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Effect of meta-Topolin on morphological, physiochemical, and molecular dynamics during in vitro regeneration of Salix tetrasperma Roxb.

Abstract

An efficient in vitro propagation protocol has been established for a valuable medicinal plant, Salix tetrasperma using mature nodal explants. The investigation aimed to observe the influence of various combinations and concentrations of cytokinins (mT, BA, and Kn) and auxins (NAA, IAA, and IBA) on regeneration potential using the Murashige and Skoog (MS) medium. Among individual cytokinin treatments, 5.0 µM mT resulted highest response of 92% with maximum shoot number (11.6 ± 0.08) per explant and shoot length (4.5 ± 0.22 cm) after 12 weeks of culture. However, synergistic treatment of mT (5.0 µM) and NAA (0.5 µM) further improved proliferation with (21.3 ± 0.40) shoots per explant and (6.9 ± 0.13 cm) shoot length in 96% cultures after 12 weeks of incubation. Rooting from in vitro raised microshoots was achieved on ½ MS medium supplemented with various concentrations of low-dose auxins. The highest number of roots (10.4 ± 0.20) per shoot with mean root length (5.7 ± 0.11 cm) with maximum rooting frequency (97%) was observed in 0.5 µM IBA, after 4 weeks of culture. The rooted plantlets achieved a remarkable 86% survivability rate, when transferred to ex vitro conditions during acclimatization. Analysis of photosynthetic parameters and their characteristics during the acclimatization phase revealed a gradual decline in photosynthetic attributes during initial weeks; however, a significant improvement was noted as the growth proceeded. SEM analysis revealed the ultra-morphological structural differences between in vivo and in vitro derived leaves of S. tetrasperma. Moreover, DPPH assay observed differential antioxidant activity of in vitro raised plantlets throughout the acclimatization period. The GC-MS analysis from leaf extracts of donor plants and in vitro derived plantlets has revealed a broad spectrum of phytochemical compounds with significant pharmacological properties. No polymorphism in the banding pattern was found when the genetic fidelity of the regenerated plants was evaluated using SCoT primers, indicating the clonal stability of micropropagated plants. This study is the first to explore the use of mT in regeneration of S. tetrasperma, showing its more effectiveness than BA and Kn.

Key message

A reliable and more advanced in vitro protocol was established for Salix tetrasperma using cytokinin (mT), a novel aromatic cytokinin. This method led to significant improvement in multiple shoot formation and elongation, root development. Moreover, the assessment of transpiration rate (E), intercellular CO2 concentrations (Ci), net photosynthetic rate (PN), stomatal conductance (gs), chlorophyll (Chl a/b), carotenoid content, phytochemical profile and total antioxidant activity during acclimatization ensures enhanced survivability of in vitro raised plants. Furthermore, the application of mT did not compromise the genetic fidelity of the regenerated plants.

Peer Review reports

Introduction

The genus Salix, generally known as ‘willows’, is the largest genus of the Salicaceae family. The genus comprises about 400–500 species inhabit through a broad range of climatic conditions from subtropical to boreal climates. Currently, numerous willow species and their hybrid varieties are being utilized for their versatile applications and notable credentials in phytoremediation [1], agroforestry [2] and bioengineering [3] purposes. Salix tetrasperma Roxb., a valuable and deciduous tree generally known as Indian willow, is found mainly on humid soils in cold as well as temperate regions of the northern hemisphere [4]. The latest studies have revealed that Salix tetrasperma has great potential for the production of biofuels and lignocellulosic biomass due to its energetic growth and developmental habit [5]. More importantly, various parts of the plant and their extracts have potential medicinal value and are reported to have analgesic, anti-inflammatory, astringent, analgesic and anti-fever properties. Aspirin, derived from the Salix plant, is widely recognized for its effective application as a non-steroidal anti-inflammatory drug (NSAID). The dried and powdered leaves, when combined with sugar, have medicinal benefits for various ailments, including swelling, epilepsy, rheumatism, infectious diseases, hemorrhoids, and gallbladder stones [6,7,8]. The phytocompounds associated with these pharamacological activities exhibit more diversified chemical composition in buds, bark and leaves than other plant component of Salix tetrasperma. Due to its ecological, economic and prominent medicinal importance, this tree becomes highly vulnerable to exploitation from its natural habitat. Although the conventional method through vegetative cuttings is the chief means of propagation but dioecious character, season specificity, slow growth and differential geographical allocation of the male and female co-partners of S. tetrasperma makes it challenging to propagate through vegetative methods. The rapid pace of urbanization, coupled with the complex and growing demands of the human population, makes it challenging for traditional propagation methods to meet the requirement for the rapid advancement of newly favoured genotypes. Plant tissue culture technology provides the ultimate solution for addressing this need in numerous woody species and other related scenarios [9, 10]. This technology offers a useful tool for the propagation and multiplication of a variety of medicinal plant species in a short amount of time to fulfil the rising demand for a range of pharmaceutical companies [11]. Moreover, micropropagation methods are employed to facilitate the global exchange of disease-free clonal material [12, 13] and to induce genetic transformation for the production of transgenic regenerants [14].

A significant challenge of in vitro regeneration technology is the high mortality rate of plants during their transition from controlled laboratory to natural conditions [15]. Upon transfer to ex vitro conditions, in vitro derived plants are exposed to changes in temperature, light intensity, and water availability. This necessitates a period of acclimatization to ensure the successful establishment and survival of the plantlets. During acclimatization, the environment is gradually adjusted to mimic ambient relative humidity and light levels [16]. This transitional phase allows for the development of morphological, anatomical and physiological characteristics that are better suited to the new conditions, thus reducing the influence of the in vitro environment.

