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Article

Biostimulatory Effects of Bacillus subtilis and Pseudomonas corrugata on Phytochemical and Antioxidant Properties of In Vitro-Propagated Plants of Nardostachys jatamansi (D. Don) DC

by
Janhvi Mishra Rawat
1,
Mrinalini Agarwal
1,
Shivani Negi
1,
Jigisha Anand
1,
Prabhakar Semwal
1,
Balwant Rawat
2,*,
Rajneesh Bhardwaj
2 and
Debasis Mitra
3
1
Department of Biotechnology, Graphic Era (Deemed to be University), Dehradun 248002, Uttarakhand, India
2
School of Agriculture, Graphic Era Hill University, Dehradun 248002, Uttarakhand, India
3
Department of Microbiology, Graphic Era Deemed to be University, Dehradun 248002, Uttarakhand, India
*
Author to whom correspondence should be addressed.
Bacteria 2025, 4(3), 38; https://doi.org/10.3390/bacteria4030038
Submission received: 21 April 2025 / Revised: 27 June 2025 / Accepted: 24 July 2025 / Published: 1 August 2025
(This article belongs to the Special Issue Harnessing of Soil Microbiome for Sustainable Agriculture)
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Abstract

Plant growth-promoting rhizobacteria (PGPRs) are well known for their capacity to enhance the growth and survival of in vitro-grown plants. However, their effect on Nardostachys jatamansi (D. Don) DC., a critically endangered medicinal plant in the Indian Himalayan Region, is still unknown. In this study, a simple, reproducible protocol for in vitro propagation of N. jatamansi was established using shoot tip explants, cultured on Murashige and Skoog (MS) medium supplemented with different plant growth regulators, including N6-benzylaminopurine, thidiazuron (TDZ), and naphthalene acetic acid (NAA). MS media supplemented with 2.0 μM TDZ and 0.5 µM NAA created a significant shoot induction with an average of 6.2 shoots per explant. These aseptically excised individual shoots produced roots on MS medium supplemented with Indole Butyric Acid or NAA within 14 days of the transfer. The PGPR, viz., Bacillus subtilis and Pseudomonas corrugata, inoculation resulted in improved growth, higher chlorophyll content, and survival of in vitro-rooted plants (94.6%) after transfer to the soil. Moreover, the PGPRs depicted a two-fold higher total phenolics (45.87 mg GAE/g DW) in plants. These results clearly demonstrate the beneficial effects of P. corrugata and B. subtilis on the growth, survival, and phytochemical content of N. jatamansi.

1. Introduction

Nardostachys jatamansi (D.Don) DC., locally known as Jatamansi, is a small, erect, hairy, perennial rhizomatous herbaceous plant belonging to the Valerianaceae family. It is distributed in the steep and open areas of the Himalayan regions of India, Nepal, and Bhutan between 3000 and 5200 above mean sea level (a.m.s.l) [1]. The most useful portion of the plant, the rhizomes, is dark brown, aromatic, tapering, and heavily covered in reddish-brown fibrous remnants of old leaf bases. This gives the rhizomes the appearance of a thick beard, hence the name “jatamansi” [2].
For many years, the traditional uses of N. jatamansi have included the stimulant, hepatoprotective, cardioprotective, sedative, neuroprotective, and antidiabetic properties of both its roots and rhizomes. The roots are also used to treat headaches, epilepsy, menopausal symptoms, intestinal colic, and flatulence [3,4,5]. According to phytochemical studies, N. jatamansi is a valuable source of essential oils that are rich in coumarins and sesquiterpenes. It also contains seselin, nardol, jatamansinone, valeranone, jatamansinol, nardosinone, jatamansic acid, nardal, nardin, oroselol, jatamansin, spirojatamol, and dihydropyranocoumarin [6].
Pharmaceutical industries mostly obtain this important plant species directly from their natural habitat in order to meet their demands. The species of N. jatamansi is becoming rare within a few habitats and has now been included in the list of Himalayan Critically Endangered (CR) plants because of the depletion of naturally growing populations caused by uncontrolled collection, unsustainable grazing, and rising raw material demand. Furthermore, according to Mulliken and Crofton (2008), N. jatamansi is among the (top seven Asian) CITES-listed plants, and as such, careful harvesting and conservation are necessary [7].
The most common methods for propagating N. jatamansi are seeds and vegetative parts. However, only 10–20% of seeds germinate under natural conditions, which is very low and is one of the primary factors in the extinction of this important species [8]. A brief reproductive phase follows a lengthy 3–4-year juvenile phase in plants. As a result, the in vitro approach can greatly aid in the mass reproduction and preservation of this extremely endangered species. Although there have been a few reports on the in vitro propagation of N. jatamansi [2,9,10,11,12,13], the effects of PGPR on phytochemical parameters, growth, and survival have not yet been investigated.
The transfer of in vitro-propagated plants during acclimatization and their survival is the most crucial step in the plant tissue culture technique. During the hardening and acclimatization of in vitro-grown plantlets, most plants are unable to tolerate the attack of soil microbes and environmental conditions. Because of this microbial attack and environmental change, 10–40% of in vitro-regenerated juvenile plants either die or are unable to compete in the market, which causes a significant loss to investors at the commercial level [14,15]. To overcome this problem, several researchers have reported the use of PGPRs during the hardening of in vitro-propagated plants, which is also known as biological hardening [16,17,18,19,20,21,22]. The effectiveness of such plant growth-promoting rhizobacteria (PGPRs) in biological hardening to enhance the growth, survival, and secondary metabolites of tissue culture-raised plants has been realized and reported by several researchers [16,17,23,24,25,26,27]. Many soil microorganisms like Bacillus, Pseudomonas, Azospirillum, etc., have been proposed as a suitable PGPR for agricultural crops, considering their phytobeneficial and abiotic stress tolerance properties [28,29,30,31,32]. In particular, the application of Bacillus subtilis and Pseudomonas corrugata has been explored for improving the acclimatization and adaptation of in vitro-regenerated plants by promoting root development, enhancing nutrient uptake, and inducing systemic resistance, thereby facilitating a more successful transition to ex vitro conditions [32,33,34].
Several studies have highlighted the plant growth-promoting potential of Bacillus subtilis and Pseudomonas corrugata, particularly in enhancing the adaptation of in vitro-propagated plants [34]. These rhizobacteria exert a wide range of PGPR activities, including phosphate solubilization, nitrogen fixation, production of indole-3-acetic acid (IAA), siderophore production, and 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity, which collectively contribute to improved plant growth and stress tolerance [35,36,37]. B. subtilis has been widely reported for its ability to enhance root architecture and nutrient uptake through IAA production and phosphate solubilization [38,39]. Similarly, P. corrugata exhibits strong siderophore production and ACC-deaminase activity, which help alleviate ethylene-induced stress during plant acclimatization [35,40]. The synergistic interaction of these traits aids in successful ex vitro establishment and overall growth promotion of tissue culture-derived plants, thereby supporting their use in biological hardening protocols.
Based on the above-mentioned points, the current study aimed to (1) develop an efficient in vitro propagation protocol of N. jatamansi from the shoot tip; (2) evaluate the effect of PGPRs, viz., Bacillus subtilis and Pseudomonas corrugata, on the growth, phytochemical parameters, and antioxidant activities of in vitro-propagated plants; and (3) analyze genetic fidelity using ISSR markers.

