Effects of Decreasing Hill Number per Unit Area Combined with Increasing Seedling Number per Hill on Grain Quality in Hybrid Rice

Abstract

Hill number per unit area and seedling number per hill are foundational agrotechnical factors shaping the growth and development of rice plants. This study aimed to determine the effects of decreasing the hill number per unit area combined with increasing the seedling number per hill on grain quality in hybrid rice. Field experiments were performed in Hengyang, Hunan Province, China, in 2022 and 2023 using the hybrid rice variety Huazheyou 261, as well as in Liuyang, Hunan Province, China, in 2023 using the hybrid rice varieties Yueyou 2646 and Zhenliangyouyuzhan. Treatments (combining hill number per unit area and seedling number per hill) encompassed a combination of 24 hills per m² and one seedling per hill (H24S1) and a combination of 14 hills per m² and three seedlings per hill (H14S3) in Hengyang, as well as a combination of 28 hills per m² and two seedlings per hill (H28S2) and a combination of 14 hills per m² and four seedlings per hill (H14S4) in Liuyang. There were no significant differences in the leaf area index at the heading stage, as well as no significant differences in canopy light transmittance during the grain-filling period between H24S1 and H14S3 in Hengyang, or between H28S2 and H14S4 in Liuyang. The differences in grain quality traits, including milling traits (brown, milled, and head rice rate), appearance traits (rice length, rice length-width ratio, chalky grain rate, and chalkiness degree), amylose and protein content, and pasting characteristics were also not significant between H24S1 and H14S3 in Hengyang, nor between H28S2 and H14S4 in Liuyang. This study indicates that the grain quality in hybrid rice is unaffected by decreasing the hill number per unit area integrated with increasing the seedling number per hill.

1. Introduction

Rice is grown in over 100 countries that produce more than 715 million tons of paddy rice or 480 million tons of milled rice annually [1]. Rice is one of the most widely consumed staple foods around the world, providing 21% of energy and 15% of protein for more than half of the global population [2]. China is the largest rice producing and consuming country in the world [3], contributing approximately 30% of the global rice production and 28% of the global rice consumption [4].

The development of hybrid rice plays a vital role in increasing rice production and ensuring rice self-sufficiency in China [5]. As living standards improve, consumers are becoming more critical of the quality of rice. Consequently, the focus of rice production is shifting from quantity to quality [6], driving the development of high-quality hybrid rice varieties [7,8]. However, the grain quality of rice is not only determined by variety but also by agrotechnical factors [9].

Hill number per unit area and seedling number per hill are foundational agrotechnical factors shaping rice plant growth and development. In recent years, large-scale farming has rapidly developed in China [10], with rice farmers favoring low hill number per unit area, as it increases seedling transplant efficiency. Additionally, to avoid possible yield losses due to an insufficient panicle number produced by a lower hill number per unit area, large-scale rice farmers typically increase seedling number per hill.

There has been a study determining the effects of decreasing the hill number per unit area combined with increasing the seedling number per hill on grain quality in hybrid rice [11]. The results of this study demonstrated that decreasing the hill number per unit area while increasing the seedling number per hill optimized canopy structure and light utilization, consequently improving rice grain quality. However, this study was performed under artificial shading conditions. We hypothesized that the results obtained under artificial shading conditions may not be applicable in natural settings.

In this study, field experiments were conducted to determine the grain quality traits of hybrid rice varieties under different combinations of hill number per unit area and seedling number per hill under natural conditions. The objective of this study was to determine the effects of decreasing hill number per unit area combined with increasing seedling number per hill on grain quality in hybrid rice.

2. Materials and Methods

2.1. Sites and Soils

Field experiments were performed in Hengyang (26°53′ N, 112°28′ E), Hunan Province, China, in 2022 and 2023, as well as in Liuyang (28°09′ N, 113°37′ E), Hunan Province, China, in 2023. The experimental field at Hengyang had purple sandy soil with the following chemical properties: pH = 6.03, organic matter = 30.2 g kg⁻¹, available N = 127 mg kg⁻¹, available P = 14.1 mg kg⁻¹, and available K = 164 mg kg⁻¹. The experimental field at Liuyang had clay soil with the following chemical properties: pH = 5.27, organic matter = 45.1 g kg⁻¹, available N = 182 mg kg⁻¹, available P = 32.9 mg kg⁻¹, and available K = 180 mg kg⁻¹.

