Physiological Responses of Pak Choi (Brassica rapa Subsp. Chinensis) Genotypes to Salt Tolerance

Abstract

Salinity stress poses a significant challenge to Pak Choi (Brassica rapa subsp. Chinensis) production. To address this limitation, we conducted an evaluation of 24 Korean native Pak Choi species to identify genotypes with resistance to salt stress. Through cluster analysis of electrolyte leakage data, we discovered ‘IT262109’, ‘IT279432’, and ‘IT185735’ as native accessions displaying the strongest salt tolerance. Additionally, our assessment of the maximum quantum yield of photosystem II revealed a 3.7% reduction in seaweed yield in the highly salt-tolerant system compared with the control group, while the weak salt-tolerant system experienced a substantial reduction rate of 45.7% to 49.4%. Notably, salt stress had a significant impact on Pak Choi growth, but the salt-tolerant genotype exhibited less growth reduction compared with the salt-sensitive genotype. Based on the electrolyte leakage and maximum quantum yield data, it was evident that ‘IT185735’ demonstrated poorer growth compared with ‘IT262109’ and ‘IT279432’. Consequently, ‘IT262109’ and ‘IT279432’ show great potential as parent varieties for enhancing salt tolerance in Pak Choi.

1. Introduction

Brassicaceae (Cruciferae) vegetables are consumed all over the world. Among them, Pak Choi (Brassica rapa subsp. Chinensis) is used worldwide in a variety of culinary dishes, from Asian stir-fries to international fusion cuisine, due to its mild, peppery flavor and crisp texture [1]. The literature suggests that many of the green leafy brassicas are also rich in essential amino acids; vitamins A, B, C, E, and K₁; and plant sugars [2]. Further, Glucosinolates, a family of highly unique secondary metabolites found in Brassicaceae vegetables, and their breakdown products, isothiocyanates, are effective in the protection against carcinogenesis as well as in the prevention of various chronic diseases, including cardiovascular diabetic nephropathy and neuropathy [3]. The leafy vegetable called Pak Choi holds significant importance in Korea and other Asian countries, such as Malaysia, Cambodia, and China [4,5]. In Korea, Pak Choi cultivation takes place in both open fields and controlled facilities, with approximately 84% of production occurring in facilities [6]. Facility cultivation allows for year-round production and controlled shipping times; however, the practice of continuous cropping, fertilization, and the prevention of rainfall inside the facilities can lead to salt accumulation in the soil [7]. Since Pak Choi is categorized as a plant with low salt tolerance, the elevated salt concentration in the soil adversely affects photosynthesis efficiency, ion absorption, and transport, ultimately resulting in decreased productivity [8,9,10]. As such, the production of Pak Choi and their composition in plants are influenced by environmental growth conditions and cultivation methods. Although several techniques have been proposed to mitigate salinization, such as green manure crop cultivation, subsoil inversion, culvert drainage pipe installation, and microbial agent treatments, practical implementation on farms remains limited [11,12]. Consequently, the development and cultivation of salt-resistant varieties emerge as the fundamental approach to mitigating direct salt damage in facility cultivation.

The ideal salt content in soils for Pak Choi cultivation depends on the specific soil type. Generally, a recommended guideline suggests maintaining electrical conductivity (EC) of less than 2 dS/m (deciSiemens per meter) for optimal growth [13,14]. However, Pak Choi’s salt tolerance, like that of other plant species, varies based on soil type. Sandy soils require lower salt levels to prevent issues like osmotic stress and hindered water uptake due to their limited salt-retention capacity, while clayey soils can tolerate slightly higher salt levels but remain vulnerable if salt levels exceed the ideal range [15,16]. Excessive soil salt can have detrimental effects, including osmotic stress, and reduced water and nutrient uptake, resulting in wilting, diminished yields, and ion toxicity due to the accumulation of harmful ions. This disrupts cellular processes, causing leaf burn, reduced photosynthesis, and potentially plant death [17]. The literature suggests that subjecting Pak Choi to low light intensity and water deficit leads to a reduction in photosynthetic capacity and biomass [18]. Additionally, drought stress, under constant nitrogen content, increases the root/shoot ratio by reducing the shoot dry weight and increasing the root dry weight [19]. However, it is unknown if salt stress influences growth and photosynthesis in Pak Choi.

Hydroponics is a type of horticulture in which plants, particularly cash crops, are grown in an aqueous solvent without the use of soil but using mineral fertilizer solutions [20]. Hydroponic systems play a pivotal role in advancing in vitro plant studies, providing a controlled and versatile environment for plant growth. These systems eliminate the variability associated with soil-based cultivation, enabling precise manipulation of nutrient composition; balanced pH, EC, and temperature; and high levels of light, carbon dioxide, and oxygen. Consequently, they facilitate comprehensive research on plant physiology, genetics, and responses to various stressors [21]. In vitro studies using hydroponics allow researchers to dissect intricate molecular mechanisms, investigate nutrient uptake and transport processes, and assess the impact of external factors on plant development without the confounding influence of soil microorganisms [22,23]. Moreover, hydroponic setups enable the production of genetically identical plant material, which is essential for experiments involving transgenic or mutant plants, ultimately contributing to the advancement of agricultural practices, crop breeding, and sustainable food production [24].

