3.1 Calcium (Ca2+)
Data observed for calcium root and shoot of Silybum marianum (L.) Gaertn, grown at two different altitudes i.e. Qta & Tbt under Cd+ 2 (500µM) toxicity statistically showed significant (P < 0.05) and non-significant (P > 0.05) respectively. The obtained results revealed that highest calcium content in root was significant (P < 0.05) and reported in H2O2 spray under cadmium stress at high altitude Qta while the lowest calcium content was observed in H2O priming under control conditions at Qta (Fig. 2).
However, the order of changes in Root calcium content at Qta was observed as: H2O2 spray Cd+ 2> SA P + FS Cd+ 2>H2O2 P + FS Cd+ 2>H2O P + FS Cd+ 2> H2O spray Cd+ 2 >SA priming Cd+ 2> H2O2 priming control > H2O2 priming control > H2O2 spray control > SA priming control > SA spray Cd+ 2>Control Cd+ 2>SA P + FS control > H2O priming Cd+ 2>H2O spray control > SA spray control > Control of control > H2O2 P + FS control > H2O P + FS control > H2O priming control.
While the order of improvement at Tbt was recorded as follows: H2O2 P + FS Cd+ 2> H2O2 priming Cd+ 2> SA priming control > SA P + FS control > SA priming Cd+ 2>SA P + FS Cd+ 2> SA spray control > H2O priming control > H2O P + FS control > H2O spray control > SA spray Cd+ 2>H2O spray Cd+ 2> H2O priming Cd+ 2> H2O2 P + FS control > H2O2 spray Cd+ 2> Control Cd+ 2>H2O P + FS Cd+ 2> H2O2 priming control > H2O2 spray control.
Furthermore, the results for shoot calcium content revealed non-significant (P > 0.05) results by showing SA Foliar spray under cadmium stress to be the best treatment in enhancing calcium content in shoot of Milk thistle while the lowest content of calcium in shoot was observed in Control under control conditions at high altitude Qta (Fig. 2).
The order of changes in shoot calcium content at Qta was observed as: SA spray Cd+ 2> SA priming Cd+ 2> H2O spray Cd+ 2> SA P + FS Cd+ 2> H2O2 spray Cd+ 2>H2O2 P + FS Cd+ 2>H2O2 priming Cd+ 2>H2O priming Cd+ 2> SA priming control > H2O spray control > SA P + FS control > H2O2 spray control > H2O P + FS Cd+ 2> Control Cd+ 2> H2O2 priming control > H2O2 P + FS control > SA spray control > H2O priming control > H2O P + FS control > Control of control.
While at low altitude Tbt, the order of improvement was observed as: SA spray control > SA priming Cd+ 2> SA priming control > H2O P + FS Cd+ 2> H2O priming control > H2O P + FS control > SA P + FS control > Control Cd+ 2>H2O2 P + FS control > H2O2 P + FS Cd+ 2> H2O2 spray Cd+ 2> H2O spray Cd+ 2> SA P + FS Cd+ 2> SA spray Cd+ 2> H2O2 priming Cd+ 2>H2O2 priming control > H2O spray control > H2O2 spray control > H2O priming Cd+ 2> Control of control.
In a nutshell, results revealed that the treatment of plants with 10 µM Hydrogen peroxide (H2O2) enhanced the root calcium content under heavy metal toxicity (Cd+ 2) however, the calcium content in shoot reported to have increased by the application of 0.25 µM Salicylic acid (SA) under cadmium stress which illustrates a positive sign of plant signaling molecules to have been playing key role in plant growth and development mechanism under stress conditions (Fig. 2).
3.2 Potassium (K)
Data obtained for Potassium (K) content of root and shoot of Milk thistle which was grown at two varying altitudes i.e. (Qta & Tbt) under heavy metal cadmium (500µM) toxicity showed statistically significant (P < 0.05) results. Data further suggested that in root of milk thistle the best treatment for the potassium was observed as H2O P + FS under cadmium stress at low altitude (Tbt) while the treatment for low potassium content was observed in H2O spray under control conditions at high altitude (Qta). Resultantly, the potassium content in Milk thistle root under both cadmium and control conditions of H2O priming and foliar spray enhanced at both altitudes (Fig. 2).
Hence, the order of changes in potassium content of root at high altitude Qta was observed as: SA spray control > H2O2 priming control > SA priming Cd+ 2>H2O2 priming Cd+ 2>SA spray Cd+ 2>H2O P + FS control > SA P + FS control > H2O P + FS Cd+ 2> H2O2 P + FS Cd+ 2> SA priming control > H2O spray Cd+ 2> H2O2 P + FS control > SA P + FS Cd+ 2> H2O2 spray Cd+ 2>H2O priming Cd+ 2> Control Cd+ 2> H2O priming control > H2O2 spray control > Control of control > H2O spray control.
