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Article

Comparative Analysis of Germination Traits and Gene Expression in Hybrid Progeny of Neo-Tetraploid Rice Under NaCl Stress Conditions

by
Peishan Huang
1,2,3,4,†,
Xinhui Xie
1,2,3,4,†,
Xiaoyu Cai
1,2,3,4,
Shihui Chen
1,2,3,4,
Yutong Zheng
1,2,3,4,
Zijuan Huang
1,2,3,4,
Muhammad Qasim Shahid
1,2,3,4,
Xiangdong Liu
1,2,3,4 and
Jinwen Wu
1,2,3,4,*
1
State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, South China Agricultural University, Guangzhou 510642, China
2
Guangdong Laboratory for Lingnan Modern Agriculture, South China Agricultural University, Guangzhou 510642, China
3
Guangdong Provincial Key Laboratory of Plant Molecular Breeding, Base Bank of Lingnan Rice Germplasm Resources, South China Agricultural University, Guangzhou 510642, China
4
College of Agriculture, South China Agricultural University, Guangzhou 510642, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agronomy 2025, 15(9), 2066; https://doi.org/10.3390/agronomy15092066
Submission received: 3 July 2025 / Revised: 11 August 2025 / Accepted: 25 August 2025 / Published: 27 August 2025
(This article belongs to the Special Issue Innovative Research on Rice Breeding and Genetics)
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Abstract

Neo-tetraploid rice is a highly fertile variety created from autotetraploid rice. It demonstrates stronger heterosis and produces stable hybrid progeny. However, there is insufficient data regarding abiotic stress in neo-tetraploid hybrid progeny, especially in relation to salt stress. Two hybrid progenies, high salt-resistance tetraploid rice hybrid progeny (HSRTH) and low salt-resistance tetraploid rice hybrid progeny (LSRTH), were generated by crossing the neo-tetraploid rice cultivars ‘Huaduo 3’ and ‘Huaduo 8’ with the autotetraploid rice Huanghuazhan-4x. Here, we assessed the germination characteristics and seedling growth of two neo-tetraploid hybrids at six NaCl concentrations: 0, 50, 100, 150, 200, and 250 mmol/L. HSRTH demonstrated a higher tolerance to salt stress, achieving a grain germination rate of 48.00 ± 2.63% compared to LSRTH, which reached only 5.00 ± 1.41% under a 250 mmol/L NaCl treatment. Cytological observations showed that the root tip differentiation zone and coleoptiles of HSRTH were less affected by NaCl stress treatment, resulting in fewer cortical cell abnormalities, decreased stele issues, and fewer rhizodermis cell problems, such as shrinkage. Gene expression analysis revealed nine genes that showed differential expression in HSRTH compared to LSRTH. Our study demonstrated that HSRTH showed strong salt stress tolerance, providing a basis for selecting salt-resistant rice germplasm and offering insights for developing salt-tolerant rice varieties using neo-tetraploid resources.

1. Introduction

Rice (Oryza sativa L.) is one of the world’s most critical staple food crops. Over half of the world’s population relies on rice as their primary source of dietary energy [1]. There is a great demand to ensure food security through stable and increased rice production. However, the growth of the global population, extreme weather, and decreased land area are the primary challenges to food security. Soil salinization is a significant environmental challenge that poses a serious threat to the sustainable development of agriculture worldwide [2,3,4]. Rice cultivation is a cost-effective and efficient method for improving and utilizing saline–alkali soils [5,6,7,8]. However, the commonly grown rice varieties typically have low salt tolerance. The physiological limitations of these rice varieties restrict the potential of using rice to improve saline–alkali land, resulting in large areas remaining underutilized and hindering the expansion of rice cultivation, as well as further increases in grain yield. The exploration and utilization of salt-tolerant rice germplasm resources, coupled with breeding elite rice varieties with enhanced salt tolerance, is an effective way to meet the challenge of soil salinization.
Rice is a moderately salt-sensitive crop [9,10,11]. Salt stress can substantially affect rice during the growth and developmental cycle [12,13,14]. Higher concentrations of salt stress can reduce the water-absorbing capacity of rice grains, prolong germination time, and decrease the germination index during the rice grain germination stage [15]. In the seedling stage, salt stress inhibited the growth and development of the rice root. The roots are much shorter and thicker under the salt stress treatment. In addition, yellowing and dwarfing of leaves, as well as diminished photosynthetic efficiency, were also detected in the rice, which hamper the rice plants’ uptake of water and nutrients [16,17,18]. During the reproductive stage, salt stress hinders rice spikelet differentiation, leading to degenerated spikelets and a decrease in the number of spikelets per panicle. These effects also decreased grain plumpness, compromising yield and quality [19,20,21,22]. Furthermore, salt stress generates a substantial amount of reactive oxygen species (ROS) within rice plants, leading to oxidative damage to biomacromolecules, including cell membranes, nucleic acids, and proteins. This exacerbates physiological dysfunctions in rice, further jeopardizing its overall performance [23,24,25].
Polyploid rice is a new type of rice resulting from chromosome doubling [26]. Compared to their diploid counterparts, autotetraploid rice exhibits significant differences in phenotype charts, physiological–ecological traits, genomic architecture, and patterns of gene expression regulation. These distinctions offer a valuable source of genetic diversity for developing new rice cultivars with desirable attributes. Neo-tetraploid rice is a highly fertile polyploid variety derived from the hybrid of autotetraploid rice [27,28]. Neo-tetraploid rice lines displayed high fertility and normal morphological traits, which showed the potential to overcome sterility in autotetraploid rice [27]. Neo-tetraploid lines showed a great potential of hybrid vigor and heterosis when crossed with autotetraploid rice lines, including the hybrid cross of Huajingxian-4x × Huaduo3, T449-4x × Huaduo1, and T485-4x × Huaduo8. In addition, neo-tetraploid rice exhibited robust biological advantages in growth vigor, stress resistance, and other agronomic traits [29]. The neo-tetraploid rice Huaduo1 demonstrated greater salt tolerance than its two autotetraploid rice parents [30]. Till now, little is known about the salt tolerance mechanisms of neo-tetraploid rice.
In the previous analysis, we verified that the neo-tetraploid rice Huaduo1 exhibited stronger resistance to salt stress [30]. Currently, no information is available on the abiotic stress faced by neo-tetraploid hybrid progeny, especially regarding salt stress. Here, we examine and evaluate the salt tolerance of hybrid progeny in neo-tetraploid rice. Two hybrid lines, named high salt-resistance tetraploid rice hybrid progeny (HSRTH) and low salt-resistance tetraploid rice hybrid progeny (LSRTH), were created from crosses between the neo-tetraploid rice cultivars ‘Huaduo 3’ [27] and ‘Huaduo 8’ [28] with the autotetraploid rice Huanghuazhan-4x. A comparative analysis of salt tolerance was performed between these two lines during germination and the seedling stages. The study evaluated germination characteristics, root structures, bud traits, and expression of salt tolerance genes to reveal the genetic variation in the hybrid progeny of neo-tetraploid rice. The results provide valuable insights and references for effectively utilizing neo-tetraploid rice resources in the development of salt-tolerant rice varieties.

