Effects of Exogenous Auxins on the in Vitro Growth and Physiological Responses of Two Unnamed Tomato (Solanum lycopersicum L.) Genotypes Under PEG-Induced Osmotic Stress

2.1 Plant materials and explant preparation

Seeds of two unnamed tomato genotypes locally grown in Iraq (Solanum lycopersicum L.) were acquired from the Iraqi Ministry of Agriculture. However, formal cultivar registration/accession information was not available to the investigators. Therefore, the materials are called genotype A and genotype B throughout the manuscript. Therefore, the results of this study are offered as preliminary screening of two unnamed tomato genotypes/accessions, and conclusions regarding the genetic background or drought tolerance of the tomato cultivars are not drawn. The seeds were washed under running tap water, surface sterilized in 70% (v/v) ethanol for 1 min, and then surface-sterilized in 2% (v/v) sodium hypochlorite for 10 min. The seeds were sterilized with sterile distilled water thrice and germinated aseptically on Murashige and Skoog (MS) medium without hormones [16]. After two weeks, healthy nodal segments of approximately 1.0–1.5 cm were excised and used as explants for the experimental treatments [17].

2.2 Culture medium and growth conditions

All explants were cultured on MS basal medium supplemented with 30 g L^-1 sucrose and solidified with 7 g L^-1 agar. About 30 mL of MS medium was added into 250 mL culture jars, and one explant was placed in each jar. The pH of the medium was adjusted to 5.8 before autoclaving at 121 ℃ for 20 min. Cultures were incubated at 25 ± 2 ℃ under a 16 h photoperiod with a light intensity of approximately 2000 lux [8].

2.3 Polyethylene glycol and auxin treatments

This is an exploratory comparison of nine treatment combinations. PEG-6000 was used to induce osmotic stress at 0%, 5%, and 7% (w/v). The osmotic potentials were estimated to be approximately -0.027 MPa for 5% PEG-6000 and -0.05 MPa for 7% PEG-6000 at 25 ℃ using the formula given by Michel and Kaufmann [6]. Within the parameters of this in vitro system, the 5% and 7% PEG treatments were considered moderate and severe osmotic stress, respectively. The experimental treatment combinations were: control (0% PEG without auxin), 5% PEG without auxin, 7% PEG without auxin, 5% PEG + IAA, 5% PEG + IBA, 5% PEG + NAA, 7% PEG + IAA, 7% PEG + IBA, and 7% PEG + NAA. Auxins were added at a single concentration of 1.0 mg L^-1 after filter sterilization. The inclusion of PEG-only treatments allowed separation of the direct osmotic-stress effect from the auxin-mediated response. Similar use of plant growth regulators in micropropagation systems has been reported for several horticultural species [10, 18]. The approximate PEG-induced osmotic potentials were calculated using the method of Michel and Kaufmann [6] as the approximate PEG induced osmotic potentials were calculated as -0.05 MPa and -0.08 MPa at 25 ℃ for 5% and 7% PEG, respectively, following the approach of Michel and Kaufmann [6]. In this study, 5% PEG and 7% PEG were considered as moderate and severe stress, respectively, in the in vitro system. for 7% PEG at 25 ℃. The 5% and 7% PEG used in this study were defined as moderate and severe stress, respectively, and these are defined relative to this in vitro system.

2.4 Experimental design

The experiment was replicated twice in a completely randomized design (CRD) with two genotypes and nine treatment combinations. Each genotype-treatment combination had five replications in each run, with one explant per culture jar. Experimental run was included in the statistical model to account for between-run variation. The interaction between experimental run and treatment was examined before determining whether data from the two runs could be combined.

Each run × treatment combination had a treatment × run interaction that was not significant (p > 0.05), and hence data were pooled across the two runs, resulting in n = 10 for each genotype × treatment combination in the pooled analysis.

2.5 Growth and physiological measurements

The number of roots per plantlet, root length (cm), and shoot length (cm) were measured after 4 weeks of culture. The lengths of roots and shoots were determined with a digital caliper. Fully expanded leaves were used to estimate the chlorophyll content with a Soil Plant Analysis Development (SPAD) relative chlorophyll meter, and chlorophyll content was reported in SPAD units. The proline content was measured by the ninhydrin method of Bates et al. [19]. Fresh leaf tissue (0.5 g) was homogenized in 3% sulfosalicylic acid and centrifuged at 10,000 rpm for 10 min. Two mL of the supernatant was combined with 2 mL acid ninhydrin and 2 mL glacial acetic acid and heated to 100 ℃ for 1 h, cooled on ice, and extracted with 4 mL of toluene. The absorbance at 520 nm was measured, and proline concentration was determined from a standard curve and reported as µmol g^-1 fresh weight.

2.6 Statistical analysis

Data are presented as mean ± standard error (SE). Two-way analysis of variance (ANOVA) was used to analyze the Genotype, treatment combination, and the interaction between these. In all cases of pairwise comparisons, p < 0.05, means were compared using Tukey's HSD test. Model assumptions were assessed using Shapiro–Wilk tests and Q–Q plots of residuals for normality and Levene's test for homogeneity of variance. Root number was additionally evaluated as a count response using a generalized linear model with an appropriate distribution based on dispersion diagnostics. Data were presented in graphs and analyzed statistically using the Statistical Package for Social Sciences (SPSS) version 26 and plotting software, such as GraphPad Prism.