While, the order of improvement at Tbt observed as: H2O P + FS Cd+ 2> H2O spray Cd+ 2> H2O2 P + FS Cd+ 2> SA P + FS Cd+ 2> H2O P + FS control > H2O2 spray Cd+ 2>SA priming control > SA P + FS Cd+ 2>H2O2 priming Cd+ 2> SA spray control > H2O2 P + FS control > H2O2 spray control > H2O priming Cd+ 2> H2O spray control > SA P + FS control > Control Cd+ 2> H2O priming control > H2O2 priming Cd+ 2> Control of control > SA spray Cd+ 2.
Apart from significant results of root potassium content, Milk thistle showed statistically significant (P < 0.05) results in shoot as well as by illustrating that the treatment H2O2 priming under Cd+ 2 stress at Qta was the best while the least treatment with potassium content was recognized as H2O priming under control conditions at the same altitude (Qta) (Fig. 2).
The order of changes in shoot potassium content at high altitude Qta was observed as: H2O2 priming Cd+ 2> H2O2 priming control > H2O2 P + FS control > SA P + FS control > SA P + FS Cd+ 2> H2O2 P + FS Cd+ 2> H2O P + FS control > H2O2 spray Cd+ 2> SA priming Cd+ 2> H2O spray control > H2O2 spray control > SA spray control > H2O spray Cd+ 2> H2O P + FS control > H2O priming Cd+ 2> SA priming control > SA spray Cd+ 2> Control of control > Control Cd+ 2> H2O priming control.
While the order of improvement at Tbt was observed as: SA P + FS Cd+ 2> SA priming Cd+ 2> H2O P + FS Cd+ 2> H2O spray Cd+ 2> SA P + FS control > SA priming control > H2O2 P + FS Cd+ 2> H2O2 spray Cd+ 2> H2O P + FS control > SA spray Cd+ 2>H2O2 priming Cd+ 2>H2O2 spray control > H2O2 spray control > H2O2 P + FS control > H2O priming Cd+ 2> H2O2 priming Cd+ 2> Control Cd+ 2> SA spray control > Control of control > H2O priming control.
Considering overall results of Milk thistle potassium content, it has been observed that plant perform well at different altitudinal variations because of their properties and different sort of adaptation. It has been reported that potassium content in Milk thistle at both altitudinal fields i.e. (Qta & Tbt) reported to have been increased due to the plant signaling molecules such as H2O2 which reported to be the best source of enhancement of plant physiological and biochemical attributes under stress of heavy metal cadmium. Thus, potassium content increase under control conditions but significantly enhanced due to the SA along with H2O2 by suppressing the negative and harmful role of cadmium (Cd+ 2) (Fig. 2).
3.3 Nitrate (NO3−)
Data reported for nitrate content in the root and shoot of Milk thistle under a concentration of 500µM Cd+ 2 stress at two varying altitudes i.e. (Qta & Tbt) statistically showed significant (P < 0.05) results. The data further revealed that in root, maximum nitrate content was observed in SA P + FS under cadmium stress at high altitude Qta while the minimum nitrate accumulation was observed in Control of control conditions at low altitude Tbt (Fig. 2).
However, the order of changes within root nitrate content at Qta was recorded as: SA P + FS Cd+ 2> SA spray Cd+ 2> SA P + FS control > H2O2 spray Cd+ 2> H2O2 P + FS Cd+ 2> H2O2 priming control > SA priming Cd+ 2> H2O2 P + FS control > H2O2 spray control > SA priming control > SA spray control > H2O spray Cd+ 2> H2O2 priming control > H2O P + FS control > H2O P + FS Cd+ 2> H2O priming Cd+ 2> H2O priming control > Control Cd+ 2> Control of control > H2O spray control.
While the order of improvement at Tbt was observed as: H2O2 priming Cd+ 2> H2O priming Cd+ 2> SA priming Cd+ 2> H2O2 spray Cd+ 2> SA spray Cd+ 2> SA P + FS Cd+ 2> H2O2 P + FS control > SA spray control > H2O2 P + FS Cd+ 2> H2O P + FS Cd+ 2> H2O2 priming control > H2O spray control > H2O priming Cd+ 2> SA P + FS control > Control Cd+ 2> H2O2 spray control > H2O priming control > H2O P + FS control > SA priming control > Control of control.
Data for shoot nitrate content in Silybum marianum (L.) Gaertn also showed statistically significant (P < 0.05) results that showed the maximum nitrate accumulation in SA P + FS under control conditions at high altitude Qta while the minimum nitrate content was reported in Control under control conditions at low altitude Tbt.
