Stress adaptive plasticity from Aegilops tauschii introgression lines improves drought and heat stress tolerance in bread wheat (Triticum aestivum L.)

Stage I: preliminary evaluation of Ae. tauschii derived ILs for agronomic traits

Total 382 germplasm lines including 369 BC₁F₆/BC₁F₇ ILs, 11 parental lines used in developing these ILs, and two commercial checks (viz., PBW725 and HD3086), were evaluated for various agronomic traits. Of the 11 parental lines, two were T. durum wheat varieties (viz., PBW114 and PDW233), six were synthetics (viz., Syn1 to Syn 6), and three were advanced breeding lines (ABLs) (viz., BWL3279, BWL3531, and BWL4444) (Table S1). All the germplasm lines have been developed and are being maintained at the Department of Plant Breeding and Genetics, Punjab Agricultural University, Ludhiana. The details of the crossing procedure and parental lines used in developing these ILs are shown in Fig. 1.

All germplasm lines were sown in the augmented block design during 2019–20 cropping season. The experimental field was divided into 10 blocks with 40 plots (which includes 38 germplasm lines and two check varieties) in each block. Therefore, the experiment included a total of 400 plots. Each plot consisted of four rows of one meter length, with a row spacing of 20 cm, and the distance between each plot was 50 cm. The sowing was done during the first week of November, which is the ideal sowing time for wheat in the Punjab, India.

The agronomic data on spike length (cm), spikelet per spike, plant height (cm), and tiller number per meter length were recorded. Furthermore, visual observations were also made on days to anthesis, days to maturity, and stay-green type phenotype during growing cycle. Of the 369, 59 ILs with good agronomic performance (such as stay green, non-lodging, and having optimum days to flowering and maturity) were selected and harvested to measure grain yield (gm/plot) and 1,000 seed weight (gm). In addition, of the total 11 parental lines, only nine parental lines contributing to these 59 ILs (except PDW233 and Syn6) were also harvested.

Stage II: Evaluation of selected ILs for heat and drought stress tolerance under growth chambers

The experimental material comprised 74 germplasm lines, including 59 ILs (selected from stage-I), nine parental lines, and six check varieties (viz., PBW343, PBW660, C306, Raj3765, PBW725, and HD3086) (Fig. 1; Table S2). The experiment was laid out in a completely randomized design (CRD) with three replications and four treatments under controlled conditions. The details of the treatments used include: T1-control (grown in distilled water at 20 °C temperature); T2-mild heat stress (grown in distilled water at 30 °C temperature); T3-severe heat stress (grown in distilled water at 35 °C temperature); and T4-drought stress (grown in 20% poly-ethylene glycol (PEG) at 20 °C temperature).

Seedling characteristics were accessed using the modified cigar roll method of seed germination (Zhu, Kaeppler & Lynch, 2005). Bold and uniform seeds from each genotype were selected and disinfected with 0.1% HgCl₂ for 20–30 min and 70% ethanol for 10–15 min. Later seeds were thoroughly washed with deionized water (diH₂O) for three times to remove the HgCl₂ and ethanol. Twenty seeds from each germplasm lines were placed horizontally on germination paper moistened with distilled water (for T1, T2, and T3) and 20% PEG solution (for T4; to ensure drought stress from sowing itself). Subsequently, germination papers were rolled and were shifted immediately to respective growth chambers (adjusted to three temperature regimes; 20 °C, 30 °C, and 35 °C) to ensure the heat stress from the beginning. Then seeds were allowed to germinate under dark conditions for first three days. Once seeds were germinated, the growth chambers were adjusted to a 16 h photoperiod (i.e., 16 h of light and 8 h of darkness) at three temperature regimes (i.e., 20 °C, 30 °C, and 35 °C) for the next 12 days. On the 12th day after germination, data on germination percentage, shoot length (cm), and root length (cm) were recorded. The seedling vigour was calculated by using the following formula: ${\displaystyle \text{Seedling vigour}=\frac{\left[\text{shoot length}\left(\mathrm{cm}\right)+\text{root length}\left(\mathrm{cm}\right)\right]\times \text{germination percentage}\left(\text{\%}\right)}{100}}$

The stress tolerance index (STI) for seedling vigour under heat (T2 and T3) and drought (T4) stresses was derived as the ratio of seedling vigour under stress to the seedling vigour under controlled conditions, multiplied by 100. Furthermore, based on seedling vigour STI, the ILs were classified as tolerant (i.e., STI ≥ 80%), moderately tolerant (i.e., STI = 50–80%), and susceptible (i.e., STI ≤ 50%).

