Source of Material and Treatment

Immature seeds were the source of explants and culture in MS media [22]. Three hormones, i.e., 2,4-D, IBA, and NAA, with five levels each (1 to 5 mg/L), were applied for callus induction. The callus was subjected to shoot and root regeneration using three levels of kinetin, i.e., 0.5 mg/L, 1 mg/L, and 1.5 mg/L, and two levels of NAA, i.e., 0.5 mg/L and 1 mg/L, respectively.

Surface Sterilization of the Explant

The immature seeds were sterilized with distilled water 3 times. The explant was disinfected by dipping it in 5% bleach for 30 min., shaking it well, and then sterilizing it with 90% ethanol for 1 min. Explant surface sterilization was performed with autoclaved distilled water 3-5 times until the sterilant was completely removed.

Callus Induction and Regeneration

Sterilized forceps and a knife were used for the removal of the immature embryo from the seed and placed on MS + callus media having five levels of the 2,4-D hormone (1 mg/L, 2 mg/L, 3 mg/L, 4 mg/L, and 5 mg/L), phytagel 2 g/L, and 30 g/L sucrose. The medium’s pH was maintained from 5.7 to 5.8, and autoclaved for 20 min. at 15 lbs/inch² pressure, and 121ºC temperature. Explants were cultured at a temperature of 20±1 to 27±1ºC throughout periods of light and darkness, and the callus induction percentage from the cultured embryo was noted. Calli were shifted to freshly prepared MS media with the same hormone to maintain callus and further propagation. Callus media was refreshed after 14 days. A similar procedure was carried out for callus induction in NAA and IBA hormones, but callus induction was not seen at any level of IBA or NAA. After callus induction in 2,4-D media, the callus was shifted to regeneration media having various concentrations of kinetin and NAA along with 30 g sucrose and 2 g/L phytagel (Fig. 1). The traits recorded for callus were callus induction and its fresh and dry weights; however, shoot and root regeneration traits were shoot length, root length, root percentage, number of shoots per plant, and their fresh and dry weights. Weights were recorded using a digital balance, and lengths were measured with the help of a measuring tape.

Statistical Analysis

All the determinations were made in three replicates from the completely randomized design (CRD). Software Statistix 8.1 assessed the significant differences among the wheat varieties at varying kinetin, NAA, and 2,4-D concentrations. MS EXCEL was used to present the data. PCA was performed to determine the correlation among wheat varieties with callus formation and root and shoot regeneration. The graphical presentation was done using ggplot2, factoextra, and factominer packages in R software (4.0.2).

Callus Formation

Different concentrations of three hormones, i.e., NAA, 2,4-D, and IBA, were added to MS media separately for callus initiation; however, callus initiation was observed by the 2,4-D hormone only after nearly 14 days, which shows that 2,4-D is an effective hormone for callus development [23]. The induction of callus was observed through visual inspection and was characterized by the appearance of an undifferentiated mass of cells. All the callus traits showed highly significant (p≤0.001) variation among wheat varieties, 2,4-D, and the interaction between varieties and 2,4-D (Table 1).

All the wheat varieties responded differently to 2,4-D concentrations in the media. Callus formation wasn’t recorded in all wheat varieties at the lowest concentration of 2,4-D, i.e., 1 mg/L. At 2 mg/L of 2,4-D, callus induction was highest in MH, followed by Arooj, Akbar, Subhani, and Dilkash varieties. The highest callus formation was recorded at 3 mg/L 2,4-D in Dilkash, MH, and Akbar. Arooj and Dilkash varieties showed the maximum callogenesis at 4 mg/L (Fig. 2a). Nawaz et al. [24] reported that callus development was negligible at 1 mg/L 2,4-D, and lower callus induction was noted at 5 mg/L. Our findings also coincide with the results of Patel et al. [25] and Mahmood et al. [26] in wheat; they recorded that 3 mg/L of 2,4-D was the best, and as the amount of 2,4-D increased, the callus initiation rate gradually decreased. The outcomes of the current study specified that the induction of callus depends not only on the different treatments of 2,4-D but also on the varietal differences present in wheat varieties, and our results are consistent with the earlier findings of [27] and [28]. The varietal differences normally result from variations in internal hormonal levels and genetic makeup [29].

Fig. 1.

Some representative pictures of wheat varieties showing a) callus formation and (b-d) root and shoot regeneration.