2. Materials and Methods

2.1. Plant Material and Explant Selection

In vitro propagation studies of N. jatamansi were carried out in the laboratory on matured seeds collected during October from the Tungnath and Chopata region of the Rudraprayag district of Uttarakhand, India, at altitudes ranging from 30°29′21.79″ N to 79°13′0.23″ E 3444 m amsl.
To obtain in vitro plants, mature seeds of N. jatamansi were surface sterilized using a 70% ethanol (v/v) solution for one minute, followed by treatment with a 0.2% mercuric chloride solution for 5 min. The treated seeds were then thoroughly rinsed with sterile water five times and left to soak in sterile distilled water for 24 h in the dark to soften the seed coat. These hydrated seeds were then placed onto Murashige and Skoog basal medium containing 0.8% w/v agar at pH 5.8, with one seed per tube [41].
Test tubes containing shoot tips (5–10 mm) with two leaf primordia from 15-day-old in vitro seedlings were transferred to the culture room. The culture room had a light intensity of 100 μEm−2 s−1, a photoperiod of 16 h per day, and a relative humidity of 70%. The explants were then used for shoot multiplication.

2.2. Induction of Shoot Buds

Multiple combinations of plant growth regulators, that is, benzylaminopurine (BAP), Indole-3-acetic acid (IAA), and thidiazuron (TDZ), at varying concentrations were tested on Basal MS medium for their ability to induce multiple shoot formation (Table 1). Each trial was replicated twice using ten explants per treatment to ensure the accuracy and reliability of the data obtained.

2.3. Rhizogenesis

Proliferating shoots, measuring approximately 3 to 4 cm in length, were inoculated on Murashige and Skoog (MS) medium supplement with different concentrations of IAA at a concentration range of 0.0–1.0 μM and Naphthaleneacetic acid (NAA) at a concentration range of 0.0–0.5 μM, either separately or in combination, for rooting (see Table 2).

2.4. Hardening of Plantlets and Inoculation Using PGPRs

N. jatamansi plants, which were raised using tissue culture, were transferred to plastic trays containing a mixture of sand, soil, and compost at a 1:1:1 ratio. To ensure proper humidity, the plants were initially covered with transparent polythene. After the second week, the covers were temporarily removed for 2–3 h each day to facilitate acclimatization. By the fourth week, the cover had been completely withdrawn.
The experimental inoculation involved the utilization of two bacterial strains: Bacillus subtilis (MTCC 8528) and Pseudomonas corrugata (ATCC 29736) [16,34]. Both the isolates were taken from the institute’s laboratory. In the present study, the inclusion of these two bacterial strains was based on their proven roles in promoting plant growth through physiological and biochemical mechanisms relevant to the goals of in vitro plant acclimatization. These bacteria have been widely reported to enhance plant development and stress resilience through various biochemical mechanisms. Specifically, B. subtilis strains are known to solubilize phosphate, fix atmospheric nitrogen, produce indole-3-acetic acid (IAA), and secrete siderophores and ACC-deaminase, all of which collectively enhance nutrient uptake, root architecture, and tolerance to environmental stresses [42,43,44]. Similarly, P. corrugata has been shown to possess high ACC-deaminase activity, siderophore production, and IAA biosynthesis, contributing significantly to plant vigor and successful adaptation, particularly under in vitro to ex vitro transition phases [16,45,46].
The inoculation process was conducted on four-week-old N. jatamansi plantlets by adding a broth solution containing 106 colony-forming units (cfu)/mL to the planting hole prior to planting, according to the designated treatment. 1 mL of a 106 CFU/mL suspension per gram of soil. This ensures a consistent and measurable concentration of microbes in the soil sample [47].
Both in vitro- and seed-grown plants were included in the analysis. Each treatment consisted of twenty-five plants grown under regular soil conditions without plant growth-promoting rhizobacteria (PGPR), which served as a control.

2.5. Data Recording

After 60 d of inoculation, plant growth was assessed in terms of plant height, leaf area, number of leaves, root length, and fresh and dry weights of shoots and roots. In addition, the chlorophyll content of the leaves was measured using the Arnon method [30] with 80% acetone extract. To determine any significant differences between the mean values, statistical analysis was conducted using Duncan’s multiple range test (DMRT) with Statistical Software v5.0 (StatSoft 1995) [48], with a significance level set at p ≤ 0.05.

2.6. Preparation of Sample Extract

The sample for analysis was prepared by mixing 1 g of the powdered sample of dried roots with 5 mL of 80% methanol, followed by thorough grinding in a mortar and pestle. The resulting homogenate was then centrifuged at 8000 rpm for 15 min, and the supernatant was carefully collected. The residue was washed with 50 mL of 80% methanol. The combined filtrate (methanol) was stored at 4 °C until required. This methanolic extract was used to analyze the levels of total phenols, alkaloids, flavonoids, tannins, and antioxidant activity.

2.7. Analysis of Total Phenol, Total Alkaloids, Tannin, and Flavonoids

Total phenolic and tannin contents were quantified according to the method outlined by Swain and Hillis [49]. The total alkaloid content was determined following the protocol established by Sreevidya and Mehrotra [50], whereas flavonoids were assessed using the method described by Kim et al. [51]. The total flavonoid, phenolic, tannin, and alkaloid content was expressed as milligrams of per gram of dry weight, milligrams of gallic acid equivalent (GAE) per gram of dry weight sample, milligrams of tannic acid quercetin equivalent (QE)equivalent (TAE) per gram of dry weight sample, and milligrams of atropine equivalent (AE) per gram of dry weight sample, respectively.

2.7.1. Antioxidant Activity

Two different in vitro assays, i.e., 2,2-diphenyl-1-picryylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP) assays, were used to estimate the antioxidant activity of each sample.

2.7.2. 2,2-Diphenyl-1-Picrylhydrazyl (DPPH) Assay

The effect of the methanolic extract of N. jatamansi on DPPH radicals was measured using a previously described method Rawat et al. [52]. The absorbance of the sample was recorded against a reference (control; solution of methanol and DPPH). The DPPH radical scavenging activity of the species was estimated at concentrations ranging from 250 μg/mL to 1000 μg/mL. The antioxidant activity results are presented in mM ascorbic acid equivalent (AAE) per g dry weight of the sample. The following formula was used to calculate the scavenging capacity of the samples:
Scavenging (%) = (absorbance of the control at 517 nm − absorbance of the sample) × 100% absorbance of the control

2.7.3. Ferric Reducing Antioxidant Power (FRAP) Assay

The ferric reducing ability of the methanolic extract was determined using the method of [53]. The absorbance of the solution (methanolic extract and FRAP reagent) was measured spectrophotometrically at 593 nm wavelength. Ascorbic acid (10–100 μg/mL) was used as a standard to determine the FRAP value, and the results were presented in mM ascorbic acid equivalent (AAE) per gram dry weight of the sample.

3. Results

3.1. Seed Germination, Shoot Induction, and Multiplication

Multiple combinations of plant growth regulators have been used to develop effective and efficient in vitro micropropagation protocols for N. jatamansi. Shoots were regenerated using shoot-tip explants from in vitro seedlings. Within one week of culture, shoot buds were induced on basal medium containing 3% sucrose (Figure 1a,b); however, shoot growth was suppressed in the absence of any plant growth regulators. Several combinations of plant growth regulators, such as IAA (0.0–0.5 µM), BAP (0.0–5.0 µM), and TDZ (0.0–4.0 µM), were used to promote shoot multiplication and elongation (Table 1). The results, recorded after 2 weeks, were further analyzed and are presented in Table 1. MS medium supplemented with 0.5 µM NAA and 2.0 µM TDZ produced the highest (6.2 shoots per explant; Figure 1c) shoot proliferation in terms of induction and elongation within 4 weeks. According to Bose et al. [9], the addition of 1.0 mg/L of meta-Topolin in the medium is effective in inducing numerous shoots, but the findings of the current study indicated that the best shoot production was achieved by combining a low concentration of NAA with high TDZ. These results substantiate previous findings [14,36,37], which demonstrated that the combination of auxin and cytokinin positively influences growth in the culture medium. The media supplied with 0.5 µM NAA and 2.0 µM TDZ was also found best for maximum shoot length; however, the medium supplemented with 4.0 µM TDZ and 0.1 µM NAA exhibited the maximum number of leaves. Earlier, Rawat et al. [53] reported the shoot proliferation (4.5 shoots) in MS media supplemented with 5.0 μM BAP; however, in this study, the use of NAA and TDZ in combination gives 6.2 shoots. These results clearly show that the auxin/cytokinin ratio in the culture medium plays an important role in the morphogenetic response of cultured tissues.

