Skip to main content
Download PDF

Phyto-safe in vitro regeneration and harnessing antimicrobial-resistant endophytes as bioinoculants for enhanced growth and secondary metabolites yield in Nilgirianthus ciliatus

BMC Plant Biology volume 24, Article number: 872 (2024) Cite this article

Abstract

Nilgirianthus ciliatus, extensively exploited for its pharmacological properties, is now classified as vulnerable. In vitro micropropagation offers a sustainable approach for ecological conservation and rational utilization of this biodiversity resource. This study aimed to reduce endophytes during in vitro propagation and isolating antimicrobial-resistant endophytes from N. ciliatus by employing various concentrations and exposure times of Plant Preservative Mixture (PPM). Optimal results were observed when nodal explants treated with 0.3% PPM for 8 h, followed by inoculation in Murashige and Skoog (MS) medium supplemented with 3 mg/L 6-benzylaminopurine (BAP) and 0.3% PPM. This protocol achieved 82% shoot regeneration with minimal endophytic contamination, suggesting that the duration of explant exposure to PPM significantly influences endophyte reduction. Two antimicrobial-resistant endophytes were isolated and identified as Bacillus cereus and Acinetobacter pittii through 16S rDNA sequencing. These endophytes exhibited plant growth-promoting characteristics, including amylolytic, proteolytic, lipolytic activities, indole-3-acetic acid production, phosphate solubilization, and stress tolerance. In vivo application of these endophytes as bioinoculants to N. ciliatus not only improved growth parameters but also significantly increased the levels of pharmacologically important compounds, squalene, and stigmasterol, as confirmed by High-performance thin-layer chromatography (HPTLC). This study demonstrates that PPM is a promising alternative for sustainable micropropagation of N. ciliatus. Furthermore, it highlights the potential of antimicrobial-resistant endophytes as bioinoculants to improve growth and medicinal value, offering a sustainable solution for conservation and large-scale cultivation of this species.

Peer Review reports

Introduction

The soil microbiota, comprising a diverse array of microorganisms, plays a pivotal role in facilitating the growth and development of wild plants in their natural habitats [1].

Among these microorganisms, endophytes are particularly significant, as they establish symbiotic relationships within the internal tissues of plants without causing harm, thereby enhancing plant resilience and productivity [2, 3]. Recent studies have shown that endophytes not only assist in plant stress tolerance but also in promoting growth under adverse environmental conditions by producing phytohormones, siderophores, and other beneficial compounds [4].

Endophytes are known to substantially increase biomass accumulation by producing essential plant growth-promoting enzymes including amylase, protease, lipase, phosphatase, and nitrate reductase, which are vital for nutrient assimilation and overall plant health [5, 6]. In addition, endophytes hold immense value in plant breeding research, as they not only enhance biomass growth but also facilitate the accumulation of secondary metabolites, which are crucial for plant’s defense mechanisms and medicinal properties [7].

In the light of challenges posed by biotic and abiotic stresses, prioritizing antimicrobial-resistant (AMR) endophytes is crucial for protecting plant health and ensuring sustained productivity. In recent years, the exploration of AMR endophytes has become a focal point in sustainable agriculture, as these microorganisms can naturally enhance plant defense mechanisms without the need for chemical interventions. Furthermore, these endophytes hold significant promise as biofertilizers, thereby contributing to sustainable agricultural practices by improving nutrient uptake and soil health in breeding experiments with medicinal plants [8]. various methods, including the application of antibiotics and fungicides, have been explored to screen for AMR endophytes in tissue culture. However, the use of commercial antibiotics often associated with drawbacks, such as reduced explant viability and regeneration potential, necessitating the search for effective alternatives [9].

One promising alternative is the Plant Preservative Mixture™ (PPM), a novel broad-spectrum biocide commercially formulated based on US Patent 5,750,402. PPM was specifically developed to mitigate microbial contamination in tissue cultures. Unlike conventional antibiotics, PPM is chemically synthesized, heat-stable and can be autoclaved with media, making it an attractive alternative to conventional antibiotics and fungicides in plant tissue culture experiments [10, 11].

