Assessing lead and cadmium tolerance of Chenopodium ambrosioides during micropropagation: an in-depth qualitative and quantitative analysis

Experimental materials

Axillary branches measuring 7–9 cm in length were carefully excised from healthy C. ambrosioides plants growing at Malakand University in Khyber Pakhtunkhwa (KP), Pakistan. These branches were subjected to a meticulous cleansing process, involving a through rinsing with tap water for 6–7 min, followed by immersion in a solution of mercuric chloride (0.05%) containing few drops of Tween-20 for 12–15 min. Subsequently, the explants were washed with sterilized distilled water and the surface sterilized explants (2–3 cm long) were then horizontally cultured in screw capped jars containing Murashige & Skoog (1962) medium, enriched with 3% sucrose. The medium was semi-solidified with agar (0.6%), and its pH was meticulously adjusted to fall within the range of 5.50–5.56. Following the adjustment, the entire setup was adjusted to autoclaving at a temperature of 121 °C under a pressure of 15 psi for 10–13 min. To provide optimal growth, all cultures were incubated within a growth chamber at a constant temperature of 26 ± 2 °C, under a lighting system that simulated a 16-hr day followed by an 8-hr night cycle.

Callus induction and subculture for shoot initiation

Explant segments, comprising nodes, leaves, and petioles with sizes ranging from 2 to 3 cm, were placed in culture media containing varying concentrations of NAA (0.5, 1.0, and 1.5 mg/l), Indole-3-acetic acid (IAA) (0.5, 1.0, and 1.5 mg/l), and BA (0.5, 1.0, and 1.5 mg/l). These components were either used individually or combined with NAA (0.5 mg/l) or IAA (0.5 mg/l) to initiate the formation of callus (Table 1). After a thirty-day period, calluses that had developed from nodes, leaves, and petioles were isolated and subsequently transferred to growth media containing different concentrations of NAA (1.0, 1.5, and 2.0 mg/l), BA (1.5 and 2.0 mg/l), and combinations of BA/NAA (1.5/1.0 and 2.0/1.0 mg/l). These conditions were utilized to induce the formation of shoots through sub-culturing.

Shoot proliferation and root induction

For shoot proliferation, the in-vitro shoots were sliced into two to three pieces and transferred onto a growth medium supplemented with varying concentrations of growth regulators. Specifically, concentrations of 1.0, 1.5, and 2.0 mg/l of BA and kinetin were used. These growth regulators were applied either individually or in combination with NAA at a concentration of 1.0 mg/l. The aim was to standardize the concentrations and combinations for shoot proliferation (Table 2). The observation took place four weeks after the initial inoculation, where the shoot multiplication percentage, as well as the number and length of shoots, were recorded.

Moving on to root induction, individual shoots that were 30 days old were carefully excised and transferred to a medium containing a combination of BA and NAA at concentrations of 1.5/1.0 mg/l for the purpose of regeneration. Subsequently, these shoots were further placed on media supplemented with NAA at concentrations of 1.0, 1.5, and 2.0 mg/l. This was done both individually and in combination with BA/NAA at 1.5/1.0 mg/l and kinetin/NAA at 1.5/1.0 mg/l. The intention here was to facilitate the induction of roots. The progress of root formation, including induction percentage, numbers, and length, were then observed and recorded on the 24th and 26th days post-inoculation.

Acclimatization

Plantlets were gradually acclimated to a non-sterile environment by gently loosening the screws of the culture containers and lastly detached the cap. The plantlets were detached from the culture media and rinsed using sterilized distilled water, effectively eliminating the residual observing medium. These cleansed plantlets were then transplanted into plastic cups filled with a sterilized mixture of soil and sand (2:1 ratio). To initiate this transition, the plantlets were initially enclosed with plastic cups and wetted with half MS medium.

