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Article

In Vitro Screening for Salinity Tolerance in Garden Pea (Pisum sativum L.)

by
Stanislava Grozeva
*,
Slavka Kalapchieva
and
Ivanka Tringovska
*
Maritsa Vegetable Crops Research Institute, Agricultural Academy, Plovdiv 4003, Bulgaria
*
Authors to whom correspondence should be addressed.
Horticulturae 2023, 9(3), 338; https://doi.org/10.3390/horticulturae9030338
Submission received: 7 February 2023 / Revised: 27 February 2023 / Accepted: 28 February 2023 / Published: 3 March 2023
(This article belongs to the Section Biotic and Abiotic Stress)
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Abstract

:
Soil salinity is one of the abiotic stress factors gaining importance in recent years due to the changing climate and rising temperatures. This possesses a serious risk to food security worldwide and a range of adaptations and mitigation strategies are required. Crop improvement through breeding is a possible solution to overcome salinity stress. In this respect, a study was designed to screen a collection from garden pea accessions to salinity tolerance in in vitro conditions. We analyzed the effects of four salinity levels (0, 50, 100, and 200 mM NaCl) on seed germination, seedling emergence, shoot and root lengths, and plant fresh weight in 22 garden pea genotypes. Data showed that more of the pea genotypes are able to tolerate 50 mM NaCl concentration. The increasing of salt levels to 100 and 200 mM NaCl caused a significant decrease in germination and reduced the length and weight of shoots and roots. Among the genotypes, varieties Uspeh 72, Paldin, and Flora 6 were highly sensitive to salinity stress, while varieties Prometey, Musala, and Zornitsa were distinguished as more tolerant. The results depicted that salinity treatments had a more negative effect on plant growth than on seed germination, which is probably due to the in vitro conditions in which the experiments were conducted. The studied accessions of garden pea were sensitive to salt stress; nevertheless, some tolerant accessions were identified.

1. Introduction

Abiotic stresses such as heat, cold, drought, and salinity are an outcome of global climate change and have a direct influence on plant growth, yield, and quality. Soil and water salinity are two of the main limitations for crop production [1]. On one hand, they are due to the change associated with temperature rise and excessive evaporation (high evapotranspiration and low rainfall). On the other hand, they are due to the introduction of soluble salts containing Na+, Cl−, SO42−, and HCO3− into soil with the irrigation water [2,3]. The negative effect of salinity causes soil erosion and disturbs the ecological balance [4]. According to FAO (2021), over 833 million hectares of the world’s land, or 10% of the cropland, is affected by salinization, which possess a major risk to food security [5].
Pea (Pisum sativum L.) is among the most important leguminous crops with agronomic benefits and numerous food and feed usages. Plants are commonly utilized in rotations with cereals facilitating disease protection, weed control, and improving soil nitrogen supply. The seeds are a good source of proteins and also contain essential amino acids, minerals, vitamins, phytochemicals, and fiber [6,7]. Pea is one of the most adaptive species. Nevertheless, it is sensitive to salinity, especially during vegetative and reproductive phases [8]. Under salt stress plants have to cope with osmotic (inhibition of the capacity of water absorption) and ionic (toxic effect of high concentration of Na+ and Cl−) stresses [9,10]. Salinity disturbs many physiological functions, as the ability of biological nitrogen fixation [11,12] causes nutritional imbalance, flower abortion, and a decrease in grain yield [13].
There are various indicators used to evaluate salt tolerance in pea. They are seed germination rate, Na+ and K+ concentrations in plant tissues, plant height, root length, leaf necrosis, plant tissue dry weight, plant productivity, etc. [14]. The complex nature of salinity tolerance and lack of a single trait for assessment makes the breeding of tolerant cultivars difficult and time-consuming [15]. Moreover, conventional breeding using hybridization and selection in segregating populations has failed to provide desirable results for a short period. In vitro techniques offer an opportunity for rapid screen of a large number of genotypes, eliminating the impact of other potential stress factors such as pathogens, temperature, and humidity [16,17,18]. They are successfully applied as an alternative and inexpensive approach for studying the effects of different abiotic stress factors such as drought [19,20], heavy metals [21], and salinity [22,23]. In addition, they are also used for the selection and development of genetically stable stress tolerant lines after somaclonal variation [23,24,25,26].
The aim of this study was in vitro screening a garden pea collection to salinity tolerance by evaluation of seed germination and plant growth.

2. Materials and Methods

2.1. Plant Material

The experiment was conducted with 22 genotypes of garden pea Pisum sativum L. that belonged to early (4), mid-early (12) and late (6) groups. The collection consisted of: local varieties (15), breeding lines (4), and introduced varieties (3) (Table 1).