Nilgirianthus ciliatus (Nees) Bremek, commonly known as Strobilanthes ciliatus Nees, is an indigenous and highly traded vulnerable undershrub endemic to the southern Western Ghats of India [12]. This plant belongs to the Acanthaceae family and is locally known as “Sinnangurinji” in Tamil, “Karimkurinji” in Malayalam, and “Karvi” in Hindi [13]. Traditionally, this plant has been extensively used in the treatment of neurological disorders, rheumatism, glandular swelling, leprosy, and deadly infections [14]. N. ciliatus has a strong reputation for traditional medicinal practices due to its extraordinary medicinal properties [15]. This highly traded shrub from tropical forests is recognized for its valuable metabolites, including lupeol, stigmasterol, betulin, phytol, amyrin, vitamin E, and pharmacologically active anticancer triterpenoids (taraxerol and squalene) [16]. N. ciliatus extracts exhibit a wide range of pharmacological activities, including antibacterial, anti-inflammatory, antioxidant, antiviral, hypoglycemic, hepatoprotective, analgesic, and anticancer properties [17,18,19].

However, unsustainable harvesting practices, including the uprooting and overexploitation of this plant from its natural habitat by traditional practitioners and pharmaceutical industries, have led to a significant decline in its population, warranting its inclusion in the RED DATA list of South Indian medicinal plants as a vulnerable species by the International Union for Conservation of Nature (IUCN) [20]. The extensive utilization of this plant by the herbal industries in Kerala, with annual consumption exceeding 200 MT, has resulted in a 40% population decrease over the last three generations, highlighting the urgent need for conservation efforts [21]. With the increasing demand for N. ciliatus biomass, definite measures are required to preserve this valuable medicinal plant. Plant tissue culture is a pivotal field of biotechnology for the large-scale production of medicinal plants with high concentrations of secondary metabolites and for screening antimicrobial-resistant endophytes present in plants, which can be used as potential biofertilizers.

Considering the multiple benefits of endophytes, this study aimed to elucidate the dynamics of endophytic microorganisms in the growth and secondary metabolites accumulation of N. ciliatus. Furthermore, this study advances our understanding of plant-microbe interactions and offers practical insights for improving plant efficiency and sustainability. To the best of our knowledge, this is the first study to report the presence of endophytes in N. ciliatus, and it pioneered the use of Bacillus cereus and Acinetobacter pittii as potential bioinoculants.

Materials and methods

Plant material and surface sterilization protocol

Fresh stocks of N. ciliatus were procured from the Foundation for Revitalization of Local Health Traditions (FRLHT), Bengaluru, Karnataka, India and maintained in a shade house at the Department of Biotechnology, Alagappa University, Karaikudi, Tamil Nadu, India. Healthy nodal segments measuring 2–3 cm in length were selected for in vitro culture initiation. Two different surface sterilization protocols (SSP1 and SSP2) were employed to eradicate epiphytes from the explants. Initially, explants were cleansed under running tap water for 20 min to remove soil and dust particles.

For SSP1, nodal explants were first treated with 0.1% Bavistin solution (prepared in sterile distilled water (SDW)) for 10 min followed by rinsing in distilled water for 10 min. Subsequently, the explants were immersed in 0.1% mercuric chloride (HgCl2) for 3 min, and washed thrice with SDW, further treated with 70% ethanol (EtOH) for 1 min and washed thrice with SDW. The SSP2 involved a 30-second ethanol wash followed by three times SDW rinse, a 15-minute 0.1% Bavistin wash, and a final treatment with 0.01% w/v mercuric chloride for 3 min, concluded by three times SDW rinse to eliminate excess HgCl2. Finally, the surface-sterilized explants were then blot-dried, and the edges exposed directly to disinfectants were trimmed. The sterility of the protocols was verified by incubating the final rinse water in Murashige and Skoog (MS) medium at 25 °C for one week to check for epiphytic contamination.