3.2. Rhizogenesis of In Vitro-Developed Shoots

To induce rhizogenesis in the regenerated shoots, 2–4 cm elongated shoots were sub-cultured on basal MS medium, with or without the addition of plant growth regulators. A total of 12 media combinations containing IAA or NAA (individually or in combination) were used, which helped in root induction in plantlets. The concentrations of both plant growth regulators played a crucial role in influencing the percentage of shoot-forming roots and the number of roots per shoot, as outlined in Table 2. The number of shoots displaying root initiation, as well as the number of roots induced per shoot, were documented after four weeks of cultivation.
Comparatively, IAA showed a better response than NAA in terms of the mean number of roots per shoot and percentage of shoot-forming roots. In contrast, the medium supplemented with 1.0 µM NAA showed maximum root length. High frequency of rhizogenesis (89.6%) in in vitro shoots was observed on media having 0.5 µM IAA (Figure 1d). The results of the current study, which showed that the presence of individual auxins (IAA or IBA) reduced the number of shoot-producing roots, are also supported by previous studies on Aconitum balforii [54,55] and Digitalis ferruginea [14].

3.3. Acclimatization with PGPR and Field Establishment

The results showed that 78.6% of the plants survived after 4 weeks of transfer to pots filled with soil, sand, and compost (1:1:1: v/v) and maintained in F conditions (controlled environment structure where temperature 25–30 °C, humidity 60–65%, and light are regulated to support the hardening and acclimatization of in vitro-cultured plants before transferring them to open fields). These tissue-cultured raised plants were utilized after four weeks to study the impact of PGPRs on growth and phytochemical parameters.
Inoculation of micropropagated plants of N. jatamansi with the two bacterial isolates was effective in enhancing the survival rate of plants after transfer to the soil (Figure 2, Table 3). The plants inoculated with P. corrugata showed the maximum survival rate (94.6%) after 60 d of inoculation. The survival rate was 85.3% in the plants inoculated with B. subtilis, whereas only 66.3% survival was observed in tissue culture-raised plants. In the control, only 18.4% of the seeds germinated and survived under natural conditions. After 60 d of transfer, the plants grew well and attained 8–10 cm in height. The acclimatized plants showed normal growth without phenotypic variation.

3.4. Effect of PGPR on Growth Parameters

Bacterial inoculation had a positive impact on various aspects of plant growth parameters, such as plant height, leaf area, number of leaves, root length, and fresh and dry weights of shoots and roots. Among the various treatments, the P. corrugata-inoculated plants showed the maximum increase in growth parameters compared to the B. subtilis-inoculated and -uninoculated plants (Table 1; Figure 1e). Singh et al. also reported an increase in aboveground growth in Coleus forskohlii plants colonized by Pseudomonas monteilii and Glomus fasciculatum [56]. The study discovered that these plants had greater aboveground biomass and higher rates of N, P, and K absorption. In another study, field tests were used by Aseri and coworkers to assess the effectiveness of PGPR, such as A. chroococcum and A. brasilense, and AM fungi, such as G. mosseae and G. fasciculatum, on the development, uptake of nutrients, and biomass production of pomegranate (Punica granatum L.) [40,57].
The chlorophyll content in the N. jatamansi plants was significantly increased by approximately 40% with PGPR inoculation compared to the uninoculated plants. The chlorophyll content was 1.12 mg/g FW and 0.94 mg/g FW in the plants inoculated with P. corrugata and B. subtilis, respectively, and 0.70 mg/g FW in the uninoculated plants. Thus, the statement indicates that an increase in chlorophyll content results in a higher photosynthetic rate, as well as improves plant biomass. A similar effect of PGPR inoculation on the chlorophyll content of Punica grantum after four months was also noted by Aseri et al. [40]. Additionally, the efficiency of plants in terms of root and shoot biomass, as well as the chlorophyll and NPK content, was significantly increased by inoculation with a single microbe [24,58,59].

3.5. Effect of PGPR on Total Phenol, Total Alkaloids, Tannin, and Flavonoids

Plant secondary metabolites are valuable resources for fragrances, food additives, flavors, pharmaceuticals, and various other industrially significant compounds. These metabolites play a crucial role in protecting plants from various threats, including herbivores, phytopathogens, and insect pests, as well as in helping plants withstand biotic and abiotic stresses. Environmental stressors, whether biotic or abiotic, increase the synthesis rate of secondary metabolites. These stressors can be microbial, physical, or chemical in origin [60,61,62]. Rhizosphere microorganisms, including PGPRs, are biotic elicitors that can induce the production of secondary metabolites [32,63,64,65].
The impact of PGPR strains P. corrugata and B. subtilis on total phenolic, total alkaloid, tannin, and flavonoid content was examined in in vitro-propagated plants of N. jatamansi. The plants inoculated with P. corrugata exhibited significantly higher levels of total phenolics, total alkaloids, tannins, and flavonoids than the uninoculated plants of N. jatamansi. Specifically, the total phenolic content was approximately two-fold higher in the P. corrugata-inoculated plants (45.87 mg GAE/g DW) than in uninoculated control plants (23.87 mg GAE/g DW). B. subtilis also displayed a stimulatory effect on the phytochemical parameters, but the values were lower than P. corrugata. The lesser effect of B. subtilis strain was attributed to its poor adaptation to root exudates and/or insufficient root colonization. Both P. corrugata and B. subtilis showed almost similar outcomes (33.89 mg AE/ g DW and 33.37 mg AE/ g DW, respectively) in the case of total alkaloid production. The increase in total phenolic, alkaloid, tannin, and flavonoid contents in response to PGPR inoculation was due to the increase in plant dry weight and growth-promoting chemicals synthesized by microbes. These substances influence plant metabolic pathways [32,63,64,65]. Palermo et al. reported a significant increase in total phenolic content exclusively in plants that were both inoculated with Bacillus velezensis strain GB03 and subjected to larval damage [49]. The study also highlighted the complex interactions among beneficial microbes, herbivores, and plant defense responses, underscoring their potential role in enhancing plant resilience and boosting the production of secondary metabolites [63,64,65]. PGPR inoculation markedly improved the growth and nutrient uptake of Cucumis sativus seedlings, especially under higher substrate moisture levels [66]. Significant increases were observed in plant height, root length, and fresh weight, with synergistic effects from PGPR and moisture resulting in up to 197% and 267% increases in height and root length, respectively [66]. Similar observations have been documented in previous studies on Hyoscyamus niger [60], Stevia rebaudiana [24], and Salvia officinalis [67]. The plants grown from the seeds displayed minimum values for all four parameters (Figure 3, Table 4).

3.6. Antioxidant Activity

Plants have different types of secondary metabolites such as tannins, phenolics, flavonoids, and alkaloids. These compounds are known to act as scavengers of free radicals and are anti-carcinogenic, anti-inflammatory, and antimutagenic [68,69,70,71,72,73], and others have reported that the powerful antioxidant activity of these phytochemicals improves human health by modulating metabolism. Furthermore, it is also useful in treating diseases that are caused by deregulation of free radical generation in cells, such as cardiovascular diseases, cancer, inflammatory disorders, and diabetes.
The total antioxidant activity, measured by DPPH and FRAP, varied significantly (p < 0.05) between the inoculated and uninoculated plants (Table 4). According to the reducing ability, both assays revealed that the inoculated plants exhibited higher antioxidant activity than the control plants (Table 4). The plants inoculated with P. corrugata and B. subtilis showed 6.76 and 5.44 mM AAE/g DW antioxidant activities, respectively, in the FRAP assay. In the DPPH assay, the antioxidant activities were 67.89 and 63.46 mM AAE/g DW for the P. corrugata- and B. subtilis-inoculated plants, respectively. Moreover, the in vitro-raised uninoculated plants also showed higher antioxidant activity than control plants. The high concentrations of secondary metabolites (tannins, polyphenols, alkaloids, and flavonoids) may be the cause of their powerful antioxidant potential. Owing to their redox characteristics and ability to generate singlet oxygen, secondary metabolites have previously been shown to have substantial antioxidant activity and function as primary antioxidants [74,75].

