Impact of Qiangdi 863 Nanosynergids Treated Water on Rice Physiology, Grain Production, and Grain Quality

Global warming, climate change, environmental stress and population increase have threatened food security and increased food production demand. Increasing Rice productions could be a better solution to solve or at least decrease the severity of the global food crisis. The modern rice cultivation techniques have been insucient for sustainable rice production. So, Qiangdi 863 nano synergid is hypothesized to enhance the early growth of rice, rice physiology, grain yield, and grain quality. The present study was carried out to determine the role of Qiangdi 863 nano synergid on the growth and yield of one Chinese rice variety Zhongzao 39 and four Pakistani rice varieties KSK 133, KS 282, Super basmati, and PK 1121 aromatic. Field experiments were conducted during the years 2017, 2018, and 2019. Appling nano-treated water enhanced the germination rate along with radicle and plumule lengths and improved the photosynthesis rate, SPAD value in rice leaves for all ve varieties. The results showed that nano-treated water improved the anti-oxidant Catalase activity (CAT), Superoxide Dismutase (SOD) Peroxide (POD) production, declined Malondialdehyde (MDA content), enhanced endogenous Salicylic acid (SA), Jasmonates (JA) Brassinosteroids (BR) hormonal concentration and yield parameters of rice in 2017, 18 and 19 years of data. These ndings indicate that nano synergids can be effectively used for rice cultivation in different environmental conditions (China and Pakistan). This is the rst reported application of Qiangdi 863 nano synergid in Pakistani rice varieties with effective outcomes highlighting signicant results in KSK 133. water and in control. Speed of emergence (SE), percentage emergence (PE) and seed emergence energy percentage (SEEP) to evaluate the impact of nano treated water on the emergence of rice seed. According to the results 24 hours and 48 hours of nano water treatment were proved non-effective nano synergid treatment for rice germination, so did not proceed for further analysis. However, 72 hours of nano water treatment showed signicant results than control. Speed of emergence (SE) and was improved for KSK 133, super basmati, and PK 1121 Aromatic than KS 282 and Zhongzoa 39. Highest SEEP was observed in KSK 133 (40% in 2017; 55% in 2018; 45% in 2019) and lowest in Zhongzoa 39 (5% in 2017; 5% in 2018; 10% in 2019) pottery trays (NPTs), high energy nanomaterials showed better rice seed germination (Jun-rong et al. 2016). seasons 2017, 2018 and 19 years after NTW application most prominent seedling emergence were Brassinosteroids (BRs) are polyhydroxylated steroidal hormones or growth regulators, which are associated with different physiological functions e.g. seed germination, cell elongation, cell divisions, root development, also respond to various biotic and abiotic stress (De Vleesschauwer et al. 2012). BR signaling genes improved rice architecture and increased grain yield (Bajguz 2011). In a present piece of work, KSK 133 showed the highest amount of BR, lowest in Zhongzoa 39 (Table 5). In previous studies claimed that BR activated the specic transcription factors which can stimulate BR- targeted genes. BR regulated the antioxidant enzyme activities, SPAD value (photosynthetic capacity), chlorophyll contents to improve plant growth (Anwar et al. 2018). In conformity with earlier reports, it was observed that BR promotes growth and act as plant immunity against Blast fungal disease (Magnaporthe grisea) in rice (Yang et al. 2013). Jasmonic acid (JA) are originated from lipid, known as α-linolenic acid, which plays important role in rice defense system from microbial infection (bacterial and fungal)(Yang et al. 2013). The lipid-derived compounds help in plant biotic and biotic stress response or protection. (Schaller and Stintzi 2009). In a recent study, JA and nano synergid showed a stimulatory effect on rice immunity. cumulative of PC 1 57.55%. CAT, chlorophyll content and 1000 grain weight grouped with positive loading on the upper side of the biplot, these parameters a positive correlation among themselves. Dry weight, JA, SA and observed in the lower side positive and SOD were correlated in biplot. PCA PC 1 45.63% and PC 2 was 18.83%. PC 2 cumulative percentage was 64.46%. Dry MDA and 1000 grain weight on the right upper side of the biplot, these parameters had a positive correlation among themselves. CAT, SOD, POD chlorophyll and on the lower side of the biplot. year PCA 3 cumulative 63.49%. PC3 rst 46.75% and second 16.75%. The upper right side of the PC3 had a positive correlation in dry weight, chlorophyll, SPADE, CAT, SOD, MDA and grain weight. All JA, SA and were on the lower side of the biplot were also positively correlated.