In vitro culture initiation

Plant regeneration from nodal explants was performed using previously established protocols [22]. For primary shoot initiation, surface-sterilized nodal explants (approximately 2 cm) were inoculated into sterile culture tubes (25 × 150 mm) containing 10 mL of MS medium supplemented with 3 mg/L BAP. Agar (8 g/L) was used as a solidifying agent. All cultures were maintained at 25 ± 2 °C under a 16 h photoperiod with 50 µmol photons m− 2 s− 1 of white light.

Effect of PPM on the growth of N. ciliatus and Screening of AMR Endophytes

Endophytic colonization was evident within 3-5 days post-inoculation. By the 7th day, microbial overgrowth had compromised the viability of the nodes (Fig. 1). To eradicate microbial contamination barriers, PPM was introduced into the medium. Surface-sterilized explants were immersed in half-strength MS liquid media supplemented with 0.1 − 0.5% PPM (S1, S2, S3, S4, and S5) for various periods ranging from 0 to 12 h (T2, T4, T6, T8, T10, and T12), followed by inoculation of explants at 2 h intervals in full-strength MS medium fortified with 3 mg/L BAP and 0.1–0.5% PPM (I1, I2, I3, I4, and I5). Fifty explants were cultured per treatment and an experiment without PPM served as a control. All cultures were evaluated for microbial contamination at weekly intervals, and culture establishment was recorded. After four weeks, well-grown plants without endophytes were transferred to multiplication medium (MS medium + 3 mg/L BAP + 0.1 mg/L IAA).

Fig. 1
figure 1

Various endophytic contamination encountered during in vitro culture initiation

Full size image

Detection and isolation of PPM-resistant endophytes

Within one week, few endophytes were observed during the incubation of the explants at the optimal PPM concentration. The endophytes were considered to be PPM-tolerant. Subsequently, distinct colonies were isolated and labeled as NCEB1 and NCEB2. The isolated endophytic bacteria were subjected to enzymatic, plant growth promoting attributes and Molecular characterization.

Characterization of AMR endophytic bacteria

Enzymatic characterization

Amylolytic activity

To evaluate the amylolytic activity of NCEB1 and NCEB2, overnight cultures were inoculated in the center of an LB agar medium supplemented with 0.8% starch solution and incubated at 28 °C for 3 days. Subsequently, 1% Gram’s iodine solution was applied to all plates to assess α-amylase-producing activity by recording the formation of clear zones around the colonies of endophytic bacteria [23].

Proteolytic activity

The proteolytic activity of NCEB1 and NCEB2 was assessed by inoculating overnight cultures on skim milk agar. After 3 days of incubation at 28 °C, the development of a clear halo zone around the colony indicated the proteolytic activity of the endophytic bacteria [24].

Lipolytic activity

The lipase-producing abilities of NCEB1 and NCEB2 were evaluated by inoculating overnight cultures on lipase agar medium. The production of lipase was indicated by the formation of a clear halo zone around the colonies after 3 days of incubation at 28 °C [24].

In vitro plant growth-promoting attributes of endophytic bacteria

Bio stimulation assay: Indole Acetic acid production

The indole acetic acid (IAA) production of isolated endophytic bacteria was evaluated using the colorimetric method described by Passari et al., 2015 [25]. Initially, the NCEB1 and NCEB2 cultures were grown in 2 mL of LB broth at 28 °C under constant agitation at 120 rpm overnight. Subsequently, 1 mL of the bacterial inoculum was added to 10 mL of LB broth supplemented with 100 µg/mL L-tryptophan and incubated under constant agitation at 120 rpm and 28 °C in the dark for 48 h. After incubation, the culture was centrifuged at 5000 rpm for 10 min, and the supernatant was collected for further analysis. IAA production was evaluated using a mixture containing 300 µL of the supernatant and 700 µL of Salkowski’s reagent (1.2% 0.5 M FeCl3 in 37% H2SO4), which was incubated at room temperature in the dark for 15 min. The absorbance of the resulting mixture was measured quantitatively using a spectrophotometer at 530 nm. IAA concentrations were estimated using a standard curve based on the known concentrations of 3-indolacetic acid (10-50 µg/mL).

