Improving grain yield and quality by enhancing accumulation of zinc in rice under subtropical condition

One of the primary abiotic factors limiting rice production is zinc (Zn) deficiency. Effective management of Zn in rice soils is crucial, as rice is a staple crop for many nations. To address this issue, a pot trial was conducted at the net house of the Department of Agronomy, Bangladesh Agricultural University, Mymensingh, from December 2019 to May 2020. The trail aimed to assess the impact of zinc on yield performance and grain zinc content of rice. The experiment employed a completely randomized design (CRD) with three replications, involving the applications of 6 different rates of Zn to 3 distinct varieties of boro rice. The result revealed that BRRI dhan28 exhibited superior yield and yield - related traits when treated with 12 kg Zn ha - 1 . Meanwhile, the application of 10 kg Zn ha - 1 resulted in the highest Zn content in the grain of BRRI dhan74, considering the quantity of Zn accumulated by the grain. Based on the findings of the study, it can be concluded that applying 12 kg of Zn ha - 1 is likely the optimal Zn management strategy to achieve outstanding performance in the rice cultivar BRRI dhan28. Furthermore, applying 10 kg of Zn ha - 1 may enhance the Zn content of the grain in BRRI dhan74.


Introduction
Rice stands out as one of the world's most widely cultivated grains, constituting a substantial share of cereal consumption and production (1). Remarkably, over 50% of the global population depends on rice as a staple food (2). In the realm of nutrition, food security and economics, no other cereal grain holds greater importance than rice. However, ensuring food security in the face of a burgeoning global population presents a formidable challenge (3). Consequently, the development of novel strategies and techniques will play a pivotal role in shaping the future of rice production (4). However, as rice cultivation takes place in an increasingly precarious environment, it faces additional challenges, including nutritional deficiencies, moisture stress, pests and diseases infections, as well as weed infestations, all of which hinder its growth and diminish production. With the rising utilization of macronutrient (NPK) fertilizers and the widespread cultivation of high-yielding rice varieties in recent years, several secondary micronutrients have been rapidly depleted from the soil, leading to shortages of these vital elements in various regions of the country (5).Micronutrient malnutrition is a global concern, and in recent years, regions practicing intensive agriculture is practiced have begun to prioritize addressing its deficiencies. Inadequate intake or deficiency of essential micronutrients disrupt plant growth by interfering with metabolic and biochemical processes, ultimately leading to reduced yield and quality (6). Significantly, malnutrition is a prevalent issue, particularly in impoverished nations like Bangladesh, where people consume primarily starchy staple foods that lacks essential micronutrients (7). Among micronutrient deficits, zinc insufficiency stands out as one of the most critical agricultural challenges worldwide. Despite being a vital microelement, the widespread shortage of zinc in both plants and human diets is cause for alarm (8,9). Due to the diverse physiological and biochemical functions that Zn serves in plants, even a minor deficiency can have detrimental effects on development, production and the Zn content in plant parts used for food. Zinc deficiency predominantly impact nations where cereals constitute a fundamental component of the diet, posing a humanitarian concern, particularly in regions where cultivation occurs in Zndeficient soils (10)(11)(12). Consequently, there has been a suggestion that increasing the Zn content in staple foods such as rice could potentially contribute to alleviating Zn deficiency (5). Numerous rice-growing regions world-wide have reported prevalent zinc inadequacy in their rice crops (13). In the early stages of development, insufficient Zn availability leads to leaf bronzing and a reduced number of tillers ,consequently retarding maturity and decreasing rice yield (14). Addressing Zn deficiency in rice through methods like fortification (adding Zn to food products during processing) or supplementation is neither costeffective nor a sustainable long-term solutions (15). However, biofortification presents a pragmatic and economical approach to enhance the bioavailability of zinc in the edible parts of plants and mitigate zinc nutritional deficits (16,17). The fact that rice lacks essential micronutrients like Zn underscores the critical importance of biofortification for this staple crop. Zinc biofortification in food crops can be achieved through either a genetic approach or agronomic method such as appropriate soil and plant fertilizer application (8). Agronomic biofortification refers to the deliberate use of mineral fertilizers to increase the content of a specific mineral in the edible part of crops, thereby enhancing the intake of the desired nutrient (18). This practice is also referred to as ferti-fortification (19). Agronomic biofortification enhances crops's ability to utilize and mobilize micronutrients, thereby promoting crop growth (9). To biofortify cereals with Zn, maintaining a sufficient quantity of available Zn in soil is essential. However, while higher zinc fertilizer rates may increase Zn uptake, this approach might not be costeffective as cultivation expenses rise. Zn fertilization not only elevates the nutritional value of these products for humans but also augments production and grain Zn content in cereal crops (20). Another strategy to improve Zn accessibility for plants in lowland environments involves selection of suitable Zn sources for soil application (21). The commonly utilized Zn source is ZnSO4, valued for its high dissolution rate and affordability. Additionally, Zn-EDTA (ethylene diamine tetra acetic acid) is recommended due to its effectiveness in enhancing Zn availability for the plant (7). Nonetheless, among various approaches to address Zn deficiency, Zn biofortification, aimed at increasing Zn content in rice grains, emerges as the most practical, enduring, and economical solution. While a majority of Zn fertilizer experiments and subsequent recommendations for rice have focused on rectifying fertilizer Zn insufficiency, there have been relatively few studies on enhancing zinc accumulation in rice through Zn application.
Given the preceding information, the current study aimed to assess the importance of zinc fertilization in augmenting zinc accumulation in rice grains.

