Drought Tolerance Evaluation and Growth Response of Chinese Cabbage Seedlings to Water Deficit Treatment

Abstract

Drought is a significant climatic factor that significantly affects the production of Chinese cabbage, a crop that is highly susceptible to drought stress. The development and cultivation of drought-tolerant varieties could be a viable strategy to minimize the damage caused by climate change and ensure stable production of Chinese cabbage. This requires the implementation of technologies for early evaluation and selection of a plethora of resources. In this study, we screened 100 varieties and breeding resources for drought tolerance under a water deficit treatment at the seedling stage. We also evaluated the growth response of Chinese cabbage varieties and breeding resources under water deficit treatment and selected drought-tolerant Chinese cabbage genotypes. We confirmed that the visual score for wilting, which evaluates the wilting response during the recovery process of Chinese cabbage seedlings through water deficit treatment and re-watering, can be used as an indicator for evaluating tolerance to drought stress. The visual score for wilting showed a high correlation with major traits representing drought tolerance. Our findings highlight the need for an integrated approach that considers various environmental conditions, varieties, and lines to select and develop drought-tolerant varieties. We selected ‘18-FH112-1’ and ‘18-FH112-1-2’ among others, and these germplasms will be useful resources for drought tolerance breeding. This study provides a foundation for future efforts to develop drought-tolerant Chinese cabbage varieties, thereby contributing to the stable production of this crucial crop.

1. Introduction

The primary ingredient of kimchi, Chinese cabbage (Brassica rapa L. ssp. pekinensis), is predominantly cultivated in autumn for kimjang (kimchi-making season). Chinese cabbage is a cool-season vegetable that thrives best in relatively cool climates, such as autumn, at temperatures of 18–20 °C. The optimal temperature for heading is 15–18 °C [1]. With advancements in cultivation technology, varieties that can be grown in summer and winter have been developed, thereby enabling year-round production. However, under high-temperature conditions exceeding the optimal growth temperature, the growth of Chinese cabbage deteriorates and physiological disorders and pest infestations occur frequently, resulting in decreased yield [2].

In the high-temperature period of summer, the cultivation of Chinese cabbage is nearly impossible because of poor growth and physiological disorders. The summer cultivation of Chinese cabbage is carried out in relatively cool areas with large diurnal temperature ranges, such as sub-alpine areas 400–600 m above sea level and alpine regions above 600 m. However, recent climate change and extreme weather conditions (heatwaves, droughts, heavy rains, etc.) have made it difficult to stabilize Chinese cabbage production, particularly during summer [2,3,4].

Chinese cabbage, which is composed of 90–95% water, grows vigorously in a short period of time and requires a relatively large amount of water. It is susceptible to drought and if drying occurs during the early growth stage, leaf development and growth are inhibited, causing a drastic decrease in yield. The period when most water is required is the initial heading stage, which is approximately 40–50 days after sowing [5].

If proper irrigation facilities are not equipped and reliance is placed on natural rainfall, or if it is difficult to secure irrigation water owing to prolonged drought, even with irrigation facilities, crops are susceptible to drought damage. Drought induces crop dehydration, soil erosion, decline in fertility, and other adverse effects [6]. This leads to a vicious cycle of decreased Chinese cabbage production, insufficient supply, and increased prices.

Drought stress caused by high temperatures and drought affects the growth and physiological responses of Chinese cabbage, thereby reducing its yield and quality [7,8]. Drought stress slows the growth rate of Chinese cabbage, decreases the size and number of leaves, and in severe cases, causes leaf yellowing, wilting, and necrosis [9]. It can easily cause physiological disorders and pest damage owing to increased levels of stress-related hormones and reactive oxygen species (ROS), decreased photosynthesis, and tipburn caused by inhibited calcium absorption and movement due to root damage [10,11]. According to a report by Lee et al. [12], when drought stress continued for two weeks during the cultivation period, the leaf tissue of Chinese cabbage collapsed. When it was continued for more than four weeks, the yield decreased by more than 60%.

The development and cultivation of drought-tolerant varieties to reduce damage caused by climate change could be one way to contribute to the stable production of various crops, including Chinese cabbage [13]. Chinese cabbage shows different mechanisms of reaction and degrees of resistance to drought stress depending on the variety [14]. It can be exposed to drought stress throughout the cultivation period, from transplanting to harvest, and the degree of response varies depending on the degree and duration of stress and growth stage.

To develop drought-tolerant Chinese cabbage varieties, it is necessary to utilize technologies for the early evaluation and selection of various resources. Some studies have identified suitable indices and traits have been identified for the selection of drought-tolerant resources [15,16]. In this study, we evaluated the drought tolerance and growth response of Chinese cabbage varieties and breeding resources under water deficit treatment and selected drought-tolerant Chinese cabbage genotypes.

2. Materials and Methods

2.1. Screening for Drought Tolerance through Water Deficit Treatment of Chinese Cabbage Seedlings

2.1.1. Plant Materials

To evaluate drought tolerance in Chinese cabbage under water deficit treatment at the seedling stage, commercial Chinese cabbage varieties and breeding resources were used. Drought tolerance evaluation was conducted twice in spring (April to June), and autumn (August to November), evaluating 50 varieties and resources each time. On 15 April and 11 October 2022, 18 seeds per variety or resource were sown. The seeds were sown in a horticultural growing media-filled 72-cell plug tray (280 mm width × 540 mm length × 48 mm height, 6 cells horizontally × 12 cells vertically, cell capacity 34 mL, Beomnong Co., Ltd., Jeongeup-si, Republic of Korea) and were watered until the growing media was sufficiently wet. The plug trays were placed on a cultivation bench in a Venlo-type glass greenhouse (38 m length × 24 m width, 4.5 m side height) and were irrigated 1–2 times daily depending on the weather and the growth stage of the crop. Starting from the third week after sowing, liquid fertilizers (for leafy vegetables, Daeyu Co., Seoul, Republic of Korea and Hyponex N-P-K = 6-10-5, Hyponex Japan Co., Ltd., Osaka, Japan) were diluted 1000 times and watered once and twice at four-day intervals. Four weeks after sowing, Chinese cabbage seedlings with 4–5 unfolded leaves were used as experimental materials for drought tolerance screening.

2.1.2. Water Deficit Treatment and Investigation for Drought Tolerance Seedling Test

A water deficit treatment was applied to four-week-old Chinese cabbage seedlings with 4–5 unfolded leaves. The period of water deficit treatment was set to seven days, adjusting the period according to the wilting response of the plant at the time of treatment. After the water deficit treatment, the degree of wilting was evaluated daily. When the degree of wilting of the varieties or resources was such that the entire plant body wilted and only the new leaves at the growing point were alive (visual score for wilting: 4) or dead (visual score for wilting: 5), watering was resumed and the degree of wilting was evaluated daily to assess the degree of recovery. In spring, a four-day water deficit treatment was conducted from 13 May to 17 May 2022, followed by a 22-day recovery response investigation until June 8 after resuming watering. In autumn, a nine-day water deficit treatment was conducted from November 7 to November 16, followed by a nine-day recovery response investigation until November 25, after resuming watering.