Stage III: Field evaluation of selected ILs for heat and drought stress tolerance

Germplasm lines used in stage-II (viz., 74 lines) were also evaluated for heat and drought tolerance under field conditions for two consecutive years (viz., 2020–21 and 2021–22) (Fig. 1; Table S2). During each cropping season, plant material was sown under four different environmental conditions viz. early (early heat stress), timely sowing (control), late sowing (terminal heat stress), and drought conditions. Details of field environments used for assessing heat and drought stress tolerance are depicted in Table 1.

The experiment was laid out in a randomized complete block design (RCBD) with two replications in all eight environments. The plots were 1.6 m² in size and separated by 50 cm. Each plot included four rows of two-meters length, with a row spacing of 20 cm. Data on days to 50% heading, spikelet number per spike, spike length (cm), plant height (cm), tiller number per meter, days to maturity, and grain yield per plot (gm) were collected.

Experimental area, agronomic practices, and weather data

Field and lab experiments were conducted in the experimental area and wheat laboratory of the Department of Plant Breeding and Genetics, Punjab Agricultural University, Ludhiana (30.9° North and 75.86° East, with a mean sea level of 244 m). The predominant soil type at the experimental site was a sandy loam with a slightly alkaline condition (pH from 7.8 to 8.5). The climate of the experimental location was sub-tropical and semi-arid with daily minimum temperatures ranging from 0 to 4 °C during December-January and the maximum temperature ranging from 39 to 45 °C during May (Sharma et al., 2021). Land preparation, including plowing, harrowing, and flanking, was done to achieve fine tilth, and the crop was raised using standard agronomic practices suggested for the Punjab region. To ensure sufficient soil moisture, the field was irrigated with a total of four irrigations (with 75 mm per irrigation) during stage-I and stage-III (for the first six environments; viz., E1–E6). However, last two environments (viz., E7 and E8) in stage-III were grown as rainfed wheat to ensure drought stress. Pre-emergence (pendimethalin; Stomp at 1.5 liter per acre) and post-emergence (weed and time-specific) herbicides were used to control the weeds. Prevailing insects, such as aphids, were controlled by spraying thiamethoxam (Actara at 20 gm per acre). Crop was harvested when it reached physiological maturity and agronomic data were collected on regular basis. Furthermore, the weekly weather data on mean maximum and minimum temperatures (°C), total rainfall (mm), mean sun-shine hours (hours/day), and mean relative humidity (%) were accessed from the Department of Climate Change and Agricultural Meteorology, Punjab Agricultural University, Ludhiana (Fig. S1).

Statistical analyses

Analysis of variance (ANOVA) for augmented block design (in stage-I), CRD (in stage-II), and RCBD (in stage-III) were done in Agricolae package built in the RStudio (R Core Team, 2020). Pearson’s correlation coefficient analysis, linear regression analysis, genotypic main effect plus genotype × environment interaction (GGE) biplot, additive main effects and multiplicative interaction (AMMI), and weighted average of absolute scores for the best linear unbiased predictions (BLUPs) of the genotype × environment interaction (WAASB) analysis were made using “Metan package” built in RStudio.

Stage I: Preliminary evaluation of ILs for agronomic traits

Data on spike length, spikelet number, plant height, and tiller number were collected from all the germplasm lines, including ILs (369), parents (11), and check verities (2). Analysis of variance (ANOVA) for augmented block design with adjusted mean showed significant differences (P-value < 0.05) among germplasm lines (Table S3). Of the 369 ILs, 59 lines having good agronomic characteristics such as compact spike (i.e., short spikes (7.8–20.7 cm) with a greater number of spikelets per spike(15–25.7) (Figs. 2A, 2B), reduced plant height (i.e., non-lodging; 67.33–114 cm) (Fig. 2C), optimum number of tillers (30–129) (Fig. 2D), optimum days to anthesis and maturity, and stay green type, were selected and harvested. The selected ILs showed significant variations for seed yield (gm per plot), with the highest yielding line, IL-134 (with 675 gm/plot) and the lowest yielding line, IL-279 (150 gm/plot) (Fig. 2E). The selected ILs also showed a significant variation for 1,000 seed weight, which ranged from 24.4 (IL-227) to 52.8 gm (IL-50) (Fig. 2F). Among the selected ILs, highest yielding line i.e., IL-134 (675 gm/plot), also showed higher seed yield over parental lines and both the check varieties i.e., PBW725 (619.35 gm/plot) and HD3086 (527.25 gm/plot). Furthermore, IL-29 (46.2 gm), IL-50 (52.8 gm), IL-42 (45.8 gm), and IL-134 (48.8 gm) showed higher 1,000 seed weight over parents and check varieties.

