SJA_42_2_1026-1034

Introduction

Food legumes are important for nutrition and food security around the world, especially in semi-arid regions, where they are a significant source of protein, fiber, and micronutrients and contribute to dietary and economic diversification (FAO, 2021). Lentil (Lens culinaris Medik.) has a unique place in Moroccan and Mediterranean settings in relation to their nutritional value and for smallholder farmers (Ghimire et al., 2020). However, much of lentil yield is limited by abiotic stresses early in the growing season including germination and seedling establishment (Pushpavalli et al., 2020).

Successful germination is necessary for the establishment of crops and determining their yield potential. The germination process of seed is determined by both genetic and environmental factors, temperature being one of the most critical. Temperature regulates seed dormancy, activates metabolism and also controls rate and synchrony of germination (Wahid et al., 2007). Every species, and often cultivar, has a well-defined optimal temperature range for optimal germination (Marcos-Filho, 2015).

Climate change is increasingly associated with more intense and frequent heat waves, which create significant new challenges when considering sustainable crop production. During the key sowing time periods in Morocco, it is now common to have high temperature events (>35°C) (IPCC, 2022). A number of studies have shown that high temperature can adversely affect germination by negatively influencing membrane stability, enzyme activity, and expression of stress responsive genes (Hasanuzzaman et al., 2013; Moghadam et al., 2021). When seed is subjected to temperatures above 30–35°C, it can result in irreversible injury to the cellular structure which may result in delayed, erratic, or reduced seedling emergence (Zafar et al., 2020; Kumar et al., 2021).

While good progress has been made in the breeding of lentils for drought tolerance, limited exploratory work has systematically characterized the response to acute heat stress during germination in Moroccan lentil germplasm. The identification of genetically diverse and heat tolerant genotypes is important for developing breeding and adaptation for increasingly unstable climates (Sita et al., 2017; Jha et al., 2020).

A preliminary assessment of the impact of three controlled temperature environments on key indicators of germination in 17 genetically diverse varieties of lentils is currently being carried out. This study aims to examine the effects of temperature and variety on germination success and mean germination time (as measured by the germination index and germination kinetic parameters) as well as evaluating the degree of genotype× environment interaction in relation to thermal stress and the possibility for improving the degree of lentil varieties tolerance to a changing climate via variety selection. Defining the thermal limits of lentil germination is an essential first step in evaluating how future increases in temperature may impact the establishment of crops in Morocco and similarly climatic regions. The study did not represent the entire global range of lentil diversity in the current variety set so these limits the conclusions derived from looking at the genetic diversity for heat tolerance at germination by the germplasm used in this experiment.

Materials and Methods

Plant material

Seventeen lentil varieties (Lens culinaris Medik.) of both national and international origin were used: ABDA, BEKRIA, BICHETTE, HAMRIA, CHAKKOUF, EXTRA, L24, ABOA, JAMAAT SHAIM, V10, V11, V12, V13, BELUGA, LAIRD, ANICIA, CLARA. Seeds were provided by INRA (Rabat and Kénitra) and by local suppliers or farmers for foreign varieties. Prior to testing, seed lots were stored under optimal humidity and temperature conditions to ensure viability.

Experimental design

The methodology for seed germination was based on protocols established in the ISTA (International Seed Testing Association, 2023). Twenty seeds for each combination of variety and temperature were placed in Petri dishes containing Whatman No. 1 filter paper, moistened with water. After being placed into climate-controlled chambers at the start of the experiment (without a separate acclimation step), the seeds were allowed to germinate at constant temperatures of either 20°C, 26°C, or 32°C , Over a 6-day period between the two treatments. The design of the experiment was a fully factorial combination of the 17 varieties by the 3 temperatures, with four replicate samples of each treatment; this was designed following the accepted practices for the implementation of germination tests developed by ISTA and ensures that there is a trade-off between the ability to conduct valuable experiments and the degree of statistical certainty of their results. Future studies will allow for increased analysis power by utilizing either larger sample sizes or by conducting a true power analysis.

Parameter definition and measurement

Germination was defined as radicle emergence of ≥2 mm. Counts were performed daily over 6 days. The parameters analyzed included:

Final germination percentage (GP):

Mean germination time (MGT): , where Niis the number of seeds germinated on day Di(Ellis and Roberts, 1981)

Germination index (GI): (Maguire, 1962)

Germination kinetics: Cumulative germination percentages were determined by counting the number of new germinations each day, then summed across the 6 observation period for each replicate and plotted against time to describe germination rate and synchrony of stages of germination within treatments. When appropriate, the time to 50% of final germination (T50) was calculated using a linear regression.