4. Discussion

This study successfully established an in vitro culture of N. jatamansi and demonstrated that inoculation with the PGPR strains Bacillus subtilis and Pseudomonas corrugata significantly enhanced secondary metabolite accumulation and antioxidant potential in plantlets. It also promotes root development, which expands the absorptive surface area, amplifying water and mineral uptake.
Even though inoculation with B. subtilis and P. corrugata increased the antioxidant potential and phytochemical constituents, more research on the plant’s essential oil is necessary to fully comprehend the role of elicitors in the synthesis of secondary metabolites. While our results confirm that B. subtilis and P. corrugata can indeed enhance phytochemical profiles and antioxidant capacity, further investigation into essential oil profiles, gene expression (transcriptomics), and metabolomic profiling would provide deeper mechanistic insights and support biotechnological applications for large-scale production. Only two strains were evaluated in this study; future research should consider a broader diversity of microbial strains, including potential synergistic consortia, to identify more effective or specialized elicitors. Furthermore, the current investigation focused on short-term in vitro responses. Long-term studies, particularly under field conditions, are essential to assess the persistence of PGPR effects on plant growth, yield, and metabolite accumulation over time.
Additionally, exploring how these biochemical PGPR traits influence key biosynthetic pathways would strengthen the understanding of plant–microbe interactions in secondary metabolism. This study may help fill the gap between the demand for and supply of raw materials and support the conservation of species in natural habitats. To further understand the production of secondary metabolites in response to PGPRs in these medicinal plants, transcriptome and metabolomics investigations should be conducted in the future.

Author Contributions

Conceptualization, J.M.R. and B.R.; methodology, M.A. and S.N.; software, J.A. and P.S.; validation, J.M.R., P.S., and R.B.; formal analysis, P.S.; investigation, J.M.R. and B.R.; resources, J.M.R.; data curation, P.S., R.B., B.R., and D.M.; writing—original draft preparation, M.A., J.M.R., and J.A.; writing—review and editing, P.S. and B.R.; visualization, B.R.; supervision, J.M.R., R.B., and B.R. All authors have read and agreed to the published version of the manuscript.

Funding

This research received external funding from the Science and Engineering Research Board, Department of Science and Technology, Govt. of India (TAR/2022/000018).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

Acknowledgments

The authors are thankful to the Vice Chancellor of the University for providing the facilities to carry out this work. Financial support to Janhvi M. Rawat from the Science and Engineering Research Board, Department of Science and Technology, Govt. of India (TAR/20222/000018), is gratefully acknowledged.

Conflicts of Interest

The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. Also, Dr Debasis Mitra, who is one of the guest editors of this issue, is not involved in the processing, review, and decision of this manuscript.

Abbreviations

The following abbreviations are used in this manuscript:
AAEAscorbic acid equivalent
AEAtropine equivalent
BAP6-Benzylaminopurine
CRCritically endangered
DMRTDuncan’s multiple range test
DPPH2,2-Diphenyl-1-picrylhydrazyl
FRAPFerric reducing antioxidant power
GAEGallic acid equivalent
IAAIndoel-3-acetic acid
IUCNInternational Union for Conservation of Nature
MSMurashige and Skoog
NAANaphthalene acetic acid
PGPRPlant growth-promoting rhizobacteria
QEQuercetin equivalent
TAETannic acid equivalent
TDZThidiazuron