2.2. In Vitro Salinity Conditions

Dry, healthy seeds were surface sterilized in 5% calcium hypochlorite for one hour and rinsed three times in sterile distilled water. The seeds were sown on four variants of basal medium (macro- and micro-salts [27], vitamins B5 [28], 20 g L−1 sucrose, and 7 g L−1 agar) differing by the concentration of NaCl (0, 50, 100, and 200 mM). All culture media was adjusted to pH = 5.8 before being autoclaved, then autoclaved at 121 °C for 20 min. The culture vessels with seeds were incubated in a growth chamber at 25 °C ± 1 °C temperature, under a 16 h photoperiod with photosynthetic proton flux density (PPFD) of 200 μmol m−2 s−1.

2.3. Evaluation

The germination rate (GR) was determined after 7 days of seed cultivation, while the seedling emergence rate (ER) was recorded after 14 days. The shoot length (SL), root length (RL) (mm), and plant fresh weight (PFW) (mg) of 14-day-old seedlings were also measured.

2.4. Data Analysis

The experiment was repeated two times in three replications each, with five seeds per replication. The results were expressed as means of six independent (biological) replications. The mean standard deviation (SD) and the percentage of trait decrease compared with non-treated control (T-C%) were calculated. Differences among accessions grown in different salinity levels were compared using Tukey HSD and two-way analysis of variance. The analyses were performed with SPSS and R.

3. Results

3.1. Effect of NaCl Concentration on Seed Germination and Seedling Emergence

The experimental results showed that in the control treatment (0 mM NaCl) seed germination was 100% or close to, while the seedling emergence rate varied from 83.3 to 100% (Figure 1 and Figure 2, Supplementary Table S1). An increase in salinity level to 50 mM NaCl caused a decrease in germination and emergence by 3.3–25.9% and 3.6–36.0%, respectively, in most of the accessions. At this salinity level, line L_1857 appeared as the most susceptible genotype, followed by the variety Uspeh 72 and line L_6100, where both germination and seedling emergence rates were decreased by 30.0–46.7% and 34.5–55.2%, respectively. In two genotypes (Prometey and Vyatovo), germination and emergence rates were almost unchanged compared with the control treatment. An increase in salinity to 100 mM NaCl caused further decreases in both indexes up to 60.0% and 82.8%, respectively. A significant decrease in germination and seedling emergence rate was established in culture medium supplemented with the highest salt level (200 mM NaCl), where the decrease in seed germination and seedling emergence rate in most of the accessions varied from 25.0% to 96.6% and from 42.9% to 100.0%, respectively. Seedling emergence was not observed in varieties Denitsa and Flora 6 at the highest dose of applied stress factor. The best performing genotype with a comparatively high level of tolerance at the 100 mM NaCl and 200 mM NaCl concentrations was the variety Prometey. Additionally, germination and seedling emergence rate of varieties Zornitsa and Musala decreased on the medium with 200 mM NaCl, but still remain about the level of 50%, so these genotypes could also be distinguished as tolerant.

3.2. Effect of NaCl Concentration on Plant Growth

The studies on seedling morphology have shown that salinity affects both shoot length and root length (Figure 2 and Figure 3). After 14 days of cultivation, the addition of 50 mM NaCl in the medium decreased shoot length from 3.8% in variety Zornitsa to 64.3% in variety Plovdiv compared with the control 0 mM NaCl (Supplementary Table S1). The shoot length was reduced from 35.8% (variety Denitsa) to 85.7% (variety Flora 6) on the medium with 100 mM NaCl, and from 68.8% (variety Zornitsa) up to 97.4% (variety Uspeh 72) on the medium containing 200 mM NaCl. A similar tendency was observed in terms of root growth (Figure 2 and Figure 4). At the lowest concentration of NaCl (50 mM), the root length was reduced by 3.8–77.0%; at 100 mM concentration of NaCl in the medium the reduction was by 35–83%. At the highest salinity level (200 mM NaCl), over 80% reduction of root length was observed.
The results from the current study clearly indicate that the increasing of NaCl concentration significantly inhibits seedling growth in all studied pea accessions, but different responses were observed according to the genotype (Figure 5, Supplementary Table S1). Some accessions (L_22/16 af., Prometey and Vyatovo) could tolerate to some extent the applied salt stress and no reduction of plant fresh weight was observed at 50 mM NaCl compared with the control. In the rest of the accessions, significant reduction of plant growth from 16.5% to 74.1% was recorded at the lowest level of salinity (50 mM NaCl). Again, with the increase of NaCl concentration, the plant fresh weight significantly decreased and reached 98.5% (variety Uspeh 72) at the level of 200 mM NaCl.
These results were supported by the two-factor analysis of variance, which showed that the concentration of NaCl in the culture medium (Factor B) had the strongest influence on all of the measured traits, followed by genotype (Factor A) and the interaction A x B, which have similar relative effect sizes (Table 2).
Cluster analysis divided the genotypes into three distinct groups according to their sensitivity to salinity based on all measured traits. The first cluster consisted of eight accessions (Eco, Kazino af., L_22/16 af., Ran 1, Musala, Zornitsa, Prometey, and Vyatovo) that could be assigned as tolerant to the applied stress. The second cluster included four accessions (L_22/16, Mira, Vechernitsa, and Denitsa) assigned as susceptible. The third cluster combined the highly susceptible ones (Skinado, Uspeh 72, Flora 6, Hebar, L_6100, Paldin, L_1857, Pl. perla, Plovdiv, and Marsi) (Figure 6).

