Polyethylene glycol, Osmotic potential, Moisture stress, Germination traits.

Indian mustard (Brassica juncea (L.) Czern and Cross) is an economically and nutritionally important oilseed crop cultivated across the Northern plains of India which covers 85-90% of the total area under the cultivation of Brassicas in India. B. juncea is cultivated in the arid and semi-arid regions of India for oil production and utilized as condiments, vegetables and fodder for cattle. These zones are under several risks like water scarcity, enormously increased population, depleted natural resource pools and above all, anthropogenic climate change. These pronounced spatial and temporal variabilities of ten threaten the survival and productivity of mustard crop. In India mustard is grown under rainfed cropping system, that limits seed germination, enhances seedling death in turn decline in yield (Rai et al., 2020).

Drought stress causes a decline in both quality and quantity in B. juncea, as it is highly susceptible to water shortage at every growth stage commencing from germination to maturity (Khanzada et al., 2020). Brassicas are negatively impacted by drought stress at several stages, including germination, seed development, grain yield and quality, as well as photosynthetic efficiency (Sujata et al., 2023). A sturdy seedling is a significant indicator that describes the seed yield of the plant over a short period of time. Drought tolerant cultivars with impermeable rooting abilities at seedling stage ameliorate the drastic effects of moisture stress under open field conditions (Ahmed et al., 2019).Drought results in decreased plant biomass, decreased seed oil and protein content, and decreased chlorophyll content due to pigment loss and damaged thylakoid membranes. Seed germination and seedling growth are critical phases often subjected to high mortality rates. Drought and salt stress inhibit and delay germination and seedling establishment (Almansouri et al., 2001). Drought stress causes a significant decline in germination and seedling growth and even complete crop failure due to the severity of the stress. Cultivar selection depends on germination percentage, germination speed and seedling growth influenced by drought stress. The best performing variety at the seedling stage could increase the production in rainfed areas (Khan et al., 2017). Therefore, screening under drought stress is imperative to identify tolerant genotypes for better germination, seedling establishment and higher yields. Polyethylene glycol (PEG-6000) creates osmotic stress and is a drought stimulant (Ashraf et al., 1996 and Ara et al., 2019) which is relatively more static, non-ionic and cell impervious than lower (PEG 4000) and higher (PEG8000) concentrations. Earlier studies demonstrated the screening of B.juncea and B.napus with PEG-induced drought stress (Geetha et al., 2012 and Jamil et al., 2019) still, the influence of seven osmotic potentials (drought stress levels) on the 82 advanced inbred lines of Brassica juncea is not available.

Based on the previous studies it was hypothesized that seven osmotic potentials/stress levels would significantly impact germination traits and rigorously evaluate the inbred lines of B.juncea. Therefore, the study was conducted for two years (2018-20) with objectives (i) To investigate the effects of negative osmotic potentials on germination traits (ii) To screen drought tolerant B. juncea inbred lines over two years based on their overall performance under osmotic potentials (iii) Identify suitable germination traits as screening tools for drought stress.

Plant material and treatments. The experiment was conducted with 82 inbred lines comprising released varieties, natural B. juncea germplasm, derived B. juncea lines, exotic Indian mustard lines, introgression material and zero erucic acid lines of B. juncea procured from the Oilseed section Department of Plant Breeding and Genetics, Punjab Agricultural University, Ludhiana, India, for consecutive two years (2018 –2019 and 2019-2020). Seven drought treatments consisting of distilled water control/ 0 Mpa and osmotic potentials (-0.20, -0.40, -0.60, -0.80, -1.00, and -1.20 MPa), which were prepared according to Michal and Kufmann (1973). Mature, healthy and uniform seeds were disinfected by immersion in a 0.1% mercuric chloride solution for one minute, then the seeds were washed three times with distilled water. Twenty seeds of each inbred line per replication were grown in each petri plate double-lined with filter paper placed in the bottom and top. The experiment was conducted in a completely randomized design (CRD) with three replications in each year. Germination was carried out in a growth chamber (CONVIRON CMP 6010, Model No. A1000) at 25 ±1°C and RH 85%. 