Heavy metals treatments and growth tolerance index (GTI)

In the context of heavy metals treatments, the in-vitro shoots were transplanted onto a growth medium enriched with specific metals, in conjunction with the optimal blend of BA/NA at a ratio of 1.5/1.0 mg/l. A series of concentrations for Pb(NO3)2 (0.5, 1.5, and 2.5 mg/l) and CdCl2 (0.5, 1.5, and 2.5 mg/l) were employed, combined with the previously mentioned optimal BA/NAA ratio (1.5/1.0 mg/l). The purpose of this study was to identify strains that exhibited a degree of tolerance. As a control group, an MS medium was employed that contained the ideal BA/NAA ratio (1.5/1.0 mg/l), yet lacked the introduction of Pb(NO3)2 and CdCl2. Subsequent to these treatments, the in-vitro shoots underwent assessment in terms of measurements and weight determination. To ascertain the dry biomass, the plant materials underwent a 24-hour exposure to 80 °C and were subsequently weighed. This facilitated the computation of the growth tolerance index (GTI) for the shoots, presented as a percentage. The GTI was calculated using the following formula: ${\displaystyle \mathbf{GTI}=\frac{\text{Dry weight of shoots developed on media with}Pb{\left(N{O}_3\right)}_2\text{or}\left(CdC{l}_2\right)}{\text{Dry weight of shoots developed on media devoid of}Pb{\left(N{O}_3\right)}_2\text{or}\left(CdC{l}_2\right)}\times 100.}$

Statistical analysis

Differences among the treatments were assessed using a single factor analysis of variance (ANOVA). Furthermore, the variances among the variables were examined by a post-hoc Tukey honest significant difference (HSD) test. Statistically significant variations were considered significant at a threshold of P < 0.05. To assess the impact of heavy metals, we conducted a correlation analysis involving multiple parameters of C. ambrosioides such as the concentration of heavy metals (HMC mg/l), the growth tolerance index (GTI %), multiplication rate (%), number of shoots, shoot length, and biomass. All the statistical analyses were performed in XLstat and OriginLab Pro-2023b (https://www.originlab.com) software’s.

Callus induction

Node, leaf and petiole of C. ambrosioides were inoculated on media containing various concentrations of NAA, IAA, BA, NAA/BA, IAA/BA and NAA/IAA as outlined in Table 1. Within a span of 10 days from inoculation, all three types of explants exhibited the development of callus. The texture of resulting callus varied, ranging from compact and soft to friable and granular. Specifically, callus formation occurred in petiole explant at a rate of 78.27%, leaf at a rate of 76.37% when treated with 1.5 mg/l NAA, and in node at a rate of 65.71% with 1.0/0.5 mg/l BA/NAA (Table 1). Notably, the callus developed from nodes, particularly under the influence of BA with or without presence of NAA, exhibited a remarkable organogenic potential. Conversely, the callus derived from petiole and leaf explants did not display any organogenic potential (Table 2). In the case of IAA, both alone and in combination with BA, the formation of callus was observed; however, their corresponding fresh weights and percentage responses remained relatively low (Table 1). Explants that were placed on a growth regulator-free medium (control) failed to exhibit any growth and eventually died after 15 days of inoculation.

Shoot from callus

Thirty days old callus from node, leaf and petiole were isolated and sub-cultured on media containing NAA, BA and BA/NAA (Table 2). Nodal explants produced compact, friable, glandular callus with a light yellowish type. In the presence of BA, either alone or with conjunction with NAA (Table 2), these nodal callus cultures displayed prolific shoot formation. The average number of shoot formed with BA ranged from 10.84 ± 1.45 to 14.83 ± 0.93, with corresponding shoot lengths ranging from 5.21 ± 0.83 to 5.42 ± 0.83. Conversely, when utilizing the combination of BA/NAA, the average number of shoots formed ranged from 9.43 ± 0.63 to 8.78 ± 0.49. Notably, the application of NAA (at concentration of 1.0–2.0 mg/l) resulted in the formation of an average of 6.25 ± 0.61 to 5.45 ± 1.73 shoots, respectively. Nevertheless, callus derived from leaf and petiole sources exhibited a lack of shoots regeneration (Table 2).

