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Chapter 3 Nanopriming technology: enhancement of germination process and starch metabolism of aged rice seeds using phytosynthesized silver nanoparticles*

Chapter Abstract

Application of nanomaterials for agriculture is relatively new as compared to their use in biomedical and industrial sectors. In order to promote sustainable nanoagriculture, biocompatible silver nanoparticles (AgNPs) have been synthesized through green route using kaffir lime leaf extract for use as nanopriming agent for enhancing seed germination of rice aged seeds. Results of various characterization techniques showed the successful formation of AgNPs which were capped with phytochemicals present in the plant extract. Rice aged seeds primed with phytosynthesized AgNPs at 5 and 10 ppm significantly improved germination performance and seedling vigor compared to unprimed control, AgNO3 priming, and conventional hydropriming. Nanopriming could enhance -amylase activity, resulting in higher soluble sugar content for supporting seedlings growth. Furthermore, nanopriming stimulated the up-regulation of aquaporin genes in germinating seeds. Meanwhile, more ROS production was observed in germinating seeds of nanopriming treatment compared to unprimed control and other priming treatments, suggesting that both ROS and aquaporins play important roles in enhancing seed germination. Different mechanisms underlying nanopriming-induced seed germination were proposed, including creation of nanopores for enhanced water uptake, rebooting ROS/antioxidant systems in seeds, generation of hydroxyl radicals for cell wall loosening, and nanocatalyst for fastening starch hydrolysis.
This chapter is based on a journal paper published in Scientific Reports.


Nanotechnology has the potential to revolutionize the agriculture and play an important role in food and crop production (Parisi et al., 2015; Servin et al., 2015). During the past decade, a number of patents and products incorporating engineered nanoparticles (NPs) into agricultural practices, e.g. nanopesticides, nanofertilizers, and nanosensors, have been developed with the collective goal to promote the efficiency and sustainability of agricultural practices requiring less input and generating less waste than conventional products and approaches (Liu and Lal, 2015; Servin et al., 2015). Silver nanoparticles (AgNPs) are the most commercialized nanomaterials widely used in antimicrobial and personal care products, building materials, water filtration, medical instruments, and in many other industrial and biomedical applications (Borase et al., 2014). Since applications of NPs in agriculture need to be economical, ecofriendly, biocompatible and non-toxic (Liu and Lal, 2015; Mahakham et al., 2016), synthesis of bioengineered NPs for agricultural purpose should be compatible with these requisites. Plant based materials seem to be the best candidates for synthesizing biocompatible NPs due to their biochemical diversity of plant extract, non-toxic phytochemical constitutes, non-pathogenicity, low cost and flexibility in reaction parameters as compared to chemical synthesis methods (Mahakham et al., 2016; Singh et al., 2016).
In commercial agriculture, rapid and uniform seed germination and seedling emergence are important determinants of successful stand establishment (Chen and Arora, 2013; Rajjou et al., 2012). Germination begins with water uptake by the mature dry seed (imbibition) and terminates with the elongation of the embryonic axis, usually the radicle, through the seed envelope, which has as a consequence, the protrusion of the root, and later of the shoot(Rajjou et al., 2012). The α-amylase is one of the key enzymes involved in degradation of starch during germination of cereal seeds and in subsequent seedling establishment(Kato-Noguchi and Macías, 2005). This is the only enzyme which initiates hydrolysis of native starch granules and is de novo synthesized during the germination of cereal seeds and catalyzes the hydrolysis of -1, 4 linked glucose polymers to release fragments that can be further broken down by other amylolytic enzymes(Mishra and Dubey, 2008). Thus, the enhancement of -amylase during seed germination is of interest in the field of carbohydrate research to promote economic plant growth.
All seeds stored under air dry conditions will have suffered a degree of deterioration(Dragicevic et al., 2013) and seeds in long-term storage will eventually lose their viability due to spontaneous biochemical damage occurring at cellular level(Butler et al., 2009), leading to natural seed aging and subsequently limiting crop productivity. Seed priming is a technique that partially hydrates seeds in natural or synthetic compounds under specific environment to a point where germination-related metabolic processes begin, but radicle emergence does not occur(Ibrahim, 2016). Seed priming has been found to be useful for enhancing seed quality, seedling establishment and crop yields as well as increasing tolerance to environmental stresses(Chen and Arora, 2013; Ibrahim, 2016). Seed priming can improve the germination of weak, damaged or aged seeds(Dragicevic et al., 2013) or even under adverse environment(Ibrahim, 2016). A number of commonly used priming agents include polyethelyne glycol, inorganic salts, nutrients, and plain water (Butler et al., 2009; Horii et al., 2007; Hussain et al., 2015). However, different priming solutions have different properties, effectiveness, and optimization of priming agents is required for each crop species (Horii et al., 2007). Therefore, there is a growing need to develop new priming agents to enhance seed germination of various crop plants.
In recent years, several metal-based NPs, e.g., AgNPs (Mohamed et al., 2017), AuNPs (Mahakham et al., 2016), CuNPs (Panyuta et al., 2016; Taran et al., 2017), FeNPs (Panyuta et al., 2016), FeS2NPs (Srivastava et al., 2014), TiO2NPs (Zheng et al., 2005), ZnNPs (Panyuta et al., 2016; Taran et al., 2017), ZnONPs (Latef et al.)) and carbon-based NPs, e.g., fullerene (Kole et al., 2013) and carbon nanotubes (Ratnikova et al., 2015)) have been applied as seed pre-treatment agents for promoting seed germination, seedling growth, and stress tolerance in some crop plants. Among these studies, only a few researchers have used seed priming strategy, in which seeds must be re-dried to their original moisture content before sowing. Thus, the mechanism behind seed nanopriming would be different from that of pre-sowing seed treatment without drying seeds. In addition, comprehensive studies on physiological and molecular mechanism of nanopriming effects on seed germination have not been elucidated, and thus there are many questions remained to be addressed, especially mechanism behind NPs-induced seed germination.
In this study, jasmine rice (Oryza sativa L. cv. KDML 105) was selected as a model plant since this cultivar is one of the world’s best quality aromatic rice cultivars which is in high demand for world’s markets, but it is sensitive to various environmental stresses, resulting in the reduction of yield (Nounjan et al., 2016). Moreover, many seed lots of jasmine rice are naturally aged seeds due to improper storage conditions under ambient temperatures by most farmers. Therefore, the overarching aims of this study are to develop green method for the synthesis of AgNPs and present a new priming technique using phytosynthesized AgNPs to enhance the germination and starch metabolic process of rice aged seeds alternative to conventional priming. The mechanism behind AgNPs induced seed germination and starch mobilization was also proposed.