Germination traits. Seedling germination was recorded daily up to 13 days after sowing (DAS). Germination percentage, germination speed and mean germination time was recorded after 7 DAS as per ISTA (2019). After the completion of experiment, seedlings were used for recording shoot length, root length, fresh weight, and dry weight and were oven-dried at 70°C until a constant weight was achieved. Formulae for germination traits are depicted in Table 1. The germplasm evaluated for drought tolerance during 2018-19 and 2019-20 Table 2.

Statistical analysis. IBM SPSS (v22) software was used to conduct statistical analysis. Data was subjected to analysis of variance (ANOVA at p<0.05), Post hoc Tukey’s, to determine the statistical differences among inbred lines, drought stress levels, and their interactions. The traits were further explained through correlation and principal component analysis (PCA) for determining the linkage between traits and inbred lines under different moisture levels (Table 4 and 5). PCs with eigenvalue >1 were considered significant through PCA. The significance of the eigenvalues, established by Kaiser (1960), was used to choose the statistically significant principal components (PCs). Only the PCs that exhibited eigenvalues higher than one were significant. Principal component (PC) analysis, a multivariate statistical analysis for examining and simplifying complex and large datasets which transforms the larger number of correlated variables into smaller ones, as explained. Biplot analysis can select variables categorized into main groups and subgroups based on homogeneity and dissimilarity. The favourable germination traits and tolerant inbred lines were selected based on the overall results.

Table 1: Description of germination traits and methodology.

PEG induced drought stress significantly affected the studied germination traits (Table 3, 4 and Fig. 1). Significant differences (p<0.05) among all the treatments represented diversity among the studied traits. Inbred lines and their interactions with PEG treatments were highly significant, implying differential responses of inbred lines in varied drought stress levels.

Germination Percentage. PEG induced drought stress had a negative impact on GP with the increased stress levels or decreased osmotic potential (Table 3 and 4). Inbred line DJ-1-2 showed a maximum GP (98.4%) while RL-1359 (40.0%) showed minimum under control. At -0.20Mpa germination percentage ranged from 23.4% in Pusa Bold to 90.0% in JC-1359-23-558 and MCP-12-3. At -0.40 and -0.60Mpa GP was minimum in JPM-2(10.0% and 0 or no germination) but maximum in PBR-97 (85.0%) and JC-1359-23-558 (77.5%). MCN-12-14 line showed a maximum GP of 60.8% while in seven inbred lines (CP-98, DJ-1-2, DJ-124-1-DT-70, ELM-151, GR-325, JPM-2, and K-9-108) germination was checked at -0.80Mpa. At -1.00 Mpa EC-564647 showed tolerance to PEG and exhibited a maximum of 49.2% while in sixteen inbred lines (CP-98, DJ-1-2, DJ-124-1-DT-28, DJ-124-1-DT-70, DJ-127, DJ-5, ELM-151, GR-325, JJ-210-5-4, JM-06-010, JPM-2, K-9-108 S62, M-305, MCP-12-606, NRCDR-2, and RL-1359 germination was checked. Osmotic potential of -1.20 Mpa checked the germination completely, but the eight inbred lines (A8-4-2×JR-049, JC-1359-23-558, MCP-12.224, NRCHB-101, PBR 357, PBR-91, PBR-97 and QM-7-196 germinated, with <20% GP except for JC-1359-23-558 which showed 23.8%. As compared to control reduction of GP was 10.8, 26.5, 41.7, 57.1 and 98.0% with increased stress levels corresponding to -0.20, -0.40, -0.60, -0.80, -1.00 and -1.20 Mpa respectively.