2.2. Experimental Design and Crop Management

The experiment conducted in Hengyang used the hybrid rice variety Huazheyou 261 (Huazhe 2A × 16T-261). Two treatments (different combinations of hill number per unit area and seedling number per hill) were utilized in the experiment, encompassing a combination of 24 hills per m² and one seedling per hill (H24S1) and a combination of 14 hills per m² and three seedlings per hill (H14S3) (Figure 1A,B). The hill spacing was 30 cm × 14 cm and 30 cm × 24 cm for H24S1 and H14S3, respectively. The experiment was arranged in a randomized block design with four replicates yearly. The plot size was 50 m².

The experiment in Liuyang employed two hybrid rice varieties: Yueliangyou 2646 (Huayue 468S × Huahui 2646) and Zhenliangyouyuzhan (Longzhen 36S × Yuzhan). Two treatments (different combinations of hill number per unit area and seedling number per hill) were utilized in the experiment, encompassing a combination of 28 hills per m² and two seedlings per hill (H28S2) and a combination of 14 hills per m² and four seedlings per hill (H14S4) (Figure 1C,D). The hill spacing was 30 cm × 12 cm and 30 cm × 24 cm for H28S2 and H14S4, respectively. The experiment was arranged in a randomized block design with three replicates for each variety. The plot size was 30 m².

The seeds were coated by a commercial coating regent, soaked in tap water at room temperature for 24 h, and then incubated at 35 °C for 12 h for germination. The germinated seeds were sown on the 8th of May, 2022 and 2023 in Hengyang, and on the 9th of May, 2023 in Liuyang. The seedlings were transplanted on the 7th of June, 2022 and 2023 in Hengyang, and on the 4th of June, 2023 in Liuyang. Chemical fertilizers were applied at rates of 180 kg of N ha⁻¹, 90 kg of P₂O₅ ha⁻¹, and 180 kg of K₂O ha⁻¹ in Hengyang and at rates of 150 kg of N ha⁻¹, 75 kg of P₂O₅ ha⁻¹, and 150 kg of K₂O ha⁻¹ in Liuyang. The N fertilizer was divided into three applications: 50% one day before transplanting, 30% seven days after transplanting, and 20% at the panicle initiation stage. All P fertilizer was applied one day prior to transplanting. The K fertilizer was divided into two applications: 50% one day before transplanting and 50% at the panicle initiation stage. A water depth of 5–10 cm was maintained in each plot from transplanting to seven days before maturity, at which point the plot was drained for harvesting. Diseases, insects, and weeds were controlled using standard agrochemicals.

2.3. Measurements

The daily mean temperature and the solar radiation during the grain-filling period (from heading to maturity) were documented using a Vantage Pro2 weather station (Davis Instruments Corp., Hayward, CA, USA) near the experimental field. At the heading stage, ten hills of rice plants were sampled to measure the leaf area using a LI-3000C leaf area meter (Li-Cor Inc., Lincoln, NE, USA). The leaf area index was calculated by dividing the leaf area by the plant coverage area. At the heading and maturity stages, photosynthetically active radiation (PAR) at the bottom of the canopy and total incident solar radiation were measured between 11:00–13:00, using a SunScan canopy analysis system (Delta-T Devices Ltd., Cambridge, UK). Canopy light transmittance at each growth stage was computed as the percentage of the PAR at the bottom of the canopy relative to the total incident solar radiation. Canopy light transmittance during the grain-filling period was defined as the average of canopy light transmittance at the heading and maturity stages.

At maturity, approximately 500 g of rough rice grains were sampled from each plot. The sampled rough rice grains were sun-dried and stored at room temperature for three months before quality analysis. A weight of 100 g of rough rice grains was de-hulled and polished utilizing a JGMJ8098 milling machine (Jiading Cereals and Oils Instrument Co., Ltd., Shanghai, China). Brown rice rate, milled rice rate, and head rice rate were calculated according to the rough rice weight. A weight of 10 g of milled rice grains was used to determine the rice length, rice length-width ratio, chalky grain rate, and chalkiness degree, using an MRS-9600TFU2L scanner (Zhongjing Technology Co., Ltd., Shanghai, China) and a SC-E image analysis software (Wanshen Detection Technology Co., Ltd., Hangzhou, China).