Features of experimental location
The investigation took place at the net house of the Department of Agronomy, Bangladesh Agricultural University, Mymensingh, using 30 Litre pots with a diameter of 35 cm and filled with 25 kg soil. The study location is situated at latitude 24°42′55′′N, longitude 90°25′ 47′′E, and elevation 19m above sea level. The test location is within the Old Brahmaputra floodplain (AEZ-9) (22) and experiences a subtropical monsoon climate with a humid environment. Prior to the experiment, composite topsoil samples (0-20 cm) were collected from the field for analysis. The physiochemical constituents of the soil that were analysed are presented in Table 1.

Experimental set up
The experimental treatment consisted of two components: six different rates of zinc viz., 0 kg ha -1 (Zn0), 8 kg ha -1 (Zn8), 10 kg ha -1 (Zn10), 12 kg ha -1 (Zn12), 14 kg ha -1 (Zn14), 16 kg ha -1 (Zn16), applied on 3 distinct boro rice varieties namely, BRRI dhan64, BRRI dhan74 and BRRI dhan28. Zn was applied as ZnSO4 and doses were converted from kg ha -1 to g pot -1 . With a total of 54(9×6) pots and 9(3×3) pots in each batch, the experiment was replicated 3 times using a completely randomized design (CRD). In each replication, nine pots were positioned next to each other with a distance of 10 to 25 cm in between them. Table 2 provides the evaluation % of the cultivars, along with some of their key qualities, in order to assess their adaptability to different attributes.

Preparation of pot and crop husbandry
Pots were placed within the net house, each containing 25 kg of dry soil, and sufficient water was added to attain an appropriate soil moisture level. Fertilizers, including urea, triple super phosphate (TSP), muriate of potash (MoP), and gypsum, were applied at rates of 1.5g, 0.75g, 0.95g and 0.45g per pot respectively. Zinc sulphate (ZnSO4) was applied according to the specifications for each treatment.
During the final pot preparation, all fertilizers except for urea were administered in full doses. Urea was divided into 3 equal portions and applied at 15-, 30-and 50-day intervals following transplantation. On January 12, 2020, seedlings specific to each cultivar, nurtured for forty days in a nursery bed, were transplanted into the pots containing 4cm of water. Additional water was supplied as needed after the initial ponded water subsided. Pond irrigation continued until 15 days before harvest. Weeds were occasionally observed during the growth period, particularly in the early stages and were manually removed. No significant insect infestations were noted during the growth phase, except for rice hispa which was managed by applying pesticides (Fenitrothion 50 EC) at the tillering stage.

Harvesting and data collection
Upon reaching maturity (90% ripened grain), the entire plant was cut at the ground level using a sickle. Each pot's harvested crop was packed and carefully marked separately. After sun drying of the plant materials,detailed records were made for yield contributing parameters, as well as for grain and straw yields.