Materials and Methods

Chemicals and materials used for AgNPs synthesis

AgNO3 (99.98%) was purchased from Merck (Germany). All aqueous solutions were prepared using double distilled deionized (DI) water. All reagents were of analytical grade. All glassware was washed with aqua regia solution and rinsed three times with DI water.

Plant materials

Kaffir lime (Citrus hystrix D.C.), a small tree belonging to family Rutaceae, has been widely used as flavoring in Southeast Asian cuisines due to its highly aromatic and unique citrus flavors and strong fragrances. Kaffir lime leaves possess some important bioactive compounds (e.g. polyphenols), and given its good antioxidant properties (Peter, 2012) we selected kaffir lime leaves (Inset of Figure 1) as natural material source for AgNPs biosynthesis.

Preparation of plant extract

Preparation of plant extract as reducing and stabilizing agents for AgNPs synthesis was done according to the method of Karthik et al.(2016) with some modifications. Briefly, the fresh leaf samples were initially rinsed with running tap water and then washed with DI water to remove the adhering dust particles and other contaminants.
After blot drying, leaves (20 g) were cut into small pieces, blended in a kitchen blender with 100 ml of DI water. The blended extract was heated at 80 ºC for 5 min and cooled at room temperature. Then, the extract was filtered through Whatman No.1 filter paper before centrifuging at 5000 rpm for 15 min to remove the heavy batteries. The filtered extract was kept in refrigerator (4 ºC) up to 1 week for it to be used as reducing and stabilizing agents.

Synthesis of silver nanoparticles (AgNPs)

In a typical reaction procedure, 3 mL of kaffir lime leaf extract was added to 12 mL of 1 mM AgNO3 aqueous solution and incubated in the dark for 1 h with gentle stirring. The solution turned to yellowish brown color after 1 h incubation and the reaction completed within 24 h. The AgNPs obtained from the solution were purified by repeated centrifugation at 10,000 g for 15 min followed by dispersion of the pellet in sterile DI water 4 times. The water-suspended nanoparticles were lyophilized for 48 h followed by characterization for the structure and composition.

Characterization of phytosynthesized AgNPs

The bioreduction of the silver ions in the solution was determined under UV-visible spectroscopy using Perkin-Elmer Lambda 2 UV198 between 300 to 800 nm. Morphological details of purified AgNPs were measured with transmission electron microscopy (TEM) at 200 kV using FEI TECNAI G2 20 equipped with selected area electron diffraction pattern (SAED). Simultaneously, the elemental compositions of the samples were also recorded using Energy Dispersive X-ray analysis. X-ray powder diffraction (XRD) using Cu Kα radiation with λ = 0.15418 nm (Bruker D2 phaser) was used to investigate the crystalline nature and particles size of the synthesized AgNPs.
Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectra of the freeze-dried samples were recorded using a Bruker Tensor 27 equipment to determine the surface functional groupsof the AgNPs.