4. Discussion

Salt stress is among the factors that affect vegetative and reproductive processes the most and has an impact on all stages of plant development, from seed germination to plant growth [29]. Seed germination is considered to be one of the most critical stages in the plant life cycle. The data in the current study showed that the germination percentage exhibited a decreasing trend with the increase in salinity level. At the salinity level 50 mM NaCl germination rate was 53.3–96.7%, while at the highest salt concentration (200 mM NaCl) it ranged from 3.3% to 70.0%. The same tendency was observed in in vitro [4] and in in vivo conditions [30]. Authors found that germination was not significantly affected at 50 and 100 mM NaCl, while it was reduced remarkably at 200 mM NaCl. The negative impact of salinity on germination is probably due to the decrease in water potential, which results in the decline of water uptake by seeds [23,31]. This leads to changes in the activity of enzymes and hormones, which could be critical for seed germination. Additionally, NaCl can inhibit embryo development, which also negatively affects seed germination. According to some researches, seed sterilization by soaking in aqueous solution for more than 30 min for in vitro cultivation may facilitate seed germination due to the preliminary water uptake [32]. The authors explain that the delay or the lack of seed germination is due to the increased water potential of the seeds, impeding the further uptake of water from an environment with lower water potential. We observed this in our study, especially at higher levels of salt.
Many investigations of Legumes showed that seedling emergence is more sensitive to salt stress than seed germination is [32,33,34]. It was observed that in doses over 150 mM NaCl the seedling emergence level rarely exceeded 50% and, in some genotypes, fell to 0 [30,35]. Salt stress significantly reduced the emergence rate in our study. Only in three out of twenty-two accessions at 200 mM NaCl did the seedling emergence rate exceed 40%. The osmotic, but not the toxic, effect is the main reason for the delayed development of the shoots, since water is needed for the processes of intensive cellular division in the growing parts [36]. The lack of a well-developed vacuole in which harmful ions could be discharged makes the dividing cells sensitive to applied stress [37].
Salinization affects the subsequent development of plants, and the effect depends on both the dose and the genotype. The influence of the stress on plant growth is often determined on the basis of the shoot and root measurements. Growth parameters such as shoot and root length and biomass are used to evaluate the influence of the stress. In the current study, we observed a decrease in shoot length, root length, and plant fresh weight with the increase in NaCl concentration in all pea accessions after two weeks of cultivation. Moreover, the data showed that root growth was more affected by salinity compared with shoot growth, but these differences were not valid at the highest concentration of NaCl. Similar results were reported in several crops subjected to salt stress in vitro, such as garden pea [38], grass pea [4,32], potato [20], and tomato [39]. At 50 mM NaCI, the root and shoot growth were not significantly reduced, but when salinity was raised to 200 mM the inhibition of growth parameters was more than 80% in the same genotype. In the salinity conditions, the growth reduction was mainly due to the osmotic effect [40]. Nevertheless, the reduction in root and shoot development and plant growth may be also a result of the toxic effects of the higher concentration of NaCl and the unbalanced nutrient uptake by the seedlings [41,42]. Physiologically, many processes are affected by salinity, but the inhibition of cell growth as a result of water deficit leads to a reduction of plant growth, leaf area, stem elongation, and biomass. These traits can be used as important and informative in respect to in vitro screening of salt-tolerant plants [31,43,44]. The suppressed growth is a response of the plant to the applied stress and, often, salt tolerance is inversely proportional to growth rate [16,24]. This allows us to identify some salt-tolerant accessions as Prometey, Musala, and Zornitsa.