Biofertilization assay: phosphate solubilization assay

The phosphate solubilization activity of the bacterial strains (NCEB1 and NCEB2) was quantitatively evaluated, using the method described by Passari et al., 2015 [25]. For each strain, 1 mL of overnight-grown bacterial suspension was inoculated into 50 mL of NBRIP medium, while the non-inoculated medium served as the control. The flasks were incubated at 30 °C for 72 h in a shaker incubator at 125 rpm. After incubation, the supernatant was collected by centrifugation at 10,000 rpm for 10 min. The soluble phosphate in the culture supernatant was determined using the ammonium molybdate blue method. Briefly, the bacterial supernatant (1 mL) was mixed with trichloroacetic acid (500 µL, 10% w/v) in a test tube, to which 4 mL ammonium molybdate reagent (1:1:1:2 ratio of 3 M H2SO4, 2.5% (w/v) ammonium molybdate, 10% (w/v) ascorbic acid, and distilled water) was added. The test tubes were incubated at room temperature for 15 min and the absorbance of the developed blue color was measured at 880 nm using a spectrophotometer. A standard curve of KH2PO4 (10-50 µg/mL) was used to determine the number of soluble phosphates produced by each bacterium, expressed in µg/mL.

Stress tolerance assay: salt and drought stress

The growth of the selected bacterial isolates was analyzed under various stress conditions following the established methodologies. To assess salt stress tolerance, 20 µL of overnight grown NCEB1 and NCEB2 cultures were inoculated into LB medium supplemented with various salt concentrations (2%, 4%, 6%, 8%, and 10%). After incubation for 24 h and 48 h, the absorbance of the culture was measured at 600 nm, with the uninoculated medium used as a blank.

To assess drought stress tolerance, 20 µL of overnight grown NCEB1 and NCEB2 cultures were inoculated into LB medium supplemented with varying PEG concentrations (2%, 4%, 6%, 8%, and 10%). After incubation for 24 h and 48 h, the absorbance of the culture was measured at 600 nm, with the uninoculated medium used as a blank.

Molecular characterization of endophytic bacteria

Molecular identification was performed based on bacterial 16S rRNA gene amplification and sequence analysis [26, 27]. Total genomic DNA from overnight grown endophytic cultures was extracted using the sucrose TE buffer method. The extracted DNA was resuspended in 20 µL of sterile Milli-Q water and used as a template for 16S rRNA gene amplification using universal primers 26 F (5′-AGAGTTTGATCCTGGCTCA-3′) and 1100R (5′-AGGGTTGCGCTCGTTG-3′). Amplification was performed in an Eppendorf master cycler ProS (Eppendorf, Germany) under the following thermal cycling conditions: initial denaturation at 95 °C for 10 min; 35 cycles of denaturation (95 °C for 45 s), annealing (55 °C for 60 s), and extension (72 °C for 90 s); and final elongation at 72 °C for 10 min. The amplified PCR products were resolved using agarose gel electrophoresis (1.5%) and visualized using a gel documentation system. Partial 16S rDNA amplicons were sequenced using an Applied Biosystems 3130 Genetic Analyzer (Applied Biosystems). The resulting 16S rRNA gene sequences were then analyzed using the Basic Local Alignment Search Tool (BLAST) at the National Center for Biotechnology Information (NCBI) to find the closest matches in the nucleotide database.

In vivo plant growth-promoting activity of the isolated endophytes

The growth-promoting activity of the identified bacterial isolates on N. ciliatus were assessed under shade house conditions. The experiments were conducted using a randomized complete block design (RCBD). Two bacterial isolates (NCEB1 and NCEB2) were grown in LB broth at 28 °C for 24 h with continuous shaking at 150 rpm. The bacterial cells were then centrifuged at 10,000 rpm for 15 min, and the resulting pellets were diluted with distilled water to obtain a final concentration of 108 CFU/ml was adjusted using OD value at OD600, which was used to treat the N. ciliatus.