Chemical analysis
Rice grains harvested from 5 plants pot -1 were thoroughly dried in oven at 65 °C. After dehusking, the dried grain samples were finely powdered using a ball mill. In a desiccator, the powdered grain was stored in zip-log polythene bags. Zn concentrations were determined by analyzing the grain powders. 200 mg of rice powder was precisely measured using a Mettler Toledo Analytical Balance, and placed into labelled digestion containers (a Velp block digester). Each digestion tube received 5 cc of nitric acid (70% RCI Premium Labscan). Nitric acid in the same volume was added to the tubes labelled as Certified Reference Material and Blanks (NMIJ CRM7501-a, Japan). Tubes were left overnight for pre digestion. Following the pre-digestion period, 2ml of 30% hydrogen peroxide was added to each vessel via pipette. Tubes were then left open for 15 minutes for outgassing. Subsequently, the vessels were placed in the block digester, and the temperature was gradually raised to 140 ˚C. A dense straw brown fume was developed inside the vessels. The digestion continued for 5 hrs until a clean white fume was visible. Finally, the block digester was switched off and left for cooling. The digested material was transferred into 50 mL centrifuge tube, ensuring the complete transfer by washing the digestion vessel with deionized water. Final volume was made adjusted to 50 mL using deionized water and the weight was recorded again to calculate dilution factor (w/w). The digested samples were subsequently analysed using an Atomic Absorption Spectrometer (AAS) at the Soil Resource Development Institute (SRDI) lab, Jamalpur to determine zinc concentration in rice grains. Finally, the rice grain zinc concentrations were reported in mg kg -1 .

Statistical analysis
The data recorded for various parameters were properly collated, tabulated and statistically analyzed. The analysis of variance was carried out using the MSTAT-C software tool. Duncan's Multiple Range Test was used to adjudge the mean differences between the treatments (23). Table 3 presents the analysis of variances for yield and yieldcontributing features. All the parameters were significantly influenced by the varietal effect. Except for PH (plant height), Zn fertilizer had a notable impact on all the parameters. The interaction between variety and Zn rate exerted a considerable impact on nearly all parameters, although it did not significantly affect the TGW (1000-grain weight). Variety also had a significant effect on the zinc content of rice grain. Additionally, Zn fertilization significantly influenced the Zn content of rice grain. Moreover, the interaction between variety and Zn fertilization demonstrated a substantial impact on Zn concentration in the rice grain.   (Table 4).

Zn fertilizer level's effect on yield and yield related traits
Significant differences in the yield and yield-related traits of rice were observed due to varying Zn fertilizer rates. Tiller production increased by 2.49% to 16 TGW varied between 21.05 g and 23.02 g, with the highest weight associated with Zn12 treatment, and the lowest with Zn10. GY significantly ranged between 25.68 g pot -1 and 31.23 g pot -1 , with a linear increase observed as zinc doses were increased up to 12 kg ha -1 . The maximum GY was achieved with Zn12 treatment, while Zn0 resulted in the lowest. Zn fertilization led to a 3.93% to 21.61% increase in grain yield compared to the control. Similarly, Zn12 treatment yielded the highest straw yield (SY) of 45.81 g pot -

1
, representing a 10.87% increase compared to the control's 41.32 g pot -1 . Biomass yield (BY) increased by 3.71% to 14.98% due to Zn fertilization, with the highest BY of 77.04 g pot -1 observed in Zn12 treatment, and the lowest of 67.00 g pot -1 from Zn0. Zn fertilization also contributed to a 4.08% to 13.35% increase in HI. The highest HI of 41.69% was recorded with Zn12, while the lowest of 36.78% was obtained from Zn0 (Table 5).