5. Conclusions

The obtained results clearly demonstrated that salinity stress induced significant reduction of seed germination, seedling emergence rate, and plant growth parameters (shoot length, root length, and plant weight) in the 22 studied garden pea genotypes. The negative effect of salt stress depended mainly on salt concentration, followed by the genotype. Most of the studied genotypes showed better seed germination, shoot emergence, and plant growth at salinity levels 0 and 50 mM NaCl. Further increase in the salt level (100 mM and 200 mM NaCl) resulted in significant decreases in plant growth parameters; in two of the genotypes, the level of seedling emergence fell to 0. The assessment based on the studied traits suggested that varieties Prometey, Musala, and Zornitsa have a higher tolerance to salt stress. The conducted in vitro screening can be used as a basis for the selection of salt-tolerant accessions. Further studies with the selected accessions are needed in in vivo salt stress to assess their performance and agronomic value.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/horticulturae9030338/s1: Table S1: In vitro seed germination, seedling emergence rates, shoot length, root length, and plant fresh weight of 22 garden pea accessions under four levels of salinity.

Author Contributions

Conceptualization, S.G., S.K., and I.T.; methodology, S.G. and I.T.; investigation, S.G.; resources, S.K.; data curation, S.G. and I.T.; writing—original draft preparation, S.G., S.K., and I.T.; writing—review and editing, S.G. and I.T.; visualization, I.T.; supervision, S.G. and I.T.; project administration, S.G. and I.T.; funding acquisition, S.G., S.K., and I.T.. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Bulgarian Ministry of Education and Science under the National Research Program “Healthy Foods for a Strong Bio-Economy and Quality of Life”, approved by DCM # 577/17.08.2018.

Data Availability Statement

Not applicable.

Acknowledgments

We would like to thank the technical staff of MVCRI’s Plant Tissue Culture Laboratory for growing experiment and data collection. We are thankful to Martin Tilev and Dimitar Mendev for English language editing of this manuscript. We sincerely appreciate all valuable comments and suggestions from the two anonymous reviewers that helped us to improve the quality of the manuscript.