An increase in callus fresh weight was noted by boosting the level of 2,4-D up to 3 mg/L. In Dilkash at 3 mg/L of 2,4-D, callus fresh weight was highest, followed by MH and Arooj. The highest values of fresh weights were recorded in MH, Arooj, and Dilkash at 4 mg/L of 2,4-D. The lowest fresh weights were noted in the Subhani variety at every level of 2,4-D. In general, the results demonstrated that raising the concentrations of 2,4-D positively affected the fresh weight up to 3 mg/L. After that, at higher levels (4 and 5 mg/L), fresh weights were reduced (Fig. 2b). Maximum callus dry weight was noted at 2 mg/L of 2,4-D in Akbar, followed by Arooj, Subhani, and Dilkash, while minimum callus dry weight was recorded in MH. At 4 mg/L, the dry weight of the callus was the highest in Akbar, followed by Arooj and Dilkash, and the lowest value was recorded in Subhani. The highest callus dry weight was observed in Arooj, and the lowest dry weight was noticed in Subhani at 3 mg/L of 2,4-D (Fig. 2c). Callus is used in various biotechnological aspects; therefore, the wheat varieties having higher values of callus fresh and dry weights possess a greater ability to grow [30]. Yadav et al. [31] showed that 2,4-D was the most potent for in vitro callogenesis in wheat among other auxins. Similarly, Kumar et al. [32] reported that 2,4-D was superior to NAA or IBA for callus formation. One of the most potent synthetic auxins, 2,4-D, is essential to several in vitro techniques, including callogenesis, embryogenesis, organogenesis, and shoot regeneration [33].

Fig. 2.

Effect of different concentrations of 2,4-D hormone on a) callus induction, b) callus fresh weight, and c) callus dry weight of 5 wheat varieties (Dilkash, Arooj, MH, Akbar, and Subhani). Values represent means ± S.E. Significant differences were measured by the least significant difference (LSD) and indicated by different lowercase letters. S.E. stands for standard error.

Role of Kinetin in Shoot Regeneration

Cytokines best promote plant cell division and cell differentiation, as they perform a major role in plant tissue culture [34]. It promotes the growth of shoots from the meristematic tissues and encourages the development of multiple shoots [35]. Kinetin, a synthetic cytokinin, was used to stimulate shoot formation. This protocol was devised to check the level of kinetin, which provides maximum results in shoot induction. All shoot regeneration traits showed highly significant (p≤0.001) results for varieties and kinetin. The interaction between varieties and kinetin represented highly significant variations (p≤0.001) for shoot length and number of shoots per plant, while significant (p≤0.05) results were obtained for fresh and dry weights (Table 2).

It was noted in all varieties that by boosting the concentration of kinetin up to 1.5 mg/L, the number of shoots formed was improved. However, Dilkash, Arooj, and MH varieties showed more formation of shoots as compared to Akbar and Subhani (Fig. 3a). Our study is contradictory to the findings of Mahmood et al. [26], who tested the greatest regeneration ability of wheat varieties at 1 mg/L kinetin and after that at 1.5 mg/L kinetin. In comparison, El Sayed et al. [36] studied the MS medium provided along with 0.5 mg/L kinetin and 0.5 mg/L BAP, which led to maximum regeneration. Rahman et al. [37] revealed that maximum regeneration in wheat was achieved at 1 mg/L kinetin. Conflicts may result from diverse explant sources, genetic material under research, external factors, and variability in cultural conditions [26, 38]. The environment can influence the callus’s ability to regenerate by changing the internal hormonal balance of the explant [38].

Shoot length and fresh weight of shoots were maximum at 1.5 mg/L in all varieties compared to the lower levels, i.e., 0.5 and 1 mg/L kinetin. Dilkash exhibited better performance in terms of shoot length and fresh weights; however, Subhani showed a poor response (Fig. 3b) and c)). The highest shoot dry weight was noted at 1.5 mg/L in Dilkash, followed by Akbar. The lowest dry weights were noted at 0.5 mg/L kinetin (Fig. 3d). Plants with better growth features show better survival potential in both normal and stressed environmental conditions [39].

Root Formation from Shoots by Different Levels of NAA

Auxins are the primary plant growth regulators that promote root growth in many plant species. The role of NAA, a synthetic auxin, in root induction at different levels was observed. All the root traits showed highly significant variations (p≤0.001) among wheat varieties and NAA levels, except root percentage, while the interaction between NAA and varieties represented highly significant variations (p≤0.001) for root fresh and dry weights (Table 3).