However, plants possess inherent resistance mechanisms that can minimize the effects of salt stress, and this ability varies across species and within species due to genetic traits [25]. Native crop resources, cultivated in specific regions for extended periods, not only adapt well to local environmental conditions but also align with the preferences of local consumers [26,27]. Consequently, selecting salt-tolerant genotypes from native resources and utilizing them as breeding materials can facilitate the development of varieties that meet the demands of Korean growers and consumers alike. However, to date, there has been no evaluation of salt tolerance in native Pak Choi species collected in Korea with the aim of breeding salt-tolerant varieties. Therefore, this study aims to identify fundamental materials for the development of highly salt-tolerant Pak Choi by examining changes in physiological and growth characteristics in Korean native Pak Choi genetic resources under salt stress conditions. In this study, salt-tolerant genotypes were identified through the comprehensive assessment of chlorophyll fluorescence, electrolyte leakage, and growth parameters.

2. Materials and Methods

2.1. Plant Material and Cultivation Environment

This study was conducted in the Smart Agricultural Fusion Laboratory of Kangwon National University, utilizing a hydroponic cultivation system. A total of 24 genotypes of Korean native Pak Choi species, preserved at the Genetic Resource Center of the Rural Development Administration, were utilized. For each genotype, 100 seeds were planted onto two sheets of filter paper in a Petri dish (90 × 15), and sterilized water was added to prevent drying. The growth chamber was maintained at a temperature of 25 ± 1 °C, at relative humidity (RH) of 60 ± 5%, and under dark conditions for a two-day germination period. After two days, the Pak Choi seedlings were transplanted into urethane sponges (2.7 × 2.7 × 0.9 cm) to protect the roots and then planted in a semi-enclosed plant grower (50 × 27 × 34 cm; SPZW-A01WU1; MDNG Inc., Taiwan, China). The seedlings were acclimatized for three days at pH 5.8, electrical conductivity (EC) of 0.8 dS·m⁻¹, and photosynthetic photon flux density (PPFD) of 100 mol·m⁻²·s⁻¹. Prior to the NaCl treatment, the plants were grown for eight days under PPFD of 250 mol·m⁻²·s⁻¹ and EC of 1.2 dS·m⁻¹, using white LED lights. Subsequently, they were exposed to PPFD of 500 mol·m⁻²·s⁻¹ and EC of 1.6 dS·m⁻¹ for another eight days. The total treatment period was 16 days. Yamasaki leafy vegetable culture (N-P-K-Ca-Mg = 6-1.5-4-2-1 me·L⁻¹) served as the culture medium, and it was replenished every four days. The pH of the culture medium was adjusted using KOH and HNO3 as necessary.

2.2. NaCl Treatment Conditions

After three weeks of sowing, 30 individuals from each of the 24 lines of domestically cultivated native Pak Choi, which were grown under uniform conditions, were selected. These selected plants were then subjected to a 7-day treatment with 100 mM NaCl (Daejung, Siheung, Republic of Korea). Following the 7-day NaCl treatment, various physiological and growth parameters (number of leaves, leaf length, electrolyte leakage, and chlorophyll fluorescence) were assessed. The NaCl treatment concentration was determined based on investigations conducted using four commercially available Pak Choi seeds. The initial growth of Pak Choi before treatment exhibited leaf lengths of 6.58 ± 0.3 cm, leaf widths of 3.37 ± 0.4 cm, and an average of 6.98 leaves per plant. These plants were subsequently treated with four different NaCl levels (50, 100, 150, and 200 mM) for a duration of 7 days. It was observed from unpublished data that as the NaCl concentration increased to 150 mM or higher, the growth was poor, and the survival rate was also low. Consequently, it was concluded that treating the 24 lines of domestically cultivated native Pak Choi with a NaCl concentration of 100 mM would be an effective approach to identifying fundamental materials for the development of Pak Choi varieties with high salt tolerance.

2.3. Electrolyte Leakage Content Test

After a 7-day NaCl treatment, the second leaves of three plants were collected from each native Pak Choi. The collected leaves were washed with deionized water and then had two 5 mm diameter holes drilled in the middle of each leaf to obtain samples for measuring electrolyte leakage. The measurement of electrolyte leakage content was conducted by modifying the method described by Julkowska, et al. [28]. The collected leaf segments were placed in a 2 mL tube containing 1 mL of distilled water with 0.01% silwet L-77 solution (Van Meeuwen Chemicals, Weesp, The Netherlands) and stirred at 100 rpm for 2 h. The initial electrical conductivity was then measured using an electrical conductivity meter (LAQUAtwin-EC-33; HORIBA, Osaka, Japan). Subsequently, the tube containing the samples was stored in an ultra-low-temperature refrigerator for 5 days to ensure complete tissue destruction. Prior to measuring the secondary electrical conductivity, the tube was slowly thawed in a refrigerator at 5 °C for a day and stirred at 100 rpm for an additional 2 h; then, the secondary electrical conductivity of the sample was measured. Electrolyte leakage was calculated as a percentage by dividing the initial measured electrical conductivity by the secondary measured electrical conductivity and multiplying the result by 100.