Background
A rapid increase in population by 2050, demands increased crop production using advanced technologies (Kromdijk et al. 2016). In the past 70 years, 2250 rice varieties were introduced but still, there is the necessity to improve rice cultivation for the ourishing world population (Ahloowalia et al. 2004). FAO forecast for worldwide rice production in 2020 stands at 508.7 million tonnes, up to 1.6% from 2019. In 2021 global trade in rice is tentatively pegged at 47.6 million tonnes, 2.8 million tonnes from 2020 (OECD. 2019). In 2019, china's rice production was estimated at 209.6 million tonnes. Rice is an economically important viable cereal cash crop of Pakistan. In 2019, Pakistan's predictable rice production was 11.5 million tonnes (OECD. 2019). During the 2017-18 area under rice, cultivation was 2,899 thousand hectares and the production of rice was 7,442 thousand tonnes. (Chandio and Yuansheng 2018). Due to diversi ed domestic needs with limited resources (water and land) for a sustainable crop, adoption of the latest agricultural technologies may ensure the improved crop production per unit area (Asfaw et al. 2012).
For sustainable agriculture, nanotechnology has been observed as an advanced technology to cope with the globally increasing population (Ditta et al. 2015). The main aspect of nanotechnology is to endorse the biological metabolism of plants. Nano-treated germinated seed can stimulate crop growth and lead towards the enhanced yield and improved quality (Tuteja and Gill 2012). Though nanotechnology in agriculture is at the initial stage, it is hypothesized that it will cause revolutionary change in crop yield and quality improvement. micro-molecule clusters. The apparatus was dipped in water to absorb and emit electromagnetic waves in speci c energies (Pudake et al. 2019). They carried a mass of kinetic energy which was 30% greater than regular water. Nano-863 has been an agricultural high-tech invention and most widely used in China (Liu et al. 2007a).
Because of acclaimed reports, the qualitative nanotechnology applications on rice were noticed. Agriculturally no reporting about nanomaterial treated water has been done on Oryza sativa. Taking into consideration, the present study was designed to examine the effects of Qiangdi nano-863 nano synergids on O. sativa. For the proposed study ve varieties; one from China and four from and Pakistan were selected.

Plant material and growth conditions
In the experiment cultivated rice (Oryza sativa L. Nano synergid treated water (NTW) and rice seed soaking For different treatments, nano-synergid treated water was prepared with a variation of the duration of soaking of the disc in water. Qiangdi nano-863 biological assistant growth apparatus (disc) was placed in a plastic bucket with 20 L water for 24 hours (one day), 48 hours (two days), and 72 hours (three days) to produce nano-treated water. Rice seed was pre-soaked in tap water for 24 hours, and then soaked in nanotreated water for 24 h and were germinated for 36 hours. In three years (2017, 2018, and 2019), each variety containing 100 rice seedlings were sowed for all the treatments in three replicate Water and fertilizer management Fertilizer application after the transplanting stage was the same as that of common rice production. Homologous nano-treated water was used to irrigate the rice seedlings originated from nano-treated seeds. The recommended doses of fertilizer NPK were 53 kg N, 16 kg P, and 33 kg K ha − 1 for rice.
Germination, Seedling Growth, and dry biomass Germination of seed was documented daily according to AOSA (association o cial seed analysis) till it became persistent. After compiling data, speed of germination (SG), nal germination percent (FGP %) and germination energy percentage (GE %) was calculated using the following formulae. Ten random seedlings/ treatment were selected for measuring the dry biomass. Shoot and root dry weights (10 seedlings) were recorded after oven drying at 70 °C for 24 hours in a drying oven (Islam et al. 2012).
Chlorophyll content/mg g −1 Fw Chlorophyll was extracted from 0.2 g of fresh leaves soaking in 25 ml of acetone and alcohol (1:1) for 24 h in the dark at room temperature.
The absorbance of the extract was measured at 663, 645, and 470 nm by using a UV-VIS spectrophotometer (UV-2600, Shimadzu, Japan) to estimate chlorophyll a, chlorophyll b, carotenoids contents and total chlorophyll content (Marschall and Proctor 2004 The activity of SOD was determined by the method described by Zheng et al. (2016). This activity is measured through inhibited photoreduction of nitro-blue tetrazolium (NBT). The reaction mixture of SOD contained 25 mmol sodium phosphate buffer (pH 7.8), 13 mmol methionine, 2 µmol ribo avin, 10 µmol EDTA-Na 2 , 75 µmol NBT, and 0.1 ml leaf extract. The total quantity of reaction mixture was 3 ml. The test tube containing reaction solutions was irrigated with light ( uorescent lamps 300 µmol m − 2 s − 1 ) for 20 min and the activity was measured at 560 nm wavelength.