The order of improvement in shoot nitrate content in Milk thistle at high altitude Qta was observed as: SA P + FS control > SA P + FS Cd+ 2> SA spray Cd+ 2> SA spray control > H2O2 spray control > H2O2 P + FS Cd+ 2> SA priming Cd+ 2>H2O2 P + FS control > SA priming control > H2O spray Cd+ 2> H2O P + FS Cd+ 2> H2O2 priming Cd+ 2> H2O2 spray Cd+ 2>H2O P + FS control > H2O spray control > H2O2 priming control > H2O priming Cd+ 2> Control Cd+ 2> H2O priming control > Control of control.
While the trend for shoot nitrate at low altitude Tbt was reported as: SA P + FS control > H2O2 P + FS Cd+ 2> SA spray Cd+ 2>H2O2 P + FS control > H2O P + FS Cd+ 2> SA P + FS Cd+ 2> H2O2 spray Cd+ 2> H2O2 spray Cd+ 2> SA spray control > H2O2 priming Cd+ 2> SA priming Cd+ 2> H2O2 spray control > H2O P + FS control > H2O priming Cd+ 2>H2O2 spray control > H2O2 priming control > SA priming control > H2O priming control > Control Cd+ 2> Control of control.
In a nutshell, it has been observed that the combination treatment of 0.25 µM salicylic acid (SA) (priming and foliar spray) was the best treatment in increasing the nitrate accumulation in root and shoot of milk thistle. (Fig. 2)
3.4 Cadmium (Cd+ 2)
Data obtained for cadmium content in both root and shoot of Milk thistle at two varying altitudes i.e. (Qta & Tbt) under the toxicity of cadmium at a concentration of 500µM that showed statistically significant (P < 0.05) and non-significant (P > 0.05) results respectively (Fig. 3). The data further revealed that Cd+ 2 concentration in root was significant (P < 0.05) by illustrating that the maximum cadmium content was observed in H2O2 Priming treatment under cadmium toxicity at high altitude Qta while the minimum content of Cd+ 2 was observed in H2O2 P + FS under control conditions at Tbt (Fig. 3).
However, the order of cadmium content in root at Qta was observed as: H2O2 priming Cd+ 2>SA spray Cd+ 2> H2O2 spray Cd+ 2> H2O P + FS Cd+ 2> SA priming Cd+ 2>H2O2 P + FS Cd+ 2>SA spray control > SA P + FS Cd+ 2> H2O spray Cd+ 2> H2O2 priming control > H2O spray control > H2O priming Cd+ 2>H2O P + FS control > SA priming control > H2O priming control > H2O2 P + FS control > H2O2 spray control > Control Cd+ 2> SA P + FS control > Control of control.
While the trend for root cadmium content at Tbt was recorded as: Control Cd+ 2> H2O priming Cd+ 2> H2O P + FS Cd+ 2> H2O2 spray Cd+ 2> SA spray Cd+ 2>H2O spray Cd+ 2> SA priming Cd+ 2>H2O2 priming Cd+ 2> SA P + FS Cd+ 2> SA spray control > SA P + FS control > H2O2 P + FS Cd+ 2> H2O2 spray control > SA priming control > H2O P + FS control > Control of control > H2O spray control > H2O2 priming control > H2O2 P + FS control.
Furthermore, the data obtained for shoot cadmium content reported to have non-significant (P > 0.05) results by showing the H2O2 priming treatment under cadmium concentration at Qta having maximum Cd+ 2 concentration while the lowest content of cadmium was reported in SA priming under control conditions at Tbt (Fig. 3).
The trend observed for shoot cadmium concentration at Qta was recorded as: H2O2 priming Cd+ 2>SA spray Cd+ 2> SA P + FS Cd+ 2> H2O2 P + FS Cd+ 2> SA P + FS control > SA priming Cd+ 2> H2O spray Cd+ 2> H2O spray control > H2O2 priming control > SA priming control > SA spray control > H2O P + FS Cd+ 2> H2O2 P + FS control > H2O2 spray Cd+ 2>H2O priming Cd+ 2> H2O2 spray control > Control Cd+ 2> H2O P + FS control > Control of control > H2O priming control.
While the trend observed for shoot Cd+ 2 at Tbt was reported as: SA spray Cd+ 2> SA spray control > H2O2 P + FS Cd+ 2> H2O2 spray Cd+ 2>H2O P + FS Cd+ 2> H2O P + FS control > H2O2 spray control > H2O2 priming Cd+ 2> Control Cd+ 2>H2O2 P + FS control > H2O priming control > H2O priming Cd+ 2>Control of control > H2O2 priming control > H2O spray Cd+ 2> H2O spray control > SA P + FS Cd+ 2> SA priming Cd+ 2> SA P + FS control > SA priming control.