Pearson’s correlation coefficient analysis, multiple linear regression analysis, and principal component analysis (PCA) based on selected ILs revealed a significant and positive association between seed yield and seed weight (p < 0.001) (Fig. S2). However, spike length showed a significant negative correlation with seed yield (p < 0.05) and seed weight (p < 0.001). In addition, seed weight showed a significant (p < 0.01) negative association with spikelet number and plant height. Moreover, seed yield has not shown a significant association with tiller number, plant height, and spikelet number.

Stage II: Evaluation of selected ILs for seedling heat and drought stress tolerance

ILs selected from the stage-I along with their parental lines and check varieties were subjected to seedling heat (i.e., 30 °C; T2 and 35 °C; T3) and drought stress (i.e, 20% PEG; T4). Heat and drought stresses significantly reduced the germination percentage, shoot length (except under T2), root length, and seedling vigour (Figs. 3A–3D; Table S4). Heat stress reduced the seedling vigour by 21.49 and 59.29 percent under T2 and T3, respectively. Exposure to drought stress also reduced the seedling vigour by 60.37 percent. Furthermore, pooled ANOVA for STI revealed the significant genotype × heat interaction (Table 2). Based on STI, 30 ILs were classified as heat tolerant under T2, three ILs were classified as heat tolerant under T3 (IL-50, IL-56 and IL-68), and two ILs (IL-42 and IL-44) were classified as drought tolerant (T4) (Fig. 4; Table 3). Three ILs that showed tolerance under severe heat stress (i.e., T3) also showed tolerance under T2. Heat (IL-50, IL-56, and IL-68) and drought tolerant ILs (IL-42 and IL-44) outperformed the parental lines and check varieties. Additionally, IL-50, a heat tolerant line also performed moderately under drought stress. In contrast, a drought tolerant IL, IL-44, performed moderately under extreme heat stress (i.e., 35 °C), whereas IL-42 showed high level of tolerance under T2 (i.e., 30 °C) (Table 3).

The pearson’s correlation coefficient analysis showed a significant and positive association for the seedling vigour under different treatments except for T3 and T4 (Fig. 3F) (Table 3). In addition, seedling vigour under, (i) T2, T3, and T4 was regressed on the seedling vigour under T1 (control condition) (Figs. 3G–3I); (ii) T3 and T4 were regressed on the seedling vigour under T2 (Figs. 3J, 3K); and (iii) T4 was regressed on the seedling vigour under T3 (Fig. 3L). For every unit increase in seedling vigour under controlled condition, corresponding seedling vigour under T2, T3, and T4 increased by a factor of 0.79, 0.41, and 0.40, respectively. Similarly, for every unit increase in seedling vigour under T2, there was an increase in seedling vigour by a factor of 0.52 and 0.5 under T3 and T4, respectively. However, seedling vigour under T3 has no influence on seedling vigour under T4. Furthermore, genotype × treatment interaction analysis identified the best performing ILs for heat (IL-50, IL-56, and IL-68) and drought (IL-42 and IL-44) stresses (Fig. 5A). It also revealed the IL-44 and IL-50 as the most stable ILs under both heat and drought stresses (Fig. 5B).

Stage III: Field evaluation of selected ILs for heat and drought stress tolerance

Field experiments were carried to evaluate the heat (E1 to E6) and drought (E7 and E8) stress tolerance in the ILs selected from stage-I along with their parents and check varieties over two cropping seasons (viz., 2020–21 and 2021–22). Heat and drought stress significantly (<0.05) reduced all the agronomic traits including grain yield (Fig. S3 and Table S5). Additionally, pooled ANOVA revealed the significant (<0.05) effect of genotype, stress (viz., heat and drought), year, and their interactions on studied traits including grain yield (Table 4). Based on mean data from both years, IL-77 (921 gm/plot) (under timely sowing), IL-41 (307.7 gm/plot) (under late sowing), and five ILs, IL-123 (565 gm/plot), IL-106 (513 gm/plot), IL-68 (486 gm/plot), IL105 (486 gm/plot), and IL-29 (482.5 gm/plot) (under drought stress) showed higher grain yield than parental lines and check varieties (Table 5). The list of high-yielding ILs identified for the specific and combination of environments is given in Table 5.