2. Materials and Methods

2.1. Experimental Materials

Two tetraploid rice hybrid progenies, high salt-resistance tetraploid rice hybrid progeny (HSRTH) and low salt-resistance tetraploid rice hybrid progeny (LSRTH), were used in this study. The above materials were hybridized and selected based on the seed set ratio over six generations at the Wushan Campus Teaching and Research Base of South China Agricultural University (SCAU). The grains of two distinct genotypes were harvested concurrently and preserved under the same conditions. The HSRTH resulted from crossing the neo-tetraploid rice line “Huaduo 3” with the autotetraploid rice Huanghuazhan-4x. The LSRTH was created by crossing the neo-tetraploid rice line “Huaduo 8” with the autotetraploid rice Huanghuazhan-4x.

2.2. Salt Stress Treatment of Experimental Materials

The consistent sizes of grains of HSRTH (11.00 ± 0.50 mm) and LSRTH (10.00 ± 0.50 mm) were soaked in distilled water after disinfection with 2.5% (v/v) sodium hypochlorite for 15 min. Then, 50 sterilized grains were selected and evenly distributed in the 9 cm diameter Petri dishes lined with two layers of filter paper. Each dish contained 20 mL of NaCl solution at concentrations of 0 mmol/L, 50 mmol/L, 100 mmol/L, 150 mmol/L, 200 mmol/L, or 250 mmol/L, respectively. All Petri dishes were incubated at 26 °C in a growth chamber with alternating light and dark conditions for 12 h each. The 33 W LED modulator served as the light source in the growth chamber. The NaCl solution was monitored and replaced every 24 h to maintain consistent treatment conditions. Three biological replicates were established for each treatment concentration in this study. Rice grains were treated following the NaCl stress protocol described by Lin et al. [30].

2.3. Measurement of Seed Germination Traits in Two Hybrid Progenies

To assess the variation in grain germination at different concentrations of NaCl solution, five germination traits were measured and used in this study for the two tetraploid rice hybrids. The standard for germinated rice grains was marked by a 1 mm radicle breakthrough and a germ length equal to half the grain length, starting from the 3rd day [31]. The germination rate of rice grains under various treatments was studied from the 3rd to the 7th day, with its value calculated by comparing the number of germinated grains on the 7th day to the total number of tested rice grains. The germination termination time is the point from when grains start absorbing water and sprouting until all germinated grains have finished germinating. Mean germination time was calculated as = Σ (number of germinated grains per day × number of days to germinate)/total number of germinated grains. The germination energy was calculated as the number of germinated grains on the fourth day divided by the total number of experimental grains. The relative vigor index (RVI) was calculated as (VI of the treatment group/VI of the control group) × 100. Here, the vigor index (VI) was determined by multiplying the germination index (GI) by S, where S represents the total length of roots and shoots grains) [32,33]. All of these samples were performed in three replications.

2.4. Measurement of the Germinated Grains and Seedlings in Two Hybrid Progenies

The relative root length and relative coleoptile length of the germinated grains on the 7th day were chosen and used to evaluate the phenotypic variation between the HSRTH and LSRTH under the different concentrations of NaCl solution. The relative root length was calculated as the root length of the treatment group divided by the root length of the control group. The relative coleoptile length was calculated as the coleoptile length of the treatment group divided by the coleoptile length of the control group.
Then, the healthy seedlings from each treatment group were chosen and placed in a 96-well hydroponic culture box for growing. Each hydroponic culture box was treated with different concentrations of NaCl solution. Three indicators were utilized to assess the seedlings subjected to salt stress: total root count, root-to-shoot ratio, and seedling moisture content. The seedling samples of HSRTH and LSRTH were collected and the moisture content and root-to-shoot ratio were measured on the 14th and 19th days, respectively. After these measurements, the seedling samples were dried in an oven at 60 °C until they reached a constant weight, allowing for the assessment of their moisture content and root-to-shoot ratio [30]. Seedling moisture content was calculated as (fresh weight minus the dry weight) divided by fresh weight, multiplied by 100%. Root-to-shoot ratio was calculated as underground fresh weight divided by over-ground fresh weight [34].

2.5. Semi-Section Analysis of Root Tip and Coleoptile Tissues Under the NaCl Treatment

To investigate the structural changes in the differentiation zone between the HSRTH and LSRTH under different NaCl concentrations, the primary root samples of germinated grains on the 7th day were collected. The root tip tissue was cut to a length of 0.5–0.6 cm from the root cap. The above samples were fixed in FAA for 48 h. Samples were subsequently rinsed with 50% (v/v) ethanol, followed by dehydration through a graded series of ethanol concentrations (70%, 80%, 90%, and 95% v/v). Samples were then embedded in the Technovit 7100 resin embedding kit (Kulzer Technik, Hanua, Germany) following the manufacturer’s infiltration, embedding, and polymerization protocol. Transverse sections (5 μm thickness) were obtained using a Leica RM2235 microtome (Leica Biosystems, Shanghai, China), stained with 1% toluidine blue O, and examined under a light microscope for structural characterization [35].
The coleoptile samples of HSRTH and LSRTH were collected to investigate the structural changes on the 7th day. The coleoptile samples were cut from 1 cm from the base of the coleoptile. Then, the samples were fixed in the FAA solution, dehydrated, embedded, and transversely sectioned, similarly to the root tip section.

2.6. Root Tip Observation Under the NaCl Stress Using the WE-CLSM Analysis

This study employed WE-CLSM (Whole Eosin B Staining and Clearing Laser Scanning Confocal Microscopy) analysis to examine the cellular differences in the differentiation zone between the HSRTH and LSRTH under varying NaCl concentrations. The germinated root tip in the primary root was collected, and on the 7th day, it was collected at a distance of 0.5–0.6 cm from the root cap. Then, the root samples were preserved in FAA solution for 48 h. Following this, the samples underwent dehydration through a graded (v/v) ethanol series and were then rinsed with 50% (v/v) ethanol. Subsequently, they were dehydrated through a graded (70%, 80%, 90%, and 95%) (v/v) ethanol series. The prepared root tips were observed using a Leica SPE confocal microscope (Leica Microsystems, Heidelberg, Germany) at an excitation wavelength of 543 nm to capture the images [35].

2.7. RT-qPCR Analysis

The root tissue samples of HSRTH and LSRTH were collected to evaluate the gene expression difference under the NaCl stress. Both the samples of HSRTH (salt-resistant material) and LSRTH (salt-sensitive material) were treated with 0 mmol/L and 150 mmol/L NaCl, and the root tissue was collected from a length of 0.5–0.6 cm from the root cap. Each sample consisted of three biological replicates, which were promptly frozen in liquid nitrogen and stored at −80 °C for RNA extraction. Total RNA was extracted using TRIzol (Accrate Biology, Changsha, China) Reagent following the manufacturer’s instructions. RNA integrity was verified using formaldehyde–agarose gel electrophoresis and quantified with a spectrophotometer. Next, cDNA was generated by performing reverse transcription experiments. Six representative salt stress genes, along with the Ubiquitin gene, were chosen for real-time PCR (RT-qPCR) analysis. The primers, with sequences detailed in Table S1, were synthesized by Sangon Bioengineering (Shanghai, China). The RT-qPCR experiment was conducted on the Roche LightCycler 480 system (Roche, Rotkreuz, Switzerland), with three technical replicates for each biological sample. Relative gene expression levels were determined and analyzed using the 2−∆∆Ct method [36].