The degree of wilting for each individual was divided into six stages: no symptoms (0), mild wilting (1), up to severe wilting (4), and death (5). The degree of drought tolerance was determined by calculating the evaluation value for each individual using the following equation:

visual score for wilting = [(0 × ‘stage 0’ plant number) + (1 × ‘stage 1’ plant number) + (2 × ‘stage 2’
plant number) + (3 × ‘stage 3’ plant number) + (4 × ‘stage 4’ plant number) + (5 × ‘stage 5’ plant
number)]/total number of treated plants

The lower the value, the stronger the drought stress tolerance. A schematic diagram of the drought tolerance seedling test is shown in Figure 1.

2.2. Water Potential and Growth of Chinese Cabbage Seedlings with Water Deficit Treatment

2.2.1. Plant Materials

Six Chinese cabbage varieties were used to evaluate drought tolerance by water deficit treatment at the seedling stage. The six varieties were four varieties developed at the National Institute of Horticultural and Herbal Science (‘Wongyo39ho’, ‘Wongyo42ho’, ‘Wongyo47ho’, ‘Wongyo49ho’) and two commercial varieties (‘Bulam3ho’ (Farm Hannong, Seoul, Republic of Korea), ‘CRmatjjang’ (Farm Hannong, Seoul, Republic of Korea)). On 26 February 2021, 72 seeds per variety were sown. The seeds were sown in a horticultural growing media-filled 72-cell plug tray (280 mm width × 540 mm length × 48 mm height, 6 cells horizontally × 12 cells vertically, cell capacity 34 mL, Beomnong Co., Ltd., Jeongeup-si, Republic of Korea) and were watered until the growing media was sufficiently wet. The plug trays were placed on a cultivation bench in a Venlo-type glass greenhouse (38 m length × 24 m width, 4.5 m side height) and were irrigated 1–2 times daily depending on the weather and the growth stage of the crop. Starting from the third week after sowing, liquid fertilizers (for leafy vegetables, Daeyu Co., Seoul, Republic of Korea and Hyponex N-P-K = 6-10-5, Hyponex Japan Co., Ltd., Osaka, Japan) were diluted 1000 times and watered once and twice at four-day intervals. Four weeks after sowing, Chinese cabbage seedlings with 4–5 unfolded leaves were used as the experimental materials.

2.2.2. Evaluation of Growing Media and Leaf Water Potential and Growth in Response to Water Deficit Treatment

For water stress treatment, four-week-old Chinese cabbage seedlings with 4–5 unfolded leaves were subjected to water stress. Water deficit treatment was conducted for five days, from 26 March to 31 March 2021, followed by re-watering. During the 5-day water deficit treatment period and for 2 days after re-watering, the YII (the effective photochemical yield of PS II) value of the Chinese cabbage leaves (2nd or 3rd leaf) was measured using a chlorophyll fluorescence meter (Mini PAM II, Heinz Walz GmbH, Effeltrich, Germany). The water potentials of the growing media and leaves were measured using a water potential meter (WP4; Decagon Devices, Inc., Pullman, WA, USA). The growing media from the plug tray cells was mixed well after removing the root and the water potential of the growing media was measured. A leaf disk (20 mm wide × 10 mm long) was collected from the middle part of the 3rd leaf of the Chinese cabbage using a razor blade, and the water potential was measured. Measurements were performed in triplicates for each treatment.

During the treatment period, three plants per treatment were collected daily, and the fresh weight of the aboveground and underground parts, number of leaves, SPAD values (using a chlorophyll meter, SPAD-502, Konica Minolta, Tokyo, Japan), and leaf area (using a leaf area meter, LI-3100, Li-cor Inc., Lincoln, NE, USA) were measured. After the investigation, the samples were dried in a hot air dryer (DS-80-3, Dasol Science, Hwaseong-si, Gyeonggi-do, Republic of Korea) set at 80 °C for more than three days, and the dry weight was measured. The functional ratio (100 − (dry weight/fresh weight × 100)), aboveground/underground ratio, and relative growth rate (RGR) were calculated using the measured fresh weight and dry weight values. During the treatment period, the degree of wilting for each individual was divided into six stages: no symptoms (0), mild wilting (1), up to severe wilting (4), and death (5). The degree of drought tolerance was determined by calculating the evaluation value for each individual using the aforementioned formula.

2.3. Growth of Chinese Cabbage under Greenhouse Cultivation with Water Deficit Treatment

2.3.1. Plant Materials

Eight Chinese cabbage varieties were used to evaluate growth under water deficit conditions during Chinese cabbage cultivation. The eight varieties were ‘Asian seedlings’ (Asian seedlings, Seoul, Republic of Korea), ‘Bulam3ho’ (Farm Hannong Co, Ltd., Seoul, Republic of Korea), ‘CRmatjjang’ (Farm Hannong Co, Ltd., Seoul, Republic of Korea), ‘Gyeoul-daejang’ (PPS, Yongin-si, Gyeonggi-do, Republic of Korea), ‘Gyeoul-wanggug’ (Nongwoo Bio, Suwon-si, Gyeonggi-do, Republic of Korea), ‘Hwipalam’ (Sakata, Seoul, Republic of Korea), ‘Smart-baechu’ (PPS, Yongin-si, Gyeonggi-do, Republic of Korea), and ‘Ssam-irang’ (Nongwoo Bio, Suwon-si, Gyeonggi-do, Republic of Korea). On 21 July 2022, 30 seeds per variety were sown in a 72-cell plug tray filled with horticultural growing media (Heungnong Bio No. 1; Farm Hannong Co., Ltd., Seoul, Republic of Korea). The growing media was watered until it was sufficiently wet. The plug trays with the sown Chinese cabbage seeds were placed on a cultivation bench in a Venlo-type greenhouse (38 m length × 24 m width, 4.5 m side height) and were irrigated 1–2 times daily depending on the weather and the growth stage of the crop. During the nursery period, liquid fertilizer (for leafy vegetables, Daeyu Co., Seoul, Republic of Korea) was diluted 1000 times and watered from the bottom according to the growth conditions. At 26 days after sowing, Chinese cabbage seedlings with 4–5 unfolded leaves were transplanted into a greenhouse.

2.3.2. Greenhouse Cultivation and Water Deficit Treatment

The research was carried out in the greenhouse at the National Institute of Horticultural and Herbal Science located in Wanju (35.8° N, 127.1° W, altitude 56 m), Jeollabuk-do, Republic of Korea. During the treatment period, the average temperature and daily light integral (DLI) in the greenhouse were 21.2 °C and 21 mol∙m⁻²∙day⁻¹. Chinese cabbage seedlings, 26 days after sowing, were cultivated by transplanting them into a single greenhouse (40 m length × 7 m width, 3.7 m height, 1.7 m side height, 280 m²) on 16 August 2022. Before transplanting, a compound fertilizer was applied and the soil was mixed well. Five rows of mounds, each with a width of 0.8 m, were made and each row was mulched with a black plastic film. Drip irrigation tape was placed under the plastic mulching film in each row. The distance between the plants within a row was 0.5 m. The middle row was left empty, and both sides were used as the control and water deficit treatment groups. The test plot layout was a randomized block design with two replicates, with each mound replicated. The space between the plant bodies was 0.5 m, and five plants per treatment were transplanted into each replicate. After transplanting, sufficient water was provided around the plant to facilitate the initial root establishment. Drip irrigation was carried out considering the weather and the growth status of the plant, and a supplementary fertilizer (liquid fertilizers for leafy vegetables, Daeyu Co., Seoul, Republic of Korea) was supplied. For the water stress treatment, water deficit treatment was carried out for 54 days from one month after transplanting (September 16) to the harvest date (November 9). In the control group, water management was performed considering the weather and growth status. The irrigation day was determined according to the condition of the soil and plant conditions, and irrigation was performed for approximately 30 min.