Multiplication of shoots

Cluster of shoots (2–3) were sub-cultured on media having various concentrations and combinations of BA, Kn or BA/NAA and Kn/NAA in order to standardized the optimal concentration and combination for shoot multiplication (Table 3). The results of shoots multiplication were recorded for all concentrations of BA and Kn, both with or without NAA (Table 3). Notably, the application of BA/NAA induced the highest number of shoots, ranging from 21.89 ± 1.94 to 18.39 ± 1.68, with their shoot lengths ranged from 5.49 ± 0.84 to 4.82 ± 0.76 cm. The supplementation of BA alone proliferated shoots in the ranged of 16.5 ± 1.03 to 14.61 ± 0.61. Interestingly, Kn either alone or in combination, produced less number of shoots compared to BA, whether applied separately or in combination (Table 3).

Root induction

The best rooting rate (76.73%) and number (12.32 ± 0.58) were recorded in the medium comprising NAA at a concentration of 1.0 mg/l. The formation of roots with BA/NAA (1.5/1.0 mg/l) resulted in a lower number (8.07 ± 0.16) but higher length (13.23 cm). However, the medium supplemented with Kn/NAA did not support roots formation (Table 4).

Acclimatization

The survival potential of the in-vitro regenerated plantlets when transferred to the ex-vitro environment was 71% (Fig. 1). Notably, there were no obvious phenotypic differences observed between the acclimatized plants and the original mother stock. Subsequently, the two month old plantlets were then transplanted into the lath house.

Heavy metals and shoot proliferation

After optimizing of culture condition, subsequent experiments were designed to identify a suitable line for metals (Pb(NO₃)₂ and CdCl₂) tolerance (Table 5). The inclusion of Pb(NO₃)₂ in the medium inhibited shoots multiplication rate. The percentage of shoot multiplication per explants dropped from 86.74% in the control treatment to 57.38% in culture treated with Pb(NO₃)₂ (Table 5). Although, Pb(NO₃)₂ containing media did not exhibit any visible symptoms of phenotypic alteration except for a reddish purple color, all shoots maintained a normal morphology characterized by broad lamina and short petiole similar to those of the mother plant. In some instances, the same explants produced both normally morphed shoots with green and reddish purple color shoots (Fig. 1C) at 0.5 mg/l of Pb(NO₃)₂ in the medium. The growth of the shoot biomass (dry weight) remained unaffected by Pb(NO₃)₂ at 0.5 mg/l in the medium and was comparable to the control, while higher concentration Pb(NO₃)₂ in the medium led to an increase biomass growth (Table 5).

The culture of C. ambrosioides vigorously reproduced in-vitro shoots proliferation in media supplemented with CdCl₂. The highest propagation percentage (75.71%) was obtained with 0.5 mg/l of CdCl₂ concentration in the medium. The shoots formed under varying concentrations of CdCl₂ ranged from 16.31 ± 1.61 to 13.89 ± 1.94 per explants. Notably, in presence of lower CdCl₂, concentrations, shoot multiplication was as high as in the control (Table 5). Shoots with 0.5 mg/l CdCl₂ displayed a normal morphology, while 1.5 and 2.5 mg/l of CdCl₂ induced morphological abnormalities in the in-vitro shoots. The abnormalities included narrow leaf lamina, elongated petioles, and reddish purple coloration (Fig. 1B). Biomass growth of shoots exhibited an increased trend with increasing concentration of CdCl₂ (Table 5).

Heavy metals and growth tolerance

The growth tolerance index (GTI) calculation revealed that shoot growth was enhanced in the presence of heavy metals (Table 6). The highest GTI value was observed in the medium comprising 2.5 mg/l of lead, reaching to 194.11%. Conversely, lower doses of lead resulted into low dry biomass production, with GTI values of 152.94% for 1.5 mg/l and 117.64% for 0.5 mg/l. In contrast, the GTI exhibited a reduction in culture grown on cadmium-containing medium compared to lead-containing medium. The GTI values for cadmium-containing media ranged from 188.23% to 264.70% as given in Table 6. Furthermore, the heavy metals concentration has a significant positive (r = 0.671; p < 0.01) effect on GTI (%), shoot length (r = 0.924; p < 0.001) and dry weight ((r = 0.651; p < 0.01) respectively (Fig. 2). In contrast, the HMC (mg/l) presence in the media exhibited an adverse effect on multiplication (r =  − 0.24; p < 0.05) and number of shoots (r =  − 0.655; p < 0.01).