There are numerous reports on in vitro culturing of willows [17,18,19]. While prior studies, such as those by Khan et al. [20, 21], have demonstrated the role of cytokinins like BA and Kn in shoot formation in Salix tetrasperma, their efficiency and broader potential remain limited. However, no report of in vitro propagation by enhanced axillary bud proliferation from nodal explant is available, employing newly introduced meta-Topolin (mT). This study pioneers the use of meta-Topolin (mT), a lesser-explored cytokinin, to establish a highly efficient in vitro propagation protocol for S. tetrasperma. The mT belongs to a novel class of naturally occurring aromatic cytokinins with the potential to induce regeneration responses in various woody plant species as reported earlier in Allamanda cathartica [22] and Feronia limonia [23]. The unique structural properties of the mT, specifically the presence of a hydroxyl group on its side chain facilitates the non-toxic and easily exchangeable of cytokinin due to the formation of O-glucoside conjugate forms. Moreover, the application of mT over other cytokinins have been found to be efficient for the induction and multiplication of shoots, reduction in physiological abnormalities, improved rooting and acclimatization in many plant species [24, 25].

Therefore efforts have been made to optimize the in vitro regeneration protocol for mass multiplication of S. tetrasperma under the influence of mT using nodal segment explants. Photosynthetic parameters and their aspects along with analysis of ultra-morphological structure were assessed during acclimatization period. Start codon targeted (SCoT) markers were employed to ensure the genetic stability of plantlets produced through current established protocol. The current study marks a significant advancement as it presents the pioneering report on comparative analysis of phytochemical composition between in vitro raised plants and mother plant of Salix tetrasperma. Moreover, the antioxidant activity of in vitro derived plants performed during acclimatization phase was evaluated using DPPH assay.

Materials and methods

Establishment of aseptic culture

During January 2020, young explants of S. tetrasperma were collected from lateral branches of a mature tree maintained at the Department of Botany, Aligarh Muslim University, Aligarh. The leaves were detached and explant material was cleaned by washing under running tap water for 20 min. Subsequently, a Labolene (Qualigens, India) was applied for duration of 5 min before washing several times with distilled water. The plant material was then subjected to surface sterilization using 0.1% (w/v) HgCl2 (Qualigens, India) for 4 min. The sterilized explants were cut into 0.5–1 cm nodal segments and aseptically cultured for shoot induction.

Media and culture conditions

The experiments were carried out using MS medium [26] supplemented with 3% (w/v) sucrose (Qualigens, India) and different concentrations of plant growth regulators (PGRs). Prior to autoclaving at 121℃ for 15 min, the medium was solidified with 0.8% (w/v) bacteriological grade agar (Qualigens, India), and the pH was adjusted to 5.8. The culture tubes were incubated under controlled conditions of 25 ± 2℃ with a 16/8 h (light/dark) cycle. Cool white fluorescent lamps (Philips, India) provided a photosynthetic photon flux density (PPFD) of 50 µmol− 2 s-1, and the humidity in the culture chamber was maintained at 60–65%.

Shoot induction and multiplication

To induce morphogenic response, nodal explants containing axillary buds were placed on MS medium supplemented with three different cytokinnis (mT, BA, and Kn) at various concentrations (1.0, 2.5, 5.0, 7.5, and 10 µM). These PGRs were used individually or in combination with auxins (NAA, IAA and IBA) at various concentrations (0.1, 0.25, 0.50, and 1.0 µM). Subsequent subculturing onto the same medium was performed every three weeks, for a total of five cycles. The frequency of explants producing shoots, the number of shoots per explant, and the length of the shoots were measured after 8 and 12 weeks of culture.

In vitro rooting

To initiate the rooting process, healthy and well-elongated shoots (4–5 cm) were selected and placed on half-strength MS medium. The rooting medium was supplemented with different concentrations (0.1, 0.5, 1.0, and 1.5 µM) of various auxins such as NAA, IAA, and IBA. Data regarding the percentage of successful root formation, the average number of roots per shoot, and the length of the roots were collected and recorded after four-week culture period.

Acclimatization

The rooted shoots were carefully removed from the culture medium and gently washed under running tap water. The fully grown plantlets were then transferred to thermocol cups filled with sterile substrates, including soilrite, garden soil, a 1:1 mixture of garden soil and vermiculite. To maintain suitable humidity levels, the potted plantlets were covered with transparent polythene bags and placed in a growth room. Regular watering was done every other day using a half-strength MS solution without any organic supplements. After two weeks, the polythene bags were removed to allow the plants to acclimatize under lab conditions. Following a four-week acclimatization period, the plants were transferred to pots containing regular garden soil and placed in a greenhouse.

Evaluation of physiological and biochemical parameters and biomass

The photosynthetic parameters and their aspects, including transpiration rate (E), intercellular CO2 concentrations (Ci), net photosynthetic rate (PN), stomatal conductance (gs), chlorophyll (Chl a/b) and carotenoid content, were assessed during the acclimatization period. By using a portable Infra-Red Gas Analyzer (IRGA, LI-COR 6400, Lincoln, NE, USA), the leaf gas-exchange characteristics were determined from completely grown intact leaves of in vitro raised plantlets at (0, 7, 14, 21, 28, 35, 42, 49, 56, and 70 days). Values for gs, PN, Ci, and E, were represented as mol (H2O) m− 2 s− 1, µmol (CO2) m− 2 s− 1, ppm, and mmol (H2O) m− 2 s− 1 respectively. The procedure demonstrated by Hussain et al. [27] was applied to the evaluation of chlorophyll (a/b) and carotenoid contents. Fresh leaf samples were taken from randomly chosen plants during the acclimation period of in vitro produced plants at various transfer days as mentioned earlier. Utilizing a mortar and pestle, the needed sample (0.5 g) was homogenized in 10 ml acetone (80%) following which the sample was purified using Whatman No. 1 filter paper. The absorbance of the final extract was assessed using a spectrophotometer (UV-1700, Shimadzu, Japan) at wavelengths (645 and 663 nm), (480 and 510 nm) for chlorophyll and carotenoids respectively and were expressed in mg g− 1 (fresh weight).