Mean Germination Time. The MGT showed an increasing trend with increased stress levels in all inbred lines except at -1.20 Mpa whereas no germination was recorded, with exception to eight inbred lines (Table 3 & 4). In control, GMCN-13 and JC-1359-23-558 took 2.9 days to germinate, while RLC-1 germinated in 5.7 days. At -0.20Mpa, GR-325 and JC-1359-23-558 took 3.0 days to germinate, whereas RL-1359 took 5.2 days. JC-1359-23-558 took 3 days, while JPM-2 took 6.2 days to germinate at -0.40Mpa. Germination of JPM-2 was checked at -0.60Mpa but DJ-1-2 showed a maximum value for MGT (6.5). Germination of seven inbred lines (CP-98, DJ-1-2, DJ-124-1-DT-70, ELM-151, GR-325, JPM-2, and K-9-108 was inhibited at -0.80Mpa but RLC-1 took 6.6 days to germinate. At -1.00Mpa, MGT ranged from 0 (CP-98, DJ-1-2, DJ-124-1-DT-28, DJ-124-1-DT-70, DJ-127, DJ-5, ELM-151, GR-325, JJ-210-5-4, JM-06-010, JPM-2, K-9-108, M-305, MCP-12-606, NRCDR-2, and RL-1359) to 7.0 days in RH-419-3.

Table 2: Germplasm evaluated for drought tolerance during 2018-19 and 2019-20.

At -1.20 Mpa, since only eight inbred lines (A8-4-2×JR-049, JC-1359-23-558, MCP-12.224, NRCHB-101, PBR 357, PBR-91, PBR-97 and QM-7-196 germinated, their MGT values ranged from 6.0 in PBR-91 to 5.4 in PBR-97. MGT increased by 5 (-0.20Mpa), 12.5 (-0.40Mpa), 20.4 (-0.60 Mpa), 22.2 (-0.80Mpa), 24% (-1.00Mpa) while at -1.20 Mpa only eight lines germinated and the germination of 74 lines was checked at this stress level.

Speed of Germination. Results of means comparison (Table 3&4) showed that speed of germination (SG) decreased with decreased osmotic potential or increased stress levels. Overall speed was highest at control as compared to other osmotic potentials. At control, RL-1359 (2.3) germinated with slower speed whereas JC-1359-23-558 (6.7) germinated with higher speed. JC-1359-23-558 showed maximum SG values at 0.2 (5.9), -0.4 (5.3) and -0.6 Mpa (4.9) whereas the lowest was noticed in M-305 (1.7) at -0.2 Mpa. RL-1359 showed SG of 0.5 at -0.4 and 0.3 at -0.6Mpa. At -0.8Mpa highest SG of 3.7 was shown by EC-564647 (S36) but germination was checked in JPM-2. At -1.00 and -1.20Mpa highest SG was noticed in EC-564647(3.0) and in JC-1359-23-558 (1.5) respectively. SG got declined by 13.3 (-0.20Mpa), 33.3 (-0.40Mpa), 51.1 (-0.60Mpa), 68.9 (-0.80 Mpa, 84.4 (-1.00 Mpa) and 97.8% at (-1.20 Mpa).

Root and shoot length. The root and shoot length varied significantly among Brassica juncea lines under control and PEG treatments (Table 3&4). In control, root and shoots were longest. The overall reduction percentage in comparison to control were 5.4, 16, 25, 33, 51, 95% for RL and 13, 26, 37, 50, 66, 97% for SL in the increasing stress levels/decreasing osmotic potentials respectively. Root and shoot lengths ranged from 3.8 (K-9-108) to16.4 cm (PLM-2) and 2.3 (CP-98) to 8.1cm (JM-06-0206). Increased stress level reduced both root and shoot length. At -0.2Mpa RL ranged from 3.5 (JPM-2) to 14.7 (MCN-12-45) and SL from 1.6 (RH-419-3) to 6.0cm (JM-06-0206). Magnitude of root and shoot length was lower inJPM-2 (3.0cm) and in CP-98 (1.2cm) but higher in MCN-12-45 (14.6cm) and JM-06-020 (5.1cm) at -0.40Mpa. Maximum RL (12.5 cm) and SL (4.5 cm) was recorded in MCN-12-45 and JM-06-0206 at -0.6Mpa. At -0.8Mpa, MCP-12-3 and MCP-12-630 (11.2cm) possessed longer root and JC-1359-23-558 (3.0cm) longer shoot. Osmotic potential of -1.00 Mpa severely affected the seedling lengths but MCN-12-45 and MCP-12-3 (10.1cm) showed higher RL and JM-06-0206 (3.7cm) maximum shoot length. Furthermore, osmotic potential of -1.20Mpa completely checked the germination, with the exception to 8 lines (A8-4-2×JR-049, JC-1359-23-558, MCP-12.224, NRCHB-101, PBR 357, PBR-91, PBR-97 and QM-7-196). The longest root of 8.6cm was in JC-1359-23-558 while longest shoot length of 2.0 cm in PBR-91.