During the water deficit treatment period, a soil moisture sensor (S-SMC-M005, Onset Computer Corp., Bourne, MA, USA) equipped with a data logger (HOBO data logger, H21-USB, Onset Computer Corp., Bourne, MA, USA) was installed near the plant (0.1 m depth from the ground) to measure soil moisture content. Data were measured and recorded at 30-min intervals and were collected. Leaf gas exchange was measured using a portable photosynthesis system (Li 6800, Li-Cor Co., Inc., Lincoln, NE, USA). Illumination was supplied to the leaves using an LED light source (red/blue = 69: 31). Before measurement, the leaves were acclimated in the leaf chamber for approximately 5 min at a constant temperature of 25 °C, CO₂ concentration of 400 μmol∙mol, and a PPF of 500 μmol∙m⁻²∙day⁻¹. Gas exchange parameters were determined for a fully expanded new leaf. Chinese cabbages cultivated for 84 d were harvested and investigated on November 9. The fresh mass of shoot and heads, number of outer and inner leaves (number of formed leaves), SPAD values (using a chlorophyll meter, SPAD-502, Konica Minolta, Tokyo, Japan), and occurrence of physiological damage (deficiency of calcium and boron) were investigated.

2.4. Environmental Data Measurement

During the treatment period, a data logger (WatchDog 1000 Series Micro Stations, Spectrum Technologies, Inc., Aurora, IL, USA) equipped with a temperature and humidity sensor, a soil temperature sensor (External (soil) temperature sensor, Spectrum Technologies, Inc., Aurora, IL, USA), and a photosynthetically active radiation (PAR) sensor (LightScout Quantum Light sensor, Spectrum Technologies, Inc., Aurora, IL, USA) were installed near the plants (0.3 m height from the ground) to measure the temperature, soil temperature, relative humidity, and light intensity inside the greenhouse. Data were measured and recorded at 30-min intervals and were collected. The light intensity measured at different times of the day was used to calculate DLI.

2.5. Statistical Analysis

The collected data were analyzed using SigmaPlot (v.11, Grafiti, Palo Alto, CA, USA) and the SAS statistical program (v.9.4, SAS Institute Inc., Cary, NC, USA). The data were subjected to one and two-way analyses of variance. Levene’s test was used for homogeneity of variances. Duncan’s multiple range test was performed at p ≤ 0.05 on each of the variables measured.

3. Results

3.1. Screening for Drought Tolerance through Water Deficit Treatment of Chinese Cabbage Seedlings

To assess the drought tolerance of Chinese cabbage, we tested marketable Chinese cabbage cultivars and resources over two seasons, spring (April–June) and autumn (August–November), through water deficit treatment at the seedling stage. Despite the presence of facilities with roofs and side ventilation, there were differences in the facility environment depending on the testing period. During the treatment period, the average temperature and DLI in the facility were 21.3 °C, 14 mol∙m⁻²∙day⁻¹ in spring, and 17.2 °C, 8 mol∙m⁻²∙day⁻¹ in autumn, respectively. The average temperature in autumn was 4.2 °C lower than in spring, and the DLI was 58% of the spring level, 6 mol∙m⁻²∙day⁻¹ lower (Figure 2).

During the seedling stage of Chinese cabbages in the spring (April to June), there were cultivars and lines such as ’Wongyo20040ho’ and ‘IT260822’ that showed severe wilting (visual score for wilting: 4) or death (visual score for wilting: 5) from the day after treatment. However, cultivars and lines such as ‘Smart Baechu’ and ‘18-FH112-1’ showed a slower wilting response (Figure 3A). By the fourth day of treatment, most of the treated cultivars and lines had either completely wilted (visual score for wilting: 4) or reached a state of death (visual score for wilting: 5). There were differences in the recovery responses between the cultivars and lines after re-watering on the fourth day of treatment. ‘Wongyo20040ho’ and ‘IT260822’ failed to recover and died, whereas ‘Smart Baechu’ and ‘18-FH112-1’ gradually recovered, with the plants slightly wilting (visual score for wilting: 1) or only a few leaves wilting (visual score for wilting: 2), and grew normally.

The degree of wilting due to the treatment varied depending on the testing period, and there were differences in the treatment period, re-watering time, and recovery response investigation period. This is thought to be due to the differences in the facility environment depending on the testing period. The wilting response appeared slower in autumn (November) when the facility temperature and light were relatively lower than those in spring (Figure 3B). Until the fourth day of drought, there were almost no wilting symptoms, which appeared on the fifth day. It was only on the ninth day of treatment, five days longer than that in spring, that the whole plant wilted or died, and the degree of wilting was lower than that in spring. The recovery speed after re-watering was faster than that in the spring. In spring, it took about 20 days for cultivars or lines with strong recovery power to recover to visual scores for wilting 1 or 2, whereas in autumn, it took about 10 days to recover to a similar level.

Figure 4 shows the distribution of the average visual score for wilting from the beginning of treatment to the end of the recovery response investigation, the visual score for wilting on the seventh day after re-watering (spring, 11 days after water deficit treatment (DAT), autumn, 15 DAT), and the end of the recovery response investigation (spring 26 DAT, autumn, 18 DAT) for the cultivars or lines tested for drought tolerance (50 in spring and 68 in autumn). In spring, most lines or cultivars died a week after treatment and re-watering. However, after another two weeks, some lines or cultivars recovered, with new leaves unfolding at the growth point (visual score for wilting 4), so the average visual score for wilting for most lines or cultivars was 4. In contrast, in autumn, most lines or cultivars recovered on the seventh and eighteenth day after treatment and re-watering, and the distribution of visual score values ranged from 0 to 4, with a maximum of 3. Strong water stress was imposed in spring rather than in autumn, and a strong selection was performed.

Table 1. The visual score for wilting of Chinese cabbage cultivars and breeding lines influenced by water deficit treatment in spring (May to June).