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

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Horticulturae 09 00338 g001a 550Horticulturae 09 00338 g001b 550
Figure 1. In vitro seed germination and seedling emergence rates boxplot for 22 garden pea genotypes under four levels of salinity: (A) 0 mM; (B) 50 mM NaCl; (C) 100 mM NaCl; (D) 200 mM NaCl.
Figure 1. In vitro seed germination and seedling emergence rates boxplot for 22 garden pea genotypes under four levels of salinity: (A) 0 mM; (B) 50 mM NaCl; (C) 100 mM NaCl; (D) 200 mM NaCl.
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Figure 2. The effect of salt stress on seed germination and seedling emergence rate of selected garden pea genotypes after 7 and 14 days in vitro cultivation of medium containing 0, 50, 100, and 200 mM NaCl.
Figure 2. The effect of salt stress on seed germination and seedling emergence rate of selected garden pea genotypes after 7 and 14 days in vitro cultivation of medium containing 0, 50, 100, and 200 mM NaCl.
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Figure 3. Shoot length (mm) of 22 garden pea genotypes as influenced by the concentration of NaCl in nutrient medium in vitro after 14 days of cultivation. (A) 0 mM NaCl; (B) 50 mM NaCl; (C) 100 mM NaCl; (D) 200 mM NaCl.
Figure 3. Shoot length (mm) of 22 garden pea genotypes as influenced by the concentration of NaCl in nutrient medium in vitro after 14 days of cultivation. (A) 0 mM NaCl; (B) 50 mM NaCl; (C) 100 mM NaCl; (D) 200 mM NaCl.
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Figure 4. Root length (mm) of 22 garden pea genotypes as influenced by the concentration of NaCl in nutrient medium in vitro after 14 days of cultivation. (A) 0 mM NaCl; (B) 50 mM NaCl; (C) 100 mM NaCl; (D) 200 mM NaCl.
Figure 4. Root length (mm) of 22 garden pea genotypes as influenced by the concentration of NaCl in nutrient medium in vitro after 14 days of cultivation. (A) 0 mM NaCl; (B) 50 mM NaCl; (C) 100 mM NaCl; (D) 200 mM NaCl.
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Figure 5. Plant fresh weight (mg) of 22 garden pea genotypes as influenced by the concentration of NaCl in nutrient media in vitro after 14 days of cultivation. (A) 0 mM NaCl; (B) 50 mM NaCl; (C) 100 mM NaCl; (D) 200 mM NaCl.
Figure 5. Plant fresh weight (mg) of 22 garden pea genotypes as influenced by the concentration of NaCl in nutrient media in vitro after 14 days of cultivation. (A) 0 mM NaCl; (B) 50 mM NaCl; (C) 100 mM NaCl; (D) 200 mM NaCl.
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Figure 6. Heatmap based on the response of 22 garden pea genotypes under four levels of salinity stress.
Figure 6. Heatmap based on the response of 22 garden pea genotypes under four levels of salinity stress.
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Table
Table 1. Description of plant material used in the experiment.
Table 1. Description of plant material used in the experiment.
GenotypeDescription
DenitsaEarly variety with wrinkled and light green seeds, TSW * 157 g
EcoIntroduced, mid-early variety, afila leaves, round, smooth, and cream seeds, TSW 187 g
Flora 6Mid-early variety with wrinkled, green seeds, TSW 126 g
HebarMid-early variety with wrinkled, light green seeds, TSW 166 g
Kazino af.Introduced, mid-early variety, afila leaves, round, smooth, and cream seeds, TSW 187 g
L_1857Mid-early breeding line, wrinkled, and dark green seeds, TSW 161 g
L_22/16Mid-early breeding line, round, smooth, and cream green seeds, weight 173 g
L_22/16 af.Mid-early breeding line, afila leaves, round, smooth, and cream green seeds, TSW 176 g
L_6100Mid-early breeding line, wrinkled, and grayish green seeds, TSW 129 g
MarsiMid-early variety with wrinkled, greenish seeds, TSW 190 g
MiraLate variety with wrinkled, greenish seeds, TSW 150 g
MusalaEarly variety with wrinkled, cream seeds, TSW 148 g
PaldinMid-early variety with wrinkled, green seeds, TSW 201 g
Pl. perlaLate variety with wrinkled, cream green seeds, TSW 168 g
PlovdivMid-early variety with wrinkled, green seeds, TSW 136 g
PrometeyLate variety with wrinkled, dark green seeds, TSW 142 g
Ran 1Early, local variety with spherical, smooth, and green seeds, TSW 213 g
SkinadoIntroduced, mid-early variety, wrinkled, and green seeds, TSW 149 g
Uspeh 72Late variety with wrinkled, cream seeds, TSW 151 g
VechernitsaLate variety with wrinkled, green seeds, TSW 102 g
VyatovoLate variety with wrinkled, green seeds, TSW 40 g
ZornitsaEarly variety with spherical, smooth, and green seeds, TSW 173 g
* TSW—Thousand seed weight.
Table
Table 2. Two-way analysis of variance for the influence of genotype (Factor A) and NaCl concentration (Factor B) on the response of 22 garden pea genotypes under four levels of salinity in vitro: seed germination (SG), emergence rate (ER), shoot length (SL), root length (RL), plantlet fresh weight (PFW).
Table 2. Two-way analysis of variance for the influence of genotype (Factor A) and NaCl concentration (Factor B) on the response of 22 garden pea genotypes under four levels of salinity in vitro: seed germination (SG), emergence rate (ER), shoot length (SL), root length (RL), plantlet fresh weight (PFW).
Source of VariationRelative Effect Size (% of Total Variance)
SGERSLRLPFW
Factor A6.78 ***7.78 ***15.30 ***19.57 ***14.56 ***
Factor B59.80 ***63.58 ***66.67 ***58.55 ***65.41 ***
A x B8.72 ***8.44 ***7.55 ***10.07 ***8.86 ***
Error24.3720.2110.4811.8011.17
*** p ≤ 0.001.
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MDPI and ACS Style

Grozeva, S.; Kalapchieva, S.; Tringovska, I. In Vitro Screening for Salinity Tolerance in Garden Pea (Pisum sativum L.). Horticulturae 2023, 9, 338. https://doi.org/10.3390/horticulturae9030338

AMA Style

Grozeva S, Kalapchieva S, Tringovska I. In Vitro Screening for Salinity Tolerance in Garden Pea (Pisum sativum L.). Horticulturae. 2023; 9(3):338. https://doi.org/10.3390/horticulturae9030338

Chicago/Turabian Style

Grozeva, Stanislava, Slavka Kalapchieva, and Ivanka Tringovska. 2023. "In Vitro Screening for Salinity Tolerance in Garden Pea (Pisum sativum L.)" Horticulturae 9, no. 3: 338. https://doi.org/10.3390/horticulturae9030338

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