2.8. Statistical Analysis

Data from stress-related NaCl experiments were collected and subsequently processed using Microsoft Excel 2021. One-way ANOVA using IBM SPSS Statistics 26.0 was employed for significance testing (p < 0.05). All results are shown as the mean ± standard deviation (SD), based on three independent biological replicates. The graphical data were generated using GraphPad Prism 8.0 software representations.

3. Results

3.1. Comparison of Rice Grain Germination Time Between HSRTH and LSRTH Under the NaCl Stress

This study aims to investigate the differences in rice grain germination time between HSRTH and LSRTH under varying concentrations of NaCl stress. Consequently, we have summarized the grain germination time observed in this research (Figure 1, Table S2). From this study, we examined germination initiation and termination times for HSRTH and LSRTH under varying NaCl treatments. As NaCl concentration increased, the times for both germination initiation and termination were extended for each material. In the HSRTH, the germination termination time at 150 mmol/L NaCl is on the 4th day, consistent with the control group (Figure 1A). In contrast, germination of LSRTH was terminated on 7th day at a NaCl concentration of 150 mmol/L (Figure 1B).
The mean germination time (MGT) was also used to assess the germination difference in this study (Table S2). The MGT of HSRTH and LSRTH showed a significant difference when the NaCl concentration reached 50 mmol/L and above. The MGT of HSRTH and LSRTH in control was 2.84 ± 0.07 days and 2.64 ± 0.62 days, respectively. With NaCl concentrations increased to 50 and 100 mmol/L, the MGT in HSRTH was 2.93 ± 0.10 and 3.43 ± 0.21 days, respectively. Their MGTs, with a lag of 0.09 and 0.59 days, respectively, compared to the control group. In contrast, the MGT of LSRTH was 3.41 ± 0.10 and 4.09 ± 0.22 days, respectively, lagging behind the control group by 0.77 and 1.45 days. As the NaCl concentration increased to more than 150 mmol/L, the MGT of HSRTH was 3.94 ± 0.23 days, 4.53 ± 0.15 days, and 5.73 ± 0.10 days under the 150, 200, and 250 mmol/L NaCl. Their MGTs, with a lag of 1.82, 2.83, and 3.53 days, respectively, compared to the control group. In contrast, the MGT of LSRTH was 4.46 ± 0.06 and 6.17 ± 0.24 days, respectively, lagging behind the control group by 1.82, 2.83, and 3.53 days. These findings showed that LSRTH was more responsive to NaCl stress, with a significant suppression of sprouting.

3.2. Comparison of Germination Characteristics Between HSRTH and LSRTH Under the NaCl Stress

This study analyzed and summarized grain germination charts to evaluate the differences in germination between HSRTH and LSRTH (Figure 2 and Figure 3, Table S3). We determined the grain germination rates of two materials under NaCl treatment. The seed germination rates of HSRTH and LSRTH decreased progressively with increasing NaCl concentrations on the 7th day (Figure 2A–H). HSRTH exhibited a stable seed germination rate, ranging from 98.00 ± 2.83% to 100%, while the NaCl concentration was 150 mmol/L and below (Figure 3A). The germination rates of HSRTH grains remained above 97.00 ± 4.24% at 200 mmol/L NaCl and 48.00 ± 2.63% at 250 mmol/L NaCl. In contrast, LSRTH grains had significantly lower germination rates, measuring only 63.00 ± 7.07% and 5.00 ± 1.41%, respectively (Figure 3A). These results indicated that seed germination rates were negatively correlated with NaCl concentration between the two materials.
We analyzed the germination energy of two materials under the NaCl stress treatment. The germination energy reflected the speed of grain germination and grain vitality. The HSRTH and LSRTH exhibited stable germination at NaCl concentrations of 50 mmol/L or lower. As the NaCl concentration increased to over 100 mmol/L, the germination energy of LSRTH decreased to 66.00 ± 8.50% and 54.00 ± 8.50% and further dropped to 10 ± 2.83% at 200 mmol/L NaCl (Figure 3B).
The relative vigor index (RVI) was also measured for the two materials under NaCl stress conditions. With the NaCl concentration increased, both HSRTH and LSRTH showed a similar pattern in their relative vigor index (Figure 3C). The relative vigor index of HSRTH at 50 mmol/L, 100 mmol/L, 150 mmol/L, and 200 mmol/L was significantly higher than that of LSRTH across different NaCl concentration levels. The relative vigor index of HSRTH at 50 and 100 mmol/L NaCl was 90.93 ± 3.31% and 62.58 ± 0.51%, while LSRTH showed only 62.60 ± 5.58% and 33.40 ± 1.78%, respectively. These results further suggest that HSRTH exhibits stronger salt tolerance.

3.3. Comparison of Phenotypic Variation Between HSRTH and LSRTH in the Seeding Stage Under the NaCl Stress

To assess the growth variation between HSRTH and LSRTH during the seedling stage under NaCl stress treatment, this study selected and investigated the root number, relative root length, and relative coleoptile length (Figure 4). The variation between the HSRTH and LSRTH was significant under NaCl stress on the 7th day (Figure 4A–C). We examined the root number of HSRTH and LSRTH in seedlings. The root number of HSRTH ranged from 9.17 ± 0.42, 8.63 ± 0.90, and 7.23 ± 0.52 at NaCl concentrations of 100 mmol/L and below (Figure 4B). In contrast to HSRTH, LSRTH showed a significant decrease in root number. The root number of LSRTH in control is 9.27 ± 1.32. With the NaCl concentration increased to 100 mmol/L, the root number of LSRTH was only 4.75 ± 1.06, which was 4.52 ± 1.06 less than that of the control (Figure 4B). When the NaCl concentration reached 150 mmol/L, the root number of HSRTH was 3.63 ± 0.71, while that of LSRTH was only 1.17 ± 0.14. The results showed that HSRTH demonstrated higher stability than LSRTH at different NaCl concentrations. The results indicated that HSRTH exhibited greater stability than LSRTH under NaCl stress treatment.
An assessment was conducted to evaluate the relative root lengths of two materials on the 7th day under varying concentrations of NaCl stress treatments. The results showed that as the NaCl concentration increased, the relative root lengths in HSRTH and LSRTH decreased. Compared with the control, the relative root lengths of HSRTH under 50 and 100 mmol/L NaCl stress treatments were 0.89 ± 0.07 and 0.72 ± 0.02, respectively. The relative root length values were reduced by 0.11 ± 0.68 and 0.28 ± 0.17, respectively (Figure 4C). Compared with the control group, the relative root lengths in LSRTH under the 50 and 100 mmol/L NaCl stress treatment groups were 0.82 ± 0.03 and 0.55 ± 0.04, respectively. The relative root length was reduced by 0.18 ± 0.03 and 0.45 ± 0.04, respectively. When the NaCl concentration reached 150 and 200 mmol/L, the relative root lengths of HSRTH were 0.43 ± 0.02 and 0.33 ± 0.01, whereas the relative root lengths of LSRTH were 0.34 ± 0.04 and 0.20 ± 0.02. The results indicated that the relative root lengths of HSRTH were significantly higher than those of LSRTH.
The relative coleoptile length of seedlings in HSRTH and LSRTH under varying NaCl stress concentrations was further analyzed. Both materials showed concentration-dependent inhibition of coleoptile length under the NaCl stress. LSRTH showed more severe suppression than HSRTH. At a NaCl concentration of 50 mmol/L, the relative coleoptile length of HSRTH was 0.92 ± 0.01, whereas that of LSRTH was 0.86 ± 0.06. Significant differences in the relative coleoptile length of HSRTH compared to that of LSRTH when the NaCl concentration was increased to 100 and 150 mmol/L. The relative coleoptile length of HSRTH was 0.78 ± 0.06 and 0.43 ± 0.47, while the relative coleoptile length of LSRTH was 0.63 ± 0.02 and 0.26 ± 0.02 (Figure 4D). These results show that HSRTH exhibits greater salt tolerance during seeding compared to LSRTH.