For the treatment, 30 day-old equal-sized stem cuttings (developed using the traditional stem cutting method) were raised in sterilized soil in plantation pots (150 mm × 150 mm). Four different experimental setups were used: (i) N. ciliatus inoculated with 10 ml of NCEB1, (ii) N. ciliatus inoculated with 10 ml of NCEB2, (iii) N. ciliatus inoculated with 5 mL NCEB1 and 5 mL NCEB2, and (iv) a control group without any endophytic bacterial treatment. The roots were irrigated once every 15 days following the method described above. Each treatment consisted of 10 pots, and all plants were well-irrigated and protected from other bacterial infections and weeds. After 60 d of treatment, five randomly selected plants from each treatment were subjected to morphological analysis (plant length, shoot length, root length, number of leaves, number of branches, number of roots, fresh weight, and dry weight).

HPTLC quantification

Squalene and stigmasterol were quantified using a CAMAG HPTLC instrument with winCATS 1.4.3 software and a Linomat 5 sample applicator. Squalene and stigmasterol standards were obtained from Sigma Aldrich. A 1 mg/mL stock solution of squalene and stigmasterol was prepared in hexane: chloroform (1:1 ratio), and from the stock, 0.3 µl, 0.6 µl, 0.9 µl, 1.2 µl, and 1.5 µl (concentrations ranging from 0.3 µg to 1.5 µg) were loaded as 6 mm bands on TLC plates coated with silica gel 60 F 254. Similarly, 5 µL of each plant extract was loaded as 6 mm bands. The mobile solvent was petroleum ether: ethyl acetate (7:3). The bands were derivatized by spraying anisaldehyde reagent onto a TLC plate. The plates were then heated at 60˚C. Densitometric scanning was performed at 520 nm using TLC Scanner 3 densitometry. Standard graphs were plotted using these standards.

Data analysis

SPSS version 27 was used to analyze the data on endophyte eradication using antibiotics and PPM™. One-way ANOVA was used to analyze the effect of antimicrobial agents on explant survival percentage and the effect of endophytes on plant growth-promoting activity, and three-way ANOVA was used to analyze the effect of PPM on explant survival percentage.

Results

Surface sterilization

The results demonstrated a clear difference in the effectiveness of the two protocols.

The water used for the final wash in SSP1 showed microbial growth in MS medium, indicating the presence of residual contaminants. This highlights the lower efficacy of SSP1 in achieving complete surface sterilization of explants. In contrast, the water used for the final wash of explants in SSP2 resulted in no microbial growth in the MS medium after seven days of incubation. This indicates that SSP2 was highly effective in eliminating all surface-borne contaminants from the explants (Fig. 2). Hence, SSP2 was significantly more effective than SSP1 in eradicating epiphytic and surface-borne contaminants from explants. These results suggest that SSP2 should be considered as the preferred method for surface sterilization in related experimental setups.

Fig. 2
figure 2

Effect of surface sterilization process on nodal explants. (A) Sterile distilled water poured on MS medium, (B) Nodal explants soaked in sterile distilled water and poured on MS medium, (C) Final rinsed water of SSP1 poured on MS medium, (D) Final rinsed water of SSP2 poured on MS medium

Full size image

Evidence supporting the presence of endophytes

Despite the stringent surface sterilization process, endophytes occurred within 3-5 d after inoculation, as shown in Fig. 1. The time-dependent growth of these endophytes and their consistent visibility at the base of the explants provide compelling evidence of their endophytic nature. The persistence of these microorganisms, even after rigorous sterilization, suggests that they reside within the internal tissues of the explants, rather than on the surface.