In a nutshell, it has been reported that both root and shoot, the concentration of cadmium toxicity was overall high but in shoot as compared to root. Cd+ 2 was high in the respective treatments that were applied with cadmium concentration of 500µM. However, the cadmium treated plants that were applied with plant signaling molecules such as H2O2 and SA reported to decrease the level of cadmium in the respective treatments at both the varying altitudes i.e. (Qta & Tbt). Overall, the concentration of cadmium by successive application of signaling molecules effectively decreased the cadmium toxicity in milk thistle plants (Fig. 3).
3.5 Weight of 100 seeds/plant
Data recorded for hundred seeds/plant of Milk thistle grown at two different altitudes i.e. (Qta, Tbt) under cadmium (500µM) toxicity showed statistically non-significant (P > 0.05) results. Data further revealed that the maximum weight of hundred seeds was observed in H2O2 priming in control of Tbt while the minimum seeds were recorded in H2O2 P + FS in control conditions at Qta which illustrates that priming and Foliar spray of the treatments with H2O2 boosted up the weight of seeds (Fig. 4).
The order of changes under control and cadmium conditions at high altitude Qta was observed as follows: H2O2 priming Cd+ 2> H2O2 P + FS control > H2O P + FS Cd+ 2> H2O P + FS control > H2O priming Cd+ 2> SA P + FS Cd+ 2> H2O2 spray Cd+ 2> SA priming Cd+ 2> SA priming control > Control Cd+ 2>SA P + FS control > H2O2 spray control > H2O spray Cd+ 2> Control of control > H2O2 spray control > H2O priming control > H2O2 P + FS Cd+ 2> H2O2 priming Cd+ 2> SA spray control > SA spray Cd+ 2.
While the order for seeds weight at low altitude Tbt was observed as: SA spray control > SA spray Cd+ 2> H2O priming Cd+ 2> SA priming Cd+ 2> Control of control > SA P + FS control > H2O2 priming control > H2O spray Cd+ 2> SA P + FS Cd+ 2> H2O spray control > H2O2 spray control > H2O2 spray Cd+ 2>H2O2 priming Cd+ 2>H2O P + FS Cd+ 2> SA priming control > H2O priming control > H2O P + FS control > H2O2 P + FS Cd+ 2> Control Cd+ 2> H2O2 P + FS control.
In a nutshell, it has been observed that hundred seeds weight was maximum for those treatments which were primed with plant signaling molecules i.e. (H2O2 and SA) that enhanced the weight of seeds but interestingly combinational treatment (Priming and Foliar spray) of H2O2 yielded healthier seeds. However, it has been observed form the results that the weight of seeds at Tbt was higher as compared to the Qta milk thistle plants. Apart from the weight of seeds, Milk thistle also reported to have the highest yield from those treatments that were applied with SA and H2O2 at both altitudes (Fig. 4).
3.6 Silymarin
Data obtained for silymarin content of seeds of Milk thistle at two different altitudes (Qta & Tbt) under Cd+ 2 (500µM) stress gave statistically significant (P < 0.05) results (Fig. 4). Further the data revealed that the highest silymarin contents was recorded in H2O2 P + FS under cadmium stress at low altitude Tbt while H2O2 Priming at low altitude Tbt showed the minimum contents of silymarin in Milk thistle seeds under control conditions (Fig. 5).
The order of improvement for silymarin contents in the seeds of Milk thistle at Qta was observed as: SA P + FS Cd+ 2>H2O2 P + FS Cd+ 2>Control Cd+ 2>H2O priming Cd+ 2>H2O2 priming Cd+ 2> H2O2 priming control > H2O P + FS Cd+ 2> SA P + FS control > H2O2 spray Cd+ 2> Control of control > SA spray Cd+ 2> H2O2 P + FS control > SA spray control > SA priming Cd+ 2> H2O priming control > H2O2 spray control > H2O P + FS control > SA spray control > H2O spray Cd+ 2> H2O spray control.
While the order of changes at low altitude Tbt was recorded as follows: H2O2 P + FS Cd+ 2> SA P + FS Cd+ 2>SA spray Cd+ 2> SA P + FS control > H2O2 P + FS control > H2O2 spray Cd+ 2>H2O P + FS Cd+ 2> SA spray control > H2O priming Cd+ 2> H2O spray Cd+ 2> H2O2 spray control > Control of control > SA priming Cd+ 2> SA priming Cd+ 2>H2O priming control > Control Cd+ 2>H2O P + FS control > H2O2 priming Cd+ 2>H2Ospray control > H2O2 priming control.
Silymarin is the best component of Silybum marianum (L.) Gaertn which is obtained from the seeds and reported to have widely been used for its medicinal properties for liver diseases. In the following results, considering overall silymarin contents of milk thistle at both varying altitudes, it has been revealed that the highest silymarin content was observed in the respective treatments which were applied with 10 µM Hydrogen peroxide (H2O2) due to its role in alleviating the negative role of heavy metal (Cd+ 2) toxicity (Fig. 5).