A weight of 10 g of head rice grains was ground into flour with an YS-02 high-speed blender (Yanshan Zhengde Machinery Equipment Co., Ltd., Beijing, China) and passed through a 100-mesh sieve for characterizing amylose and protein content and pasting properties. The amylose content was determined using the iodine colorimetric method [12]. The protein content was determined by multiplying the nitrogen content by a conversion factor of 5.95 [13], in which the nitrogen content was identified using a Skalar SAN Plus segmented flow analyzer (Skalar Inc., Breda, The Netherlands). The pasting properties (peak, trough, breakdown, final, setback, consistency viscosities, and paste temperature) of milled rice flour were identified using an RVA-Super 4 rapid viscosity analyzer (Newport Scientific Pty Ltd., Warriewood, NSW, Australia) following the standard procedure.

2.4. Statistical Analysis

The data from Hengyang and Liuyang were independently analyzed using the analysis of variance (ANOVA) in Statistix 8.0 (Analytical Software Inc., Tallahassee, FL, USA). The statistical model of the ANOVA included replicate, treatment, year, and the interaction between treatment and year for the Hengyang site and included replicate, treatment, variety, and the interaction between treatment and variety for the Liuyang site.

3. Results

3.1. Temperature and Solar Radiation

The average daily mean temperature during the grain-filling period was 29.8 °C in 2022 and 28.7 °C in 2023 for Huazheyou 261 in Hengyang, and was 27.5 °C and 26.2 °C in 2023 for Yueliangyou 2646 and Zhenliangyouyuzhan in Liuyang, respectively (Figure 2A,B). The average daily solar radiation during the grain-filling period was 20.3 MJ m⁻² in 2022 and 17.2 MJ m⁻² in 2023 for Huazheyou 261 in Hengyang, and 14.6 MJ m⁻² and 12.4 MJ m⁻² in 2023 for Yueliangyou 2646 and Zhenliangyouyuzhan in Liuyang, respectively (Figure 2C,D).

3.2. Leaf Area Index and Canopy Light Transmittance

The leaf area index at the heading and the canopy light transmittance during the grain-filling period were not significantly impacted by the treatment (different combinations of hill number per unit area and seedling number per hill), the year, or their interactions at the Hengyang site (Figure 3A,B). In Liuyang, the leaf area index at the heading stage was not significantly affected by the treatment or the interaction between the treatment and variety but was significantly affected by the variety (Figure 4A). Yueliangyou 2646 had an 11% higher leaf area index at the heading stage than Zhenliangyouyuzhan. The canopy light transmittance during the grain-filling period was not significantly impacted by the treatment, variety, or their interaction at the Liuyang site (Figure 4B).

3.3. Milling and Appearance Traits

None of the milling traits (encompassing brow rice rate, milled rice rate, and head rice rate) or appearance traits (including rice length, rice length-width ratio, chalky grain rate, and chalkiness degree) were significantly influenced by the treatment in Hengyang (

Table 1. Milling and appearance traits of the hybrid rice variety Huazheyou 261 grown under two treatments (combinations of hill number per unit area and seedling number per hill) in Hengyang in 2022 and 2023.

Treatment	Brown Rice Rate (%)	Milled Rice Rate (%)	Head Rice Rate (%)	Rice Length (mm)	Rice Length-Width Ratio	Chalky Grain Rate (%)	Chalkiness Degree (%)
2022
H24S1	80.6	70.2	64.7	6.15	3.56	11.6	3.14
H14S3	80.3	70.3	62.6	6.14	3.54	10.2	3.04
Mean	80.4	70.2	63.6	6.14	3.55	10.9	3.09
2023
H24S1	78.8	64.5	48.2	6.20	3.41	6.3	1.97
H14S3	78.9	64.6	52.1	6.23	3.45	5.4	1.92
Mean	78.8	64.5	50.1	6.21	3.43	5.9	1.94
Analysis of variance (F-value)
Treatment (T)	0.11 NS	0.06 NS	0.72 NS	0.17 NS	0.38 NS	3.27 NS	0.07 NS
Year (Y)	12.02 **	164.23 **	157.99 **	20.00 **	150.78 **	59.29 **	14.39 **
T × Y	0.19 NS	0.00 NS	7.88 *	1.91 NS	11.22 **	0.18 NS	0.01 NS

H24S1: a combination of 24 hills per m² and one seedling per hill; H14S3: a combination of 14 hills per m² and three seedlings per hill. ^NS denotes non-significance at the 0.05 probability level. * and ** denote significance at the 0.05 and 0.01 probability levels, respectively.