3.4. Effects of NaCl Stress on Root/Shoot Ratio and Moisture Content in HSRTH and LSRTH

To study the growth dynamics of HSRTH and LSRTH during the seedling stage under salt stress, we examined their root-to-shoot ratios and moisture content (Figure 5 and Figures S1 and S2). As the NaCl concentration increased, the root-to-shoot ratio increased for each material. The root/shoot ratio of HSRTH in the control group is 0.45 ± 0.07, and its value increased by 0.14 ± 0.06, 0.60, 0.57 ± 0.17, and 0.50 ± 0.01, respectively, under the NaCl concentration treatments of 50 mmol/L, 100 mmol/L, 150 mmol/L, and 200 mmol/L (Figure 5A). In contrast, the root-to-shoot ratio value of LSRTH in the control group is 0.44 ± 0.04. This value increased by 0.19 ± 0.02, 0.42, 0.41 ± 0.01, and 0.43 ± 0.02, respectively, under the NaCl concentration treatments of 50 mmol/L, 100 mmol/L, 150 mmol/L, and 200 mmol/L (Figure 5A).
This study also examined the moisture content of seedlings between the HSRTH and LSRTH. As NaCl concentration increased, the moisture content in HSRTH and LSRTH decreased. The moisture content of HSRTH in the control is 84.89 ± 0.04%, decreasing by 2.23 ± 0.88%, 1.70 ± 0.94%, 3.58 ± 0.38%, and 3.04 ± 0.93% under 50 mmol/L, 100 mmol/L, 150 mmol/L, and 200 mmol/L NaCl treatments, respectively (Figure 5B). The moisture content of LSRTH increased by 0.11 ± 0.58% under the 50 mmol/L NaCl concentration treatment, compared to the control value of 83.92 ± 0.25%. Furthermore, the moisture content of LSRTH decreased by 3.17 ± 0.18%, 1.50 ± 0.12%, and 3.02 ± 1.67% under NaCl concentration treatments of 100 mmol/L, 150 mmol/L, and 200 mmol/L, respectively (Figure 5B). These results indicate enhanced stability in HSRTH under salt stress, as evidenced by its more consistent water retention capacity in contrast to the fluctuating response of LSRTH.

3.5. Effects of NaCl Stress on Root Tissue in HSRTH and LSRTH

To investigate the structural alterations in root apical differentiation zone cells of HSRTH and LSRTH under varying intensities of NaCl stress, this study employed semi-thin sectioning and WE-CLSM analysis (Figure 6 and Figure 7). Both materials exhibited no significant difference at a 50 mmol/L concentration of NaCl compared to the control (Figure 6B,G). In comparison to the LSRTH, the HSRTH showed less noticeable stele region cell deformation and preserved a clearer cellular structure architecture (Figure 6B1,G1). With the increased concentration of NaCl, two materials showed significant structural changes under 100 mmol/L NaCl compared to the controls (Figure 6C,H). In LSRTH, we observed epidermal cell hypertrophy, characterized by increased cortical cell layers and distortion of the stele, indicating more severe deformation of the stele (Figure 6C1,H1). As the NaCl concentration exceeded 150 mmol/L, the root tip differentiation zone of HSRTH and LSRTH exhibited noticeable cytological responses (Figure 6D,I). HSRTH exhibited increased cortical cell layers while showing only slight changes to the stele (Figure 6D1). In comparison, the differentiation zone of LSRTH displayed cortical hyperplasia, characterized by thick, loosely organized cells of varying sizes, and significant alterations in the necrosis of the stele cells (Figure 6I1). These findings emphasize the genotype-specific adaptive strategies in response to moderate to high osmotic stress.
The WE-CLSM analyses further indicated morphological changes in root apical cells that depend on the concentration of NaCl during stress (Figure 7). Both materials were tightly arranged and showed the normal cell morphology. The root tissues were indistinguishable from those of the controls at 50 mmol/L NaCl (Figure 7B,G). As the NaCl concentration increased to 100 mmol/L, HSRTH maintained a relatively stable cellular structure, while LSRTH showed significant cellular contraction (Figure 7C,C1,H,H1). As the NaCl concentration rose to 150 and 200 mmol/L, the root tissues of the two materials showed an increase in the number of cortical cell layers, a decrease in cell volume, and a change in shape from rectangular to round or elliptical, accompanied by loosened cellular structure arrangements (Figure 7D,E,I,J). These pathological alterations were consistently more evident in LSRTH (Figure 7I1,J1). These results show that the root cells from HSRTH exhibit greater structural stability in saline environments, resulting in less cellular damage at all tested NaCl concentrations compared to those from LSRTH.

3.6. Effects of NaCl Stress on Coleoptiles in HSRTH and LSRTH

To evaluate how salt stress affects rice coleoptile development, we examined the cellular structure of coleoptiles at various NaCl levels on the seventh day of germination. In the control groups for HSRTH and LSRTH, the coleoptile cells exhibited one underdeveloped leaf and two fully mature leaves (Figure 8A,C). The developing, immature, and mature leaves contained large air cavities and more prominent vein cells (Figure 8A1,C1). The outer germinal sheath in HSRTH and LSRTH develops normally and is shed naturally, following the usual process (Figure 8A,C).
At a NaCl concentration of 200 mmol/L, the first complete leaf appeared in both HSRTH and LSRTH (Figure 8B,D). The air cavities in the incomplete leaf were smaller compared to the control samples (Figure 8B1,D1). These findings indicate that salt stress affected coleoptile development in both HSRTH and LSRTH, with HSRTH being less impacted than LSRTH.