2.4. Measurement of the Maximum Quantum Yield of Photosystem II (Fv/Fm)

At 6:00 a.m., chlorophyll fluorescence measurements were conducted using a chlorophyll fluorescence reaction meter (Junior-pam chlorophyll fluorometer; Heinz Walz GmbH, Effeltrich, Germany). The second leaves of 10 plants from each native Pak Choi were targeted for measurement after a 7-day NaCl treatment. After 20 min adaptation to darkness, a blue LED was used to irradiate the leaves at a saturated light intensity of 2800 mol·m⁻²·s⁻¹, specifically at a wavelength of 445 nm. The fluorometer provided readings for the minimum fluorescence value (Fo), maximum fluorescence value (Fm), and transient fluorescence value (Fv = Fm − Fo). Based on these measurements, the maximum quantum yield of photosystem II (Fv/Fm = (Fm − Fo)/Fm) was calculated.

2.5. Measurement of Growth Parameters

To assess the growth patterns under salt stress conditions, several parameters were measured, including leaf area and number, live weight of above- and below-ground parts, as well as dry weight after drying. Leaf area was determined using Easy Leaf Area software, following the method employed by Kim, Jang, Zebro and Heo [7], once the first leaf was collected. The number of leaves was calculated by counting the total number of leaves after a 7-day NaCl treatment. Fresh weight of the leaf was determined by weighing the sample in its fresh state, maintaining its integrity for live-weight measurements. Dry weight was measured by drying the samples for 3 days in a hot air dryer (Eyela, Tokyo, Japan) at 70 °C, and the values were presented similarly to the live-weight measurements. All growth-related traits were measured in the same manner as the maximum quantum yield of photosystem II, with each measurement being performed 10 times.

2.6. Statistical Analyses

All statistical results were reported as means and standard errors, calculated using the SPSS program (ver. 18.0; SPSS Inc., Chicago, IL, USA) for each strain. Furthermore, a hierarchical clustering analysis was conducted based on the electrolyte leakage content measurements to classify subgroups according to the response of the accessions to salt stress. The centroid linkage method, which is less influenced by outliers, was utilized for the cluster analysis, and the similarity distances between groups were determined and expressed as squared Euclidean distances. The significance of each group was tested using Duncan’s multiple range test at a significance level of p < 0.05.

3. Results

3.1. Changes in Electrolyte Leakage Content by NaCl Treatment

The study employed electrolyte leakage analysis to assess the salt tolerance of Pak Choi plants. The results clearly indicated that as the concentration of NaCl treatment increased, there was a corresponding rise in electrolyte leakage. Specifically, in the 24 genotypes, the electrolyte leakage content was 9.9~14.9% without NaCl treatment, but it increased to 26.2~49.6% with the application of 100 mM NaCl (

Table 1. Effects of different salt concentrations on electrolyte leakage and measurement of maximum quantum yield of photosystem II (Fv/Fm) in Pak Choi (Brassica rapa subsp. chinensis) landraces from Korea.