Ten randomly selected plantlets (mT, BA and Kn -derived) were instantly measured for fresh weight production after which they were dried for 48 h in the shade. The shade-dried plantlets were then placed in a hot oven set at 60 °C for 24 h in order to eliminate any moisture that may have remained. To determine the amount of dry matter produced, dried plants were weighed, and the average values were calculated in mg/plantlet.

Scanning electron microscopy (SEM) analysis

Fresh leaf samples were obtained from two sources: in vivo plant and 4-week-old acclimatized plantlets. These samples were prepared for SEM analysis. Firstly, the leaf samples were fixed in a solution containing 2.5% glutaraldehyde, 2% paraformaldehyde, and sodium cacodylate (0.1 M) buffer for a duration of 2 h. Subsequently, post-fixation was conducted using 1% osmium tetraoxide. The samples were then dehydrated using a graded series of ethanol concentrations (50%, 70%, 95%, and 100%). The dried samples were coated with a thin layer of gold-palladium using a sputter coater (JEOL JFC-1600). Finally, the prepared samples were observed under a SEM (JEOL JSM-6510LV) at resolutions of 500X and 4000X, with an accelerating voltage of 15 Kv.

Genomic DNA isolation and genetic fidelity analysis

Fresh leaves (0.5 g) from nine randomly chosen in vitro regenerated plants were used to isolate and purify genomic DNA using a slightly modified version of the DNA isolation process as described by Deshmukh et al. [28]. The adaptations entailed two additional steps: proteinase-K treatment and the use of a phenyl: chloroform: isoamyl alcohol mixture (in a ratio of 25:24:1 v/v). The quality and quantity of the isolated DNA were evaluated using a Nanodrop™ Spectrophotometer (Implen, Germany), with absorbance measurements conducted at 260 nm and 280 nm. To assess the genetic fidelity of the in vitro propagated plants, the PCR-based SCoT technique [29] was employed. An initial screening was performed using ten specific SCoT primers, and the PCR reactions were conducted using a Biometra thermocycler (Germany). Each PCR reaction comprised a 20 µl total volume, including 2 µl of 10X buffer, 1.2 µl of 25 mM MgCl2, 0.4 µl of 10 mM dNTPs, 0.2 µl of Taq DNA polymerase (1 U), 1 µl of primer (1 U), 1.5 µl of DNA (25 ng/µl), and 14.2 µl of deionized water. The PCR amplification consisted of a series of 45 cycles, with a denaturation step lasting 5 min at a temperature of 94 °C, followed by an annealing step of 1 min at 55 °C, and an elongation step of 1 min at 72 °C. Subsequently, a final extension step was conducted for 10 min at 72 °C. The amplified DNA fragments were separated via electrophoresis on a 1% agarose gel (w/v) stained with 5 µl of ethidium bromide (ETBR) in 1X TAE buffer (pH 8.0) and electrophoresed at 60 V for 2 h. Visualization and scanning of the DNA banding pattern were conducted using a UVI gel documentation system (Bio-Rad, USA). The experiment was repeated five times and only significant and reproducible bands were considered to ensure originality.

Gas chromatography–mass spectrometry (GC-MS) analysis and identification of bioactive compounds

To prepare samples for GC-MS analysis, fresh leaves were acquired from both 4-week-old acclimatized plants and the mother plant. The collected samples were subjected to shade dry for 1 week after washing with sterilized double distilled water. The dried leaves were crushed into a fine powder using a mortar and pestle. 250 mg of this powdered material was dissolved in 50 mL of HPLC grade methanol, and the mixture was allowed to be extracted for 48 h. Afterwards, the methanolic leaf extract was centrifuged at 10,000 rpm for 10 min, and the resulting supernatant was filtered using a 0.22 μm aminigen syringe filter (Micro-por, Genetix Biotech Asia Pvt. Ltd., New Delhi, India) to remove any remaining residues. After filtering, approximately 10 mL of the filtrate was collected in separate culture tubes. Finally, the tubes were left uncovered allowing the solvent to evaporate at room temperature until the final volume reached 2 mL, rendering it suitable for subsequent phytochemical profiling. In the GC-MS analysis, a two-microliter aliquot of the methanolic leaf extract was manually injected into an RTX-5 column of a GCMS instrument (QP-2010 Ultra, Shimadzu, Kyoto, Japan) at a split ratio 20:1. Helium gas (99.999%) was used as the carrier gas at an inlet pressure of 173 kPa. The injector and ion source temperatures were maintained at 280 °C and 260 °C, respectively. Mass spectra were acquired at 70 eV ionization energy, with a scan interval of 0.5 s, covering fragments within the 40 to 450 Da range. To determine the relative percentage of each compound, their average peak area was compared to the total area. Identification and confirmation of bioactive compounds were performed using the National Institute of Standards and Technology (NIST) database and the Wiley Library for mass spectra, facilitating the determination of their molecular weight (MW).