ILs showed higher grain yield under early sowing (E1 and E2) (840.57 gm/plot) than in the timely sowing (E3 and E4) (608.53 gm/plot) (Fig. 6). This is owing to the availability of extended grain filling duration in early sowing as it experiences less terminal heat stress during anthesis and grain filling stages (Figs. 6A–6G). In contrast, grain yield under late sowing (228.72 gm/plot) was greatly reduced due to increased temperature at anthesis and grain filling stages (Fig. 6). affected due to increased values of several climatic co-variates (viz., mean maximum temperature, number of days with >30 °C, and number of days with >35 °C) during anthesis and grain filling stages (Fig. 6). For instance, one-degree Celsius increase in the mean growing season temperature (from sowing to maturity), the grain yield was reduced by 583 gm/plot/°C (Fig. 6; Table S6). The mean maximum temperature during the grain filling stage (i.e., flowering to maturity) was more severe (with a yield reduction of 72.72 gm/plot/°C) than that of anthesis (with a yield reduction of 62.84 gm/plot/°C) (Fig. 6; Table S6). In contrast, the number of days with more than 30 °C and 35 °C had the maximum impact during anthesis stage (with a yield reduction of 71.98 and 611.85 gm/plot/day, respectively) than during the grain filling stage (with yield reduction of 50.99 and 53.21 gm/plot/day, respectively). Drought stress also reduces the grain yield by 48.70 percent (with a yield reduction of 9.88 gm/plot/cm of irrigation water) (Fig. 6H).

We also carried the multiple linear regression analysis to compare the association among the environments (Fig. 7). This revealed a weak positive association among sowing dates (viz., early, timely, and late) except for timely sowing with late sowing (R² = 0.12; p-value < 0.01) (Fig. 7). Similarly, we observed weak association between sowing dates with drought stress, except for the timely sowing and drought stress (R² = 0.11; p-value < 0.01) (Fig. 7).

Stability analysis

Even though there was a significant genotype × year interaction; the years 2020-21 (E1, E3, E5, and E7) and 2021–22 (E2, E4, E6, and E8) differentiated ILs in a similar manner for grain yield, as depicted by biplots (Fig. 8). The which-won-where view of GGE-biplot (Fig. 8A), AMMI analysis (Fig. 8B), and nominal yield based on WAASB (Fig. 8C) identified specifically adapted ILs for early sowing (E1 and E2), timely sowing (E3 and E4), late sowing (E5 and E6), and drought stress (E7 and E8) (Table 5).

Classifying genotypes based on interaction-PCA1 (IPCA1) (57.76%) have identified IL-98 as the most stable IL (due to smallest IPCA1 score of 0.01) (Fig. 8C; Table S7). The joint interpretation of mean yield vs stability biplot (GGE biplot) based on PC1 (51.44%) and PC2 (27.32%) identified IL-259 as the stable, high-yielding genotype (Fig. 8D). However, of the total, only 57.76 (IPCA1) and 78.76 percent (PCA1 and PCA2) of the genotype × environment interaction (GEI) variation was explained by AMMI and GGE biplots, respectively, whereas the remaining variation was not included by these biplots (Figs. 8C, 8D). Therefore, to include the maximum GEI variation, WAASB scores were derived by retaining different number of IPCAs (Fig. S4). Plotting grain yield vs. WAASB scores identified IL-98 as the most stable genotype (due to lowest WAASB score i.e., 0.16) (Fig. 8E; Table S6). Whereas, IL-30, IL-259, and IL-364, were considered as high-yielding genotypes with less stability (Fig. 8E). Even though WAASB includes all significant IPCA (P-value < 0.01) axes, it will not give weightage to stability and yield performance while giving ranks to genotypes. Therefore, to overcome this, WAASBY scores (also known as superiority index) were calculated by assigning different weights to stability (100 to 0) and yield performance (0 to 100) (Fig. S5).

The WAASBY scores were calculated by giving 65% weightage to grain yield and 35% weightage to stability (Fig. 9). This helped in identifying IL-47, IL-51, and IL-259 as the high-performing stable ILs under heat and drought stress.

Comparisons among the stages

Comparing the different screening methods (i.e., stages) based on yield was not possible as stage-II includes only seedling evaluation. Therefore, to compare the efficiency of three stages, we used yield data from stage-I and stage-III (early, timely, late, and drought sown) and seedling vigour from stage-II (T1, T2, T3, and T4). The Pearson’s correlation coefficient analysis revealed a weak association between seedling vigour and grain yield (in stage-I and stage-III), except for seedling vigour under T2 and yield under drought (P value < 0.01) (Table 6). The grain yield under stage-I has a significant positive association with grain yield under timely sown (<0.01) and drought (<0.01) conditions of stage-III.

Comparing the effectiveness of different stages

For comparing different screening techniques, we used seedling vigour from stage-II and seed yield from stage-I and stage-III. Studies reported a lack of correlation between screening methods involving heat and drought stress at seedling stage and anthesis and grain filling stage (Poorter et al., 2016). Similarly, no significant correlation was observed between seedling vigour under growth chambers and seed yield under field conditions. Seed yield under field screening is a complex interaction of several climatic factors (Hede et al., 1999). Therefore, the genotypes showing higher seedling vigour did not showed higher seed yield in field experiments. Therefore, the advanced screening methods, such as heat chambers or moving shelters, need to be included in future studies.