Genotype	Electrolyte Leakage (%)			Fv/Fm
	Control	100 mM Salt	Fold Change	Control	100 mM Salt	%
IT112271(1)	11.3 ± 1.2	44.0 ± 4.7	3.89	0.81 ± 0.1	0.54 ± 2.7	−33.3
IT135403(2)	14.7 ± 2.3	38.4 ± 3.3	2.61	0.79 ± 0.4	0.71 ± 1.8	−10.1
IT135406(3)	11.2 ± 1.1	55.7 ± 4.2	4.97	0.81 ± 0.1	0.44 ± 1.9	−45.7
IT135441(4)	13.5 ± 1.1	37.1 ± 3.3	2.75	0.81 ± 0.1	0.73 ± 1.6	−9.9
IT166982(5)	14.9 ± 0.8	43.7 ± 2.1	2.93	0.81 ± 0.1	0.49 ± 0.5	−39.5
IT166983(6)	11.5 ± 1.8	35.7 ± 2.6	3.10	0.81 ± 0.3	0.68 ± 0.4	−16.1
IT185735(7)	10.1 ± 2.4	28.3 ± 1.4	2.80	0.81 ± 0.1	0.78 ± 0.2	−3.7
IT188180(8)	10.5 ± 0.9	49.6 ± 4.2	4.72	0.79 ± 0.2	0.40 ± 0.9	−49.4
IT188181(9)	14.5 ± 0.1	36.5 ± 2.7	2.52	0.79 ± 0.1	0.64 ± 1.2	−18.9
IT210064(10)	14.3 ± 0.8	38.3 ± 4.2	2.68	0.80 ± 0.3	0.74 ± 1.8	−7.5
IT262109(11)	12.3 ± 1.2	27.0 ± 2.3	2.20	0.82 ± 0.3	0.79 ± 0.2	−3.7
IT279432(12)	11.0 ± 0.9	26.2 ± 2.0	2.38	0.81 ± 0.1	0.78 ± 0.1	−3.7
IT280392(13)	13.4 ± 2.0	39.2 ± 3.5	2.93	0.81 ± 0.1	0.69 ± 0.4	−14.8
IT280393(14)	14.9 ± 1.3	39.9 ± 3.6	2.68	0.83 ± 0.5	0.71 ± 0.3	−14.5
IT280394(15)	13.5 ± 2.2	34.4 ± 2.3	2.55	0.79 ± 0.2	0.72 ± 0.0	−8.9
IT280395(16)	10.4 ± 0.4	44.5 ± 3.4	4.28	0.82 ± 0.2	0.48 ± 0.5	−41.5
IT280396(17)	9.9 ± 0.9	35.7 ± 4.7	3.61	0.83 ± 0.2	0.71 ± 0.3	−14.5
IT280397(18)	13.2 ± 2.1	37.4 ± 3.1	2.83	0.81 ± 0.1	0.70 ± 0.3	−13.6
IT280398(19)	11.7 ± 0.3	37.2 ± 3.8	3.18	0.80 ± 0.3	0.72 ± 0.2	−10.0
IT293143(20)	12.4 ± 1.7	35.4 ± 2.7	2.85	0.81 ± 0.4	0.73 ± 0.1	−9.9
IT293144(21)	12.5 ± 1.6	32.7 ± 0.9	2.62	0.81 ± 0.3	0.69 ± 0.2	−14.8
IT301979(22)	14.2 ± 1.1	40.7 ± 1.3	2.87	0.80 ± 0.2	0.64 ± 0.4	−20.0
IT301980(23)	10.4 ± 0.8	39.5 ± 6.3	3.80	0.81 ± 0.3	0.62 ± 0.4	−23.5
IT304044(24)	13.3 ± 2.2	33.8 ± 3.4	2.54	0.81 ± 0.1	0.76 ± 1.6	−6.2
Average	12.5 ± 1.3	38.0 ± 3.2	3.10	0.81 ± 0.2	0.66 ± 0.75	−18.1

). Additionally, the fold change was a minimum of 2.20 and a maximum of 4.97, showing differences depending on the genotypes. In a nutshell, the average electrolyte leakage content was 12.5% without NaCl treatment, but it increased to 38.0% with the application of 100 mM NaCl. To differentiate the salt tolerance levels among the systems used in this experiment, we conducted cluster analysis based on the measurements of electrolyte leakage content. This analysis unveiled the existence of four distinct clusters within the section subjected to 100 mM NaCl treatment (Figure 1). Within these clusters, IT262109, IT279432, and IT185735 were grouped together in the first cluster, demonstrating the highest degree of salt stress tolerance among the examined accessions. In contrast, IT188180 and IT135406 were categorized into the final cluster, indicating the lowest salt stress tolerance.

3.2. Changes in the Maximum Quantum Yield of Photosystem II According to NaCl Treatment

In this study, we assessed salt tolerance by examining the maximum quantum yield of photosystem II (PSII) in response to NaCl concentrations. The results consistently indicated that as the NaCl concentration increased, there was a corresponding decrease in PSII maximum quantum yield. For the highly salt-tolerant accessions, including IT262109, IT279432, and ITIT185735, the maximum quantum yield of photosystem II in plants subjected to the 100 mM NaCl treatment ranged from 0.78 to 0.79 (Table 1). Remarkably, these accessions displayed only a slight 3.7% reduction in PSII yield under the 100 mM NaCl treatment. In contrast, the weakly salt-tolerant accessions, IT188180 and IT135406, exhibited a maximum quantum yield of photosystem II ranging from 0.44 to 0.40. This corresponded to a more substantial decrease, ranging from 45.7% to 49.4%, compared with the untreated control (Table 1). This observation is significant, as it highlights the sensitivity of Fm, which reflects the maximum fluorescence value obtained in the dark-adapted state of the photosystem II reaction center. It serves as a sensitive indicator of the electron transfer level of PSII and can detect the impact of various abiotic and biotic stresses on plant photosynthetic function. The results underscore the clear correlation between salt tolerance and PSII performance, with highly salt-tolerant accessions maintaining their PSII function to a greater extent under salt stress compared with weakly salt-tolerant accessions.