Fresh weights.  Data recorded for the fresh weights of 10 mustard seedlings varied significantly (Table 3&4). The magnitude of reduction of fresh weights ranged from 14-99% for FWR and 15-98% for FWS in the increased PEG treatments as compared to control. The highest decline was noticed at OP -0.60Mpa i.e., 51 and 48% for FWS and FWR. FWR ranged from 36.8 (ELM-123) to 285.0mg (PBR-97) and FWS from 159.1 (RH-419-3) to 529.2mg (DJ-124-1-DT-62) under control. At -0.20Mpa maximum FWR was observed in NLM-13 (252.0mg) and maximum FWS in DJ-124-1-DT-62 (481.0mg) but ELM-123 (131.2mg) showed minimum FWR and RH-419-3 (136.3mg) showed lowest FWS. At -0.40Mpa, FWR varied from 20.3 (A8-4-2×JR-049) to 228.8 (NLM-13) and FWS from 70.7 (A8-4-2×JR-049) to 412.3mg (DJ-124-1-DT-62). The declining trend of FWR and FWS continues and at -0.6Mpa germination of JPM-2 (S59) was inhibited. Higher FWR was recorded in DJ-124-1-DT-62 (149.3mg) and higher FWS in MCP-12-630 (330.1mg). At -0.8Mpa germination was checked in seven lines however PBR 357 (118.7mg) showed higher FWR and DJ-124-1-DT-62 (254.2mg) higher FWS. At the osmotic potential of-1.00Mpa, maximum FWR was recorded in RH-419-3 (77.2mg) and maximum FWS in MCP-12-227 (176.0mg). At -1.20Mpa, maximum FWR and FWS were recorded in PBR 357 (19.5mg) and PBR-91 (84.2mg) respectively.

Dry weights. The dry weight of mustard seedlings was significantly influenced by drought stress caused by the PEG 6000 treatments (Table 3 & 4). Among the germination traits, the impact of induced drought stress was higher on dry weights. Compared to control, the magnitude of reduction was in the increasing order of 23, 46, 68, 83, 94, and 99% for DWR and 18, 38, 58, 74, 88, and 94% for DWS corresponding to PEG levels. DWR at 0Mpa/control ranged from 7.7 (ELM-151) to 47.7mg (DJ-124-1- DT-31). Similarly, DWS varied from 13.8 (ELM-151) to 65.7mg (PBR-97). The higher values were exhibited by DJ-124-1- DT-31 (40.4mg DWR) and PLM-2 (50.2 mg DWS) but lower byJM-06-0206 (5.7mg DWR) and ELM-151(11.0mg DWS) at -0.2Mpa. Root and shoot growth declined with increased PEG treatments and at osmotic potential -0.4Mpa the range was 1.3 (JPM-2) to 27.7 mg (RLM-619) for DWR and 4.5 (JPM-2) to 38.9mg (RLM-619) for DWS. JPM-2 (S59) couldn’t resist the severity of stress and at -0.6Mpa its germination was checked but PBR-97 exhibited higher DWR (17.7mg) and DWS (36.8 mg). Osmotic potential of -0.8Mpa extremely reduced the dry weights but PBR-97 resisted the stress and showed higher magnitudes of 11.5mg for DWR and 28.0mg for DWS. At -1.00 Mpa the mean dry matter was very less (2.7mg of DWR in PBR-97 and 13.8mg of DWS in RLM-619). As the germination was completely checked at -1.20Mpa with the exception to eight lines with higher DWR in PBR-91 and PBR-97 (1.5mg) and maximum DWS in MCP-12.224 (7.0mg).

