and maintaining physiological functions (Sharma et.al. 2016). The presence of increased levels of ascorbic acid contributes to improved stress tolerance and overall plant health in nanoparticle-treated Gram cultivars. Ascorbic acid is a potent antioxidant that helps plants cope with oxidative stress. It scavenges ROS and protects cellular components from damage, thereby maintaining cell integrity and function. Elevated levels of ascorbic acid indicate improved antioxidant defense mechanisms in the plant, which can enhance stress tolerance and overall plant health. The biochemical changes observed in Gram cultivars following treatment with ZnO and TiO₂ nanoparticles involve enhancements in photosynthetic pigments (chlorophyll a, b, and carotenoids) and antioxidant compounds (ascorbic acid). These changes collectively contribute to improved photosynthetic efficiency, photoprotection, and stress tolerance, ultimately leading to enhanced growth and biomass production in Gram cultivars. Present study conducted for assessment of effectiveness of nanoparticles. the higher ascorbic acid was noted in treatment 50ppm TiO₂ (3.88 mg/g fresh leaf) while ZnO showed least increment of ascorbic acid content as compared to control. Age wise ascorbic acid content was increased and enhance level of ascorbic acid content was seen at 20 DAS than 10 DAS in all treatments (Fig. 4).

The world's food problem is partly caused by the enormous hurdles that the growing global population presents to agricultural output. Through the activation of nutrients and energy in plant cells, nanotechnology emerges as a promising green technology for improving agricultural productivity and fortifying soil. Therefore, present study conducted to assess effectiveness of ZnO and TiO₂ nanoparticle on Gram seed germination, root length, and shoot length and fresh weight and dry weight of plants. Results demonstrated that TiO₂ is more effective in seed germination than ZnO nanoparticles and showed 100% rate at 48-h observation. All nanomaterials stimulated root elongation and promoted shoot growth and biomass of plants at 50 ppm concentrations. The purpose of this study is to evaluate the mechanisms and efficacy of nanoparticles in promoting seed germination. The knowledge gathered from this research is essential for the creation of eco-friendly nanotechnologies, which will ultimately help to lessen the world's food crisis. According to the study, additional field research is necessary to have a more comprehensive understanding, especially in order to completely comprehend the impacts of NPs on the growth, improvement, and health of agricultural plants.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

Authors are grateful to the Department of Environmental Science, Savitribai Phule Pune, University, Pune Maharashtra.

Data availability:

Not applicable

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The authors declare no competing interests.

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Role of ZnO and TiO₂ nanoparticles in improving seed germination, growth and biomass accumulation in Green gram (Cicer arietinum L.) cultivar

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Abstract

Figures

1. Introduction

2. Materials and methods

2.1. Experimental design:

2.2 Selection of seed and nanoparticles

2.3 Seed germination:

3.4 Plant analysis:

2.3 Growth and biomass analysis:

2.4. Biochemical analysis:

2.4. 1 Total chlorophyll and carotenoids:

2.4.2 Estimation of ascorbic acid:

3. Result and Discussion

3.1 Effectiveness of Nanoparticles on Seed Germination and Vigorous Index:

3.2 Effectiveness of Nanoparticles on growth and developments

3.2.1 Root Length:

3.3 Effectiveness of Nanoparticles on Biomass

3.4 Biochemical changes

3.4.1 Chlorophyll a, b & total chlorophyll

3.4.2. Carotenoids:

3.4.3. Ascorbic acid:

4. Conclusion

Declarations

Declaration of competing interest

Acknowledgements

Data availability:

References

Additional Declarations

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