3.3. Changes in Growth Characteristics According to NaCl Treatment

In this study, we investigated various growth characteristics in response to salt treatment. Specifically, the average above-ground live weight was 25.18 g without NaCl treatment, but it decreased to 19.63 g with the application of 100 mM NaCl. Additionally, the average below-ground live weight was 3.85 g without NaCl treatment, but it decreased to 2.95 g with the application of 100 mM NaCl. These figures represented decreases of 23.5% and 25.3% compared with the untreated group. Additionally, the average weight during drying for above-ground parts was 2.05 g without NaCl treatment, but it decreased to 1.63 g with the application of 100 mM NaCl. Moreover, the average weight during drying for below-ground parts was 0.20 g without NaCl treatment, but it decreased to 0.11 g with the application of 100 mM NaCl, indicating reductions of 21.8% and 49.3%, respectively, compared with the untreated control (

Table 2. Effects of different salt concentrations on fresh weight in Pak Choi (Brassica rapa subsp. chinensis) landraces from Korea.

Genotype	Fresh Weight (g)
	Scion			Rootstock
	Control	100 mM Salt	%	Control	100 mM Salt	%
IT112271(1)	23.19 ± 0.4	17.98 ± 0.4	−22.5	2.79 ± 0.6	2.28 ± 0.5	−18.3
IT135403(2)	9.23 ± 0.6	5.26 ± 0.7	−43.0	1.71 ± 0.4	1.17 ± 0.5	−31.6
IT135406(3)	25.72 ± 1.0	16.74 ± 0.4	−34.9	2.56 ± 1.0	1.84 ± 0.7	−28.1
IT135441(4)	14.81 ± 0.9	10.56 ± 0.9	−28.7	1.38 ± 0.5	0.99 ± 0.7	−28.3
IT166982(5)	34.79 ± 0.4	29.44 ± 0.4	−15.4	4.84 ± 0.6	3.78 ± 0.5	−21.9
IT166983(6)	15.76 ± 0.9	11.66 ± 0.7	−26.0	3.27 ± 1.0	2.16 ± 0.8	−33.9
IT185735(7)	31.14 ± 0.7	27.84 ± 0.4	−10.6	4.68 ± 0.7	3.73 ± 0.5	−20.3
IT188180(8)	39.40 ± 0.8	21.91 ± 0.9	−44.4	4.99 ± 0.7	3.45 ± 0.8	−30.9
IT188181(9)	55.52 ± 0.5	46.78 ± 0.5	−15.7	11.78 ± 0.3	8.84 ± 0.4	−25.0
IT210064(10)	32.79 ± 0.5	28.43 ± 0.6	−13.3	4.92 ± 0.5	3.84 ± 0.5	−22.0
IT262109(11)	25.9 ± 1.2	24.70 ± 1.0	−4.6	6.31 ± 1.1	5.43 ± 1.0	−13.9
IT279432(12)	12.23 ± 0.5	12.07 ± 0.6	−1.3	2.04 ± 0.3	1.96 ± 0.5	−3.9
IT280392(13)	37.72 ± 0.5	33.49 ± 0.5	−11.2	4.48 ± 0.5	3.87 ± 0.5	−13.6
IT280393(14)	14.95 ± 0.6	8.99 ± 0.7	−39.9	3.49 ± 0.7	2.39 ± 0.7	−31.5
IT280394(15)	18.68 ± 0.3	13.38 ± 0.4	−28.4	2.82 ± 0.3	2.34 ± 0.4	−17.0
IT280395(16)	17.56 ± 0.5	13.14 ± 0.4	−25.2	1.89 ± 0.4	0.87 ± 0.4	−54.0
IT280396(17)	11.79 ± 0.6	7.72 ± 0.8	−34.5	1.57 ± 0.8	0.87 ± 0.8	−44.6
IT280397(18)	31.75 ± 0.6	22.94 ± 0.5	−27.7	4.85 ± 0.7	3.84 ± 0.6	−20.8
IT280398(19)	11.84 ± 0.5	9.88 ± 0.7	−16.6	2.19 ± 0.5	1.78 ± 0.6	−18.7
IT293143(20)	25.51 ± 0.8	20.16 ± 0.9	−21.0	4.03 ± 1.0	3.05 ± 0.9	−24.3
IT293144(21)	16.21 ± 0.7	13.97 ± 0.6	−13.8	2.97 ± 0.5	2.51 ± 0.5	−15.5
IT301979(22)	15.70 ± 0.7	8.84 ± 0.8	−43.7	2.26 ± 0.7	1.01 ± 0.8	−55.3
IT301980(23)	43.78 ± 0.8	37.89 ± 0.8	−13.5	5.38 ± 0.7	4.76 ± 0.7	−11.5
IT304044(24)	38.42 ± 0.9	27.23 ± 0.6	−29.1	5.13 ± 0.7	3.97 ± 0.6	−22.6
Average	25.18 ± 0.7	19.63 ± 0.6	−23.5	3.85 ± 0.6	2.95 ± 0.6	−25.3

and

Table 3. Effects of different salt concentrations on dry weight in Pak Choi (Brassica rapa subsp. chinensis) landraces from Korea.