References

  1. Chauhan, R.S.; Nautiyal, M.C. Commercial viability of cultivation of an endangered medicinal herb Nardostachys jatamansi at three different agroclimatic zones. Curr. Sci. 2005, 89, 1481–1488. [Google Scholar]
  2. Katara, A.; Pandey, G.; Rastogi, S.; Rawat, A.K. An important Indian traditional drug of Ayurveda jatamansi and its substitute bhootkeshi: Chemical profiling and antioxidant activity. Evid.-Based Complement. Altern. Med. 2013, 2013, 142517. [Google Scholar]
  3. Chen, K.K.; Mukerji, B. Pharmacology of Oriental Plants: Proceedings of the First International Pharmacological Meeting, Stockholm, Sweden, 22–25 August, 1961; Elsevier: Amsterdam, The Netherlands, 2013; pp. 51–60. [Google Scholar]
  4. Chaudhary, S.; Chandrashekar, K.S.; Pai, K.S.; Setty, M.M.; Devkar, R.A.; Reddy, N.D.; Shoja, M.H. Evaluation of antioxidant and anticancer activity of extract and fractions of Nardostachys jatamansi DC in breast carcinoma. BMC Complement. Altern. Med. 2015, 15, 50–63. [Google Scholar] [CrossRef]
  5. Purnima, M.B.; Kothiyal, P. A review article on phytochemistry and pharmacological profiles of Nardostachys jatamansi DC medicinal herb. J. Pharmacogn. Phytochem. 2015, 3, 102–106. [Google Scholar]
  6. Rekha, K.; Rao, R.R.; Pandey, R.; Prasad, K.R.; Babu, K.S.; Vangala, J.R.; Kalivendi, S.V.; Rao, J.M. Two new sesquiterpenoids from the rhizomes of Nardostachys jatamansi. J. Asian Nat. Prod. Res. 2013, 15, 111–116. [Google Scholar] [CrossRef] [PubMed]
  7. Mulliken, T.; Crofton, P. Review of the Status, Harvest, Trade and Management of Seven Asian CITES-Listed Medicinal and Aromatic Plant Species; BfN-Skripten, Federal Agency for Nature Conservation: Bonn, Germany, 2008; pp. 41–60. [Google Scholar]
  8. Nautiyal, B.P.; Chauhan, R.S.; Prakash, V.; Purohit, H.; Nautiyal, M.C. Population studies for the evaluation of germplasm and threat status of the alpine medicinal herb, Nardostachys jatamansi. Plant Genet. Resour. Newsl. 2003, 136, 34–39. [Google Scholar]
  9. Bose, B.; Kumaria, S.; Choudhury, H.; Tandon, P. Assessment of genetic homogeneity and analysis of phytomedicinal potential in micropropagated plants of Nardostachys jatamansi, a critically endangered, medicinal plant of alpine Himalayas. Plant Cell Tissue Organ Cult. 2016, 124, 331–349. [Google Scholar] [CrossRef]
  10. Rawat, V.; Ghildiyal, A.; Singh, L.; Jugran, A.K.; Bhatt, I.D.; Nandi, S.K.; Pande, V. Methyl jasmonate induced polyphenols and antioxidant production in callus suspension culture of Nardostachys jatamansi. Plant Biosyst. 2020, 154, 851–859. [Google Scholar] [CrossRef]
  11. Pant, H.C.; Pant, H.V.; Kumar, A.; Tomar, H.; Sharma, M.D.; Gaurav, N. In Vitro clonal propagation of Nardostachys jatamansi: A traditional Himalayan medicinal plant. J. Mountain Res. 2021, 16, 87–98. [Google Scholar] [CrossRef]
  12. Dhiman, N.; Devi, K.; Bhattacharya, A. Development of low cost micropropagation protocol for Nardostachys jatamansi: A critically endangered medicinal herb of Himalayas. S. Afr. J. Bot. 2021, 140, 468–477. [Google Scholar] [CrossRef]
  13. Pant, H.C.; Rautela, I.; Pant, H.V.; Kumar, A.; Kumar, P.; Fatima, K.; Gaurav, N. Comparison of antioxidant properties and flavonoid of natural and in vitro cultivated Nardostachys jatamansi. Agric. Sci. Dig. 2024, 44, 406–413. [Google Scholar] [CrossRef]
  14. Verma, S.K.; Yucesan, B.; Sahin, G.; Gurel, E. Embryogenesis, plant regeneration and cardiac glycoside determination in Digitalis ferruginea subsp. ferruginea L. Plant Cell Tissue Organ Cult. 2014, 119, 625–634. [Google Scholar] [CrossRef]
  15. Cassells, A.C.; Mark, G.L.; Periappuram, C. Establishment of arbuscular mycorrhizal fungi in autotrophic strawberry cultures in vitro: Comparison with inoculation of microplants in vivo. Agronomie 1996, 16, 625–632. [Google Scholar] [CrossRef]
  16. Trivedi, P.; Pandey, A. Biological hardening of micropropagated Picrorhiza kurrooa Royel ex Benth., an endangered species of medical importance. World J. Microbiol. Biotechnol. 2007, 23, 877–878. [Google Scholar] [CrossRef]
  17. Mathur, A.; Mathur, A.K.; Verma, P.; Yadav, S.; Gupta, M.L.; Darokar, M.P. Biological hardening and genetic fidelity testing of micro-cloned progeny of Chlorophytum borivilianum. Afr. J. Biotechnol. 2008, 7, 1046–1053. [Google Scholar]
  18. Bagde, U.S.; Prasad, R.; Varma, A. Interaction of mycobiont: Piriformospora indica with medicinal plants and plants of economic importance. Afr. J. Biotechnol. 2010, 9, 9214–9226. [Google Scholar]
  19. Das, A.; Tripathi, S.; Varma, A. Use of Piriformospora indica as a potential biological hardening agent for endangered micropropagated Picrorhiza kurrooa Royel ex Benth. Proc. Nat. Acad. Sci. India Sect. B Biol. Sci. 2017, 87, 799–805. [Google Scholar] [CrossRef]
  20. Sun, W.; Shahrajabian, M.H.; Soleymani, A. The roles of plant-growth-promoting rhizobacteria (PGPR)-based biostimulants for agricultural production systems. Plants 2024, 13, 613. [Google Scholar] [CrossRef]
  21. Igiehon, B.C.; Babaloloa, O.O.; Hassen, A.I. Rhizosphere competence and applications of plant growth-promoting rhizobacteria in food production—A review. Sci. Afr. 2024, 23, e02081. [Google Scholar] [CrossRef]
  22. Gharib, F.A.E.L.; Osama, K.; El-Sattar, A.M.A.; Ahmed, E.Z. Impact of Chlorella vulgaris, Nannochloropsis salina, and Arthrospira platensis as bio-stimulants on common bean plant growth, yield, and antioxidant capacity. Sci. Rep. 2024, 14, 1398. [Google Scholar] [CrossRef]
  23. Hao, G.; Du, X.; Zhao, F.; Ji, H. Fungal endophytes-induced abscisic acid is required for flavonoid accumulation in suspension cells of Ginkgo biloba. Biotechnol. Lett. 2010, 32, 305–314. [Google Scholar] [CrossRef]
  24. Vafadara, F.; Amooaghaiea, R.; Otroshyb, M. Effects of plant-growth-promoting rhizobacteria and arbuscular mycorrhizal fungus on plant growth, stevioside, NPK, and chlorophyll content of Stevia rebaudiana. J. Plant Interact. 2014, 9, 128–136. [Google Scholar] [CrossRef]
  25. Sureshbabu, K.; Amaresan, N.; Kumar, K. Amazing multiple function properties of plant growth-promoting rhizobacteria in the rhizosphere soil. Int. J. Curr. Microbiol. App. Sci. 2016, 5, 661–683. [Google Scholar] [CrossRef]
  26. Swamy, M.K.; Akhtar, M.S.; Sinniah, U.R. Response of PGPR and AM fungi toward growth and secondary metabolite production in medicinal and aromatic plants. In Plant, Soil and Microbes: Mechanisms and Molecular Interactions; Springer: Cham, Switzerland, 2016; Volume 2, pp. 145–168. [Google Scholar]
  27. Yilmaz, A.; Karik, Ü. AMF and PGPR enhance yield and secondary metabolite profile of basil (Ocimum basilicum L.). Ind. Crops Prod. 2022, 176, 114327. [Google Scholar] [CrossRef]
  28. Tiwari, S.; Lata, C.; Chauhan, P.S.; Nautiyal, C.S. Pseudomonas putida attunes morphophysiological, biochemical and molecular responses in Cicer arietinum L. during drought stress and recovery. Plant Physiol. Biochem. 2016, 99, 108–117. [Google Scholar] [CrossRef] [PubMed]
  29. Vimal, S.R.; Singh, J.S.; Arora, N.K.; Singh, D.P. PGPR: An effective bio-agent in stress agricultural management. In Microbial Empowerment in Agriculture: A Key to Sustainability and Crop Productivity; Sarma, B.K., Jain, A., Eds.; Biotech Books: New Delhi, India, 2016; pp. 81–108. [Google Scholar]
  30. Vimal, S.R.; Singh, J.S.; Arora, N.K.; Singh, S. Soil-plant-microbe interactions in stressed agriculture management: A review. Pedosphere 2017, 27, 177–192. [Google Scholar] [CrossRef]
  31. Tiwari, P.; Singh, J.S. A plant growth promoting rhizospheric Pseudomonas aeruginosa strain inhibits seed germination in Triticum aestivum (L) and Zea mays (L). Microbiol. Res. 2017, 8, 7233. [Google Scholar] [CrossRef]
  32. Tiwari, S.; Prasad, V.; Lata, C. Bacillus: Plant Growth Promoting Bacteria for Sustainable Agriculture and Environment. In New and Future Developments in Microbial Biotechnology and Bioengineering; Singh, J.S., Singh, D.P., Eds.; Elsevier: Amsterdam, The Netherlands, 2019; pp. 43–55. [Google Scholar]
  33. Pataczek, L.; Armas, J.C.B.; Petsch, T.; Hilger, T.; Ahmad, M.; Schafleitner, R.; Zahir, Z.A.; Cadisch, G. Single-Strain Inoculation of Bacillus subtilis and Rhizobium phaseoli Affects Nitrogen Acquisition of an Improved Mungbean Cultivar. J. Soil. Sci. Plant Nutr. 2024, 24, 6746–6759. [Google Scholar] [CrossRef]
  34. Kumar, B.; Trivedi, P.; Pandey, A. Pseudomonas corrugata: A suitable bacterial inoculant for maize grown under rainfed conditions of Himalayan region. Soil Biol. Biochem. 2007, 39, 3093–3100. [Google Scholar] [CrossRef]
  35. Trivedi, P.; Pandey, A.; Palni, L.M.S. In vitro evaluation of antagonistic properties of Pseudomonas corrugata. Microbiol. Res. 2008, 163, 329–336. [Google Scholar] [CrossRef]