Preparation of priming solutions

The phytosynthesized AgNPs were used for seed priming test. AgNPs concentrations at 10 and 20 mg L 1, designated as AgNPs10 and AgNPs20 nanopriming agents, respectively, were freshly prepared by dispersing the particles in deionized water using ultrasonic vibration (100 w, 40 kHz) for 30 min. Similarly, for silver nitrate (AgNO3) priming solutions, two concentrations of AgNO3 at 10 and 20 ppm designated as AgNO310 and AgNO320, respectively, were dissolved in deionized water and kept in dark bottle. Deionized water was used as hydropriming.

Seed priming method

Seeds of jasmine rice (Oryza sativa L. cv. KDML105) were obtained from Khon Kaen Rice Research Centre, Thailand. These were naturally aged seeds due to long period of storage under ambient temperature (25-30 ºC) for 3 years. The initial seed moisture content (MC) was 8.9% (on dry weight basis). Healthy seeds were selected from the same seed lot and used for all the experiments. For seed priming method, seeds were surface-sterilized in 3% H2O2 and then rinsed with deionized water. Seeds were then soaked in SNP or SN priming solutions for 24 h with continuous aeration. The ratio of seed weight to solution volume was 1:4 (g mL 1). Seeds soaked in deionized water were defined as hydropriming (HP) and were washed three times with deionized water (3 min) and surface-dried on paper towel. Seeds were dried back to their original moisture content in shed at room temperature (25 ± 2 °C), sealed in polythene bags and stored at 4 °C until further use. The untreated seeds were used as the unprimed control (UP).

Germination assay and growth measurement

Germination tests on the primed and unprimed seeds were performed in triplicates. Briefly, a piece of sterile filter paper (Whatman no.1) was placed on a sterile plastic Petri dish (90 × 15 mm) and moistened with 5.0 ml deionized water. Ten sterilized seeds were allowed to equilibrate under room temperature for 24 hrs, then placed in each plate, covered with lid and sealed with Parafilm M® to avoid moisture loss. All Petri plates were kept in an incubator under dark at 25 °C.
After initiation of germination assay, seed germination was monitored daily for 6 days and seeds were considered germinated when the radicle was extended to more than 5 mm. Besides, at the end of germination experiment, length and weight of seedlings (roots and shoots) were measured. The data on germination percentage (GP), germination index (GI), germination rate (GR), and seedling vigour index (SVI) were calculated according to the equations provided elsewhere (Ellis and Roberts, 1981; Feizi et al., 2013).

Seed water uptake and dehydrogenase activity

The water uptake by seeds during imbibition at 4 and 24 h was determined following the method described elsewhere (Bhardwaj et al., 2012). Dehydrogenase activity of the root tips was determined according to the method given by Singh et al. (2009)

Table of Contents
Co-Authorship forms
Table of Contents
List of Figures
List of Tables
Chapter 1 Introduction and Background
Chapter Abstract
1.1 Introduction to nanoparticles
1.2 Synthesis of nanoparticles (NPs)
1.3 Chemical synthesis of metallic NPs
1.4 Green synthesis of metallic NPs
1.5 Biological synthesis of metallic NPs using plants
1.6 Characterization techniques
1.7 Interaction of engineered NPs with plants
1.8 Effects of carbon nanotubes (CNTs) on plant growth and Development
1.9 Potential use of CNTs as fertilizer
1.10 Rationale of the research
1.11 Objectives of the research
1.12 Overview of the thesis
Chapter 2 Ecofriendly and green synthesis of phytochemicals-capped gold nanoparticles and their application as nanopriming agent for promoting germination of crop seeds: A sustainable agriculture approach
Chapter Abstract
2.1. Introduction
2.2. Materials and Methods
2.3 Results and Discussion
2.4. Conclusion
Chapter 3 Nanopriming technology for enhancing germination and starch metabolism of aged rice seeds using phytosynthesized silver nanoparticles
3.1 Introduction
3.2 Materials and Methods
3.3. Results
3.4 Discussion
3.5. Conclusion
Chapter 4 Environmental Assessments of the Effects of Carbon Nanotubes on Maize Plants and Synchrotron Based micro-X-ray florescence Imaging of Nutrients in Kernels
4.1 Introduction
4.2 Materials and Methods
4.3 Results and Discussion
4.4. Conclusion
Chapter 5 General Conclusion
Engineered Nanoparticles for Agricultural and Environmental Prospective: Eco-friendly Synthesis, Characterisation, and Interaction with Model Crop Plants

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