). However, all the milled and appearance traits were significantly impacted by the year. In particular, the milled rice rate and head rice rate were 8% and 21% lower in 2023 than in 2022, respectively. The chalky grain rate and chalkiness degree in 2023 were 46% and 37% higher than those in 2022, respectively. Significant interaction effects between treatment and year on milled and appearance traits were observed for head rice rate and rice length-width ratio.

The main impacts of the treatment on milled and appearance traits were not significant in Liuyang (

Table 2. Milling and appearance traits of the hybrid rice varieties Yueliangyou 2646 and Zhenliangyouyuzhan grown under two treatments (combinations of hill number per unit area and seedling number per hill) in Liuyang in 2023.

Treatment	Brown Rice Rate (%)	Milled Rice Rate (%)	Head Rice Rate (%)	Rice Length (mm)	Rice Length-Width Ratio	Chalky Grain Rate (%)	Chalkiness Degree (%)
Yueliangyou 2646
H28S2	79.0	67.5	60.2	6.33	3.40	5.1	1.62
H14S4	78.8	67.4	59.3	6.34	3.42	3.1	0.88
Mean	78.9	67.4	59.8	6.34	3.41	4.1	1.25
Zhenliangyouyuzhan
H28S2	80.6	67.5	58.0	6.81	3.44	4.7	1.14
H14S4	80.6	68.1	57.1	6.82	3.41	5.4	1.37
Mean	80.6	67.8	57.5	6.82	3.43	5.1	1.26
Analysis of variance (F-value)
Treatment (T)	0.80 NS	0.60 NS	0.39 NS	0.16 NS	0.07 NS	0.58 NS	0.52 NS
Year (Y)	104.35 **	2.01 NS	2.61 NS	531.98 **	0.62 NS	1.32 NS	0.00 NS
T × Y	0.48 NS	2.39 NS	0.00 NS	0.01 NS	2.22 NS	2.49 NS	1.92 NS

H28S2: a combination of 28 hills per m² and two seedlings per hill; H14S4: a combination of 14 hills per m² and four seedlings per hill. ^NS denotes non-significance at the 0.05 probability level. ** denotes significance at the 0.01 probability level.

). Significant effects of variety on milled and appearance traits were found for brown rice rate and rice length. Zhenliangyouyuzhan had a slightly (2%) higher brown rice rate and an 8% higher rice length than Yueliangyou 2646. None of the milled or appearance traits were significantly impacted by the interaction effect between treatment and variety.

3.4. Pasting Properties and Amylose and Protein Content

The pasting properties and amylose and protein content were not significantly altered by treatment in Hengyang (

Table 3. Pasting properties and amylose and protein content of milled rice flour of the hybrid rice variety Huazheyou 261 grown under two treatments (combinations of hill number per unit area and seedling number per hill) in Hengyang in 2022 and 2023.

Treatment	Viscosity (cP)						Pasting Temperature (°C)	Amylose Content (%)	Protein Content (%)
	Peak	Trough	Breakdown	Final	Setback	Consistency
2022
H24S1	3687	1747	1941	2854	−834	1107	76.6	14.8	7.32
H14S3	3747	1782	1965	2861	−885	1079	76.4	15.1	7.47
Mean	3717	1764	1953	2857	−860	1093	76.5	14.9	7.40
2023
H24S1	3622	1820	1803	2848	−774	1028	76.2	12.7	6.42
H14S3	3431	1741	1690	2778	−653	1037	76.4	12.9	7.01
Mean	3527	1781	1746	2813	−714	1033	76.3	12.8	6.71
Analysis of variance (F-value)
Treatment (T)	0.63 NS	0.26 NS	0.37 NS	0.55 NS	0.32 NS	0.41 NS	0.00 NS	1.56 NS	7.44 NS
Year (Y)	5.20 *	0.15 NS	7.98 *	1.12 NS	5.52 *	16.13 **	0.61 NS	103.73 **	25.41 **
T × Y	2.25 NS	1.81 NS	0.87 NS	0.86 NS	1.93 NS	1.44 NS	1.09 NS	0.29 NS	2.58 NS

H24S1: a combination of 24 hills per m² and one seedling per hill; H14S3: a combination of 14 hills per m² and three seedlings per hill. ^NS denotes non-significance at the 0.05 probability level. * and ** denote significance at the 0.05 and 0.01 probability levels, respectively.