Cultivar/Breeding Line	Company/Organization	Visual Score for Wilting
		26 DAT z	11 DAT	Average
Smart-baechu	PPS	1.28	3.28	3.20
Ssam-ilang	Nongwoo Bio	1.61	3.44	3.49
18-FH112-1	NIHHS	1.89	3.33	3.26
AVRDC-KJH-1985-100390	NAAS	3.33	4.39	4.46
Chunssamhwang 51 Cabbage	Asian seedlings	3.72	4.33	4.06
Wongyo20037ho	NIHHS	3.77	4.31	4.11
Sangjang-gun	Sakata Korea	3.83	4.61	4.34
13-2-10	NIHHS	3.94	4.61	4.44
15-CDY-B3-1	NIHHS	3.94	4.22	4.19
CRmatjjang	Farm Hannong	4.06	4.44	4.25
Dodam-baechu	The Kiban	4.20	5.00	4.79
Summer Star-baechu	Farm Hannong	4.22	4.67	4.53
12-2-3	NIHHS	4.28	5.00	4.65
Hwipalam	Sakata Korea	4.28	4.67	4.32
Gaeul-daejang	PPS	4.29	4.89	4.49
18-BD85	NIHHS	4.33	4.83	4.56
Asia-ppuli-baechu	Asian seedlings	4.33	4.89	4.88
Wongyo20042ho	NIHHS	4.33	5.00	4.81
Bulam3ho	Farm Hannong	4.44	4.72	4.46
Bom-wang-gug	Nongwoo Bio	4.56	5.00	4.73
Lyeoggang	Nongwoo Bio	4.62	5.00	4.82
Gaeul-yangban	Dana Seed	4.67	5.00	4.64
Wongyo20038ho	NIHHS	4.67	5.00	4.96
Wongyo20039ho	NIHHS	4.72	5.00	4.86
Wongyo20044ho	NIHHS	4.72	4.94	4.85
Nolang-gimchi-baechu	Dana Seed	4.89	5.00	4.86
Cheongna	Dana Seed	4.89	5.00	4.77
Cheongnam	Dana Seed	4.89	5.00	4.75
Cheongsaem-baechu	Heungnong Seed	4.89	5.00	4.88
Matnan-beta	The Kiban	4.94	5.00	4.83
Wongyo20049ho	NIHHS	4.94	5.00	4.75
12-2-36	NIHHS	5.00	5.00	4.78
CHN-YAAS-2010-10	NAAS	5.00	5.00	5.00
Gyeoul-daejang	PPS	5.00	5.00	5.00
Gyeoul-wanggug	Nongwoo Bio	5.00	5.00	5.00
Geumbich-cheonglog	The Kiban	5.00	5.00	4.84
Namdogain	The Kiban	5.00	5.00	4.89
Yeong-gwang	Sakata Korea	5.00	5.00	4.89
Yeongung-sidae	The Kiban	5.00	5.00	4.89
Wongyo20040ho	NIHHS	5.00	5.00	4.89
Wongyo20047ho	NIHHS	5.00	5.00	4.87
Josaeng-wanggug	Nongwoo Bio	5.00	5.00	4.89
Jongga-bom	Nongwoo Bio	5.00	5.00	4.89
Chungkwang	Dana Seed	5.00	5.00	4.78
Chuseol	Koregon	5.00	5.00	4.89
Chupoong	Sakata Korea	5.00	5.00	4.88
Paldo-janggun	Asian seedlings	5.00	5.00	4.89
Highstar-baechu	Farm Hannong	5.00	5.00	4.89
Homerun-baechu	PPS	5.00	5.00	4.89

^z days after water deficit treatment.

and

Table 2. The visual score of Chinese cabbage cultivars and breeding lines for wilting influenced by water deficit treatment in autumn (November).

Cultivar/Breeding Line	Company/Organization	Visual Rates for Wilting
		18 DAT z	15 DAT	Average
Wongyo20042ho	NIHHS	0.19	0.63	1.91
22-DA88	NIHHS	0.28	0.28	2.46
18-FH112-1-2	NIHHS	0.31	0.29	1.53
22-DA91	NIHHS	0.31	0.31	2.07
22-DA72	NIHHS	0.50	0.61	2.81
22-DA83	NIHHS	0.50	1.06	2.08
18-FH112-1	NIHHS	0.56	0.56	2.20
18-FH112-1-1	NIHHS	0.56	0.63	1.64
18-FH112-1-3	NIHHS	0.72	0.63	1.86
22-DA84	NIHHS	0.83	0.56	2.09
22-DA87	NIHHS	1.00	1.00	2.61
22-DA82	NIHHS	1.39	1.39	2.70
22-DA92	NIHHS	1.39	1.78	2.77
22-DA90	NIHHS	1.89	2.17	3.13
22-DA68	NIHHS	2.06	2.28	3.74
22-DA85	NIHHS	2.11	2.22	3.43
22-DA86	NIHHS	2.17	2.39	3.19
22-DA89	NIHHS	2.24	3.29	3.79
22-DA100	NIHHS	2.35	2.47	3.44
22-DA95	NIHHS	2.39	2.29	3.59
Wongyo20039ho	NIHHS	2.50	2.50	3.73
Wongyo20037ho	NIHHS	2.56	2.67	3.34
Wongyo20049ho	NIHHS	2.56	2.44	3.81
Gyeouldaejang	PPS	2.65	2.94	3.90
15-CDY-B3-1-1	NIHHS	2.83	2.83	4.04
15-CDY-B3-1-2	NIHHS	2.83	2.83	3.91
Wongyo20047ho	NIHHS	2.88	3.59	4.06
22-DA93	NIHHS	3.06	3.22	4.06
Wongyo20044ho	NIHHS	3.11	3.00	3.74
22-DA94	NIHHS	3.19	3.44	3.93
22-DA101	NIHHS	3.29	3.29	3.95
22-DA64	NIHHS	3.39	4.00	4.26
22-DA61	NIHHS	3.67	3.78	4.37
Hwipalam	NIHHS	3.67	3.94	4.02
Ssamilang	Nongwoo Bio	3.67	3.83	4.35
22-DA96	NIHHS	3.72	3.83	4.21
22-DA97	NIHHS	3.72	4.11	4.30
15-CDY-B3-1-3	NIHHS	3.83	3.94	4.19
Wongyo20040ho	NIHHS	3.83	4.33	4.36
22DA99	NIHHS	3.89	3.17	4.10
22DA63	NIHHS	4.00	3.67	4.33
Gyeoulwanggug	Nongwoo Bio	4.00	4.22	4.42
Smartbaechu	PPS	4.00	4.00	4.27
Asiappulibaechu	Asian seedlings	4.22	4.50	4.40
Wongyo20038ho	NIHHS	4.22	4.44	4.52
18-BD85-1	NIHHS	4.33	4.39	4.52
22DA69	NIHHS	4.33	4.44	4.48
18-BD85-2	NIHHS	4.44	4.50	4.61
22-DA67	NIHHS	4.44	4.72	4.60
22-DA54	NIHHS	4.56	4.61	4.61
22-DA73	NIHHS	4.56	4.67	4.64
Bulam3ho	Farm Hannong	4.56	4.56	4.43
22-DA62	NIHHS	4.67	4.67	4.64
22-DA70	NIHHS	4.67	4.78	4.68
22-DA71	NIHHS	4.67	4.83	4.67
22-DA98	NIHHS	4.72	4.67	4.56
22-DA65	NIHHS	4.78	5.00	4.66
CRmatjjang	Farm Hannong	4.79	4.78	4.52
22-DA66	NIHHS	5.00	5.00	4.75

^z days after water deficit treatment.

show the average visual score for wilting from the beginning of the treatment to the end of the recovery response investigation, the visual score for wilting on the seventh day after re-watering (spring, 11 DAT, autumn, 15 DAT), and the end of the recovery response investigation (spring, 26 DAT, autumn, 18 DAT) for the cultivars and lines tested for drought tolerance (50 in spring and 68 in autumn). The visual score for wilting in spring was in the range of 1.28~5.00 on treatment day 26, 3.28~5.00 on treatment day 11, and 3.20~4.89 on average during the treatment period. The visual score for wilting in autumn ranged from 0.19 to 5.00 on treatment day 18, 0.63 to 5.00 on treatment day 15, and 1.91 to 4.89 on average, during the treatment period. There were differences in visual scores at the time of investigation and on average, more recovery occurred as the recovery period increased, but the wilting response was similar between cultivars and lines regardless of the testing period. It is thought that it is appropriate to judge the degree of drought tolerance based on the visual score value investigated seven days after re-watering.