Effect of PPM on the growth of N. ciliatus and Screening of AMR Endophytes

The effect of PPM on the growth of N. ciliatus and the screening of antibiotic-resistant (AMR) endophytes were evaluated based on the survival percentage of the explants inoculated in the medium. A significant difference (p < 0.05) in explant survival rates were observed among the various treatment conditions. Overall, explants soaked in 0.3% (v/v) PPM for up to 8 h resulted in the highest survival percentage. Beyond this period, the survival rate began to decrease. The highest survival rate (90.66 ± 2.3%) was recorded for nodal explants soaked in half-strength MS medium augmented with 0.3% (v/v) PPM for 8 h, followed by those inoculated in MS medium supplemented with 3 mg/L BAP and 0.3% (v/v) PPM. Furthermore, nodal explants soaked in half-strength MS medium with 0.3% (v/v) PPM for 8 h followed by inoculation in MS medium with 3 mg/L BAP and 0.2% (v/v) PPM exhibited a survival rate of 82 ± 2%. Soaking the explants at a higher concentration of 0.5% (v/v) PPM for different durations resulted in the lowest survival rates. The explants became brown and proved to be fatal. The detailed survival percentage of explants upon PPM treatment was shown in Fig. 3, Supplementary Table 1.

Fig. 3
figure 3

Influence of plant preservative mixture on the survival percentage of N. ciliatus regeneration. Inoculation and soaking medium containing 0.1 to 0.5% of PPM in MS medium

Full size image

In vitro propagation of N. ciliatus using nodal culture

After one week of culture, green juvenile primary shoot buds were successfully developed using the optimized protocol. These primary shoots exhibited healthy growth and were subsequently transferred to Murashige and Skoog (MS) medium supplemented with 3 mg/L benzyl aminopurine (BAP) and 0.1 mg/L indole-3-acetic acid (IAA) to induce multiple shoots. After one month, the N. ciliatus shoots exhibited significant growth and were then transferred to half-strength MS medium fortified with 1.0 mg/L indole-3-butyric acid (IBA) to promote in vitro rooting. Fully developed plants were then acclimatized by transferring them to a plant growth chamber before being moved to a shade house for further growth and development (Fig. 4).

Fig. 4
figure 4

In vitro multiple shoot induction and rooting of N. ciliatus using nodal explants. (A) primary shoots induction on MS medium with optimized PPM™ protocol; (B) Multiple shoots induced on MS medium supplemented with BA (3.0 mg/l) in combination with IAA (0.1 mg/l); (C) & (D) In vitro rooting of N. ciliatus on half-strength MS medium supplemented with 1.0 mg/L IBA

Full size image

Detection and isolation of PPM tolerant endophytic bacteria

Despite PPM treatment, bacterial growth was observed in some explants inoculated in MS medium. Two PPM-tolerant endophytic bacteria were successfully screened and isolated from the explants. These isolates were labeled as NCEB1 and NCEB2 for subsequent experiments (Fig. 5).

Fig. 5
figure 5

PPM-resistant bacteria isolated from the nodal explants of N. ciliatus. (A) NCEB1; (B) NCEB2

Full size image

Molecular characterization of bacterial endophytes

BLAST search results revealed that the bacterial isolate NCEB1 had high similarity to Bacillus cereus, while NCEB2 was similar to Acinetobacter pittii. These identifications were based on the highest sequence alignment scores and percentage identities with the known bacterial sequences in the database. To ensure the availability and accessibility of these sequences for future research, the nucleotide sequences of NCEB1 and NCEB2 were deposited in the GenBank database with accession numbers PP237779 and PP238070, respectively. To further validate the phylogenetic placement of these isolates, a Neighbor-Joining tree was constructed based on the sequence alignments. This tree, which illustrates the evolutionary relationships of NCEB1, NCEB2, and their closest matches in the NCBI database, is provided as Supplementary Data FigS1and 2. The molecular characterization of these bacterial endophytes provides critical insights into the AMR microbes associated with N. ciliatus, facilitating further studies on their functional roles in plant growth promotion and stress tolerance, as well as their potential applications in the development of bioinoculums.