); however, these characteristics, encompassing peak, breakdown, setback, and consistency viscosities as well as amylose and protein content, were significantly influenced by year. The peak, breakdown, and consistency viscosities as well as the amylose and protein content were 5–14% lower in 2023 than in 2022, while the setback viscosity was 17% higher in 2023 compared to 2022. The interaction effects between the treatment and year on the pasting properties and amylose and protein content were not significant.

The main impacts of the treatment on all the pasting properties and amylose and protein content were not significant in Liuyang (

Table 4. Pasting properties and amylose and protein content of milled rice flour of hybrid rice varieties Yueliangyou 2646 and Zhenliangyouyuzhan grown under two treatments (combinations of hill number per unit area and seedling number per hill) in Liuyang in 2023.

Treatment	Viscosity (cP)						Pasting Temperature (°C)	Amylose Content (%)	Protein Content (%)
	Peak	Trough	Breakdown	Final	Setback	Consistency
Yueliangyou 2646
H28S2	3565	1897	1668	3086	−478	1189	75.8	14.0	6.89
H14S4	3770	1988	1781	3157	−613	1169	75.5	14.1	6.64
Mean	3667	1943	1725	3122	−546	1179	75.6	14.1	6.77
Zhenliangyouyuzhan
H28S2	4152	1843	2309	2861	−1291	1018	81.5	11.6	7.25
H14S4	4056	1840	2215	2862	−1193	1022	81.6	11.7	7.43
Mean	4104	1842	2262	2862	−1242	1020	81.5	11.6	7.34
Analysis of variance (F-value)
Treatment (T)	1.34 NS	1.15 NS	0.02 NS	0.82 NS	0.09 NS	1.81 NS	0.02 NS	0.23 NS	0.09 NS
Year (Y)	86.01 **	5.96 NS	66.88 **	42.62 **	126.28 **	660.62 **	273.07 **	129.60 **	27.01 **
T × Y	10.22 *	1.29 NS	2.48 NS	0.76 NS	3.49 NS	3.97 NS	0.18 NS	0.00 NS	3.81 NS

H28S2: a combination of 28 hills per m² and two seedlings per hill; H14S4: a combination of 14 hills per m² and four seedlings per hill. ^NS denotes non-significance at the 0.05 probability level. * and ** denote significance at the 0.05 and 0.01 probability levels, respectively.

). However, the pasting properties (outside of the peak viscosity) and amylose and protein content were significantly impacted by variety. The final, setback, and consistency viscosities and the amylose content were 8%, 127%, 13%, and 17% lower in Zhengliangyouyuzhan than in Yueliangyou 2646, respectively. In contrast, the peak and breakdown viscosities, the pasting temperature, and the protein content were 12%, 31%, 8%, and 8% higher in Zhengliangyouyuzhan than in Yueliangyou 2646, respectively. All the pasting properties except the peak viscosity, as well as the amylose and protein content, were not significantly influenced by the interaction between treatment and variety.

4. Discussion

Both the experiments in Hengyang and Liuyang demonstrated that decreasing the hill number per unit area combined with increasing the seedling number per hill did not impact the leaf area index at the heading, the canopy light transmittance during the grain-filling period, or the grain quality traits in hybrid rice. These findings are not aligned with the previous study by Deng et al. [11], which observed that decreasing the hill number per unit area integrated with increasing the seedling number per hill enhanced the canopy light transmittance, increased the leaf net photosynthetic rates, and improved the grain quality (increased the head rice rate and decreased the chalky grain rate and chalkiness degree) in rice. This alteration could be partially attributed to different experimental conditions between this study and the study conducted by Deng et al. [11]. The present study was conducted under natural conditions, whereas the study of Deng et al. [11] was performed under artificial shading conditions. The different results between the two studies support our hypothesis and also suggest that there may be overlapping effects between shading and the integration of hill number per unit area and seedling number per hill on grain quality. This underscores the requirement for further investigations to determine the impact of combinations of hill number per unit area and seedling number per hill on rice grain quality between various light conditions.