Peroxidase (POD) U g-1 FW
The POD activity was based on the determination of guaiacol oxidation at 470 nm by H 2 O 2 and was expressed as U g-1 FW. The change in absorbance at 470 nm was recorded for every 20 s by spectrophotometer. One unit of POD activity is the amount of enzyme that will cause the decomposition of 1 µg substrate at 470 nm (HITACHI U-3900) for 1 min in 1 g fresh sample at 37 °C Zheng et al. (2016).

Malondialdehyde (MDA)
The MDA content level was determined by the method of enzyme extracted solution (2 ml) was added in 1 ml 20% (v/v) trichloroacetic acid and 0.5 ml (v/v) thiobarbituric acid (Chun and WANG 2003). The mixture was heated in a pre-heated water bath at 95℃ for 20 min and cooled at room temperature. The solution was centrifuged at 10,000 rpm × g for 10 min after cooling. The lipid peroxidation absorbance was measured at 450, 532 and 600 nm by spectrophotometer (UV-VS Spectrophotometer-2600 Shimadzu), its content was expressed as µmol g − 1 FW.
Effect of nano-treated water on seedling emergence characteristics Seed germination is a fast activity in the plant life cycle and mainly tests offer numerous bene ts like ease, sensitivity, low cost and suitability for unstable chemicals or samples (Wang et al. 2005). In this study, germination was observed for 24 hours (one day), 48 hours (two days) and 72 hours (three days) nano treated water and in control. Speed of emergence (SE), percentage emergence (PE) and seed emergence energy percentage (SEEP) were considered to evaluate the impact of nano treated water on the emergence of rice seed. According to the results 24 hours and 48 hours of nano water treatment were proved non-effective nano synergid treatment for rice germination, so did not proceed for further analysis. However, 72 hours of nano water treatment showed signi cant results than control. Speed of emergence (SE) and was improved for KSK 133, super basmati, and PK 1121 Aromatic than KS 282 and Zhongzoa 39.  Table 2). According to the present experimental observation, a nano synergid is a good tool for the enhancement of germination and growth.   Effect of nanotechnology on rice seedlings (root and shoot lengths) before transplanting Nanotechnologies or nano-materials are a double-edged weapon because they have both bene cial and negative consequences as well (Service 2004). Therefore, crop treating with nano-material, the speci c nano synergids, or nanotechnologies with suitable energies should be selected to reduce negative effects. Nano synergid cannot penetrate plant cells; nano synergid had speci c energies which only took part in the breakage of water molecules. So, these energies were destroyed after the formation of activated water and reduced the chances of nanotoxicity effects on plant cells (He 2005). There was no recent data based on nano synergid so the present study considers some nanomaterial examples. Recent data collection showed whole plant lengths, root, shoot length was signi cantly increased due to nano synergid treated water in all rice varieties, respectively ( Fig. 2: Table 4). Earlier studies revealed that nano fertilizers may have a synergistic effect for improved nutrient uptake by plant cells, resulted in optimal growth (Morteza et al. 2013).  Chlorophyll Content (Chl a, b, Carotenoids and total chlorophyll) In the present study, we speculated that chlorophyll a, b, and total chlorophyll content enhanced by nano-treated water. It can be closely related to photochemical reaction activity. The effect of nano-TiO 2 has experimented on photosynthetic rate, showed improved photochemical reaction activity like absorbance of light, the transformation of light energy to electron energy, photophosphorylation e cacy and oxygen progression (Hong et al. 2005). Total chlorophyll content (CHL a, b, carotenoids) decreased with control and increased in NTW observed in different rice varieties (Table 5). Though nano-materials and nanotechnologies make a positive effect on the plant seed germination and growth, should address some serious challenges like nanomaterial reaction lower the photosynthetic activity, phytotoxicity (Nair et al. 2010).   (Table 5) Salicylic acid (SA) is an important phenolic compound present in plants at various levels e.g. rice contains high basal SA levels (5000-30,000 ng g − 1 fresh weight). SA is produced from benzoic acid. SA is present in leaves as the free acid. In rice maintain a high level of SA because of free SA in leaves and shoot and lesser in roots (Hu et al. 2017). SA helps in control the redox reactions, protects from oxidative stress and biotic and abiotic stress as well (Hu et al. 2017). The present study increased antioxidant, photosynthetic activity, and increased level of endogenous SA in rice leaves (Table 5).
Endogenous SA displays a vital antioxidant role in defending rice from oxidative stress. So a high amount of SA can directly be related to trigging antioxidant responses, modulate redox balance and scavenge ROS (Grant and Loake 2000). In a recent study increased SA showed in The cross-talk of plant hormones is the best way to respond to plant stress. SA and JA are resistant factors and BR responsible for aboveground plant growth. So, the present study stated that endogenous hormones play an important in growth, the developmental process, and plant immunity (Fig. 7). The same observation expressed in previous studies like BR and JA pathways have involved a balance between growth and defense, SA controls early defense gene expressions and JA tempts late defense based gene expressions (He et al. 2017).