Enzymatic characterization of endophytic bacteria

The enzymatic activities of the isolated endophytic bacteria, NCEB1 (Bacillus cereus) and NCEB2 (Acinetobacter pittii), were evaluated to understand their potential roles in promoting plant growth and health. Amylolytic, proteolytic, and lipolytic activities of the bacteria were tested using specific substrate media. Both NCEB1 and NCEB2 exhibited significant amylolytic and proteolytic activity, as indicated by the clear zones observed in their respective media. However, their lipolytic activity was limited as evidenced by the formation of faint zones (Fig. 6). The suitable positive control has been used to validate the enzyme producing activities of the isolates. Bacillus amyloliquefaciens was used as positive control for amylolytic activity, Bacillus subtilis were used as positive control for proteolytic and lipolytic activities.

Fig. 6
figure 6

Enzymatic analysis of PPM tolerant endophytes. (A) Amylolytic assay; (B) Proteolytic assay; (C) Lipolytic activity

Full size image

In vitro plant growth-promoting attributes of endophytic bacteria

The plant growth-promoting attributes of the endophytic bacteria NCEB1 (Bacillus cereus) and NCEB2 (Acinetobacter pittii) were evaluated based on their production of indole acetic acid (IAA), phosphate solubilization potential, and tolerance to drought and salt stress. NCEB1 exhibited higher IAA production (28.814 ± 1.2 µg) than NCEB2 (17.934 ± 0.874 µg). NCEB2 demonstrated a higher phosphate solubilization potential, measuring 22.658 ± 1.762 µg, whereas NCEB1 showed a solubilization potential of 17.211 ± 2.387 µg.

The tolerance of NCEB1 and NCEB2 to drought and salt stress was tested to evaluate their potential resilience under adverse environmental conditions. NCEB1 showed significant tolerance to both drought and salt stress, up to 8% and 6%, respectively, even after 48 h of exposure. In contrast, NCEB2 tolerated up to 4% of both drought and salt stress under the same conditions. These results suggest that NCEB1 possesses a higher capacity to endure stressful environments, which can be advantageous for plant growth under abiotic stress conditions. Figure 7 illustrates the comparative IAA production, phosphate solubilization capacities, and stress tolerance levels of NCEB1 and NCEB2, highlighting their distinct and valuable attributes.

Fig. 7
figure 7

In vitro Assessment of Plant Growth-Promoting Traits of Endophytic Bacteria: (A) Analysis of Indole Acetic Acid Production and Phosphate Solubilization, (B) Influence of Endophytic Bacteria on Salt Stress, (C) Impact of Endophytic Bacteria on Drought Stress

Full size image

In vivo plant growth-promoting activity of the isolated endophytes

The growth of N. ciliatus was significantly influenced by treatment with different bacterial inoculants, as detailed growth attributes are presented in Fig. 8. The inoculation of N. ciliatus with B. cereus resulted in a notable increase in shoot height and leaf number compared to the control group, indicating a positive impact on the aerial growth of the plant. Conversely, plants treated with A. pittii exhibited well-developed root systems, suggesting that A. pittii primarily enhances root growth and development. The most pronounced growth improvements were observed in plants treated with the combination of B. cereus and A. pittii. These plants demonstrated significant increases in several growth parameters, including the number of branches, shoot height, root height, and the number of new leaves formed, compared to those treated with individual bacterial strains and the control group. These findings suggest that the combined application of B. cereus and A. pittii has a synergistic effect on N. ciliatus growth, underscoring the potential of these bacterial strains as effective bioinoculants to enhance various aspects of plant growth.

Fig. 8
figure 8

In vivo plant growth promoting activity of endophytic bacteria on N. ciliatus. (A) NCEB1 and NCEB2 treatment on N. ciliatus; (B) Biometric parameters of N. ciliatus on endophytes treatment

Full size image

HPTLC quantification of squalene and stigmasterol

HPTLC quantification of squalene and stigmasterol in N. ciliatus plants treated with NCEB1, NCEB2, and the combination of NCEB1 and NCEB2 revealed significant variations in the levels of these compounds. The retention factors for squalene and stigmasterol were determined to be 0.76 and 0.39, respectively (Fig. 9).