Total antioxidant activity (DPPH)

The free radical scavenging activity of methanol extracts obtained from Salix tetrapserma was analyzed using the DPPH assay, following a standard protocol of Rahman et al. [30]. Briefly, to initiate the assay, a solution of 2,2-diphenyl-1-picrylhydrazyl (DPPH) in methanol was prepared at a concentration of 0.1 mM. Afterwards, 2.4 mL of DPPH solution was mixed with different volumes (25, 50, 75, and 100 µL) of the methanol extract to make a final volume of 4 mL. The mixture was gently agitated and allowed to stand undisturbed for 30 min in the dark. Then absorbance of the samples was measured at 517 nm using a UV–visible spectrophotometer, with a control reading taken. The percentage of DPPH free radical scavenging activity was determined using the following formula.

Percent inhibition of DPPH scavenging activity = (A0 − At)/A0 × 100)

Whereas A0 represents the OD of the control (without extract) and

At represents the OD of the sample (with extract).

Statistical analysis

The experiments were conducted following a completely randomized block design (RBD), with five replicates assigned to per treatment and each experiment was repeated three times. Statistical analysis was performed using one-way Analysis of Variance (ANOVA) in SPSS (Chicago, USA) to analyze the data. Differences among means were evaluated using Duncan’s multiple range test (DMRT) at a significance level of P ≤ 0.05. The results were presented as the mean ± standard error (SE) derived from five repeated experiments.

Results and discussions

Effect of single cytokinins on shoot initiation and multiplication

In the present study, the effectiveness of three different cytokinins (BA, mT, and Kn) was investigated for inducing bud breakage and multiple shoot formation. The addition of growth hormones to MS medium resulted in rapid initiation of bud break and the development of multiple shoots from nodal explants. Among the various cytokinins investigated, the application of mT exhibited the highest success rate in terms of shoot induction (Fig. 1A-C). Specifically, mT at 5.0 µM concentration resulted in the maximum production of 11.6 ± 0.08 shoots per explant with an average shoot length of 4.5 ± 0.22 cm and a regeneration response of 92% after 12 weeks of incubation (Table 1; Fig. 1B.). Additionally, BA (5.0 µM) and Kn (5.0 µM) also demonstrated significant but comparatively lower regenerative effects, with 8.2 ± 0.07 and 6.1 ± 0.06 shoots per explant with individual shoot length 3.8 ± 0.15 cm and 3.2 ± 0.11 cm and regeneration responses of 84% and 75%, respectively, after 12 weeks of culture (Table 1). However, explants cultured on basal MS medium without any cytokinins failed to produce shoots even after six weeks of culture, highlighting role of cytokinins in the induction of multiple shoots. The levels of endogenously produced plant hormones can vary among species, developmental stages, genotypes, explant types, and cultural conditions in tissue cultures [31]. Consequently, these variations may contribute to the different responses observed in various species when exposed to the same culture media supplemented with identical set PGRs. Furthermore, in addition to the differences in hormone types, the concentrations of cytokinins also play a significant role in axillary shoot development and the induction of in vitro cultures [32]. Cytokinins are reportedly to function as key tasks in cell division and DNA synthesis, which might be the source for the multiple shoot induction [33].

Fig. 1
figure 1

A. Nodal segment (bud break), induction of shoots on MS + mT (5.0 µM) B. Shoot proliferation on MS + mT (5.0 µM) after 12 weeks of culture C. Shoot proliferation on MS + mT (5.0 µM) + NAA (0.5 µM) after 12 weeks of culture

Table 1 Effect of single cytokinin treatments on multiple shoot induction from nodal explants, after 8 and 12 weeks of culture

The results of the study demonstrated a positive outcome, indicating that mT showed superior effectiveness compared to BA and Kn for this specific plant species. Lower regeneration frequencies were associated with reduced productivity in terms of shoot length, leaf area, and leaf size as the hormone concentrations were manipulated. Upon increasing the concentration of mT beyond the optimal level did not result in any improvement in shoot induction. Instead, a reverse trend was observed in all the evaluated parameters. The decrease in regeneration capabilities may be attributed to the detrimental effects of high hormone concentration on cultured cells, leading to the formation of vegetative buds. Interestingly, the effectiveness of mT has been reported in other plant species, such as Oxystelma esculentum [34] and Allamanda cathartica [35]. Various studies conducted on different species, including Ipomoea batatas [36], and Syzygium cumini [25], have also demonstrated the superior performance of mT compared to other cytokinins in terms of morphogenic response.

Effect of combination of cytokinins and auxins

A synergistic effect for large-scale shoot multiplication rate was observed when optimized concentrations of the tested cytokinins (mT, BA or Kn) were combined with various auxin (NAA, IAA or IBA,) concentrations. When compared to a single dose of cytokinin treatment, this method has proved to be quite beneficial since all combinations resulted in a high shoot yield as well as greener and healthier plantlets. Among various combinational treatments, the best outcome was obtained on MS medium supplemented with 5.0 µM mT and 0.5 µM NAA (Fig. 1C). This combination resulted maximum (96%) regeneration frequency of the cultures exhibiting an average of 21.3 ± 0.40 shoots per explant with shoot length of 6.9 ± 0.13 cm after 12 weeks of culture (Table 2; Fig. 1C). Additional combinations of BA and Kn with different concentrations of auxins also showed promising results, although with slightly lower effectiveness. Particularly, the combination of 5.0 µM BA and 0.5 µM NAA produced an average of 17.4 ± 0.33 shoots with a mean length of 6.4 ± 0.12 cm in 92% cultures after 12 weeks (Table 3). Similarly, the combination of 5.0 µM Kn and 0.5 µM NAA resulted in a mean shoot number of 14.6 ± 0.27 and a mean length of 5.7 ± 0.10 cm with maximum response of 84% after 12 weeks of cultures (Table 4). Auxins are commonly used in plant tissue culture to induce indirect organogenesis through callus formation or to promote rooting. However, when combined with cytokinins, auxins have shown promising results in accelerating the in vitro shoot development of Erythrina variegate [37]. In contrast, individual cytokinin treatments alone have been sufficient to achieve high rates of shoot multiplication, as reported in numerous cases [38, 39]. Auxins and cytokinins have the capacity to interact synergistically, additively, or antagonistically at different levels and in various plant species, leading to the modulation of physiological responses for optimal growth and development [40].