3.7. Gene Expression of NaCl Stress on Root Tissues in HSRTH and LSRTH

HSRTH exhibited greater stability than LSRTH under NaCl stress treatment. Therefore, we further conducted RT-qPCR analysis to compare the gene expression levels of root tissues between the HSRTH and LSRTH under 0 mmol/L and 150 mmol/L NaCl concentrations. Here, a total of six genes, named LOC_Os11g26790, LOC_Os06g41010, LOC_Os02g52780, LOC_Os03g20090, LOC_Os04g32920, and LOC_Os05g25770 were selected and verified in this study (Figure 9A). These genes have been functionally characterized and verified to be associated with salt stress [37,38,39,40,41,42]. Among these genes, LOC_Os11g26790, LOC_Os06g41010, LOC_Os02g52780, and LOC_Os03g20090 are salt-tolerance related genes; LOC_Os04g32920 and LOC_Os05g25770 are salt-sensitive related genes.
The RT-qPCR results confirmed the differential expression of six genes between HSRTH and LSRTH. After 7 days of treatment with 150 mmol/L NaCl, the expression levels of LOC_Os11g26790, LOC_Os06g41010, and LOC_Os02g52780 in root tip tissues of both HSRTH and LSRTH showed significant increases compared to their respective controls (Figure 9B–D). Notably, the expression levels of LOC_Os11g26790 and LOC_Os02g52780 in HSRTH were more up-regulated than in LSRTH under 150 mmol/L NaCl (Figure 9B,C). In addition, we found that the expression levels of LOC_Os03g20090 were significantly decreased in both down-regulation and LSRTH after 7 days under 150 mmol/L NaCl (Figure 9E). The gene expression level in HSRTH was less affected and down-regulated in the HSRTH. We also verified that the gene expression levels of two salt-sensitive genes LOC_Os04g32920 and LOC_Os05g25770 were significantly reduced in the root tip tissues of both HSRTH and LSRTH plants when subjected to 150 mmol/L NaCl.
These findings demonstrated the reliability and accuracy of the salt stress-related gene, which was also differentially expressed in the HSRTH compared with the LSRTH (Figure 9B–G).

4. Discussion

4.1. Germination Characteristics of Neo-Tetraploid Rice Hybrids

Soil salinization affects morphological, physiological, biochemical, and molecular processes. It can notably decrease rice yield, germination, growth rates, and tillering [43,44]. Polyploid rice is considered a valuable germplasm resource to meet the environmental challenges [29]. Neo-tetraploid rice shows high yield potential and is a promising approach for environmental stress adaptation challenges [27,28,29]. Little research has focused on its salt tolerance during germination and the seedling stages, making it important to examine its characteristics in hybrids.
In this study, we selected two tetraploid rice hybrids to assess their resistance to environmental salt stress. NaCl is frequently used to evaluate salt stress in rice [45]. Rice germination was significantly influenced by NaCl concentration in a salt stress environment [46]. In this study, we utilized six varying gradients of NaCl solutions to replicate salt stress conditions. When comparing LSRTH with HSRTH, the latter exhibited a greater seed germination rate. The germination rates for HSRTH were 97.00 ± 4.24% at a 200 mmol/L NaCl concentration and 48.00 ± 2.63% at a 250 mmol/L NaCl concentration. In contrast to HSRTH, LSRTH has a germination rate of just 63.00 ± 7.07 and 5.00 ± 1.41%. The germination energy characteristics indicate that HSRTH demonstrates higher salt tolerance than LSRTH. The relative vigor index in HSRTH was higher than that of LSRTH in each concentration treatment. These results indicated that HSRTH exhibited stronger salt tolerance than LSRTH.
The seedling stage is a crucial phase for plant development under salt stress. This study investigated the differences between the two materials in terms of root number, relative root length, relative coleoptile length, root-to-shoot ratios, and moisture content. The root-to-shoot ratio is a crucial indicator of salt stress tolerance. The salt-tolerant japonica varieties Zhejiang Rice 78 and Xiushui 134 showed a strong ability to recover under salt stress, with their root lengths after salt treatment exceeding those of the salt-sensitive Japonica varieties [47]. Our research demonstrates that different concentrations of NaCl reduce the number of roots, relative root length, and relative coleoptile length under the NaCl stress treatment. The root number of HSRTH demonstrated greater stability, with only minor effects on relative root and coleoptile lengths under salt stress, compared to LSRTH. These findings were similar to those of Huaduo1, which can survive salt stress [30]. These findings suggest that HSRTH exhibits greater stability than LSRTH when exposed to salt stress treatment.

4.2. Stable Adaptability in Root Tissue Likely Causes Salt Resistance in Neo-Tetraploid Rice Hybrids

Soil salinization poses a significant challenge to rice production [48]. Root tissue typically responds to environmental stress. When subjected to salt stress, the sclerenchyma cell layer in the root decreases, while the suberization of the endodermis increases [49]. It has been shown that the root length, root surface area, and average root diameter of different salt-tolerant rice varieties decreased significantly with increasing salt concentration during the critical period of rice fertility. Salt-tolerant rice varieties showed better root morphology compared to salt-sensitive varieties [50]. Therefore, we examined the cellular structure in the root tip differentiation zone at different NaCl solution concentrations. Compared to LSRTH, HSRTH exhibited a greater ability to resist salt at higher NaCl solution concentrations and with extended treatment times.
Root tissue plays a crucial role in mitigating salt stress. The epidermal cells and stele are the primary components of root tissue, which are essential for water absorption and transport. The smaller cortical cells and increased cortical layers were observed in HSRTH under various concentrations of NaCl treatment. This study also revealed that the apical differentiation zone of LSRTH showed easily deformed and irregularly shaped cortical and stele cells under 100–200 NaCl treatments. These results were similar to those of the rice material Huaduo1 under salt stress. The root tissue of Huaduo1 under salt stress was significantly contracted and distorted compared with the no-treatment group [30]. Additionally, our WE-CLSM findings indicated that the stability of epidermal and outer skin cells in HSRTH adjusts to salt stress conditions, enhancing the root system’s capacity for water absorption. These observations demonstrated that salt stress impacted the cortical cells and the number of cortical layers in the apical differentiation zone. Moreover, the findings confirmed that treatment concentrations of 200 and 250 mmol/L NaCl are suitable for evaluating salt stress and identifying salt-resistant varieties.
Root tissue is generally responsive to salt stress [16,23]. To date, numerous salt stress-related genes have been identified as playing a role in plant adaptation to salt stress [37,38,39,40,41,42,51]. To reveal the reason for the enhanced salt resistance of HSRTH, we selected six salt stress-related genes based on the results from neo-tetraploid rice [28,30]. These genes have been functionally characterized and verified to be associated with salt stress [37,38,39,40,41,42]. In the present work, we confirmed the gene expression levels of salt-tolerance to the HSRTH and LSRTH under salt stress. OsRAB16A (LOC_Os11g26790) encodes a glycine-rich protein. It shows increased salt tolerance when the gene is overexpressed in rice plants [37]. OsbZIP23 (LOC_Os02g52780) is a bZIP transcription factor and functions as a pivotal regulator in abscisic acid (ABA)-dependent drought and salt stress responses [39]. The expression levels of these two genes showed significant increases compared to their respective controls. Notably, HSRTH was more up-regulated than in LSRTH under 150 mmol/L NaCl. These results indicated that salt-tolerance related genes in HSRTH were more up-regulated than in LSRTH, and they may play important role in salt stress resistant ability.
In this study, we also verified the gene expression levels of salt-sensitive related genes. OsHAK1 (LOC_Os04g32920) is a high-affinity potassium transporter, and the oshak1 mutant was more sensitive to salt stress compared with the wild type [41]. Our result indicated that this gene was significantly decreased in HSRTH and LSRTH compared to their respective controls. However, the gene expression level of OsHAK1 is more stable in HSRTH than the LSRTH under 150 mmol/L NaCl treatment. OsWRKY45 (LOC_Os05g25770) is the other rice salt-sensitive related gene and is the WRKY-like transcription factor with two alleles. One of its transcript, OsWRKY45-2, is negatively regulated by the response to salt stress [42]. Here, we proved that the expression level of OsWRKY45-2 was significantly decreased in HSRTH and LSRTH compared to their respective controls. The gene expression level of OsWRKY45-2 is significantly lower in HSRTH compared with LSRTH under 150 mmol/L NaCl. The lower gene expression level of OsWRKY45-2 in HSRTH suggests that it may have a less salt-sensitive ability compared to LSRTH. All of these results indicated that salt stress-related genes may also play an important role in salt stress resistance ability, which might explain why HSRTH shows strong salt stress adaptability and tolerance. Our results offer a foundation for understanding and uncovering the regulatory mechanism of salt tolerance in HSRTH.