We selected three cultivars and lines with a visual score of less than 4.00 in spring and nine cultivars and lines with a visual score of less than 1.00 in autumn as drought-tolerant resources. ‘18-FH112-1’ was confirmed to be drought-tolerant in both spring and autumn, while ‘Wongyo20040ho’ was confirmed to be drought-sensitive in both spring and autumn water deficit treatments. There were differences in wilting and recovery responses to drought stress depending on the cultivar, line, and period.

3.2. Changes in Growing Media and Leaf Water Potential and Growth Response of Chinese Cabbage Seedlings to Water Deficit Treatment

We investigated the changes in the water potential of the growing media and Chinese cabbage seedling leaves, along with their growth during the water deficit treatment and recovery periods. Chinese cabbage seedlings, grown for four weeks, were subjected to five days of water deficit treatment, followed by re-watering on the fifth day. During the treatment and recovery periods, the average temperature and DLI in the facility were 21.9 °C, and 31 mol∙m⁻²∙day⁻¹, respectively. The water potential of the growing media and leaves of Chinese cabbage seedlings decreased sharply as the duration of the water deficit treatment increased, and the degree of decrease varied depending on the type of Chinese cabbage (Figure 5). On the fifth day of water deficit treatment, the water potential of the growing media ranged from −23.8 to −83.3 MPa, while that of the Chinese cabbage seedling leaves ranged from −44.08 to −86.8 MPa (Figure 6). The decrease in the water potential of the growing media showed a similar trend to the decrease in the leaf water potential; thus, the treatments with lower water potential in the growing media also had lower leaf water potential.

On the fifth day of the water deficit treatment, the leaf area, fresh weight, and water content of the Chinese cabbage seedlings tended to decrease compared to the control, with significant differences observed in ‘CRmatjjang’, ‘Wongyo20047ho’, and ‘Wongyo20049ho’ (Figure 7). Particularly, ‘CRmatjjang’ and ‘Wongyo47ho’, which had leaf area and fresh weight values 20–30% higher than other varieties, experienced a decrease in these values to 20–30% of the control group by the water deficit treatment. The water content was significantly lower (72%) than that of the control, which had an average of 89%. On the other hand, ‘Wongyo20039ho’ and ‘Wongyo20042ho’ did not show a significant decrease following the water deficit treatment.The SPAD value and root-shoot ratio tended to increase following water deficit treatment, with ‘CRmatjjang’, ‘Wongyo20047ho’, and ‘Wongyo20049ho’ showing a significant increase. However, ‘Bulam3ho’, ‘Wongyo20039ho’, and ‘Wongyo20042ho’ did not show a significant increase following water deficit treatment. There were no differences in leaf number, dry weight, or Y(II) values between treatments.

Growth was investigated on the second day of re-watering, two days after the five-day water deficit treatment, to evaluate the recovery response after water deficit treatment (Figure 8). Leaf area, fresh weight, and water content, which were significantly reduced by water deficit treatment, recovered to levels similar to those of the untreated control because of the water supply from re-watering. Consequently, the SPAD value decreased to a level similar to that of the untreated controls. However, ‘CRmatjjang’ and ‘Wongyo20047ho’ did not recover to the control level, showing significantly lower values for leaf area, fresh weight, and water content. These two varieties had 48% and 70% leaf area, 47% and 69% fresh weight, and 93% and 98% water content, respectively, compared to the control.

When correlation analysis was conducted between the visual score for wilting and the growth indicators, negative correlations were observed between leaf area, SPAD value, root fresh weight, total fresh weight, aboveground dry weight, and total dry weight. Lower visual scores (healthier plants) were associated with higher values of leaf area, SPAD value (chlorophyll content, leaf color), fresh weight, and dry weight (Figure 9). The RGR during the seven days from the start of water deficit treatment to two days after re-watering decreased to 33.8–63.9% level in the water deficit treatment group, depending on the variety (Figure 10).

3.3. Growth of Chinese Cabbage in Greenhouse Cultivation under Water Deficit Treatment

To compare the results of the drought stress evaluation of Chinese cabbage seedlings with the actual response during field cultivation, we evaluated the growth of Chinese cabbage after water deficit treatment during greenhouse cultivation. After transplanting, we managed the crop as per normal practices (agricultural technology guide) and started water deficit treatment one month after transplanting. The volumetric water content of the soil due to the water deficit treatment is shown in Figure 11. During the 54-day water deficit treatment period, the volumetric water content of the soil in the treatment group continuously decreased and maintained a level of 0.1 m³·m⁻³. In contrast, in the control group, watering was performed 10 times, with volumetric water content increasing to 0.3 m³·m⁻³ after watering and then gradually decreasing to 0.1 m³·m⁻³.

On the 12th day of the water deficit treatment, the net assimilation rate, stomatal conductance, and transpiration rate of Chinese cabbage leaves were as shown in

Table 3. The photosynthetic performance of Chinese cabbage cultivars 12 days after water deficit treatment (42 days after transplanting).

Cultivar (A)	Treatment (B)	Net Assimilation Rate (μmol∙m−2∙s−1)	Stomatal Conductance (mmol∙m−2∙s−1)	Transpiration Rate (mmol∙m−2∙s−1)
Gyeoul-daejang	WDT	7.7 ± 0.18 z	192.0 ± 47.0	2.3 ± 0.5
	Cont.	8.2 ± 0.64	262.8 ± 73.5	3.1 ± 0.6
Gyeoul-wanggug	WDT	8.3 ± 0.23	282.5 ± 2.6	3.1 ± 0.1
	Cont.	8.0 ± 0.34	252.4 ± 40.8	3.0 ± 0.4
CRmatjjang	WDT	8.6 ± 0.33	310.9 ± 24.1	3.5 ± 0.2
	Cont.	8.4 ± 0.40	311.4 ± 10.2	3.3 ± 0.2
Hwipalam	WDT	8.6 ± 0.36	318.9 ± 32.9	3.6 ± 0.3
	Cont.	8.3 ± 0.44	309.7 ± 76.5	3.4 ± 0.7
Bulam3ho	WDT	7.8 ± 0.18	271.8 ± 38.6	3.2 ± 0.4
	Cont.	9.0 ± 0.19	286.7 ± 22.6	3.3 ± 0.1
Smart-baechu	WDT	7.7 ± 0.34	221.4 ± 24.0	2.6 ± 0.3
	Cont.	8.0 ± 0.63	233.7 ± 30.6	2.9 ± 0.4
Ssam-ilang	WDT	9.0 ± 0.29	367.7 ± 10.1	3.8 ± 0.1
	Cont.	8.7 ± 0.12	364.1 ± 29.4	3.9 ± 0.2
Asia-ppuli-baechu	WDT	8.0 ± 0.86	303.4 ± 11.9	3.4 ± 0.0
	Cont.	6.5 ± 0.93	234.7 ± 52.1	2.9 ± 0.5
F value
A		0.0006	<0.0001	<0.0001
B		0.7051	0.8858	0.6547
A × B		0.0110	0.1810	0.1223

^z mean ± SD.