Table 2 Effect of different auxins treated with optimal concentration of mT (5.0 µM) in MS medium on shoot regeneration from nodal explants after 8 and 12 weeks of culture
Table 3 Effect of different auxins treated with optimal concentration of BA (5.0 µM) in MS medium on shoot regeneration from nodal explants after 8 and 12 weeks of culture
Table 4 Effect of different auxins treated with optimal concentration of Kn (5.0 µM) in MS medium on shoot regeneration from nodal explants after 8 and 12 weeks of culture

Therefore, the relative effectiveness of auxins in combination with cytokinins (mT, BA, and Kn) can be ranked as NAA ≥ IAA or IBA for large-scale multiplication of shoots. Such significant effects of auxin and cytokinin hormone combinations are well aided by previous research on Vitex negundo [41], Pterocarpus marsupium [24], and Crinum brachynema [42].

In vitro rooting

In the current study in vitro derived healthy and well-developed microshoots were transferred to media containing ½ MS supplemented with various concentrations of auxins (IBA, IAA, and NAA) to induce root formation. Among the different auxin treatments, the most effective for in vitro rooting was observed with ½ MS medium augmented with 0.5 µM IBA. This treatment resulted in the highest number (10.4 ± 0.20) of roots per shootlet and mean individual root length (5.7 ± 0.11 cm) with maximum rooting frequency of 97% after four weeks of transfer (Table 5; Fig. 2A). However, NAA and IAA at the same concentration observed 8.3 ± 0.13 and 6.2 ± 0.12 number of roots with rhizogenic response in 84% and 76% cultures, respectively. Therefore, three can be ranked in terms of their effectiveness as IBA ≥ NAA or IAA (Table 5). The initiation of root development in in vitro cultured shoot clusters is a crucial step in micropropagation systems. The shoots growing on Kn supplemented media were comparatively thinner as compared to those growing on BA and mT. Moreover, the physical strength of the growth medium (half or full strength) has a significant impact on the frequency of root growth and shoot initiation in various species of willows. Although most Salix species are observed to have natural vegetative regeneration, some of the genre members, such as Salix caprea particularly male plants are hard to root induction using traditional cutting propagation procedures [43]. The frequency of rooting is highly influenced with the age of source explants; inducing rooting at the seedling origin of the explants is easier as compared to elder ones [44]. Root formation in willow shoots grown without PGRs was reported by Chalupa [45], during the first two weeks of culture. In case of P. stocksii, it was observed that the utilization of a half-strength MS medium demonstrated enhanced efficacy in promoting root development compared to a full-strength MS medium [46]. In another study, Singh et al. [47] examined various media (¼, ½, ¾ and full), and experienced maximum rooting on ½ MS medium; induction in D. hamiltonii and D. asper. The capability of adventitious roots to grow on basal media without the addition of auxin can be attributed to the endogenous production of salicylic acid, which plays a vital role in the growth and development of plants [48]. Furthermore, the salicylic acid also facilitates the successful establishment of in vitro root formation [49].

Table 5 Effect of different concentrations of various auxins augmented with half strength MS media on root induction from in vitro raised microshoots on optimal medium (MS + mT (5.0 µM) + NAA (0.5 µM)) after 4 weeks of culture
Fig. 2
figure 2

A. In vitro rooting in microshoots on ½ MS containing IBA (0.5 µM) after 4 weeks of culture B. Acclimatized plantlets after 4 weeks of transfer

Hardening and acclimatization

The potential of regenerated plantlets to thrive in natural environment determines the efficiency of in vitro propagation protocol [25]. The immediate transplantation of regenerants from controlled culture condition to uncontrolled natural conditions is not encouraged; therefore in vitro derived plantlets are gradually allowed to face changing environmental conditions [50]. The well grown plantlets were then transferred to thermacol cups containing sterilized substrate for hardening inside the culture room for four weeks (Fig. 2B). Every other day, the plants were watered with ½ MS salt solutions. The substrates that were employed had a direct impact on the survival rate of plants. In comparison to vermiculite and garden soil, porosity of soilrite increased its ability to hold onto water, which enables better root development during acclimatization. After 4 weeks of acclimatization, soilrite had the best survival rate (86%) among the various potting materials used for acclimatization, while garden soil had the lowest survival rate (58%) for mT derived plantlets (Table 6). Additionally, during the acclimation period of regenerated plants various photosynthetic features and related properties were assessed at different time intervals. The in vitro raised plantlets were eventually moved to natural conditions, where they exhibited robust growth and thrived with normal morphology under full sunlight.