Statistical analyses

Data was analyzed with a two-way ANOVA (factors: temperature, variety). Assumptions of normality and homogeneity were tested (Shapiro-Wilk, Levene’s tests). Tukey’s HSD will be used for mean separation starting with p < 0.05 as the designated alpha. Analysis was done using SPSS v25.0. Means + SD are provided in the results.

Results

Final germination percentage (GP)

All lentil genotypes reached their final germination percentage (GP) at optimal levels (90–100%) at both 20°C and 26°C and did not significantly differ at either temperature. Conversely, when the seed was exposed to 32°C, all genotypes exhibited a drastic and statistically significant decline in GP, with rates ranging from a low of 1.25% (CHAKKOUF) to a high of 12.5% (ABDA, BEKRIA). Given the shared response observed among genotypes, 32°C can be defined as lethal for lentils, regardless of specific variety (Figure 1).

Mean germination time (MGT)

As detailed in Figure 2, MGT remained similar at 20°C and 26°C (approximately -1.5 days) at both temperatures without delay and sequenced germination. Although at 32°C, MGT is lower in some genotypes (~ 1.0–1.2 days), these differences were likely due to statistical error. Since very limited germination occurred within the first day, the mean was altered and interfered with any clear interpretation of MGT parameters within severe temperature extremes.

Germination Index (GI)

GI an integrated measure of germination speed and proportion was high (12–17) at 20°C and 26°C for all varieties, signifying vigorous, synchronized germination. At 32°C, GI decreased dramatically to <2.5 across all genotypes, confirming the severe inhibition of both the rate and proportion of germination (Figure 3).

Table 1: Descriptive statistics for germination parameters (Mean ± SD) of 17 lentil genotypes under three temperature regimes.

Variety	GP (20°C)	GP (26°C)	GP (32°C)	MGT (20°C)	MGT (26°C)	MGT (32°C)	GI (20°C)	GI (26°C)	GI (32°C)
ABDA	100±0 a	100±0 a	12.5±2 b	1.5±0.2 a	1.7±0.3 a	1.0±0.2 b	16±0.9 a	15±1.1 a	2.3±0.4 b
BEKRIA	100±0 a	95±2 a	12.5±2 b	1.6±0.3 a	1.6±0.2 a	1.1±0.2 b	15±1.0 a	16±1.4 a	2.1±0.5 b
BICHETTE	98±3 a	98±2 a	10.0±1.8 b	1.4±0.2 a	1.7±0.2 a	1.2±0.2 b	15±1.2 a	13±1.6 a	1.8±0.3 b
HAMRIA	99±1 a	96±2 a	10.5±2.0 b	1.6±0.2 a	1.5±0.3 a	1.0±0.1 b	14±1.2 a	15±1.4 a	2.0±0.4 b
CHAKKOUF	98±3 a	97±2 a	1.3±0.5 c	1.3±0.2 a	1.8±0.2 a	1.2±0.3 b	14±1.2 a	13±1.1 a	1.1±0.2 c
EXTRA	97±2 a	99±1 a	6.0±1.4 c	1.5±0.2 a	1.5±0.2 a	1.0±0.2 b	15±0.8 a	15±1.0 a	1.5±0.2 c
L24	100±0 a	100±0 a	7.6±1.2 c	1.4±0.2 a	1.6±0.2 a	1.2±0.2 b	16±0.7 a	16±1.2 a	1.7±0.2 c
ABOA	98±2 a	99±1 a	8.4±1.7 c	1.5±0.3 a	1.6±0.2 a	1.1±0.2 b	14±1.0 a	15±1.0 a	1.8±0.2 c
JAMAAT SHAIM	99±1 a	97±2 a	8.1±1.6 c	1.5±0.2 a	1.7±0.2 a	1.2±0.2 b	15±0.9 a	13±0.9 a	1.7±0.3 c
V10	98±3 a	97±2 a	9.2±1.7 c	1.4±0.2 a	1.6±0.3 a	1.1±0.2 b	14±1.1 a	14±1.0 a	1.6±0.2 c
V11	99±1 a	98±1 a	7.4±1.1 c	1.5±0.2 a	1.6±0.2 a	1.2±0.2 b	15±0.8 a	15±0.8 a	1.7±0.3 c
V12	100±0 a	98±1 a	9.8±1.5 c	1.3±0.2 a	1.5±0.2 a	1.1±0.2 b	16±0.8 a	15±0.8 a	1.8±0.2 c
V13	97±2 a	99±1 a	10.1±1.6c	1.6±0.2 a	1.7±0.2 a	1.2±0.2 b	15±1.1 a	14±1.2 a	1.7±0.2 c
BELUGA	99±1 a	98±1 a	6.9±1.1 c	1.5±0.3 a	1.6±0.2 a	1.3±0.3 b	15±0.9 a	15±0.9 a	1.6±0.4 c
LAIRD	97±3 a	98±2 a	5.5±1.3 c	1.4±0.3 a	1.6±0.2 a	1.2±0.3 b	14±1.1 a	15±1.2 a	1.4±0.3 c
ANICIA	99±1 a	95±3 a	8.0±1.2 c	1.6±0.2 a	1.5±0.2 a	1.0±0.2 b	15±0.7 a	15±0.6 a	1.9±0.3 c
CLARA	98±2 a	97±2 a	7.2±1.0 c	1.5±0.3 a	1.5±0.3 a	1.1±0.2 b	14±1.1 a	13±1.2 a	1.6±0.2 c