The experiment in Hengyang demonstrated that a lower milling quality (lower brown, milled, and head rice rates) but a higher appearance quality (lower chalky grain rate and chalkiness degree) took place in 2023 relative to 2022. A lower daily temperature during the grain-filling period could account for the higher appearance quality but could not explain the lower milling quality. The lower milling quality observed in 2023 might be attributable to a lower daily solar radiation during the grain-filling period because a lower daily solar radiation may reduce plant photosynthetic capacity and result in insufficient grain development and reduced milling quality in rice [14].

Additionally, the experiment in Hengyang also indicated that there were differences in eating, cooking, and nutritional quality between 2022 and 2023. Notably, both the amylose and the protein content were lower in 2023 than in 2022. The lower amylose content in 2023 relative to 2022 might not be due to a lower daily mean temperature but potentially due to a lower daily solar radiation during the grain-filling period, given that (1) a lower temperature during the ripening period may increase the activity of granule-bound starch synthase (an enzyme responsible for amylose biosynthesis), leading to an increase in the amylose content in rice [15]; and (2) a lower daily solar radiation during grain filling may enhance the activity of a soluble starch branching enzyme causing a decrease in the amylose content [14]. The reduced protein content in 2023 compared to 2022 could be explained by a lower daily mean temperature during the grain-filling period. This mirrors the study of Li et al. [16], which observed a lower protein content in rice grown under natural temperature conditions compared to a high temperature and identified that this difference in protein content might be partially attributable to alterations in the activities of the enzymes linked to protein synthesis, including glutamine synthetase, glutamic oxalo-acetic transaminase, and glutamate pyruvate transaminase. The findings of this study highlight the need to better understand the quality stability across various environments and the associated eco-physiological processes in hybrid rice.

The experiment performed in Liuyang indicated a significant difference in the leaf area index at heading between the two varieties, but this difference in the leaf area index did not result in a significant difference in the canopy light transmittance during the grain-filling period. This could be explained by the fact that canopy light transmittance is governed not only by the leaf area index but also by other canopy structural traits like leaf angle [17]. The experiment conducted in Liuyang also indicated that there were differences in grain quality traits between the two varieties. Zhenliangyouyuzhan had a higher brown rice rate, rice length, and protein content but a lower amylose content relative to Yueliangyou 2646.

Moreover, significant disparities were observed in the pasting properties between the two varieties in Liuyang. It has been well-documented that pasting properties are closely linked to amylose content in rice [18,19]. In contrast to the general belief that the lower the amylose content, the lower the pasting temperature [19], Zhenliangyouyuzhan exhibited a lower amylose content but a higher pasting temperature than Yueliangyou 2646 in this study. It has been demonstrated that there was a positive relationship between the pasting temperature and the protein content in cereals like sorghum [20], and this relationship could be linked to the fact that more proteins surround the starch granule, making starch gelatinization more challenging [21]. Consequently, in this study, the higher protein content might be responsible for the higher pasting temperature in Zhenliangyouyuzhan compared to Yueliangyou 2646.

There are two limitations that need to be acknowledged. First, the experiment in Liuyang was conducted in only one year. However, this limitation does not affect the conclusion of this study, because the results of the experiment in Liuyang are consistent with those of the experiment in Hengyang over two years. Second, only the protein content was determined in terms of nutritional quality. Besides the protein content, the mineral content is also an important aspect of nutritional quality in rice [22]. The deficiency of micronutrients such as zinc and iron, known as hidden hunger, is a serious global health problem that is widespread in developing countries [23]. This highlights that further investigations are required to determine the effects of decreasing hill number per unit area combined with increasing seedling number on the micronutrient content in grains of hybrid rice.

5. Conclusions

Decreasing the hill number per unit area combined with increasing the seedling number per hill does not alter the milling quality (brown, milled, and head rice rate), appearance quality (rice length, rice length-width ratio, chalky grain rate, and chalkiness degree), cooking and eating quality (pasting properties and amylose content), and nutritional quality (protein) in hybrid rice.