Table 6 Comparative analysis of survival rate and biomass content of 3-months-old in vitro raised plantlets of S. tetrasperma

Scanning electron microscope

The SEM examination revealed a significant difference in ultra-morphology between the in vitro and in vivo derived leave samples of Salix tetrasperma (Fig. 3). Stomata were only observable on the abaxial surface of the leaf in both cases; however the surface morphology of in vivo leaves observed heavy deposition of epicuticular wax. A similar result has also been achieved in superficial studies of mature carob leaves [51]. The in vitro dehydrated leaves displayed a smooth cuticle surface with almost no surface wax deposition (Fig. 3.1). They also had numerous and well-defined open stomata having clear aperture with elliptical in shape (Fig. 3.1a) and in other cases stomata were prominent but tightly closed (Fig. 3.1b). Moreover, some micrographs observed constricted leaf surface with deformed stomatal aperture and unhealthy guard cells (Fig. 3.1c). The presence of both open and closed stomata indicates the normal gaseous exchange of in vitro raised plantlets during acclimatization, whereas the consistent open and closed stomata suggests that plant is under stress conditions. The size, shape, density and function of stomata vary with the transfer from in vitro to ex vitro conditions [52]. Studies on Phelleodendron amurense and Aralia elata have shown that there are physiological and structural differences between in vitro and ex vitro derived plants. These differences are due to the fact that in vitro plants are grown in a controlled environment and are completely dependent on heterotrophic conditions [53].

Fig. 3
figure 3

(1) SEM examination of in vitro raised leaves, abaxial surfaces of S. tetrasperma: (1a) wide open stomata with narrow aperture, uniform guard cells; (1b) closed stomata with inner wall of guard cells; (1c) deformed stomata, uneven inner wall of guard cells. (2) The abaxial surface of in vivo leaves showing distribution of closed (red arrow) and open (blue arrow) stomata with uniform heavy deposition of epicuticular wax (2a) an open stomata covered with star shaped epicuticular wax; (2b) an closed stomata showing star shaped waxy depositions near the guard cells; (2c) deformed stomata showing star shaped waxy depositions near the guard cells

The surface characterization of in vivo plants revealed significantly increased wax deposition on both abaxial and adaxial surfaces [54]. During acclimatization an increase in stomatal density resulted with a reduction in pore size indicated a gradual stabilization of in vivo plantlets. The deposition of elongated wax particles are dispersed throughout the adaxial surface could also be seen clumped together in some places (Fig. 3.2). These results are in parallel agreements with the recent findings conducted on Aerva lanata [55]. According to compositional analyses of wax, various plant species revealed the presence of primary or secondary alcohols, fatty acids, ketones and aldehydes [56]. The SEM analysis of adaxial surface of in vivo leaf showed numerous stomata, having clear aperture with prominent guard cells (Fig. 3.2). The micrographs showed the presence of both open stomata (Fig. 3.2a) and closed type (Fig. 3.2b) with wide pore aperture. Additionally, electron micrographs observed constricted leaf surface containing few stomata having deformed aperture and unhealthy guard cells (Fig. 3.2c). Similar results were reported in a study conducted on Leucospermum cultivars [57].

Photosynthetic parameters and biomass content

When tissue culture generated plants are transplanted from in vitro to ex vitro circumstances, one of the main issues they face is adjusting to high transpiration rate, varied initial CO2 conductivity, low relative humidity, and abnormal stomatal movement [58,59,60]. Consequently, it is essential to assess different physio-biochemical characteristics while conducting the transplantation and acclimatization procedures for in vitro cultured plantlets. The assessment of photosynthetic parameters and their attributes observed a downward tendency during the first 14 days of transplantation in soilrite. After that, all the parameters significantly increased up to 28 days of acclimatization (Fig. 4A-F). Conversely, some parameters, including gs, PN, Ci and E, decreased dramatically after 28 days up to 35 days of acclimatization of regenerated plants (Fig. 4A-D). However, no apparent decrease was observed in the Chl a/b and carotenoid contents (Fig. 4E-F). The decreased trend was attributed due to change of transplanting material from and light irradiance (100 PPFD). It may be also suggested that underdeveloped photosynthetic system responsible for maintaining the internal water balance have contributed to the inadequate functioning of physio-biochemical processes [61]. Consequently, all photosynthetic parameters considerably increased after 35 days; afterwards no significant change was resulted between 56 and 70 days of acclimatization. Several studied conducted earlier have also reported the sudden reduction in photosynthetic pigment levels over the first few weeks, followed by a steady rise during the acclimatization of micropropagated plants [62]. The enhanced intake of water and nutrients following the development of new roots in microshoots improved all physiological functions, indicating the early underdeveloped in vitro roots were now developed and functional. The maximum values of physiological and biochemical attributes shown by in vitro raised plants included 0.062, 40.24, 303, 4.00, 2.31, 0.96, and 0.31 for gs, PN, Ci, E, Chl a, Chl b and carotenoid content respectively at 70 days of acclimatization (Fig. 4A-F). The enhanced carotenoid content suggests that the plants have effectively endured light-induced stress by safeguarding chlorophyll pigments from photo-oxidation throughout the stress period, as reported by Kenneth et al. [63]. The observed changes in photosynthetic parameters in the current investigation are consistent with earlier research findings [64, 65].