Identification and categorization. Critical analysis of PEG induced drought stress for screening and evaluation of 82 inbred lines over the years (represented as pooled mean of two years 2018-20) under lab conditions for various germination traits led to identification and characterisation into 13 highly tolerant strains, 29 moderately tolerant,17 highly susceptible and 23 moderately susceptible strains. Further for each trait ranges and mean values are also tabulated for all highly tolerant strains and two strains for each other category (Table 3).

Table 3: Range and mean of germination traits of identified and categorised as highly tolerant, moderately susceptible and highly susceptible lines.

“0.0” in the ranges and “-” indicates no germination

Table 4: Range, mean, standard error and least significant differences of germination traits in control and PEG6000 induced six osmotic potentials.

OP Osmotic potential, DW (Distilled water grown) GP germination percentage, MGT mean germination time, SG speed of germination, RL root length (cm), SL shoot length (cm), FWR fresh weight root (mg/10 roots), FWS fresh weight shoot (mg/10 shoots), DWR dry weight root (mg/10 roots), DWR dry weight shoot (mg/10shoots), Letters superscripted by small alphabets differ significantly in a column for a given germination trait. Significance p≤0.05 (T= treatments, G= inbred lines, G×T= interactions), Value 0.0 indicates no germination

Fig. 1. Frequency distribution graphs showing cumulative germination percentage (GP), mean germination time (MGT), speed of germination (SG), root length (RL cm), shoot length (SL cm), fresh weight root (FWR mg/10roots), fresh weight shoot (FWS mg/10shoots), dry weight root (DWR mg/10roots), and dry weight shoot (DWS mg/10shoots).

Correlation analysis of germination traits under PEG induced drought stress.  Highly significant and positive correlation existed among the germination traits under PEG6000 induced drought stress (Fig. 2).

Fig. 2. Correlation heat map of germination traits under PEG 6000 induced drought stress in Brassica juncea advanced inbred lines. GP germination percentage, MGT mean germination time, SG speed of germination, RL root length, SL shoot length, FWR fresh weight root, FWS fresh weight shoot, DWR dry weight root, DWR dry weight shoot. Correlation is significant (p=0.01).

High positive correlation of GP with other germination traits was in the order of SG (r=0.98) > DWS (r=0.85) > FWS (r=0.83) > SL (r=0.81), FWR (r=0.74) > RL (r=0.72) > DWR (r=0.68) > MGT (r=0.28). MGT showed weak associations with all germination traits (r <0.5). RL and SL were strongly correlated with all studied traits (r ≥0.5).

Principal Component Analysis. Patterns of variation were studied for 82 Brassica juncea inbred lines using principal component analysis (PCA established by Kaiser 1960) based on the correlation matrix to evaluate the diversity of the germplasm and the association of germination traits under PEG 6000 concentrations. Out of 9 principal components (Table 5), the first two PCs exhibited eigenvalues higher than one (significant) under PEG 6000 concentrations. The other seven PCs exhibited non-significant variation and were not earnest of further interpretation (eigenvalues less than one). The first two PCs showed 69.9% and 82.3% cumulative proportion of variation in the studied cultivars. The PC1 accounted for 69.9% of the variance, and the PC2 accounted for 12.5%. The first PC was highly related to GP (0.94), SG (0.94), FWS (0.92) and DWS (0.91). PC2 was in strongly associated with MGT (0.94), RL (0.73) and SL (0.63) depicted in Table 6.

Table 5: Eigenvalues, variability, and cumulative of germination traits under PEG 6000 induced drought stress.

PC = principal components PC1 to PC9 of studied mustard germination traits, S= selected component (PC1 and PC2)

Table 6: Principal component analysis (loading factor) of germination traits under PEG 6000 concentrations.

GP germination percentage, MGT mean germination time, SG speed of germination, RL root length (cm), SL shoot length cm), FWR fresh weight root, FWS fresh weight shoot, DWR dry weight root, DWR dry weight shoot 

PCA loading plots for germination traits under PEG 6000 induced drought stress. The smaller angles between the traits indicate the strong and positive association of germination traits, while the enlarged arches designate a negative association. The projection of traits on PC1 and PC2 under PEG 6000 induce drought stress revealed that the root length (RL) and shoot length (SL) are highly correlated and as well as with mean germination time (MGT). MGT exhibited a negative association with GP, SG, FWR, FWS, DWR and DWS. But the germination traits (GP, SG, FWR, FWS, DWR and DWS) were strongly associated with each other (Fig. 3). 