Temperature as the predominant factor

Two-way ANOVA confirmed that temperature is the principal determinant of all germination parameters (p < 0.001), whereas neither variety nor the varietal interaction with temperature contributed significantly. Tukey’s post-hoc test objectively grouped means within each temperature regime (a, b, c), as shown in Table 1.

Germination kinetics

Cumulative germination (Figure 4) revealed steep and synchronous increases at 20°C and 26°C with all

viable seeds germinating by days 3–4. At 32°C, the curve remained flat for all genotypes, reflecting not only the low success rate but also the lack of spread in germination timing.

Discussion

Final germination percentage showed high sensitivity to the temperature regimes studied. All varieties maintained high final germination percentages (90–100%) at 20°C and 26°C, indicating that these temperatures lie within the well-defined optimal range for lentil germination and early metabolic activation (Marcos-Filho, 2015). The sharp decline in germination percentage for every variety at 32°C, down to values as low as 1.25%, demonstrates that this higher temperature is severely inhibitory for germination in the tested material. This pattern is consistent with previous studies showing that exposure of seeds to temperatures above 30–35°C can cause irreversible damage to cellular structures, impair membrane integrity, and disrupt metabolic activation, leading to reduced or erratic seedling emergence (Bhandari et al., 2020; Zafar et al., 2020; Kumar et al., 2021).

Mean germination time was consistently low (about 1.5 days) for all varieties at both 20°C and 26°C, which reflects rapid and uniform emergence under favourable thermal conditions. At 32°C, the apparent reduction in mean germination time for some varieties (around 1.0–1.2 days) is likely a statistical artefact, because only a few seeds germinated very early while most remained ungerminated, thus biasing the mean towards the earliest events. This illustrates that mean germination time alone may not be a reliable indicator of stress under extreme temperatures, and it should be interpreted with caution when germination percentages are very low (Fahad et al., 2017).

As a composite indicator that integrates both the rate and the proportion of germination, the

germination index provided a more robust assessment of temperature effects than mean germination time alone. Germination index values were high (12–17) at 20°C and 26°C for all varieties, confirming vigorous and synchronized germination under these conditions, but dropped to below 2.5 at 32°C for every variety, indicating severe inhibition of both the speed and extent of germination. These findings support the use of germination index as a sensitive parameter for screening heat stress effects during germination and suggest that it should be prioritized over mean germination time for phenotyping heat responses in lentil germplasm (Maguire, 1962; Fahad et al., 2017). Cumulative germination curves further highlighted these differences: at 20°C and 26°C, germination progressed steeply and synchronously, with most seeds germinating within about four days, whereas at 32°C the curves remained flat and low, reflecting slow or absent emergence and loss of synchrony across all varieties (Bewley et al., 2013).

From a physiological perspective, the strong inhibition observed at 32°C is in line with known effects of high temperature on seed tissues and early seedling development. Elevated temperatures can compromise membrane stability, alter enzyme activity, and interfere with the reactivation of respiration and reserve mobilization that are required for radicle protrusion (Hasanuzzaman et al., 2013; Moghadam et al., 2021). Prolonged exposure to such thermal stress may lead to irreversible cellular damage, which explains the very low germination percentages and germination index values recorded at 32°C in all varieties. While we did not quantify biochemical or molecular responses directly, our results fit well within this framework and highlight the vulnerability of lentil seeds to acute heat stress during germination.

Two-way ANOVA confirmed that temperature was the principal factor determining all germination parameters, whereas the main effect of variety and the temperature × variety interaction were not significant. The similar collapse in germination percentage and germination index at 32°C in both Moroccan and international varieties therefore indicates an absence of detectable genetic variation for heat tolerance at the germination stage within the set of 17 varieties evaluated. However, this conclusion must be interpreted in light of the genetic scope of the study, since the panel used here does not capture the full diversity of lentil, particularly from hotter and more arid regions (Ghimire et al., 2020). Expanding future screenings to larger and more diverse germplasm collections could reveal rare or underutilized varieties that express greater tolerance to high temperatures during germination (Sita et al., 2017; Jha et al., 2020; Sinha et al., 2022).