5. Conclusions

In this study, we evaluated the salt stress tolerance of neo-tetraploid hybrid progenies, named the HSRTH and LSRTH. The results demonstrated that HSRTH has higher salt tolerance, with a germination rate over 48.00 ± 2.63%, even at NaCl concentrations exceeding 250 mmol/L. Our cytological and gene expression analyses indicated that the NaCl treatment showed a negligible effect on the roots and coleoptiles of HSRTH. Furthermore, six salt stress-related genes reflect genetic variation between the two tetraploid rice hybrids. These findings indicate that HSRTH is a strong candidate for rice breeding and may be a novel salt-tolerant variety.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy15092066/s1, Figure S1: comparison of seedlings in two materials under different concentrations of NaCl on the 19th day; Figure S2: comparison of the roots and shoots of HSRTH and LSRTH on the 19th day of growth under different NaCl concentration treatments. Table S1. List of primers used for RT-qPCR analysis. Table S2. Comparison of average germination time between the two materials under different concentrations of NaCl. Table S3. Comparison of the germination rate of the two materials at different concentrations of NaCl.

Author Contributions

Conceptualization, J.W.; Data Curation, P.H., X.X. and X.C.; Formal Analysis, P.H., X.X., S.C., Y.Z., Z.H. and M.Q.S.; Funding Acquisition, J.W. and X.L.; Investigation, P.H., X.X., X.C., S.C., Y.Z. and Z.H.; Methodology, Z.H., P.H. and X.X.; Project Administration, J.W. and X.L.; Resources J.W., X.L. and M.Q.S.; Supervision: J.W. and X.L.; Validation: P.H., X.X., X.C., S.C., Y.Z. and Z.H.; Visualization, P.H., X.X., Z.H. and M.Q.S.; Writing—Original Draft Preparation, P.H., X.X., X.C., S.C., Y.Z., Z.H. and J.W.; Writing—Review and Editing, J.W., X.L. and M.Q.S. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Guangdong Basic and Applied Basic Research Foundation (2023A1515012672), the Base Bank of Guangdong Rice Germplasm Resources Project (2025), the Base Bank of Lingnan Rice Germplasm Resources Project (2024B1212060009), the Guangdong Basic and Applied Basic Research Foundation (2021A1515010748) and the Laboratory of Lingnan Modern Agriculture Project.

Data Availability Statement

The data are contained within the article and its Supplementary Materials.

Acknowledgments

The authors thank Shuhong Yu and other lab members for their assistance.

Conflicts of Interest

The authors have declared that no competing interests exist.