Fig. 4
figure 4

Changes in levels of various photosynthetic parameters during acclimation (0–70 days) of in vitro rooted plantlets of S. tetrasperma. A Stomatal conductance (gs), B Net photosynthetic rate (PN), C Intercellular CO2 concentration (Ci), D Transpiration rate (E), E Chlorophyll content (Chl a/b), F Carotenoid content

The application of mT not only improved the shoot and root number, photosynthetic traits and quality but also had positive influence on the biomass content of in vitro raised plantlets. The current investigation demonstrated a significant increase in both fresh and dry weight of biomass in plantlets derived from mT treatment in comparison to plantlets derived from BA and Kn treatments. The maximum production of biomass content of 3-month old mT derived plantlet was observed as 403.5 ± 7.68 mg/plantlet and 63.7 ± 1.21 mg/plantlet for sFW and sDW respectively (Table 6). Similarly Root biomass content showed maximum mean values of 118.4 ± 2.25 mg/plantlet and 25.4 ± 0.48 mg/plantlet for rFW and rDW respectively (Table 6). The involvement of mT in optimizing photosynthetic rate, delaying leaf senescence [66], enhancing chlorophyll content [67], and changing the sink-source distribution [68] may be the possible explanation for better effect of mT over BA and Kn treated plantlets. Therefore, the overall biomass content of the in vitro raised plantlets appears to have improved as a result of these features.

Genetic fidelity

In the present study the genetic stability of nine randomly selected in vitro plants was confirmed by SCoT primers. A total of 10 different SCoT primers were assessed in the screening process, resulting in the production of 74 distinct and reproducible amplified bands (Table 7). The size of these bands ranged from 500 to 2500 base pairs (bp). Notably, the monomorphic banding profile exhibited considerable variation, with SCoT33 generating the highest number of 10 bands per primer, while SCoT20 yielded a lower count of 04 bands (Table 7; Supplementary file 1). The banding pattern observed from SCoT18 and SCoT33 primers are shown in Supplementary file 1. Thus the genetic fidelity analysis between parental plant and in vitro raised plantlets showed a homologous and consistent banding pattern. Also, the results did not show any variation in morphology or polymorphism responsible for epigenetic variation. Furthermore, the analysis of the proposed dendrogram revealed an exceptional level of similarity, exhibiting over 99% resemblance between the mother plant and nine randomly chosen in vitro plantlets (Supplementary file 1). Therefore, the present study concluded that in vitro raised plantlets exhibited absolute genetic uniformity, thus confirming this method is appropriate for producing true-to-type plants. In vitro propagation is an effective tool for the stability and conservation of plants holding significant medicinal and economical value. However, the occurrence of genetic variation between regenerants and elite parental line is a possible disadvantage during in vitro propagation technique [69]. Genetic stability can possibly be impacted by the repeated subculture of sub-clones for an extended period of time in tissue culture medium and the administration of plant hormones at higher concentrations [24]. The genetic stability of sub-clones is an essential requirement to uphold and preserve the superior characteristics of the parental line [70]. According to Kuzminsky et al. [71], a small amount of genomic DNA from in vitro raised plant can be used to identify DNA-based molecular markers at any stage of plant development, and they are not impacted by environmental factors. The genetic fidelity analysis using different molecular markers of many species has been thoroughly established by Javed et al. [69] in Erythrina variegate, Ahmed et al. [72] in Cassia alata and Fatima et al. [73] in Withania somnifera using various explant sources. The uniform banding pattern confirming the complete genetic stability between sub-clones of the parent plant as shown in our results are in parallel agreement with the reports demonstrated from Erythrina variegata [74] and Clerodendrum thomsoniae [75].

Table 7 Start codon targeted (SCoT) primers used to assess genetic fidelity of in vitro-raised plantlets of Salix Tetrasperma

GC-MS analysis

In present study, a total of 80 bioactive compounds were identified in the leaf extracts of in vitro-cultured plants and mother (control) plant, utilizing methanol as the solvent. Remarkably, 24 of these compounds were consistently found in both sets of extracts, indicating shared chemical components. Furthermore, analysis revealed various phytocompounds in both types of leaf extracts, evidenced by distinct chromatographic peaks. Specifically, 43 peaks were detected in the methanolic extracts from in vitro-cultured plants (Fig. 5), while the control plant observed a slightly lower count of 37 peaks (Fig. 6), suggesting subtle differences in their chemical profiles. Compounds such as catechol, 3-amino-2-oxazolidinone, benzoic acid, salicyl alcohol, ethyl .alpha.-d-glucopyranoside, 3-hydroxy-.beta.-damascone, 4-vinylphenol, n-hexadecanoic acid, phytol as well as the principle compound salicin were found in higher quantities. The compounds identified in methanolic extract of in vitro and mother plant is represented in Table 8. Gas chromatography–mass spectrometry (GC-MS) is a highly precise and qualitative method for identifying bioactive compounds within plant extracts [76]. Previous studies documented by El-Wakil et al. [77] and Mostafa et al. [78] have examined the phytochemical composition of Salix tetrasperma using different chromatographic techniques. However, the current study marks a significant advancement as it presents the inaugural comparative analysis between in vitro raised plants and mother plant of Salix tetrasperma. This approach is instrumental in assessing chemical uniformity and facilitates the identification of pharmacologically relevant metabolites through the application of GC-MS.

Fig. 5
figure 5

GC-MS chromatogram of methanolic leaf extract of in vitro raised plants

Fig. 6
figure 6

GC-MS chromatogram of methanolic leaf extract of control plant

Table 8 Compounds identified through gas chromatography–mass spectrometry (GCMS) analysis of methanolic leaf extract from the in vitro raised plants and mother plant of S. Tetrasperma

The increased production of secondary metabolites in in vitro plants may be due to multiple factors including explant sources, prevailing culture conditions, composition of the growth medium, and the presence of growth regulators [79,80,81]. Other studies have also suggested the higher concentration of compounds in vitro plants might be attributed to the stress associated with in vitro culture [82, 83]. Our study is in agreement with the comparative analysis study conducted by Hussain et al. [25], who reported enhanced metabolite production in in vitro derived plants as compared to mother plant extracts. However, contrary to our study, Pathak et al. [84] reported that mother plant had a higher quantity of bioactive compounds than in vitro regenerated plants of Vanda cristata Wall. Similar to our results Mamgain et al. [85] in Pluchea lanceolata and Riahi et al. [86] in Artemisia arborescens have reported the presence of various secondary metabolites from the GCMS analysis of in vitro and mother plant extracts. Additional research is needed to elucidate the functions and potential applications of these novel compounds, which could introduce new approaches to disease management.