The current study provides evidence that the osmotic stress caused by PEG had a specific effect on tomato plantlets. Root number, root length, shoot length, and chlorophyll content were significantly decreased by severe stress at 7% PEG, indicating that water deficit negatively affects morphogenesis and photosynthetic potential in vitro. Generally, the responses are in line with plant physiology, which includes a decrease in water potential, a decrease in CO₂ assimilation, and an increase in oxidative stress on plant cells under drought conditions [3, 4]. PEG-based in vitro screening is therefore useful as it provides a controlled reduction of water availability to plantlets, as the soil is not a source of variability [6, 7]. Some root traits showed a mild stimulatory response under 5% PEG; in particular, under the provision of IBA. This reaction may be considered to be low-intensity stress adaptation, not necessarily better performance in all growth parameters. In plants, environmental hormesis is defined as an effect in which low-intensity levels of stress induce protective or growth-related responses but higher intensity levels cause inhibitory ones [20]. In this situation, the hormetic effect was most pronounced in root number and length, and not in shoot length and chlorophyll content; this highlights the need to consider each trait individually.

IBA (1.0 mg L^-1) gave the maximum rooting response in comparison to the other two auxins under the 5% PEG condition. This agrees with the auxin function in the initiation of adventitious and lateral roots, meristem activity, and root elongation [9, 10]. Due to moderate osmotic pressure caused by PEG + IBA, root number and root length were increased over the unstressed control under PEG + IBA, thus indicating that IBA has a role in directing plantlet growth towards root development. The root improvement is agronomically important since a better root system is one of the primary adaptive traits determining water deficit tolerance [5]. Recent research on auxin metabolism also reinforces the idea that auxin signaling plays a role in developmental plasticity in response to stress [12]. Root traits were not the same as shoot length. The control had the highest shoot length, with the lowest values under severe PEG treatment. Though IBA enhanced rooting, there was no enhancement in shoot length above control, suggesting shoot growth remained sensitive to PEG-induced osmotic limitation. This difference in root/shoot responses is significant since drought-assisted plantlets could invest more resources into the root than the shoot under stress. Such reorganization of growth allocation has been reported for plants facing drought stress where water-saving mechanisms (smaller shoots) are coupled with maintenance or stimulation of root-related attributes [21].

The greater rooting response seen with IBA compared to IAA or NAA might be due to differences in auxin stability, auxin transport, and metabolism, and not necessarily due to the general superiority of IBA. IAA is metabolized or conjugated at a relatively high rate, while IBA may be more stable and serve as a precursor for the formation of IAA, thus maintaining auxin activity in the root-forming tissues [11, 12]. Such differences can change the nature of the auxin signal and/or receptor-mediated responses. In addition, the higher level of proline when PEG + IBA (auxin–ABA crosstalk in osmotic stress signaling) may also be due to this reason, but a measurement of ABA was not performed here; thus, it is still a hypothesis [12, 21]. The effects on root traits were more pronounced than on shoot length or chlorophyll, which were still limited by PEG-induced water deficit, indicating direct sensitivity of these processes to auxin. Since no unstressed control (auxin only) was used, stress-specific auxin responses were not distinguished from the auxin effects under non-stress conditions in this experiment. The decrease in chlorophyll content with increasing PEG concentration suggests a negative effect of PEG on photosynthetic status under stress. This reduction is consistent with reports that drought and PEG-induced osmotic stress reduce chlorophyll metabolism and photosynthetic performance [22, 23]. IAA- and IBA-treated plantlets generally showed higher SPAD values than NAA-treated plantlets at the same PEG level, but their values remained below those of the unstressed control. Thus, the protective action of auxins against the loss of chlorophyll can be summarized as "partial" instead of "total". Broad phenotyping efforts also show that chlorophyll-related traits are highly responsive to drought and can have large inter-genotype variation, as shown by Denes et al. [24] and Grozeva et al. [5]. The accumulation of proline was significantly higher under PEG stress and was highest for 7% PEG + IBA. This pattern is consistent with the proposed functions of proline as an osmoprotectant in osmotic adjustment, as a protein stabilizer, as a protectant of membranes, and as a redox buffer [13, 19]. A similar increase in proline has been mentioned for tomato cultivars under drought stress, and the proline content in tolerant cultivars is higher than in sensitive cultivars [14, 15].

In a preliminary screening, genotype B had higher proline values in in vitro conditions, but this is not the only evidence of superior drought tolerance.

In general, the IBA effects were more pronounced on root traits and proline accumulation, while effects were less pronounced on shoot length and chlorophyll content. This separation would enhance the biological interpretation of the experiment: IBA did not remove PEG-induced stress, but it supported plantlets' adaptive responses that are vital to plant development, such as root development and osmotic adjustment. These results could help optimize the in vitro system for testing tomatoes under water deficit conditions. Additional studies should incorporate molecular markers, antioxidant enzyme assays, and acclimatization or greenhouse testing to determine if the in vitro responses observed will result in drought tolerance in the whole plant.