  36. Rodríguez, H.; Fraga, R. Phosphate-solubilising bacteria and their role in plant growth promotion. Biotechnol. Adv. 1999, 17, 319–339. [Google Scholar] [CrossRef]
  37. Timofeeva, A.M.; Galyamova, M.R.; Sedykh, S.E. How Do Plant Growth-Promoting Bacteria Use Plant Hormones to Regulate Stress Reactions? Plants 2024, 13, 2371. [Google Scholar] [CrossRef]
  38. Zaidi, A.; Ahmad, E.; Khan, M.S.; Saif, S.; Rizvi, A. Role of plant growth promoting rhizobacteria in sustainable production of vegetables: Current perspective. Sci. Hortic. 2015, 193, 231–239. [Google Scholar] [CrossRef]
  39. Idris, E.E.S.; Iglesias, D.J.; Talon, M.; Borriss, R. Tryptophan-dependent production of indole-3-acetic acid (IAA) affects level of plant growth promotion by Bacillus amyloliquefaciens FZB42. Mol. Plant-Microbe Interact. 2007, 20, 619–626. [Google Scholar] [CrossRef]
  40. Mercado-Blanco, J.; van der Drift, K.M.G.M.; Olsson, P.A.; Thomas-Oates, J.E.; van Loon, L.C.; Bakker, P.A.H.M. Analysis of the pqqC gene for phosphate solubilization and ACC deaminase activity in Pseudomonas spp. Soil Biol. Biochem. 2004, 36, 255–265. [Google Scholar] [CrossRef]
  41. Murashige, T.; Skoog, F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 1962, 15, 473–497. [Google Scholar] [CrossRef]
  42. Sun, B.; Gu, L.; Bao, L.; Zhang, S.; Wei, Y.; Bai, Z.; Zhuang, G.; Zhuang, X. Application of biofertilizer containing Bacillus subtilis reduced the nitrogen loss in agricultural soil. Soil. Biol. Biochem. 2020, 148, 107911. [Google Scholar] [CrossRef]
  43. Patel, M.; Islam, S.; Husain, F.M.; Yadav, V.K.; Park, H.-K.; Yadav, K.K.; Bagatharia, S.; Joshi, M.; Jeon, B.-H.; Patel, A. Bacillus subtilis ER-08, a multifunctional plant growth-promoting rhizobacterium, promotes the growth of fenugreek (Trigonella foenum-graecum L.) plants under salt and drought stress. Front. Microbiol. 2023, 14, 1208743. [Google Scholar] [CrossRef]
  44. Ikeuchi, T.; Ishida, A.; Tajifi, M.; Nagata, S. Induction of salt tolerance in Bacillus subtilis IFO 3025. J. Biosci. Bioeng. 2003, 96, 184–186. [Google Scholar] [CrossRef]
  45. Yadav, A.N. Plant Microbiomes for Sustainable Agriculture: Current Research and Future Challenges. In Plant Microbiomes for Sustainable Agriculture. Sustainable Development and Biodiversity; Yadav, A., Singh, J., Rastegari, A., Yadav, N., Eds.; Springer: Cham, Switzerland, 2020; Volume 25. [Google Scholar] [CrossRef]
  46. Catara, V. Pseudomonas corrugata: Plant pathogen and/or biological resource? Mol. Plant Pathol. 2007, 8, 233–244. [Google Scholar] [CrossRef]
  47. Papin, M.; Philippot, L.; Breuil, M.C.; Bru, D.; Dreux-Zigha, A.; Mounier, A.; Roux, X.L.; Rouard, N.; Spor, A. Survival of a microbial inoculant in soil after recurrent inoculations. Sci. Rep. 2024, 14, 4177. [Google Scholar] [CrossRef]
  48. StatSoft Inc. STATISTICA for Windows (Computer Program Manual); StatSoft Inc.: Tulsa, OK, USA, 1995. [Google Scholar]
  49. Swain, T.; Hillis, W.E. The phenolic constituents of Prunus domestica L.—The quantitative analysis of phenolic constituents. J. Sci. Food Agric. 1959, 10, 63–68. [Google Scholar] [CrossRef]
  50. Sreevidya, N.; Mehrotra, S. Spectrophotometric method for estimation of alkaloids precipitable with Dragendorff’s reagent in plant materials. J. AOAC Int. 2003, 86, 1124–1127. [Google Scholar] [CrossRef]
  51. Kim, D.O.; Chun, O.K.; Kim, Y.J.; Moon, H.Y.; Lee, C.Y. Quantification of polyphenolics and their antioxidant capacity in fresh plums. J. Agric. Food Chem. 2003, 51, 6509–6515. [Google Scholar] [CrossRef] [PubMed]
  52. Rawat, J.M.; Pandey, S.; Rawat, B.; Purohit, S.; Anand, J.; Negi, A.S.; Thakur, A.; Mahmoud, M.H.; El-Gazzar, A.M.; El-Saber Batiha, G. In vitro production of steroidal saponin, total phenols, and antioxidant activity in callus suspension culture of Paris polyphylla Smith: An important Himalayan medicinal plant. Front. Plant Sci. 2023, 14, 1225612. [Google Scholar] [CrossRef]
  53. Rawat, J.M.; Bhandari, A.; Rajneesh; Karki, K.; Raturi, M.; Rawat, B.; Dhakad, A.K.; Thakur, A.; Chandra, A. RAPD based genetic fidelity analysis of invitro regenerated plants of Nardostachys jatamansi. Int. J. Biol. Sci. 2017, 8, 141–147. [Google Scholar]
  54. Rawat, J.M.; Rawat, B.; Agnihotri, R.K.; Chandra, A.; Nautiyal, S. In vitro propagation, genetic and secondary metabolite analysis of Aconitum violaceum Jacq.—A threatened medicinal herb. Acta Physiol. Plant. 2013, 35, 2589–2599. [Google Scholar] [CrossRef]
  55. Sharma, E.; Gaur, K.; Punetha, H.; Gaur, A.K. In vitro regeneration of Aconitum balforii Stapf: A rare medicinal herb from Himalayan alpine through root explant. Res. J. Med. Plant 2012, 6, 318–325. [Google Scholar]
  56. Singh, R.; Soni, S.K.; Kalra, A. Synergy between Glomus fasciculatum and a beneficial Pseudomonas in reducing root diseases and improving yield and forskolin content in Coleus forskohlii Briq. under organic field conditions. Mycorrhiza 2012, 23, 35–44. [Google Scholar] [CrossRef]
  57. Aseri, G.K.; Jain, N.; Panwar, J.; Rao, A.V.; Meghwal, P.R. Biofertilizers improve plant growth, fruit yield, nutrition, metabolism, and rhizosphere enzyme activities of pomegranate (Punica granatum L.) in Indian Thar desert. Scientia Hortic. 2008, 117, 130–135. [Google Scholar] [CrossRef]
  58. Salehnia Sammak, A.; Anvari, M.; Matinizadeh, M.; Mirza, M. Evaluation of inoculation Pseudomonas fluorescens and arbuscular mycorrhizal fungus on growth, morphological characteristics, and essential oil percentage of Thymus kotschyanus. J. Med. Plants By-Prod. 2022, 11, 181–189. [Google Scholar]
  59. Zhang, M.; Hua, M.; Guo, D.; Xue, Y.; Chen, X.; Rui, L.; Zhou, N. Effects of plant growth-promoting rhizobacteria on growth indicators and physiological characteristics of Peucedanum praeruptorum Dunn leaves. Plant Signal. Behav. 2023, 18, 2203571. [Google Scholar] [CrossRef]
  60. Ghorbanpour, M.; Hatami, M.; Khavazi, K. Role of plant growth promoting rhizobacteria on antioxidant enzyme activities and tropane alkaloid production of Hyoscyamus niger under water deficit stress. Turk. J. Biol. 2013, 37, 350–360. [Google Scholar] [CrossRef]
  61. Ghorbanpour, M.; Khavazi, K.; Ghafarzadegan, R.; Hatami, M. Two main tropane alkaloids variations of black henbane (Hyoscyamus niger) under PGPRs inoculation and water deficit stress induction at flowering stage. J. Med. Plants 2013, 45, 29–42. [Google Scholar]
  62. Chen, C.; Belanger, R.; Benhamou, N.; Paulitz, T.C. Defense enzymes induced in cucumber roots by treatment with plant growth promoting rhizobacteria (PGPR) and Pythium aphanidermatum. Physiol. Mol. Plant Pathol. 2000, 56, 13–23. [Google Scholar] [CrossRef]
  63. Dinesh, R.; Srinivasan, V.; Praveena, R.; Subila, K.P.; George, P.; Das, A.; Shajina, O.; Anees, K.; Leela, N.K.; Haritha, P. Exploring the potential of P-solubilizing rhizobacteria for enhanced yield and quality in turmeric (Curcuma longa L.). Ind. Crops Prod. 2022, 189, 115826. [Google Scholar] [CrossRef]
  64. Jagtap, R.R.; Mali, G.V.; Waghmare, S.R.; Nadaf, N.H.; Nimbalkar, M.S.; Sonawane, K.D. Impact of plant growth promoting rhizobacteria Serratia nematodiphila RGK and Pseudomonas plecoglossicida RGK on secondary metabolites of turmeric rhizome. Biocatal. Agric. Biotechnol. 2023, 47, 102622. [Google Scholar] [CrossRef]
  65. Palermo, J.S.; Palermo, T.B.; Cappellari, L.D.R.; Balcke, G.U.; Tissier, A.; Giordano, W.; Banchio, E. Influence of plant growth-promoting rhizobacteria (PGPR) inoculation on phenolic content and key biosynthesis-related processes in Ocimum basilicum under Spodoptera frugiperda herbivory. Plants 2025, 14, 857. [Google Scholar] [CrossRef]
  66. Zapata-Sifuentes, G.; Fortis-Hernández, M.; Sáenz-Mata, J.; Silva-Martínez, C.; Lara-Capistran, L.; Preciado-Rangel, P.; Hernández-Montiel, L.G. Effect of plant growth promoting rhizobacteria on the development and biochemical composition of cucumber under different substrate moisture levels. Microbiol. Res. 2024, 15, 1505–1515. [Google Scholar] [CrossRef]
  67. Ghorbanpour, M.; Hatami, M.; Kariman, K.; Khavazi, K. Enhanced efficiency of medicinal and aromatic plants by PGPRs. In Plant Growth Promoting Rhizobacteria (PGPR) and Medicinal Plants; Egamberdieva, D., Shrivastava, S., Varma, A., Eds.; Springer: Cham, Switzerland, 2015; Volume 42, pp. 43–70. [Google Scholar] [CrossRef]
  68. Rice-Evans, C.; Miller, N.; Paganga, G. Antioxidant properties of phenolic compounds. Trends Plant Sci. 1997, 2, 152–159. [Google Scholar] [CrossRef]
  69. Pietta, P.G. Flavonoids as antioxidants. J. Nat. Prod. 2000, 63, 1035–1042. [Google Scholar] [CrossRef] [PubMed]