Genotype	Dry Weight (g)
	Scion			Rootstock
	Control	100 mM Salt	%	Control	100 mM Salt	%
IT112271(1)	1.86 ± 1.7	1.31 ± 2.6	−29.6	0.19 ± 2.7	0.08 ± 2.7	−57.9
IT135403(2)	0.98 ± 1.6	0.53 ± 3.3	−45.9	0.17 ± 3.6	0.07 ± 3.4	−58.8
IT135406(3)	1.89 ± 4.0	1.28 ± 4.3	−32.3	0.19 ± 3.6	0.04 ± 4.4	−78.9
IT135441(4)	1.44 ± 1.6	0.99 ± 3.9	−31.3	0.15 ± 3.8	0.03 ± 4.3	−80.0
IT166982(5)	3.13 ± 2.2	2.57 ± 3.7	−17.9	0.25 ± 3.0	0.13 ± 3.4	−48.0
IT166983(6)	1.29 ± 4.0	1.05 ± 3.5	−18.6	0.18 ± 3.7	0.09 ± 4.6	−50.0
IT185735(7)	2.98 ± 1.5	2.53 ± 3.6	−15.1	0.45 ± 4.7	0.32 ± 4.5	−28.9
IT188180(8)	3.67 ± 1.2	2.91 ± 2.3	−20.7	0.19 ± 3.8	0.06 ± 3.5	−68.4
IT188181(9)	4.29 ± 3.1	3.64 ± 4.3	−15.2	0.23 ± 3.8	0.13 ± 4.7	−43.5
IT210064(10)	2.08 ± 2.3	1.87 ± 3.7	−10.1	0.25 ± 2.9	0.11 ± 3.3	−56.0
IT262109(11)	2.15 ± 1.5	2.04 ± 3.3	−5.1	0.31 ± 2.7	0.28 ± 4.6	−9.7
IT279432(12)	0.93 ± 4.4	0.84 ± 4.3	−9.7	0.19 ± 1.7	0.15 ± 4.2	−21.1
IT280392(13)	2.52 ± 3.0	2.24 ± 4.5	−11.1	0.18 ± 3.2	0.12 ± 3.8	−33.3
IT280393(14)	1.11 ± 3.4	0.77 ± 4.2	−30.6	0.18 ± 3.4	0.08 ± 4.8	−55.6
IT280394(15)	1.41 ± 1.2	1.08 ± 2.5	−23.4	0.14 ± 2.5	0.09 ± 2.5	−35.7
IT280395(16)	1.35 ± 2.7	1.08 ± 4.7	−20.0	0.09 ± 4.9	0.02 ± 4.8	−77.8
IT280396(17)	0.88 ± 1.6	0.72 ± 4.9	−18.2	0.10 ± 3.1	0.05 ± 3.9	−50.0
IT280397(18)	2.42 ± 1.6	1.98 ± 2.6	−18.2	0.19 ± 3.6	0.08 ± 3.1	−57.9
IT280398(19)	1.10 ± 1.1	0.87 ± 3.1	−20.9	0.17 ± 3.5	0.08 ± 4.3	−52.9
IT293143(20)	1.81 ± 2.1	1.31 ± 4.6	−27.6	0.22 ± 3.3	0.17 ± 4.4	−22.7
IT293144(21)	1.21 ± 3.0	1.00 ± 3.9	−17.4	0.14 ± 5.0	0.09 ± 4.5	−35.7
IT301979(22)	1.25 ± 1.3	0.76 ± 1.9	−39.2	0.12 ± 4.0	0.08 ± 2.9	−33.3
IT301980(23)	3.94 ± 5.0	3.21 ± 4.2	−18.5	0.26 ± 3.3	0.11 ± 3.7	−57.7
IT304044(24)	3.57 ± 2.7	2.63 ± 4.4	−26.3	0.27 ± 3.6	0.08 ± 3.8	−70.4
Average	2.05 ± 2.4	1.63 ± 3.7	−21.8	0.20 ± 3.48	0.11 ± 3.9	−49.3

).

Furthermore, it was observed that the reduction in live weight varied significantly among different accessions under salt stress. For example, above-ground highly salt-tolerant accessions such as IT262109 and IT279432 exhibited only slight reductions of 4.6% and 1.3%, respectively, in live weight compared with the untreated control under 100 mM salt stress, indicating a relatively lower reduction rate. Conversely, weakly salt-tolerant accessions like IT188180 and IT135406 experienced substantial decreases of 34.9% and 44.4%, respectively, in live weight, indicating a higher reduction rate. Additionally, below-ground highly salt-tolerant accessions such as IT262109 and IT279432 exhibited only slight reductions of 13.9% and 3.9%, respectively, in live weight compared with the untreated control under 100 mM salt stress, indicating a relatively lower reduction rate. Conversely, weakly salt-tolerant accessions like IT188180 and IT135406 experienced substantial decreases of 30.9% and 28.1%, respectively, in live weight, indicating a higher reduction rate. Likewise, it was observed that the reduction in dry weight varied significantly among different accessions under salt stress. For example, highly salt-tolerant accessions such as IT262109 and IT279432 exhibited only slight reductions of 5.1% and 9.7%, respectively, in live weight compared with the untreated control under 100 mM salt stress, indicating a relatively lower reduction rate. Conversely, weakly salt-tolerant accessions like IT188180 and IT135406 experienced substantial decreases of 20.7% and 32.3%, respectively, in dry weight, indicating a higher reduction rate.