PCA scores of advanced inbred lines under PEG6000 induced moisture stress. PCA score plots of 82 inbred lines of Brassica juncea in the first two PCA’s, PCA1 and PCA2, were computed on X and Y squared distances for each inbred line. These scores were plotted on the 2D score plot (Fig. 4). A scrutiny of these results revealed that the inbred lines RLM-619, PBR97, Pusa Bold, DJ-101-DT-A-4, DJ-113 DT-3, DJ-124-1-DT-3, DJ-124-1-DT-62, MCN 2-14, EC-564647, JM-06-0206, JC-1359-23-558, MCP-12-224 and QM-7-196 fall on the same square box, indicating their diversity and high tolerance to drought stress. The inbred lines in the adjacent box showed the relatedness and moderate tolerance to drought stress were considered moderately tolerant. The lines A8-4-2×JR-049, CP-98, DJ-1-2(DT-5), DJ-124-1-DT-28, DJ-124-1-DT-70, DJ-127 (DT-35), DJ-5, ELM-151, GR-325, JM-06022, JPM-2, K-9-108, M-305, NRCDR-2, PRG-901 and RL-1359 present in the upper right-hand square boxes are highly susceptible to PEG-induced drought stress. In contrast, the square box on the upper left side was considered moderately susceptible.

Fig. 3. Loading plot showing the germination traits of 82 inbred lines of Brassica junceaon PC1 and PC2.

Fig. 4. Score plot of 82 inbred lines of B.juncea on PC1 and PC2 represented by codes (Refer Table 2 for strain name).

Fig. 5. Cluster dendrogram of 82 inbred lines of B.juncea represented by codes (Refer to table for strain name).

Hierarchical clustering. Germination traits of 82 inbred lines of mustard were subjected to hierarchical clustering using Ward’s linkage (Fig. 5). The complete set was categorized into four categories (highly tolerant HT, moderately tolerant MT, moderately susceptible MS, and highly susceptible HS). There were 13 HT, 29 MT, 23 MS and 17HS inbred lines.

DISCUSSION

Seed germination and establishment are the two critical stages in the life cycle of a crop. Mortality leads to yield loss due to decreased plant density; thus, screening inbred lines at the early seedling stage is an important criterion for selecting drought stress-tolerant varieties. PEG 6000 is used to create osmotic potentials which fits well for testing seed germination (El-Deeb et al., 2018). Drought stress significantly affected all the studied germination traits with a significant reduction in germination and speed of germination concomitantly with the increase in the stress level, which agree with the findings of Toosi et al. (2014) and Channaoui et al. (2017) and Radhouane et al. (2007). Mean germination time increased with the increased stress levels. Comparable results have been reported by Toosi et al. (2014) and Hellal et al. (1999). Low hydraulic conductivity due to PEG was the probable reason for progressive decline in the GP. In the PEG induced treatments water is not available for imbibition to seeds (Radhoune et al., 2007) which is fundamental for germination (Sharma et al., 2013).

Different concentrations of PEG inhibited the developmental processes by negatively affecting all the germination traits. In the present study, lengths of root and shoot and their respective dry weight was reduced significantly at all the six stress levels with respect to control. Earlier in Brassica juncea (Toosi et al., 2014), Brassica napus (Jamil et al., 2019) and sunflower (Geeta et al., 2012) have reported similar reduction in lengths and dry weights. Root system has a prominent role in moisture stress tolerance (Chun et al., 2021) and is mainly deployed for water and mineral uptake (Jajarmi et al., 2014). In the current study, root and shoot growth decreased with the concomitant increase in PEG concentrations. Root length was less affected than shoot length, but the roots were relatively thin and delicate. Similarly, reduction in the dry weight of roots and shoots were observed in all inbred lines with the increase in drought stress levels/negative osmotic potentials. Our results are endorsed by Jamil et al. (2019) and Channaoui et al. (2017). Fresh and dry weights were reduced significantly at negative osmotic potentials over control. Our results are in concordance with the results of Jamil et al. (2019) (Brassica napus), Radhoune et al. (2007) (Pearl millet), Song et al. (2021) (castor bean) and Yagmur et al. 2008 (triticale) where PEG 6000 induced drought stress reduced fresh and dry weights of roots and shoots.