Zn accumulation
The zinc quantity present in rice grains was notably influenced by variety. Zn content in rice grains ranged from 25.53 mg kg -1 to 37.25 mg kg -1 , with the highest value observed in BRRI dhan74, which was statistically equivalent to BRRI dhan64, while the lowest value was recorded in BRRI dhan28 (Fig. 1). Zn accumulation in rice grains exhibited significant variations due to Zn fertilization. In comparison to the control, Zn fertilization led to a substantial increase in Zn accumulation, ranging from 16.39% to 42.76%. Specifically, Zn content in rice grains rose from 27.20 mg kg -1 in the control treatment to 38.83 mg kg -1 by Zn fertilization. An increase in Zn accumulation was observed up to 10 kg Zn ha -1 , but further increases in Zn dose resulted in diminished accumulation. Notably, Zn content saw a 42.76% increase with the application of Zn10, while only 16.39% increase was recorded with Zn16 (Fig. 2). The interaction between varieties and Zn fertilization was found to significantly influence Zn content in rice grain. Compared to the control treatment, the application of Zn fertilizer substantially raised Zn content in rice grains of different varieties by 5.00% to 45.79%. The finding revealed that the combination of BRRI dhan74 with Zn10 yielded the highest Zn content (45.22 mg kg -1 ) compared to other interactions. In contrast, BRRI dhan28 with Zn0 exhibited poor Zn accumulation performance, registering the lowest Zn content (21.38 mg kg -1 ) with this combination (Fig. 3).     (24). The optimization of Zn levels is essential to achieve increased yield and higher Zn concentration in rice grains.
The experimental finding indicates that cultivars exhibited varying performance across different Zn rates, with these differences being linked to yield and yield related characteristics. BRRI dhan28, in comparison to BRRI dhan64 and BRRI dhan74, demonstrated superior performance. The performance of a crop's variety is primarily influenced by its genetic makeup. This conclusion is supported by several studies (25)(26)(27).
Zinc fertilizer aims to enhance plant Zn absorption and elevate grain Zn content. Our current investigation demonstrates that the treatment of 12 kg Zn ha -1 yielded the highest values across all attributes, while the absence of zinc application yielded the lowest. This can likely be attributed to an ample supply of zinc, which may have improved the availability and absorption of other essential minerals, consequently enhancing crop performance. In contrast to the zero zinc treatment, it was observed that zinc concentrations of 1% and 2% significantly increased the height of rice plants (28). Numerous studies have shown that applying zinc to rice plants enhances traits such as TT, ET and GP (29)(30)(31). In this experiment, an initial increase in GY was observed up to 12 kg ha -1 Zn, followed by a decline with higher Zn rates, indicating that excessive Zn rates did not confer additional benefits in terms of GY.  While the 12 kg Zn ha -1 treatment resulted in the highest rice yield, it did not substantially differ from the 10 kg Zn ha -1 treatment. The crop's positive response to zinc application suggests that 12kg Zn ha -1 may be the optimal quantity for rice crops, as diminishing trend was observed beyond this point. Zn is essential, but an excess can be detrimental to plants by generating excessive reactive oxygen species (ROS), leading to oxidative damage (32). Using Zn fertilizer has been documented to potentially increase rice grain production by approximately 0.3%-13.0% up to a certain zinc level (33). The control group exhibited a lower rice yield, likely due to zinc deficiency, whereas the higher rice yield resulting from zinc application could be attributed to a synergy of yieldrelated factors, including ET, GP and WTG. The rise in chlorophyll content, leading to improved photosynthesis, the production of growth-promoting-compounds, metabolites, and enhanced plant development, may contribute to the increased productivity due to Zn fertilization (34). Zn treatment consistently promoted photosynthate translocation, contributing to higher GY and SY (35). Similarly, when BRRI dhan28 was treated with 12kg Zn ha -1 , this combination outperformed others in terms of PH, number of TT and ET, PL, number of GP, GY, SY, BY and HI. However, the lowest readings came from BRRI dhan64 with 0 kg Zn ha -1 . Conversely, the lowest readings were recorded for BRRI dhan64 with 0kg Zn ha -1 . The interaction between zinc fertilizer and rice variety was found to significantly affect the number of GP and GY (36,37).
The results of the trial regarding the Zn accumulation in rice grains reveal that BRRI dhan74 exhibited the highest Zn accumulation in its grains compared to other varieties. Additionally, the application of 10 kg Zn ha -1 contributed to an elevated grain Zn content. Similarly, the combination of BRRI dhan74 and 10 kg Zn ha -1 demonstrated the highest zinc content in rice grains. In contrast, while the cultivar BRRI dhan28 displayed a higher grain yield than BRRI dhan74, the Zn concentration in both rice varieties showed an inverse trend. This discrepancy might be attributed to the greater potential of BRRI dhan74 to translocate Zn to its reproductive part under the influence of 10kg Zn ha -1 . Previous research has indicated that soil application of 10kg Zn ha -1 enhanced rice grain Zn content and bioavailability (38). Furthermore, a pot experiment demonstrated that a gradual rise in Zn levels up to 15mg Zn kg -1 soil led to a significant enhancement in Zn content in brown rice (39).

Conclusion
In conclusion, BRRI dhan28 treated with 12 kg ha -1 of Zn demonstrated the most favorable outcomes in terms of yield and yield-related traits, while BRRI dhan74 with 10 kg ha -1 Zn, exhibited the highest Zn accumulation. To optimize both zinc content in the rice variety and achieve a satisfactory yield, this study recommends the application of zinc fertilizer at a rate of 12 kg ha -1 .