Salt stress had a detrimental effect on the development of Pak Choi, leading to a decrease in leaf number and leaf area (

Table 4. Effects of different salt concentrations on number of leaves and leaf area in Pak Choi (Brassica rapa subsp. chinensis) landraces from Korea.

Genotype	Number of Leaves			Leaf Area (mm2)
	Control	100 mM Salt	%	Control	100 mM Salt	%
IT112271(1)	9.3 ± 0.5	7.8 ± 0.6	−16.1	123.7 ± 6.9	84.7 ± 5.4	−31.5
IT135403(2)	7.6 ± 0.5	6.3 ± 0.3	−17.1	108.3 ± 7.9	65.5 ± 3.6	−39.5
IT135406(3)	11.2 ± 0.3	8.9 ± 0.3	−20.5	139.5 ± 6.7	75.1 ± 3.2	−46.2
IT135441(4)	8.4 ± 1.0	7.6 ± 0.5	−9.5	177.6 ± 12.9	160.9 ± 7.9	−9.4
IT166982(5)	12.5 ± 1.0	11.3 ± 1.0	−9.6	154.2 ± 10.1	137.5 ± 5.8	−10.8
IT166983(6)	9.7 ± 0.3	7.9 ± 0.3	−18.6	166.1 ± 6.7	110.9 ± 5.8	−33.2
IT185735(7)	7.9 ± 0.5	6.8 ± 0.5	−13.9	123.7 ± 9.7	94.2 ± 5.8	−23.8
IT188180(8)	13.4 ± 0.6	9.2 ± 0.3	−31.3	173.3 ± 13.6	96.7 ± 6.4	−44.2
IT188181(9)	11.6 ± 0.3	10.7 ± 0.3	−7.8	215.6 ± 4.0	200.9 ± 6.6	−6.8
IT210064(10)	8.4 ± 1.0	7.3 ± 0.5	−13.1	182.5 ± 3.6	153.6 ± 5.8	−15.8
IT262109(11)	10.1 ± 0.9	9.8 ± 0.3	−2.97	148.3 ± 10.9	138.1 ± 5.8	−6.9
IT279432(12)	9.5 ± 0.5	9.2 ± 0.3	−3.16	117.8 ± 10.5	109.3 ± 5.5	−7.2
IT280392(13)	11.3 ± 0.5	9.9 ± 0.7	−12.4	163.3 ± 10.2	148.0 ± 6.0	−9.4
IT280393(14)	9.5 ± 0.7	7.8 ± 1.1	−17.9	127.4 ± 5.5	75.5 ± 4.4	−40.7
IT280394(15)	12.3 ± 0.8	10.7 ± 0.6	−13.0	112.9 ± 10.9	86.8 ± 3.6	−23.1
IT280395(16)	13.1 ± 0.5	11.3 ± 0.4	−13.7	109.4 ± 14.3	81.9 ± 4.5	−25.1
IT280396(17)	9.2 ± 0.6	7.9 ± 0.3	−14.1	106.5 ± 11.6	73.6 ± 8.0	−30.9
IT280397(18)	14.8 ± 0.6	12.6 ± 0.6	−14.9	119.5 ± 6.8	80.8 ± 5.0	−32.4
IT280398(19)	13.2 ± 0.4	11.9 ± 0.7	−9.9	86.1 ± 2.4	68.8 ± 9.6	−20.1
IT293143(20)	10.7 ± 0.5	9.6 ± 0.5	−10.3	134.2 ± 19.3	109.2 ± 3.6	−18.6
IT293144(21)	9.7 ± 0.5	8.4 ± 0.3	−13.4	175.7 ± 5.9	144.2 ± 8.0	−17.9
IT301979(22)	8.6 ± 0.2	7.3 ± 0.3	−15.1	138.6 ± 8.8	87.4 ± 8.6	−36.9
IT301980(23)	11.8 ± 0.2	10.7 ± 0.3	−9.3	189.2 ± 8.1	172.2 ± 3.9	−9.0
IT304044(24)	12.6 ± 0.2	10.6 ± 0.3	−15.9	167.4 ± 6.4	115.7 ± 4.3	−30.9
Average	10.7 ± 0.5	9.23 ± 0.5	−13.5	144.2 ± 8.9	111.31 ± 5.7	−23.8

). On average, the number of leaves in the 100 mM NaCl treatment group decreased by 13.5%, while the leaf area decreased by 23.8% compared with the untreated condition. Interestingly, similar to the findings on live weight, the decrease in leaf number and area exhibited a wider range of variation among different accessions (Table 4). Under salt stress conditions, the decrease in leaf number for salt-tolerant accessions (IT262109 and IT279432) ranged from 2.97% to 3.16%. Conversely, in weakly resistant accessions (IT188180 and IT135406), the range of decrease was relatively large, ranging from 20.5% to 31.3%. A similar trend was observed in leaf area, with stronger salt-tolerant accessions showing less change in leaf area (

Table 5. Changes in measurements of maximum quantum yield of photosystem II (Fv/Fm) and growth response according to clusters separated with cluster analysis under salt concentration. The significance of each group was tested using Duncan’s multiple range test at a sig-nificance level of p < 0.05.