Imposed drought stress negatively and significantly impacted all the traits, but the responses varied with different stress levels. Maximum decline was observed at -0.6 Mpa and henceforth. The reduction may be attributed to the highly reduced osmotic potential (Yagmur et al., 2008) and the activity of α-amylase, β- amylase, and α-glucosidase enzymes involved in growth and germination (Channaoui et al., 2019). Accumulation of reactive oxygen species (ROS) in response to drought boosted the transcription of NADPH oxidase genes, thus affecting the developmental processes (Luo et al., 2021). Cross-talk between drought and ABA involved germination and seedling growth.

Principal component analysis can select variables that can be categorized into main groups and subgroups based on homogeneity and dissimilarity (Neeru et al., 2015 and Sisodia et al., 2017). The PC1 absorbed and described for maximum (69.9%) proportion of total variability in the set of all 9 PCs and the remaining ones accounted for a progressively lesser and lesser amount of variation. Our results have concordance with the findings of Zada et al. (2013) in B. carinata, Avtar et al. (2014) in toria and Neeru et al. (2015) in B. juncea. As per our study, inbred lines were categorised into four groups: highly tolerant (13), moderately tolerant (29), moderately susceptible (23), and highly susceptible (17). Saima et al. (2012) in wheat and Saleem et al. (2017) in Indian mustard have reported similar results.

As PEG induced drought stress affected germination traits of all Brassica juncea inbred lines, but the thirteen lines including three varieties (RLM-619, PBR97 and Pusa Bold, five natural B.juncea germplasm DJ-101-DT-A-4, DJ-113 DT-3, DJ-124-1-DT-3, DJ-124-1-DT-62, MCN 2-14, two exotic mustard lines EC-564647 and JM-06-0206, two introgression lines JC-1359-23-558  and MCP-12-224 and one zero erucic acid line QM-7-196 exhibited better performance for all germination traits viz., GP, SG, MGT, root and shoot lengths, fresh and dry weights of seedlings at the six osmotic potentials. Tolerance in the promising inbred lines were further confirmed by PCA and hierarchical clustering. Performance of Brassica juncea inbred lines under seven osmotic potentials designated that seedling germination and growth is the most reliable and efficient phase for studying drought stress. Moreover, this study also revealed that all the germination traits (GP, SG, MGT, RL, SL, FWR, FWS, DWR and DWS) can be considered as best indicators for studying moisture stress invitro. The genetic potential of elite inbred lines revealed better germination traits conferring drought tolerance, however field studies will endorse the performance of the selected lines for stress tolerance.

Screening large germplasm for drought tolerance in-vitro is simple, economical, and time-effective methodology.  Therefore, the performance of B. juncea inbred lines were evaluated with PEG induced drought stress levels. Based on the lesser decline in the germination traits, confirmed through PCA and hierarchical clustering, three varieties RLM-619, PBR97 and Pusa Bold, five natural B. juncea germplasm lines DJ-101-DT-A-4, DJ-113 DT-3, DJ-124-1-DT-31, DJ-124-1-DT-62, MCN 2-14, two exotic B. juncea EC-564647 and JM-06-0206, two introgression lines JC-1359-23-558 and MCP-12-224  and one zero erucic acid line QM-7-196 (S106) were found promising.

The PEG induced screening and evaluation of 82 inbred lines helped to identify and categorize the germplasm for further evaluation under field conditions for drought stress. Considering the degree and impacts of drought stress on the productivity of Brassicas, present study may have practical implications for farmers and agricultural policy makers.

Saleem Jahangir Dar, Pushp Sharma, Rajni Sharma and S.S. Banga  (2024). Evaluation and Characterization of Germination Traits in Brassica juncea ((L.) Czern Cross) Inbred Lines through PEG Induced Simulated Osmotic Stress. Biological Forum – An International Journal, 16(3): 136-146.