Yield of rice
The nano synergid application had a signi cant impact on yield -attributing characters i.e., Plant height (cm), Remaining biomass (R.B), Branch weight without seeds (g), Panicle weight (g), No of Panicle, Total no of seeds per panicle, Filled grain per panicle, un lled grain per panicle and 1000 grain weight as compared to the control (Table 6). In previous studies, root shoots and grains were improved by ZnO nanoparticles which act as nano fertilizer (Bala et al. 2019) same observations were made in recent studies. The yield parameters were signi cantly higher with nano synergid in KSK 133. Earlier studies showed that seed primed by Qiangdi nano-863 can achieve good yield in japonica rice (Liu et al. 2007a). As pointed out previously, nano synergid enhanced grain yield in rice, is already used in agriculture due to their lack of toxicity,

Conclusion
Nano synergid Qiangdi 863 has a great potential for application in cash crops, but there is still a long way down to reach the eld. It is impossible to know all the details about how Nano synergid work in a particular crop but need to start the eld experiments solving some problems like mounting the production and avoiding hazardous and toxic materials. The present study deals with nano synergid Qiangdi 863 in a eld experiment and they exhibited prolong effective nutrient supply; involve all the steps of the crop cycle; from sowing to transplanting and harvest. The rst time reported the potential of Qiangdi 863 nano synergid treated water to improve yield e ciency of Pakistani varieties (KS 133, Super basmati, KS 282 and 1121 aromatic). These efforts led to nano synergid based design and development. Nanotechnologybased agriculture could be more effective than fertilizer counterparts.
Nano synergid emits electromagnetic waves that generated high energy (resonance) between water molecules. The nano synergid treated water enhanced the light absorption at a speci c wavelength that changes the water molecules' structure and energy. This alteration of water structure and energy is also called activated water. Activated water absorbed by the seed and it enhanced amylase activity, continuously strikes the cell for germination. The cell energy is activated and their function is stimulated which enhances the metabolism of rice seedling.
Nano synergid treated water enters into an oxidative enzyme system like SOD is a main superoxide scavenger exchange negatively charged oxygen molecule − O 2 into H 2 O 2 , Catalase (CAT) and Peroxidase (POD) transform H 2 O 2 into water and positively charged oxygen molecule.
These oxidative enzymes reduce the toxicity of reactive oxygen species (ROS) in kerb cycle, citric acid cycle in cytosol and mitochondria.
Malondialdehyde (MDA) is polyunsaturated fatty acids in the membrane that undergo oxidation by an accumulation of free oxygen radicals.
MDA was negatively correlated with the activities of ROS scavenging enzymes. So, oxidative enzymes lower the MDA content in rice. The rice performance is improved and their immune competencies are enhanced. Plant endogenous hormones Jasmonates (JA), Salicylic acid (SA) and Brassinosteroids (BR) also play an important role in growth, development and rice immunity (protect from biotic and abiotic stress). Nano synergid treated water will induce a higher germination rate, improved vegetative growth, increase no of productive panicles and a good harvest can be achieved (Fig. 7).      Qiangdi nano-863 nano synergid release electromagnetic waves which break the water macro-molecules into micro-molecules. Micromolecules of water enter into seed activates the hormone (GA) to amylase and speed up the germination process. Oxidative enzymes activation (SOD, CAT) and riddance of reactive oxidation species (ROS) lower the production of MDA and maintain the redox reactions in different subcellular structures. H2O2 is generated in normal metabolism via the different organelle electron transport chain in mitochondria, chloroplasts PS-1 and PS II and cytosol. SA and JA also helped in oxidative response and rice immunity. BR promotes growth and antioxidant activity. Rice plant will have faster germination, establishing root system, enhanced tillers, owering and full lled grains.