100 mM Salt	Fv/Fm (%)	Fresh Weight (%)		Dry Weight (%)		Number of Leaves (%)	Leaf Area (%)
		Scion	Rootstock	Scion	Rootstock
Cluster 1	3.70 c	5.50 b	12.70 b	9.97 b	19.90 c	6.68 c	112.63 b
Cluster 2	11.73 b	25.47 a	26.32 ab	21.92 a	44.08 b	14.22 ab	25.77 ab
Cluster 3	14.28 b	25.33 a	25.83 ab	24.10 a	52.90 ab	12.70 bc	22.00 ab
Cluster 4	43.13 a	31.13 a	30.68 a	23.53 a	66.57 a	20.42 a	33.67

).

4. Discussion

Electrolyte leakage analysis serves as a valuable tool for assessing plant stress responses [29,30,31,32]. This study has effectively demonstrated its utility in evaluating salt tolerance in Pak Choi. The observed increase in electrolyte leakage at higher NaCl concentrations indicates that salt stress caused damage to the cell membranes of the plants. The categorization of accessions into four clusters based on electrolyte leakage in relation to salt tolerance offers valuable insights into the genetic diversity within Pak Choi populations. This information can be harnessed in breeding programs aimed at developing salt-tolerant varieties of Pak Choi. Moreover, this variability in salt tolerance can be attributed to genotype-specific differences in lethal salt concentrations [33].

The change in Fv/Fm, a measure of photosystem II (PSII) photochemical efficiency, can serve as a predictive indicator of its impact on the photosynthetic machinery. Fv/Fm is widely recognized as a vital gauge for evaluating a plant’s stress response to environmental conditions, with typical Fv/Fm values ranging between 0.80 and 0.84 in healthy plants [34,35]. The decrease in PSII maximum quantum yield under salt stress highlights the adverse effects of elevated NaCl concentrations on photosynthetic efficiency. Photosynthesis is a fundamental process crucial for plant growth and productivity, and any disruption in this process can significantly affect overall plant health. The varying degrees of PSII yield reduction observed among different accessions in the current study underscore the genetic diversity in salt tolerance. This valuable information can guide the identification and utilization of genotypes demonstrating superior salt tolerance in agricultural practices. Interestingly, previous studies on cucumber and tomato plants subjected to salt stress have also reported significant decreases in Fv/Fm, indicating impaired photosynthetic function due to high salt concentrations. These findings align with our study results [36,37,38], further emphasizing the detrimental effect of salt stress on photosynthesis.

This study’s examination of growth characteristics under salt stress highlights the diverse impact of salt on plant development. Notably, below-ground dry weight experienced a significant impact, while above-ground live weight, below-ground live weight, and weight changes during drying were less affected. Salt stress responses in plants involve intricate physiological and chemical interactions, making it challenging to assess salt tolerance solely based on external plant characteristics after salt treatment. Nevertheless, prior research has consistently shown negative correlations between increasing NaCl concentrations and changes in leaf area, leaf number, above-ground and below-ground fresh weight, and dry weight [39,40,41]. In our study, a similar pattern emerged regarding leaf area, with more salt-tolerant accessions exhibiting less change in leaf area. These findings align with previous studies documenting an overall reduction in growth in various plant species, including both food and horticultural crops, when exposed to salt stress [42,43,44]. The variation in response among accessions underscores the critical role of genetic diversity in salt tolerance. The decreases in leaf number, leaf area, and leaf weight further illustrate the detrimental effects of salt stress on overall plant growth. The identification of highly salt-tolerant accessions, such as IT262109 and IT279432, presents valuable genetic resources for breeding programs aimed at developing salt-tolerant Pak Choi varieties. High salt concentrations can impede water absorption due to intracellular osmotic pressure, lead to stomatal closure, disrupt cell division and elongation, and result in reduced leaf area. Consequently, these factors can hinder growth by inhibiting efficient water absorption [43,45,46].

5. Conclusions

Salt stress is a significant limiting factor that affects both the yield and quality of Pak Choi. This study elucidates the diverse responses of Pak Choi plants to salt stress and identifies promising genetic resources (‘IT262109’, ‘IT279432’, and ‘IT185735’) for breeding salt-tolerant varieties. The combination of electrolyte leakage analysis, PSII yield measurements, and growth characteristic assessments provides a comprehensive understanding of salt tolerance in this crop, contributing to the development of resilient agricultural practices in salt-affected regions.