Total antioxidant activity (DPPH)

In the present study, the antioxidant activity of the plant (in vitro) extracts was evaluated using the DPPH radical scavenging assay. The DPPH activity of various methanolic concentrations obtained from S. tetrasperma at different acclimatization periods is represented in Fig. 9. The results demonstrated the excellent DPPH activity after 14 days of transfer. The maximum activity, 56% was shown at 100 µL concentration when compared to 0 days of transfer. Similarly the remaining extracts at 75 µL, 50 µL, and 25 µL observed 51, 44 and 42% DPPH activity, respectively, compared to their respective controls (Fig. 7). The cellular well-being is highly dependent on maintaining redox homeostasis in plants, which is primarily maintained by antioxidant molecules and enzymes [87]. These antioxidants effectively neutralize the potentially harmful effects of reactive and unstable free radicals within the cells [30].

Fig. 7
figure 7

DPPH radical scavenging activity of in vitro raised plantlets of S. tetrasperma during acclimatization periods (0–70 days)

The significant antioxidant activity is likely attributed to the phenolic and flavonoid compounds identified in GCMS analysis of methanolic leaf extracts. These phytochemicals, especially the phenolics, may have actively donated hydrogen molecules during the process, resulting in a dose-dependent reduction in color from purple to brown, indicating the formation of diphenylpicryl hydrazine [88]. The results demonstrated in our study align with the phytochemical analysis of S. tetrasperma, wherein the methanolic extract exhibited a substantial presence of phenolic and flavonoid constituents [89]. These compounds were found to be instrumental in determining the radical scavenging potential of the plant. In a previous study, the antioxidant activity of S. alba bark extract was determined using the DPPH assay, and the recorded activity was slightly lower with an EC50 value of 19.1 µg/mL [90].

Our study demonstrated the first report on evaluating the antioxidant potential using leaf extract of S. tetrasperma (Indian willow). Comparatively, the leaf extracts from other Salix species, such as S. aegyptiaca and S. subserrata [91, 92], exhibited lower antioxidant activity in the DPPH assay when compared to the leaf extract of S. tetrasperma in our investigation. The observed variations in antioxidant capacity, even within the same species and under the same assay conditions, may be attributed to differences in the phytochemical profile, harvesting time, or extraction methods employed for obtaining the plant materials.

Conclusions

The present study demonstrated a superior effect of mT in achieving multiple shoot formation from nodal explants of Salix tetrasperma in comparison to BA and Kn. The optimal shoot growth with no abnormalities was observed on MS medium containing 5.0 µM mT and 0.5 µM NAA. In vitro regenerated plants on being transferred to ex vitro conditions are exposed to conditions that are inconsistent with development under greenhouse or field environment. The results demonstrated changes observed in physiological parameters, leaf morphology, and antioxidant activity during the acclimatization phase. Significant changes in photosynthetic parameters and epicuticular wax deposition on leaves were observed as the acclimatization progressed. The antioxidant activity in regenerants was significantly higher during the initial days of acclimatization. However, after that it decreased gradually, reflecting successful acclimatization and stabilization of in vitro derived plants. The genetic fidelity assessment of nine regenerated plantlets using SCoT primers exhibited a consistent monomorphic banding pattern, confirming the production of true-to-type plantlets. The GC-MS analysis of leaf extracts from donor plants and in vitro raised plantlets of Salix tetrasperma revealed prominent qualitative and quantitative differences in phytochemical profiles. In vitro derived plantlets exhibited a broader spectrum of bioactive compounds with enhanced concentrations of certain pharmacologically significant metabolites compared to in vivo plants. The study emphasizes the importance of a well-managed acclimatization process in ensuring the survival and healthy development of micropropagated plants. The established method could facilitate large-scale production of S. tetrasperma with an enhanced phytochemical profile.

Data availability

The original contributions made in the study are incorporated in the article. Additional inquiries can be directed to corresponding authors. No third party data has been used in the article.

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Acknowledgements

The Authors would like to extend their sincere appreciation to the researchers supporting Project number (RSP2025R729) at King Saud University, Riyadh, Saudi Arabia. Zubair Altaf Reshi acknowledges financial support from CSIR-UGC (Council of Scientific & Industrial Research, University Grants Commission) New Delhi, India as Junior Research fellowship under Ref. No. (560/CSIR-UGC NET) at Aligarh Muslim University.

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Z.A.R: Study-conception, data collection, statistical analysis, drafted the original manuscript. F.M.H, M.NK: Reviewing and editing. S.B.J: Supervised and edited the final version of the manuscript and figures. All authors reviewed and agreed on the final version of the manuscript.

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Correspondence to Saad Bin Javed.

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Reshi, Z.A., Husain, F.M., Khanam, M.N. et al. Effect of meta-Topolin on morphological, physiochemical, and molecular dynamics during in vitro regeneration of Salix tetrasperma Roxb.. BMC Plant Biol 25, 121 (2025). https://doi.org/10.1186/s12870-025-06095-8

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