. The net assimilation rate, stomatal conductance, and transpiration rate varied by water deficit treatment and variety, ranging from 6.5 to 9.0 mol∙m⁻²∙s⁻¹, 192.0 to 367.7 mmol∙m⁻²∙s⁻¹, and 2.3 to 3.9 mmol∙m⁻²∙s⁻¹, respectively. Although there were differences depending on the variety, there were no significant changes owing to the water deficit treatment.

The growth of cabbage harvested after 54 d of water deficit treatment is shown in Figure 11 and Table 3. The fresh mass and leaf number of cabbage significantly decreased under drought treatment, and the extent of this decrease varied depending on Chinese cabbage variety. The average fresh mass of the shoot and heads in the control was 7.2 kg and 3.2 kg, respectively, but decreased by 12% and 14% to 6.4 kg and 2.8 kg, respectively, due to drought treatment. The average number of leaves, which was 83, decreased by 12% to 75 due to drought treatment. There was no change in the number of inner leaves after drought treatment, but the number of outer leaves decreased significantly.

The growth of Chinese cabbage harvested after 54 days of water deficit treatment is shown in Figure 12 and

Table 4. The fresh mass and number of leaves of Chinese cabbage cultivars 54 days after water deficit treatment (84 days after transplanting).

Cultivar (A)	Treatment (B)	Fresh Mass (kg)		Number of Leaves
		Shoot	Head	Inner z	Outer y	Total
Gyeoul-daejang	WDT	6.4 ± 0.6 (83) x	2.7 ± 0.4 (81)	52 ± 6 (111)	24 ± 4 (106)	76 ± 5 (109)
	Cont.	7.7 ± 1.3	3.4 ± 0.8	47 ± 4	23 ± 5	70 ± 3
Gyeoul-wanggug	WDT	7.6 ± 1.6 (87)	3.1 ± 1.3 (86)	44 ± 2 (90)	23 ± 4 (87)	67 ± 5 (89)
	Cont.	8.7 ± 1.6	3.7 ± 1.6	49 ± 5	26 ± 5	75 ± 8
CRmatjjang	WDT	6.7 ± 0.6 (96)	3.0 ± 0.6 (93)	59 ± 5 (89)	27 ± 6 (89)	86 ± 10 (89)
	Cont.	7.0 ± 1.2	3.2 ± 1.2	66 ± 8	30 ± 9	96 ± 11
Hwipalam	WDT	7.4 ± 1.9 (79)	2.6 ± 0.5 (73)	67 ± 6 (99)	32 ± 6 (82)	99 ± 9 (93)
	Cont.	9.4 ± 3.1	3.5 ± 1.0	68 ± 6	39 ± 8	107 ± 8
Bulam3ho	WDT	6.9 ± 0.9 (97)	3.0 ± 0.4 (111)	67 ± 6 (92)	25 ± 4 (74)	91 ± 8 (86)
	Cont.	7.1 ± 0.1	2.7 ± 0.6	72 ± 10	33 ± 4	106 ± 12
Smart-baechu	WDT	5.9 ± 1.1 (94)	3.1 ± 0.5 (92)	65 ± 3 (98)	19 ± 3 (83)	84 ± 4 (94)
	Cont.	6.3 ± 1.5	3.4 ± 0.8	66 ± 7	23 ± 1	89 ± 7
Ssam-ilang	WDT	6.2 ± 0.8 (84)	3.7 ± 0.7 (88)	77 ± 10 (100)	22 ± 2 (74)	100 ± 10 (93)
	Cont.	7.3 ± 1.4	4.2 ± 1.2	76 ± 9	30 ± 4	108 ± 6
Asia-ppuli-baechu	WDT	3.8 ± 1.1 (102)	0.8 ± 0.2 (72)	-	-	-
	Cont.	3.8 ± 1.7	1.1 ± 0.3	-	-	-
F value
A		<0.0001	<0.0001	<0.0001	<0.0001	<0.0001
B		0.0472	0.0266	0.1379	0.0003	0.0006
A × B		0.8279	0.6962	0.4189	0.3482	0.1123
Treatment (B)
WDT		6.4b (88)	2.8b (86)	61a (94)	24b (83)	75b (88)
Cont.		7.2a	3.2a	65a	29a	85a

^z The number of leaves within the head. ^y The number of leaves outside the head. ^x mean ± SD (percent value for the control). Different letters show significant differences between water deficit treatment and control according to least significant difference test (p < 0.05).

. Due to the water deficit treatment, the yield and leaf number of Chinese cabbage significantly decreased, and the degree of decrease varied depending on the Chinese cabbage variety. The weight of the head decreased by 12%, and the weight of the core decreased by 14%. The number of leaves decreased by 88%. Although there was no significant difference in the number of inner leaves, the number of outer leaves decreased significantly.

4. Discussion

4.1. Screening for Drought Tolerance through Water Deficit Treatment of Chinese Cabbage Seedlings

Chinese cabbage, a cold-tolerant crop, has been primarily cultivated in the fall in the past. However, owing to the recent year-round increase in demand, it is now being cultivated from spring to winter. Chinese cabbage, which is mostly grown in open fields, is exposed to stresses such as high temperatures and drought, and the frequent occurrence of climate change and abnormal weather in recent years has accelerated these stresses. It is expected that the frequency and severity of these stresses will increase in many regions in the future owing to decreased precipitation and increased evaporation caused by global climate change.

Drought is the most damaging climate hazard to global populations [16]. Drought stress disrupts water balance in plants, deteriorates their overall growth status, and significantly reduces plant growth and productivity [17]. The effect of stress on plant growth depends on the growth stage as well as the exposure intensity and duration.

In particular, Chinese cabbage is known to be highly sensitive to drought stress, which not only leads to serious growth degradation, but can also develop into more serious problems [18]. For instance, drought stress lowers stomatal conductivity in Chinese cabbage leaves and closes the stomata, reducing the water absorption capacity and decreasing leaf photosynthesis and chlorophyll content, which can eventually lead to a decrease in yield.

Drought tolerance is the ability of a plant to maintain growth and productivity in a dry environment, which can enhance agricultural productivity. Resistance to drought stress is an important factor in increasing agricultural productivity. Therefore, the development of drought-resistant varieties of Chinese cabbage is important for improving agricultural productivity.