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Figure 1. Comparison of seed germination trends between the HSRTH and LSRTH under differential NaCl stress concentrations. (A) Germination trends of HSRTH grains from Day 1 to Day 7 under differential NaCl stress concentrations. (B) Germination trends of LSRTH grains from Day 1 to Day 7 under different NaCl stress concentrations.
Figure 1. Comparison of seed germination trends between the HSRTH and LSRTH under differential NaCl stress concentrations. (A) Germination trends of HSRTH grains from Day 1 to Day 7 under differential NaCl stress concentrations. (B) Germination trends of LSRTH grains from Day 1 to Day 7 under different NaCl stress concentrations.
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Figure 2. Comparison of germinated grains between the HSRTH and LSRTH under the different NaCl stress concentrations on the 7th day. (A) The germinated grains on the 7th day of HSRTH with no NaCl served as the control. (B) The germinated grains were on the 7th day of HSRTH under 150 mmol/L NaCl. (C) The germinated grains were on the 7th day of HSRTH under 200 mmol/L NaCl. (D) The germinated grains were on the 7th day of HSRTH under 250 mmol/L NaCl. (E) The germinated grains on the 7th day of LSRTH with no NaCl served as the control. (F) The germinated grains were on the 7th day of LSRTH under 150 mmol/L NaCl. (G) The germinated grains were on the 7th day of LSRTH under 200 mmol/L NaCl. (H) The germinated grains were on the 7th day of LSRTH under 250 mmol/L NaCl. Scale bars = 1 cm.
Figure 2. Comparison of germinated grains between the HSRTH and LSRTH under the different NaCl stress concentrations on the 7th day. (A) The germinated grains on the 7th day of HSRTH with no NaCl served as the control. (B) The germinated grains were on the 7th day of HSRTH under 150 mmol/L NaCl. (C) The germinated grains were on the 7th day of HSRTH under 200 mmol/L NaCl. (D) The germinated grains were on the 7th day of HSRTH under 250 mmol/L NaCl. (E) The germinated grains on the 7th day of LSRTH with no NaCl served as the control. (F) The germinated grains were on the 7th day of LSRTH under 150 mmol/L NaCl. (G) The germinated grains were on the 7th day of LSRTH under 200 mmol/L NaCl. (H) The germinated grains were on the 7th day of LSRTH under 250 mmol/L NaCl. Scale bars = 1 cm.
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Figure 3. Comparative analysis of germination traits for HSRTH and LSRTH under varying NaCl stress concentrations. (A) Comparison of germination rates on the 7th day between HSRTH and LSRTH under different NaCl stress concentrations. (B) Comparison of germination energy between HSRTH and LSRTH under different NaCl stress concentrations. (C) Comparison of the relative vigor index between HSRTH and LSRTH under different NaCl stress concentrations. * and ** represent significant and highly significant differences at p < 0.05 and p < 0.01, respectively.
Figure 3. Comparative analysis of germination traits for HSRTH and LSRTH under varying NaCl stress concentrations. (A) Comparison of germination rates on the 7th day between HSRTH and LSRTH under different NaCl stress concentrations. (B) Comparison of germination energy between HSRTH and LSRTH under different NaCl stress concentrations. (C) Comparison of the relative vigor index between HSRTH and LSRTH under different NaCl stress concentrations. * and ** represent significant and highly significant differences at p < 0.05 and p < 0.01, respectively.
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Figure 4. Comparison of the seeding stage between the HSRTH and LSRTH under differential NaCl stress concentrations on the 7th day. (A) Comparison of the seeding stage between the HSRTH and LSRTH under differential NaCl stress concentrations. Bars = 1 cm. (B) Comparison of the total number of roots between the HSRTH and LSRTH under the different concentrations of NaCl stress. (C) Comparison of the relative root length between the HSRTH and LSRTH under the different concentrations of NaCl stress. (D) Comparison of the relative coleoptile length between the HSRTH and LSRTH under the different concentrations of NaCl stress. * and ** represent significant and highly significant differences at p < 0.05 and p < 0.01, respectively.
Figure 4. Comparison of the seeding stage between the HSRTH and LSRTH under differential NaCl stress concentrations on the 7th day. (A) Comparison of the seeding stage between the HSRTH and LSRTH under differential NaCl stress concentrations. Bars = 1 cm. (B) Comparison of the total number of roots between the HSRTH and LSRTH under the different concentrations of NaCl stress. (C) Comparison of the relative root length between the HSRTH and LSRTH under the different concentrations of NaCl stress. (D) Comparison of the relative coleoptile length between the HSRTH and LSRTH under the different concentrations of NaCl stress. * and ** represent significant and highly significant differences at p < 0.05 and p < 0.01, respectively.
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Figure 5. Comparison of the root/shoot ratio and moisture content under the different concentrations of NaCl in the HSRTH and LSRTH. (A) Comparison of the root/shoot ratio under the different concentrations of NaCl in the HSRTH and LSRTH. (B) Comparison of the moisture content under the different concentrations of NaCl in the HSRTH and LSRTH. * represents significant difference at p < 0.05.
Figure 5. Comparison of the root/shoot ratio and moisture content under the different concentrations of NaCl in the HSRTH and LSRTH. (A) Comparison of the root/shoot ratio under the different concentrations of NaCl in the HSRTH and LSRTH. (B) Comparison of the moisture content under the different concentrations of NaCl in the HSRTH and LSRTH. * represents significant difference at p < 0.05.
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Figure 6. Comparison of root tip differentiation zone cells from HSRTH and LSRTH on the 7th day post-germination under varying NaCl stress concentrations. (A–E) Root tip cells in HSRTH under the different concentrations of NaCl stress treatment. (A) Root tip transection at 0 mmol/L NaCl on the 7th day. (B) Root tip transection at 50 mmol/L NaCl on the 7th day. (C) Root tip transection at 100 mmol/L NaCl on the 7th day. (D) Root tip transection at 150 mmol/L NaCl on the 7th day. (E) Root tip transection at 200 mmol/L NaCl on the 7th day. (F–J) Root tip cells in LSRTH under the different concentrations of PEG6000 treatment. (F) Root tip transection at 0 mmol/L NaCl on the 7th day. (G) Root tip transection at 50 mmol/L NaCl on the 7th day. (H) Root tip transection at 100 mmol/L NaCl on the 7th day. (I) Root tip transection at 150 mmol/L NaCl on the 7th day. (J) Root tip transection at 200 mmol/L NaCl on the 7th day. The red dotted box indicates structural change in the stele of the root tip differentiation zone. (A1–J1) Detailed diagram of the stele in the root tip elongation zone corresponding to the red dotted boxes of (A–J). Bars = 20 μm in (A–J), bars = 100 μm in (A1–J1). (Rh: Rhizodermis; Ex: Exodermis; Sc: Sclerenchyma; Co: Cortex).
Figure 6. Comparison of root tip differentiation zone cells from HSRTH and LSRTH on the 7th day post-germination under varying NaCl stress concentrations. (A–E) Root tip cells in HSRTH under the different concentrations of NaCl stress treatment. (A) Root tip transection at 0 mmol/L NaCl on the 7th day. (B) Root tip transection at 50 mmol/L NaCl on the 7th day. (C) Root tip transection at 100 mmol/L NaCl on the 7th day. (D) Root tip transection at 150 mmol/L NaCl on the 7th day. (E) Root tip transection at 200 mmol/L NaCl on the 7th day. (F–J) Root tip cells in LSRTH under the different concentrations of PEG6000 treatment. (F) Root tip transection at 0 mmol/L NaCl on the 7th day. (G) Root tip transection at 50 mmol/L NaCl on the 7th day. (H) Root tip transection at 100 mmol/L NaCl on the 7th day. (I) Root tip transection at 150 mmol/L NaCl on the 7th day. (J) Root tip transection at 200 mmol/L NaCl on the 7th day. The red dotted box indicates structural change in the stele of the root tip differentiation zone. (A1–J1) Detailed diagram of the stele in the root tip elongation zone corresponding to the red dotted boxes of (A–J). Bars = 20 μm in (A–J), bars = 100 μm in (A1–J1). (Rh: Rhizodermis; Ex: Exodermis; Sc: Sclerenchyma; Co: Cortex).
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Figure 7. Comparison of root tip differentiation zone cells in seedlings from HSRTH and LSRTH under varying concentrations of NaCl stress using the WE-CLSM analysis. (A–E) Root tip cells in HSRTH under varying concentrations of NaCl stress treatment. (A) Root tip cells without NaCl stress treatment. (B) Root tip cells were treated with 50 mmol/L NaCl stress. (C) Root tip cells were treated with 100 mmol/L NaCl stress. (D) Root tip cells were treated with 150 mmol/L NaCl stress. (E) Root tip cells were treated with 200 mmol/L NaCl stress. (F–J) Root tip cells in LSRTH under the different concentrations of NaCl stress treatment. (F) Root tip cells with no NaCl stress treatment. (G) Root tip cells under the treatment of 50 mmol/L NaCl stress. (H) Root tip cells under the treatment of 100 mmol/L NaCl stress. (I) Root tip cells under the treatment of 150 mmol/L NaCl stress. (J) Root tip cells under the treatment of 200 mmol/L NaCl stress. (A1–J1) Detailed diagram of the root tip differentiation zone corresponding to (A–J). The white dotted boxes indicate that the deformed cells in the root tip differentiation zone of HSRTH appear deformed. The yellow arrows indicate the areas of increased cortex of the root tip differentiation zone in HSRTH. Bars = 40 μm in (A–J,A1–J1).
Figure 7. Comparison of root tip differentiation zone cells in seedlings from HSRTH and LSRTH under varying concentrations of NaCl stress using the WE-CLSM analysis. (A–E) Root tip cells in HSRTH under varying concentrations of NaCl stress treatment. (A) Root tip cells without NaCl stress treatment. (B) Root tip cells were treated with 50 mmol/L NaCl stress. (C) Root tip cells were treated with 100 mmol/L NaCl stress. (D) Root tip cells were treated with 150 mmol/L NaCl stress. (E) Root tip cells were treated with 200 mmol/L NaCl stress. (F–J) Root tip cells in LSRTH under the different concentrations of NaCl stress treatment. (F) Root tip cells with no NaCl stress treatment. (G) Root tip cells under the treatment of 50 mmol/L NaCl stress. (H) Root tip cells under the treatment of 100 mmol/L NaCl stress. (I) Root tip cells under the treatment of 150 mmol/L NaCl stress. (J) Root tip cells under the treatment of 200 mmol/L NaCl stress. (A1–J1) Detailed diagram of the root tip differentiation zone corresponding to (A–J). The white dotted boxes indicate that the deformed cells in the root tip differentiation zone of HSRTH appear deformed. The yellow arrows indicate the areas of increased cortex of the root tip differentiation zone in HSRTH. Bars = 40 μm in (A–J,A1–J1).
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Figure 8. Comparison of coleoptile cells from HSRTH and LSRTH on the 7th day post-germination under varying NaCl stress concentrations. (A,B) Coleoptile cells in HSRTH under the different concentrations of NaCl stress treatment. (A) Coleoptile transection at 0 mmol/L NaCl on the 7th day. (B) Coleoptile transection at 200 mmol/L NaCl on the 7th day. (C,D) Coleoptile cells in LSRTH under the different concentrations of NaCl stress treatment. (C) Coleoptile transection at 0 mmol/L NaCl on the 7th day. (D) Coleoptile transection at 200 mmol/L NaCl on the 7th day. The red dotted box indicates the internal structure during coleoptile development. The green star indicates air cavity tissue. (A1–D1) Detailed diagram of the developing cell structure of the coleoptile corresponding to (A–D). Bars = 100 μm in (A–D,A1–D1). Cl: Complete leaf; Il: Incomplete leaf; C: Coleoptile.
Figure 8. Comparison of coleoptile cells from HSRTH and LSRTH on the 7th day post-germination under varying NaCl stress concentrations. (A,B) Coleoptile cells in HSRTH under the different concentrations of NaCl stress treatment. (A) Coleoptile transection at 0 mmol/L NaCl on the 7th day. (B) Coleoptile transection at 200 mmol/L NaCl on the 7th day. (C,D) Coleoptile cells in LSRTH under the different concentrations of NaCl stress treatment. (C) Coleoptile transection at 0 mmol/L NaCl on the 7th day. (D) Coleoptile transection at 200 mmol/L NaCl on the 7th day. The red dotted box indicates the internal structure during coleoptile development. The green star indicates air cavity tissue. (A1–D1) Detailed diagram of the developing cell structure of the coleoptile corresponding to (A–D). Bars = 100 μm in (A–D,A1–D1). Cl: Complete leaf; Il: Incomplete leaf; C: Coleoptile.
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Figure 9. The RT-qPCR confirmation of salt-related genes in HSRTH and LSRTH between 0 mmol/L and 150 mmol/L NaCl. (A) Annotation of six salt-related genes. (B) The RT-qPCR confirmation of LOC_Os11g26790 in HSRTH and LSRTH between the 0 mmol/L and 150 mmol/L NaCl. (C) The RT-qPCR confirmation of LOC_Os06g41010 in HSRTH and LSRTH between the 0 mmol/L and 150 mmol/L NaCl. (D) The RT-qPCR confirmation of LOC_Os02g52780 in HSRTH and LSRTH between the 0 mmol/L and 150 mmol/L NaCl. (E) The RT-qPCR confirmation of LOC_Os04g32920 in HSRTH and LSRTH between the 0 mmol/L and 150 mmol/L NaCl. (F) The RT-qPCR confirmation of LOC_Os04g32920 in HSRTH and LSRTH between the 0 mmol/L and 150 mmol/L NaCl. (G) The RT-qPCR confirmation of LOC_Os05g25770 in HSRTH and LSRTH between the 0 mmol/L and 150 mmol/L NaCl. * and ** represent significant and highly significant differences at p < 0.05 and p < 0.01, respectively; NA represents no significant differences.
Figure 9. The RT-qPCR confirmation of salt-related genes in HSRTH and LSRTH between 0 mmol/L and 150 mmol/L NaCl. (A) Annotation of six salt-related genes. (B) The RT-qPCR confirmation of LOC_Os11g26790 in HSRTH and LSRTH between the 0 mmol/L and 150 mmol/L NaCl. (C) The RT-qPCR confirmation of LOC_Os06g41010 in HSRTH and LSRTH between the 0 mmol/L and 150 mmol/L NaCl. (D) The RT-qPCR confirmation of LOC_Os02g52780 in HSRTH and LSRTH between the 0 mmol/L and 150 mmol/L NaCl. (E) The RT-qPCR confirmation of LOC_Os04g32920 in HSRTH and LSRTH between the 0 mmol/L and 150 mmol/L NaCl. (F) The RT-qPCR confirmation of LOC_Os04g32920 in HSRTH and LSRTH between the 0 mmol/L and 150 mmol/L NaCl. (G) The RT-qPCR confirmation of LOC_Os05g25770 in HSRTH and LSRTH between the 0 mmol/L and 150 mmol/L NaCl. * and ** represent significant and highly significant differences at p < 0.05 and p < 0.01, respectively; NA represents no significant differences.
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MDPI and ACS Style