  70. Giri, L.; Dhyani, P.; Rawat, S.; Bhatta, I.D.; Nandi, S.K.; Rawal, R.S.; Pande, V. In vitro production of phenolic compounds and antioxidant activity in callus suspension cultures of Habenaria edgeworthii: A rare Himalayan medicinal orchid. Ind. Crop. Prod. 2012, 39, 1–6. [Google Scholar] [CrossRef]
  71. Singh, R.P.; Dhanalakshmi, S.; Agarwal, R. Phytochemicals as cell cycle modulators: A less toxic approach in halting human cancers. Cell Cycle 2002, 1, 155–160. [Google Scholar] [CrossRef]
  72. Alothman, M.; Bhat, R.; Karim, A.A. Antioxidant capacity and phenolic content of selected tropical fruits from Malaysia, extracted with different solvents. Food Chem. 2009, 115, 785–788. [Google Scholar] [CrossRef]
  73. Bahadoran, Z.; Mirmiran, P.; Azizi, F. Dietary polyphenols as potential nutraceuticals in management of diabetes: A review. J. Diabetes Metab. Disord. 2013, 12, 43–52. [Google Scholar] [CrossRef]
  74. Rice-Evans, C.; Miller, N.; Paganga, G. Structure-antioxidant activity relationships of flavonoids and phenolic acids. Free Radic. Biol. Med. 1996, 20, 933–956. [Google Scholar] [CrossRef]
  75. Halliwell, B. Are polyphenols antioxidants or pro-oxidants? What do we learn from cell culture and in vivo studies? Arch. Biochem. Biophys. 2008, 476, 107–112. [Google Scholar] [CrossRef]
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Figure 1. In vitro propagation of Nardostachys jatamansi (a,b) seed germination on basal MS medium. (c) Elongation and multiplication of shoots on MS media supplemented with 2.0 µM TDZ and 0.5 µM NAA. (d) Rooting in MS medium supplemented with 0.5 µM IAA. (e) Comparison of inoculated and uninoculated plants after 60 days of inoculation; bar = 1 cm.
Figure 1. In vitro propagation of Nardostachys jatamansi (a,b) seed germination on basal MS medium. (c) Elongation and multiplication of shoots on MS media supplemented with 2.0 µM TDZ and 0.5 µM NAA. (d) Rooting in MS medium supplemented with 0.5 µM IAA. (e) Comparison of inoculated and uninoculated plants after 60 days of inoculation; bar = 1 cm.
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Figure 2. Impact of PGPR on plant height (A), survival percent (B), and chlorophyll content (C) of N. jatamansi after 60 days of inoculation with B. sabtilis and P. corrugata.
Figure 2. Impact of PGPR on plant height (A), survival percent (B), and chlorophyll content (C) of N. jatamansi after 60 days of inoculation with B. sabtilis and P. corrugata.
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Bacteria 04 00038 g003
Figure 3. Graphical representation of total phenol, alkaloids, tannin, flavonoids (A), and antioxidant activity (B) of control, in vitro-raised plants, and PGPR-inoculated plants of N. jatamansi.
Figure 3. Graphical representation of total phenol, alkaloids, tannin, flavonoids (A), and antioxidant activity (B) of control, in vitro-raised plants, and PGPR-inoculated plants of N. jatamansi.
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Table 1. Effect of different concentrations of plant growth regulators on in vitro regeneration from rhizome explants of N. jatamansi after 28 days of culture. Values represent mean ± SE of 10 replicates/treatment, and all the experiments were repeated twice. Means followed by the same letter in the column are not significantly different as indicated by Duncan’s multiple range test (p ≤ 0.05).
Table 1. Effect of different concentrations of plant growth regulators on in vitro regeneration from rhizome explants of N. jatamansi after 28 days of culture. Values represent mean ± SE of 10 replicates/treatment, and all the experiments were repeated twice. Means followed by the same letter in the column are not significantly different as indicated by Duncan’s multiple range test (p ≤ 0.05).
Plant Growth Regulators (µM)No. of ShootsShoot Length (cm)No. of Leaves
BAPTDZNAA
0.00.00.01.6 ± 0.2 j2.8 ± 0.1 f1.6 ± 0.2 i
1.00.00.12.1 ± 0.2 i3.3 ± 0.2 e1.8 ± 0.3 h
2.50.00.12.3 ± 0.2 h3.6 ± 0.2 d2.4 ± 0.2 g
5.00.00.12.1 ± 0.2 i2.7 ± 0.3 g2.3 ± 0.4 g
1.00.00.51.2 ± 0.6 k4.1 ± 0.6 ab3.2 ± 0.3 e
2.50.00.53.3 ± 0.3 f3.7 ± 0.5 c3.2 ± 0.7 e
5.00.00.53.8 ± 0.2 e2.9 ± 0.2 f2.8 ± 0.4 f
0.01.00.12.8 ± 0.4 g4.0 ± 0.7 ab3.3 ± 0.3 e
0.02.00.14.5 ± 0.3 c3.8 ± 0.6 bc3.8 ± 0.5 d
0.04.00.15.8 ± 0.4 b4.1 ± 0.4 ab4.4 ± 0.4 a
0.01.00.54.2 ± 0.2 d4.1 ± 0.5 ab4.2 ± 0.6 b
0.02.00.56.2 ± 0.3 a4.2 ± 0.4 a4.1 ± 0.5 bc
0.04.00.54.2 ± 0.2 d3.9 ± 0.4 b4.1 ± 0.4 bc
Table 2. Effect of auxin supplementation on rooting response of N. jatamansi. Values represent mean ± SE of 10 replicates/treatment, and all the experiments were repeated twice. Means followed by the same letter in the column are not significantly different as indicated by Duncan’s multiple range test (p ≤ 0.05).
Table 2. Effect of auxin supplementation on rooting response of N. jatamansi. Values represent mean ± SE of 10 replicates/treatment, and all the experiments were repeated twice. Means followed by the same letter in the column are not significantly different as indicated by Duncan’s multiple range test (p ≤ 0.05).
Growth Regulator + 1/2 MSShoot Forming Roots (%)Number of Days to RootNumber of Roots/ShootsRoot Length (cm)
IAA (µM)NAA (µM)
0.00.021.3 ± 0.8 j14–211.1 ± 0.2 i2.8 ± 0.4 e
0.10.053.6 ± 0.8 f15–202.3 ± 0.3 g2.9 ± 0.3 d
0.50.089.6 ± 1.2 a15–203.6 ± 0.7 a3.4 ± 0.4 b
0.00.151.2 ± 0.7 g10–212.1 ± 0.4 h3.6 ± 0.5 a
0.10.166.6 ± 1.0 d10–212.2 ± 0.6 gh2.8 ± 0.4 e
0.50.164.6 ± 1.3 e18–243.4 ± 0.4 b2.6 ± 0.6 f
0.00.573.4 ± 0.7 c10–212.1 ± 0.2 h3.1 ± 0.4 cd
0.10.542.3 ± 1.1 i12–183.1 ± 0.6 cd3.4 ± 0.6 b
0.50.566.6 ± 1.1 d12–182.9 ± 0.2 e3.2 ± 0.6 c
0.01.073.7 ± 1.4 c14–213.2 ± 0.8 c3.6 ± 0.5 a
0.11.048.3 ± 0.7 h10–213.4 ± 0.6 b3.4 ± 0.9 b
0.51.078.2 ± 0.9 b12–182.6 ± 0.6 f3.2 ± 0.8 c
Table 3. Effect of PGPR on growth, survival, and chlorophyll content of in vitro-raised plants and inoculated plants of N. jatamansi after 60 days of inoculation. Values represent mean ± SE of 25 replicates/treatment. Means followed by the same letter in the column are not significantly different as indicated by Duncan’s multiple range test (p ≤ 0.05).
Table 3. Effect of PGPR on growth, survival, and chlorophyll content of in vitro-raised plants and inoculated plants of N. jatamansi after 60 days of inoculation. Values represent mean ± SE of 25 replicates/treatment. Means followed by the same letter in the column are not significantly different as indicated by Duncan’s multiple range test (p ≤ 0.05).
Plant SampleGrowth ParametersSurvival %Chlorophyll Content
Root LengthRootShootLeaf AreaNo. of LeavesPlant Height
FWDWFWDW
Control (seed germinated)3.5 ± 0.2 c0.83 ± 0.02 d0.11 ± 0.01 d1.62 ± 0.2 c0.43 ± 0.1 d2.42 ± 0.1 d2.3 ± 0.2 d4.4 ± 0.6 d18.4 ± 1.2 d0.66 ± 0.1 c
Tissue culture-raised 3.6 ± 0.5 c1.02 ± 0.2 c0.23 ± 0.01 c2.42 ± 0.4 b0.84 ± 0.1 c3.65 ± 0.2 c3.2 ± 0.6 c5.8 ± 0.6 c66.3 ± 2.2 c0.70 ± 0.2 c
Inoculated with B. subtilis3.9 ± 0.6 b1.15 ± 0.4 b0.31 ± 0.01 b2.64 ± 0.3 b0.96 ± 0.1 b4.45 ± 0.7 b3.8 ± 0.8 b6.4 ± 0.8 b85.3 ± 3.6 b0.94 ± 0.7 b
Inoculated with P.corrugata4.8 ± 0.4 a1.43 ± 0.3 a0.36 ± 0.01 a3.22 ± 0.7 a1.16 ± 0.2 a4.83 ± 0.7 a4.6 ± 0.6 a7.2 ± 0.8 a94.6 ± 4.3 a1.12 ± 0.6 a
Table 4. Comparison of total phenol, alkaloids, tannin, flavonoids, and antioxidant activity of the in vitro-raised plants and inoculated plants of N. jatamansi. Values represent mean ± SE of 25 replicates/treatment. Means followed by the same letter in the column are not significantly different as indicated by Duncan’s multiple range test (p ≤ 0.05).
Table 4. Comparison of total phenol, alkaloids, tannin, flavonoids, and antioxidant activity of the in vitro-raised plants and inoculated plants of N. jatamansi. Values represent mean ± SE of 25 replicates/treatment. Means followed by the same letter in the column are not significantly different as indicated by Duncan’s multiple range test (p ≤ 0.05).
Plant SampleTotal Phenol (mg GAE/g DW)Alkaloids
(mg AE/g DW)		Tannin
(mg TAE/g DW)		Flavonoids
(mg QE/g DW)		Antioxidant Activity
(mM AAE/g DW)
DPPHFRAP
Control (seed germinated)23.87 ± 2.18 d21.87 ± 3.12 c11.71 ± 1.13 d24.12 ± 2.63 d43.13 ± 4.8 d2.11 ± 0.3 d
Tissue culture-raised29.87 ± 2.18 c25.81 ± 3.88 b16.33 ± 2.23 c29.33 ± 3.13 c55.88 ± 5.1 c3.88 ± 0.9 c
Inoculated with B. subtilis31.36 ± 2.45 b33.37 ± 3.44 a21.76 ± 2.66 b32.66 ± 3.42 b63.46 ± 5.3 b5.44 ± 0.9 b
Inoculated with P.corrugata45.87 ± 3.18 a33.89 ± 3.98 a28.39 ± 3.12 a39.67 ± 3.54 a67.89 ± 5.3 a6.76 ± 1.0 a
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Rawat, J.M.; Agarwal, M.; Negi, S.; Anand, J.; Semwal, P.; Rawat, B.; Bhardwaj, R.; Mitra, D. Biostimulatory Effects of Bacillus subtilis and Pseudomonas corrugata on Phytochemical and Antioxidant Properties of In Vitro-Propagated Plants of Nardostachys jatamansi (D. Don) DC. Bacteria 2025, 4, 38. https://doi.org/10.3390/bacteria4030038