An important limitation of the present work is that it was conducted under constant temperature conditions in climate-controlled chambers, whereas field environments are characterized by pronounced diurnal and seasonal variability. In agricultural systems, seeds and seedlings are often exposed to fluctuating day–night temperatures, short heat episodes, and gradual warming or cooling trends, which may modulate the response to thermal stress compared with strictly constant regimes (Lobell and Gourdji, 2012). Our results should therefore be regarded as baseline indicators of the intrinsic thermal sensitivity of lentil germination rather than direct predictions of field performance, and complementary field trials under fluctuating temperature conditions are needed to validate the thresholds identified here.

In addition, the present study intentionally isolated temperature as the primary environmental factor, while other abiotic stresses commonly co-occur in farmers’ fields. In Mediterranean and semi-arid environments, lentils frequently face combinations of heat, intermittent drought, soil salinity, and suboptimal soil fertility, and these combined stresses can exert additive or synergistic impacts on seedling establishment (Hasanuzzaman et al., 2013; Fahad et al., 2017; Pushpavalli et al., 2020). Incorporating combined stress treatments such as heat × drought or heat × salinity into future germination and seedling experiments, and validating the findings across multiple field environments with variable soil moisture and biotic pressures, will be crucial to design varieties and management strategies that are more resilient to climate change (IPCC, 2022).

Our work deliberately focused on the germination phase, because successful and timely germination is a prerequisite for uniform crop establishment and yield potential. Nevertheless, thermal stress can also affect post-germination stages, including seedling emergence, root and shoot growth, and later vegetative and reproductive development (Gaur et al., 2015; Bhandari et al., 2020; Sinha et al., 2022). Future studies should therefore follow the same varieties beyond germination, under both controlled and field conditions, to evaluate how early thermal sensitivity relates to seedling vigour and subsequent performance under heat stress. Such multi-stage assessments would provide a more comprehensive understanding of where in the life cycle genetic variation for heat tolerance is most expressed and most useful for breeding.

The lack of detectable genetic variation for heat tolerance at the germination stage in the present panel suggests that breeding and management strategies might need to emphasize later developmental stages, where genotypic differences under stress have already been reported in lentil and other grain legumes (Gaur et al., 2015; Bhandari et al., 2020; Sinha et al., 2022). Future research should include screening larger and genetically diverse collections for thermal tolerance, integrating detailed phenotyping of germination and seedling traits with physiological measurements and modern genomic tools (Varshney et al., 2019; Jha et al., 2020). Marker-assisted selection, genomic selection, and, where appropriate, targeted biotechnological approaches could accelerate the identification and deployment of lentil varieties with improved adaptation to rising temperatures and increasingly frequent heat waves. Evaluating these varieties under combined abiotic stresses and across multiple field environments in Morocco and similar regions will be essential to translate laboratory findings into practical climate-resilient solutions for farmers.

Conclusions and Recommendations

Lentil germination is sensitive to temperature; all of the 17 lentil varieties evaluated germinated quickly and almost completely at both 20°C and 26°C, but at 32°C both the percentage of germination and the germination index were severely reduced. The decline in germination performance at 32°C was consistent and relatively uniform among both Moroccan and international lentil varieties, indicating that there is likely no genetic variation for heat tolerance among the lentil germplasm tested. These results indicate a thermal threshold related to lentil establishment and further suggest that acute heat stress during germination is very likely to become a significant limitation to the establishment of lentils in the future as climate change scenarios predict increased frequency and intensity of extreme heat events. The restricted number of lentil varieties tested and the fact that all germination trials were conducted at constant laboratory temperatures indicates that the thresholds identified in this research will require verification under the more variable temperature conditions found in the field and ideally with a larger and more diverse collection of lentil germplasm. Future research should utilize expanding lentil germplasm screening, multi-location field trials, and current breeding techniques to develop lentil varieties and management practices that are better suited to tolerate the impact of climate change in Morocco as well as in other regions of the world.

Acknowledgements

The authors declare that there are no acknowledgements for this work.

Novelty Statement

This study is the first to systematically evaluate the thermal limits of lentil germination using 17 diverse Moroccan and international varieties, demonstrating a universal and severe inhibition at 32°C. The lack of genetic variability at this developmental stage highlights the urgent need for breeding efforts targeting heat tolerance in later phenological phases.

Author’s Contribution

Soukaina Njili: Conducted research, data collection and write-up

Hassna Leknizi: study design and provided general supervision.

Siham Belkacemi: interpretation of data

Safaa Rhaimi: Conceptualization, statistical analysis

Aicha Rossafi: Analysis of data and arrangement of tabulated data

Mohammed Ouhssine: Supervision, guiding the structuring of the study, assisting with manuscript preparation, and validation of the manuscript.

Generative AI and AI-assisted technology statement

The authors declare that no generative AI and AI assisted technology was used in the creation of this manuscript.

Conflict of interest

The authors have declared no conflict of interest.

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