Huang, P.; Xie, X.; Cai, X.; Chen, S.; Zheng, Y.; Huang, Z.; Shahid, M.Q.; Liu, X.; Wu, J. Comparative Analysis of Germination Traits and Gene Expression in Hybrid Progeny of Neo-Tetraploid Rice Under NaCl Stress Conditions. Agronomy 2025, 15, 2066. https://doi.org/10.3390/agronomy15092066

AMA Style

Huang P, Xie X, Cai X, Chen S, Zheng Y, Huang Z, Shahid MQ, Liu X, Wu J. Comparative Analysis of Germination Traits and Gene Expression in Hybrid Progeny of Neo-Tetraploid Rice Under NaCl Stress Conditions. Agronomy. 2025; 15(9):2066. https://doi.org/10.3390/agronomy15092066

Chicago/Turabian Style

Huang, Peishan, Xinhui Xie, Xiaoyu Cai, Shihui Chen, Yutong Zheng, Zijuan Huang, Muhammad Qasim Shahid, Xiangdong Liu, and Jinwen Wu. 2025. "Comparative Analysis of Germination Traits and Gene Expression in Hybrid Progeny of Neo-Tetraploid Rice Under NaCl Stress Conditions" Agronomy 15, no. 9: 2066. https://doi.org/10.3390/agronomy15092066

APA Style

Huang, P., Xie, X., Cai, X., Chen, S., Zheng, Y., Huang, Z., Shahid, M. Q., Liu, X., & Wu, J. (2025). Comparative Analysis of Germination Traits and Gene Expression in Hybrid Progeny of Neo-Tetraploid Rice Under NaCl Stress Conditions. Agronomy, 15(9), 2066. https://doi.org/10.3390/agronomy15092066

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