Maximum root formation was noted at 1 mg/L NAA (Fig. 4a). Moreover, according to Pathak et al. [40], NAA at 1 mg/L provided the best rooting response. Maximum root length was recorded in Dilkash at both levels of NAA; however, minimum root length was observed in Arooj at both levels of NAA (Fig. 4b). The highest root fresh weight was recorded in Dilkash at 1 mg/L NAA, while in Arooj and MH intermediate root fresh weight was noted, whereas Subhani and Akbar showed the lowest root fresh weight at 1 mg/L (Fig. 4c). The highest root dry weight was recorded in Dilkash and MH at both levels, i.e., 0.5 mg/L and 1 mg/L NAA, while the root dry weight was the lowest in Subhani at these levels (Fig. 4d). NAA is the most effective plant growth hormone for inducing root formation in several plants [37, 41]. Multiple investigations have shown that lower NAA levels of 0.5-3 mg/L are required for attaining optimum rooting [24, 42]. Waseem et al. [43] reported that the lowest concentration of NAA (0.5 mg L^-1) produced the greatest number of shoots per explant, leaves, and nodes per shoot when applied alone, suggesting its superiority over all other concentrations of NAA. It was also noted in Chrysanthemum that a higher NAA concentration in MS media lowered the rate of multiplication [44]. NAA treatment boosted root production in azalea cultivars, but the roots were short and fragile because high concentrations of NAA may hinder root elongation [45].

Principal Component Analysis (PCA)

The PCA demonstrated a significant impact of the 2,4-D hormone in wheat varieties for callus parameters (Fig. 5a) and b)). The PCA components, i.e., Dim1 and Dim2, exhibited a 97% total variation. Dim1 contributed 80.9% and Dim2 16.1% of the variation. Akbar reflected the highest value of CDW and the lowest CFW. Subhani showed lower values for all parameters than the others. Most of the influence of CFW was observed in Dilkash and Arooj (Fig. 5a). CFW was significantly influenced at a 3 mg/L concentration with positive eigenvalues; however, for CI, all levels of 2,4-D contributed equally except 5 mg/L 2,4-D (Fig. 5b)). Dim1 explained 80.9% of the total obtained variation; the first dimension defined CFW, CDW, and CI, so Dim1 alone should be a good indicator of the effect of the 2,4-D hormone.

Fig. 3.

Effect of different concentrations of the kinetin hormone on a) number of shoots per explant, b) shoot length, c) shoot fresh weight, and d) shoot dry weight of 5 wheat varieties (Dilkash, Arooj, MH, Akbar, and Subhani). Values represent means ± S.E. Significant differences were measured by the least significant difference (LSD) and indicated by different lowercase letters. S.E. stands for standard error.

Shoot regeneration PCA demonstrated the role of different levels of kinetin on wheat varieties in which Dim1 and Dim2 exhibited 89.2% and 5.8% variations, respectively (Fig. 5c) and d)). Akbar and Subhani showed different results regarding SDW and NOS, as Akbar has relatively higher SFW, SDW, and SL values, while Subhani possesses lower values for all parameters (Fig. 5c). The lowest scores for all parameters were shown at 0.5 mg/L kinetin. 1.5 mg/L kinetin showed a higher value on SFW, SDW, NOS, and SL. The strongest positive correlation exists between SFW and SL; however, a negative correlation was noticed between SDW and NOS (Fig. 5d).

Root regeneration PCA demonstrated the influence of different concentrations of NAA hormone on wheat varieties, in which Dim1 and Dim2 contributed 86.6% and 6.4% variations (Fig. 5e) and f)). A 1 mg/L concentration of NAA exhibited higher values of RDW with positive eigenvalues and RFW and RL with negative eigenvalues. However, no major contributor was found under 0.5 mg/L NAA treatment. A strong positive correlation exists between RL and RFW, and a negative correlation was noted between RDW and RFW.

Fig. 4.

Effect of different concentrations of NAA hormone on a) root percentage, b) root length, c) root fresh weight, and d) root dry weight of 5 wheat varieties (Dilkash, Arooj, MH, Akbar, and Subhani). Values represent means ± S.E. Significant differences were measured by the least significant difference (LSD) and indicated by different lowercase letters. S.E. stands for standard error.

Fig. 5.

Principal Component Analysis (PCA) of 5 wheat varieties for callus formation and root and shoot regeneration; a) and b): PCA represents the effect of 2,4-D treatment on 5 wheat varieties for callus induction (CI), callus fresh weight (CFW), and callus dry weight (CDW); c) and d): PCA plot of five wheat varieties represent the effect of kinetin on shoot regeneration parameters, i.e., shoot dry weight (SDW), shoot fresh weight (SFW), shoot length (SL), and number of shoots (NOS); e) and f): PCA plot represent NAA effect on root regeneration parameters, i.e., root dry weight (RDW), root fresh weight (RFW), root length (RL), and root percentage (RP).