AMA Style

Rawat JM, Agarwal M, Negi S, Anand J, Semwal P, Rawat B, Bhardwaj R, Mitra D. Biostimulatory Effects of Bacillus subtilis and Pseudomonas corrugata on Phytochemical and Antioxidant Properties of In Vitro-Propagated Plants of Nardostachys jatamansi (D. Don) DC. Bacteria. 2025; 4(3):38. https://doi.org/10.3390/bacteria4030038

Chicago/Turabian Style

Rawat, Janhvi Mishra, Mrinalini Agarwal, Shivani Negi, Jigisha Anand, Prabhakar Semwal, Balwant Rawat, Rajneesh Bhardwaj, and Debasis Mitra. 2025. "Biostimulatory Effects of Bacillus subtilis and Pseudomonas corrugata on Phytochemical and Antioxidant Properties of In Vitro-Propagated Plants of Nardostachys jatamansi (D. Don) DC" Bacteria 4, no. 3: 38. https://doi.org/10.3390/bacteria4030038

APA Style

Rawat, J. M., Agarwal, M., Negi, S., Anand, J., Semwal, P., Rawat, B., Bhardwaj, R., & Mitra, D. (2025). Biostimulatory Effects of Bacillus subtilis and Pseudomonas corrugata on Phytochemical and Antioxidant Properties of In Vitro-Propagated Plants of Nardostachys jatamansi (D. Don) DC. Bacteria, 4(3), 38. https://doi.org/10.3390/bacteria4030038

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