Evaluation of drought tolerance is an important process for assessing how well plants adapt to drought stress. The evaluation process of drought tolerance can be largely divided into: (1) preparation of plant material, (2) drought stress treatment, and (3) evaluation of response to drought stress. In this study, we prepared plant materials by sowing more than 100 Chinese cabbage breeding lines and varieties in a 72-cell plug tray filled with horticultural soil in a 34 mL volume cell and growing them for 4 weeks until 4–5 leaves fully expanded. Seedling evaluation has the advantages of being rich in treatable materials, cost-saving, space-saving, and time-saving [19,20,21]. Another advantage is that it can control experimental errors caused by morphological diversity when selecting strong and weak varieties under seedling stress. When conducting experiments in a culture room or laboratory, plants have similar external shapes; therefore, they can quickly detect developmental abnormalities during the early stages of growth [22]. In addition, seedlings react more sensitively to stress, making it easier to screen for drought tolerance than adults it is in [23].

Various methods have been proposed to treat drought stress and to evaluate crop drought tolerance [24]. These include withholding water [25], adding the osmotically active substance polyethylene glycol (PEG) to the medium [23,26,27,28], reducing water pressure in microporous tubes [29], connecting a vacuum pump to the pot [30], and using a solid column with low water permeability to separate the root area from the surface [24]. PEG treatment allows for uniform moisture potential treatment, but it can limit oxygen diffusion to the roots [31] and interfere with ion absorption [26].

In the present study, drought stress was induced by withholding water. The period of withholding water was set at seven days, and the period was adjusted according to the plant’s wilting response at the time of treatment. When the degree of wilting or death of the test variety or line by withholding water was such that the whole plant wilted and only new leaves at the growth point were alive (visual score for wilting: 4) or died (visual score for wilting: 5), we re-watered and investigated the degree of wilting every day to visually evaluate the degree of recovery (Figure 1).

The evaluation of plant responses to drought stress involves investigating growth and physiological indicators such as biomass, dry weight, water content, SPAD value, and photosynthetic characteristics after a certain period of water deficit treatment [28,32], or using formulae to index these values for evaluation [23,27,33]. In some cases, the recovery response, a type of drought adaptation, is evaluated by re-watering after a certain period of water deficit treatment [34,35,36]. Ahmed et al. (2022) evaluated the tolerance index by evaluating the period until wilting occurs and the degree of wilting during water deficit treatment of wheat seedlings and obtained the recovery index by evaluating the survival rate, the period until regrowth, and the amount of regrowth during the recovery phase after re-watering [34]. The drought tolerance index (DTI) was calculated using these indices. They confirmed that DTI can be a good indicator for evaluating drought tolerance as it has a high correlation with phenotypic and genotypic traits related to drought tolerance and recovery. In this study, we numerically transformed the degree of wilting of each individual during the period of withholding water and recovery after re-watering into a ‘visual score for wilting’.

As a result of screening for drought tolerance by withholding water and re-watering, there were differences in the response to drought stress and recovery ability depending on the variety or line. Some varieties quickly wilted or died after the water deficit treatment, whereas others showed a slow wilting response and gradually recovered to normal growth after re-watering. Drought-tolerant resources were selected based on the degree of wilting after 7 days of re-watering. In particular, ‘18-FH112-1’ was confirmed to be resistant to drought in both spring and fall (Figure 3, Table 1 and Table 2).

In the present, seedling tests were conducted over two seasons (spring and fall) to evaluate the drought tolerance of Chinese cabbage. The difference in the environmental conditions between spring and fall had a significant effect on the response of Chinese cabbage to drought stress. In particular, in the fall, the average temperature is lower and the amount of daily accumulated sunlight is less than that in spring; therefore, the drought stress response appears slowly and recovery tends to occur quickly (Figure 2 and Figure 3). It was confirmed that seasonal differences in environmental conditions affect the response of Chinese cabbage to drought stress, and it is thought to act as an important factor affecting the drought tolerance of plants as well as the differences between varieties or lines.

In general, the severity of drought depends on various factors such as the amount of watering, duration of water deficit, irrigation method, soil properties, environmental temperature, and light [28,37,38,39]. A plant’s response to the co-occurrence of drought, high temperature, and light stress is further complexed by its prioritization for more serious stress [40]. Under drought conditions, stomata are closed prematurely by the plant to prevent water loss, whereas under high temperature and light conditions, stomatal conductance increases to cool down the temperature of leaves through transpiration [41]. The optimal growth temperature for Chinese cabbage was 18–20 °C, and during the treatment period, the temperature conditions were higher than the optimal range, with an average daily maximum of 26.6 °C and an average daily maximum temperature of 29.0 °C. The DLI value was nearly twice as high in spring as in fall (Figure 2). The high temperature and high light conditions in spring are believed to accelerate moisture stress, resulting in a faster wilting onset and slower recovery than in fall.

Many studies have observed spatial and temporal variation in drought tolerance assessment because drought stress is usually difficult to replicate between locations and years, and many efforts have been made to develop uniform, stable, and reliable drought conditions [28]. Improvements in drought-tolerance testing technologies are required to reduce environmental influences and achieve consistent results. It is necessary to prepare uniform and reproducible plant materials, apply moisture stress and re-watering, and induce recovery responses under conditions that are suitable for drought tolerance screening. In addition, it is necessary to develop evaluation technologies such as image analysis technology for objective and consistent mass and high-speed (high-throughput) drought tolerance evaluation [42]. High-throughput phenotyping methods appear to be a solution to compensate for the otherwise labor-intensive and time-consuming classic methods of systematic plant phenotyping [43,44].

4.2. Changes in Growing Media and Leaf Water Potential and Growth Response of Chinese Cabbage Seedlings to Drought Stress

Therefore, controlling the water status of plants under dry conditions is crucial. To perform photosynthesis to produce assimilated products, gas exchange must occur through the open stomata, which leads to water loss. Therefore, plants must balance growth and survival under drought stress conditions. When plants face short-term deficits (hours to days), they react by minimizing water loss or exhibiting metabolic protection such as stomatal closure, decreased C assimilation and growth, xylem hydraulic changes, and osmotic adjustment [40].

Typical leaf water potential values for C3 plants are between −1 and −2 MPa, down to −4 MPa in species in arid zones, and as low as −10 MPa in extreme cases [45]. Plant growth and metabolism are significantly inhibited at moisture potentials below −2 MPa [46]. In this study, the cell capacity of the plug tray used was 34 mL, which required at least one watering per day during seedling cultivation. After stopping watering, the moisture in the growing media decreased rapidly, and the degree of decrease varied depending on the variety. On the fifth day of the water deficit treatment, the growing media water potential of ‘Wongyo20039ho’ was about −20 MPa, while that of ‘Wongyo20049ho’ decreased fourfold to −80 MPa (Figure 5 and Figure 6). The variation in growing media water potential among varieties is likely due to differences in water absorption and evaporation rates, and is believed to be an avoidance response to drought stress, such as stomatal closure.

Under conditions of limited water supply, as the growing media water potential decreased, the leaf water potential also decreased, showing a trend similar to that of the growing media water potential. Therefore, different varieties are exposed to different levels of water stress owing to differences in water absorption and evaporation rates, which are believed to cause differences in their responses to drought stress and, thus, their drought tolerance. As the water deficit treatment period increased, the water potential of the growing media and Chinese cabbage seedling leaves decreased rapidly. This indicates that drought stress has a serious impact on the plant water balance. However, the degree of the decrease varied depending on the variety.

Leaf and shoot fresh and dry weight, relative water content, SPAD, and Fv/Fm are major traits that indicate drought tolerance [28]. In barley, the reduction of fresh and dry mass and water content are the traits that are widely used for the screening of drought stress. In this study, the fresh weight, relative water content, and leaf area of Chinese cabbage seedlings decreased after 5 days of water deficit treatment (Figure 7). On the second day of re-watering after 5 days of water deficit treatment, the fresh weight, dry weight, and relative water content also decreased compared to the control, depending on the variety (Figure 8). However, in varieties such as ‘Wongyo20039ho’ and ‘Wongyo20042ho’, there were no significant changes due to water deficit treatment and the decrease in growth was not large. This shows the differences in drought stress tolerance among the varieties (Figure 8).

After five days of a single irrigation treatment, when the water supply was resumed through re-watering, the SPAD value, which increased due to the water deficit treatment, reached a level similar to that of the control. In the case of ‘Bulam3ho’, the leaf area and fresh weight recovered to a level similar to that of the control after re-watering. However, some varieties such as ‘Wongyo20047ho’ did not fully recover in terms of fresh weight, relative water content, and leaf area. The drought-treated group of ‘CRmatjjang’ showed lower values than the control, not only in terms of fresh weight, relative water content, and leaf area but also in terms of dry weight. This shows the differences in recovery abilities among the varieties. As a result, the degree of damage and recovery ability due to drought stress varies depending on the variety, and this can serve as an important indicator for evaluating drought tolerance.

However, these growth indicators are destructive methods that do not allow outstanding individuals to be tracked in the long term and are later used in drought-tolerant breeding programs. Therefore, other simple, rapid, and non-destructive methods are required for the large-scale screening of drought tolerance [28]. Correlation analysis between growth indicators, such as fresh weight and visual score for wilting, revealed significant correlations (Figure 9). The lower the degree of wilting (the healthier the plant), the higher is the growth indicator value. Therefore, the visual score for wilting could be used as an important indicator of drought tolerance. The results of this study show that the response and recovery ability of plants to drought stress varies depending on the variety and that the visual score for wilting can be useful in evaluating this. This provides important information for improving the drought tolerance of plants.

Under normal management conditions, the RGR of Chinese cabbage seedlings ranged from a maximum of 0.087 g∙g⁻¹∙day⁻¹ (CRmatjjang) to a minimum of 0.035 g∙g⁻¹∙day⁻¹ (Bulam3ho), depending on the variety, and this value decreased because of the water deficit treatment. On the second day of re-watering after water deficit treatment, the RGR of ‘CRmatjjang’, ‘Wongyo47ho’, and ‘Wongyo49ho’ was 0.044 to 0.046 g∙g⁻¹∙day⁻¹, while ‘Bulam3ho’, ‘Wongyo39ho’, and ‘Wongyo42ho’ were 0.018 to 0.026 g∙g⁻¹∙day⁻¹. According to a report by Xu et al. (2009), the recovery of plant biomass following re-watering was lower for the plants that had experienced previous drought compared to the controls, and the extent of recovery was proportional to the intensity of soil drought [47]. However, the RGR, leaf photosynthesis, and light use potential of the plant were markedly stimulated by the previous drought, depending on the drought intensity. They considered that an alternative functional state may overcompensate for the limitation of plant growth and metabolic activity due to previous droughts. The high RGR values of ‘CRmatjjang’, ‘Wongyo47ho’, and ‘Wongyo49ho’ suggest that their degree of drought stress was relatively greater than that of ‘Bulam3ho’, ‘Wongyo39ho’, and ‘Wongyo42ho’.

4.3. Growth of Chinese Cabbage in Greenhouse Cultivation under Water Deficit Treatment

Consistent genotypic evaluation is a prerequisite for the development of stress-tolerant cultivars. Shah et al. (2020) reported that the traits of early growth stages did not reflect drought tolerance at terminal growth stages and also did not confer high yield [27]. In this study, Chinese cabbage growth under water deficit treatment during greenhouse cultivation was evaluated to compare the drought tolerance response at the seedling stage.

In nature, plants can either be subjected to slow-developing water shortages (within days to weeks or months) or face short-term water deficits (hours to days) [40]. In the cool autumn with an average temperature of approximately 21 °C, the soil volumetric water content of the areas that received drought treatment under greenhouse cultivation conditions gradually decreased and remained at 0.1 m³∙m⁻³. Consequently, the response to the water deficit treatment appeared slow, and there were no differences in the net assimilation rate, stomatal conductance, and transpiration rate of Chinese cabbage leaves on the 12th day of treatment. Stomatal conductance and photosynthetic parameters are key traits for screening drought tolerance, along with biomass production, relative water content, and yield formation [28]. Stomatal conductance and photosynthesis typically decline [40].

As the water deficit treatment continued until harvest, the yield and leaf number of Chinese cabbages decreased significantly. This indicates that drought stress can have a severe impact on the growth of Chinese cabbage. However, the degree of decrease varied depending on the variety of Chinese cabbage. While the fresh mass of shoots and heads of ‘Hwipalam’ and ‘Gyeoul-daejang’ decreased to less than 85% of the control, varieties such as ‘Bulam3ho’ and ‘Smart-baechu’ had a relatively lower decrease, maintaining about 90% of the control group. This was somewhat consistent with the drought tolerance response observed at the seedling stage. In particular, the results of the drought tolerance evaluation at the seedling stage are thought to be highly related to the drought tolerance immediately after transplanting in the field. Reflecting on the actual situation during cultivation, will help to enhance the understanding of drought stress management in the agricultural field.

5. Conclusions

The visual score for wilting, which evaluates the wilting and recovery responses of Chinese cabbage seedlings to water deficit treatment and re-watering, can be used as an indicator for evaluating tolerance to drought stress. It showed a high correlation with major traits representing drought tolerance, such as leaf and shoot fresh and dry weights, relative water content, and SPAD value. This was also consistent with the field cultivation results. This demonstrates that the response and recovery ability of Chinese cabbage to drought stress varies by variety or germplasm and that a visual score for wilting can be useful in evaluating drought tolerance. However, differences in environmental conditions depending on the season during the drought tolerance seedling testing process affected the drought stress response of Chinese cabbage. This suggests that an integrated approach that considers various environmental conditions, varieties, and breeding lines is required for the selection and development of drought-tolerant varieties. It also proposes the need for the preparation of uniform and reproducible plant materials, water deficit treatment and re-watering, induction of recovery response, and objective and consistent evaluation. To this end, we suggest a method of preparing plant materials under certain conditions using a growth chamber capable of environmental control and evaluating the water deficit treatment, re-watering, and recovery response under these conditions. These methods will make a significant contribution to the accurate evaluation of the plant response to drought stress and in selecting varieties that are strong against drought stress. Through the evaluation of drought tolerance under a water deficit treatment at the seedling stage, we selected ‘18-FH112-1’ and ‘18-FH112-1-2’, among others, and these germplasms will be useful resources for drought-tolerant Chinese cabbage breeding.