Fig. 9
figure 9

High-Performance Thin-Layer Chromatography (HPTLC) Fingerprinting of control and endophytes treated Nilgirianthus ciliatus Extracts. Lane 1–5: Squalene and Stigmasterol Standards, Lane 6: Control N. ciliatus, Lane 7: N. ciliatus treated with Bacillus cereus, Lane 8: N. ciliatus treated with Acinetobacter pittii, Lane 9: N. ciliatus treated with a combination of Bacillus cereus and Acinetobacter pittii

Full size image

Squalene Content

The squalene content in the control group was 0.103 µg. Upon treatment with the bacterial strain NCEB1, the squalene content nearly doubled to 0.203 µg, indicating significant enhancement. The NCEB2 treatment resulted in a slight increase in squalene content to 0.108 µg, which was relatively close to the control value. However, the combined treatment with NCEB1 and NCEB2 resulted in a substantial increase in squalene content, reaching 0.361 µg. This indicated a synergistic effect of the combined bacterial treatment, leading to a threefold increase compared to the control.

Stigmasterol content

The Stigmasterol content in the control group was 0.242 µg. Treatment with the bacterial strain NCEB1 resulted in an increase of 0.323 µg, demonstrating significant enhancement. NCEB2 treatment also led to an increase in stigmasterol content to 0.277 µg, which was higher than that of the control, but lower than that of the NCEB1 treatment. The combined NCEB1 and NCEB2 treatment showed the highest increase in stigmasterol content, reaching 0.578 µg. This was more than double the amount found in the control group, indicating a notable synergistic effect of the combined bacterial treatments.

Discussion

N. ciliatus is one of India’s most lucrative medicinal plants, however, it is currently facing extinction in the wild due to overexploitation by the pharmaceutical industry. The seeds of this plant are notoriously difficult to propagate, and stem cutting and grafting methods often result in poor rooting success and high mortality rates [22]. As a result, micropropagation has been identified as the most promising alternative method for the mass production of this plant.

Plant Material and Surface sterilization of explants

Microbial contamination is a significant limiting factor for the success of plant tissue cultures and commercial units. Effective surface sterilization of explants is essential to minimize contamination and ensure the success of tissue culture techniques [28]. Generally, the use of individual chemicals for disinfection does not effectively eliminate epiphytic contaminants [29, 30]. Therefore, combinations of different chemicals were used to optimize the protocol for the removal of epiphytic contaminants from N. ciliatus. Our findings demonstrate that treatment with SSP2 was more effective than SSP1 in eradicating epiphytic contaminants. These findings align with previous research that highlights the importance of stringent surface sterilization protocols in achieving better contamination control [31, 32].

Evidence supporting the Presence of endophytes

Despite rigorous surface sterilization procedures, endophytic contamination persisted within a few days of inoculation, indicating the remarkable resilience of the endophytic communities and suggests that they are deeply integrated into the plant tissues. The time-dependent growth of these endophytes further supports the notion of their potential endophytic nature, characterized by well-established symbiotic relationships with the host plant, and underscores the difficulties inherent in achieving complete sterilization of the plant material [3].

The presence of endophytes in tissue culture systems can adversely affect the growth and development of plants. This phenomenon underscores the challenges associated with achieving axenic cultures and highlights the need for alternative strategies to mitigate endophytic contamination in tissue culture systems [33, 34]. The present study emphasized that, the endophytic contamination frequently occurred within a week of inoculation, resulting in the death of 90% of explants. This finding consistent with previous reports on the failure of surface sterilization in vitro walnut shoot regeneration [35]. These results indicate the need for further research to develop effective methods for eradicating endophytic contamination in explants.

Alternative to antimicrobials: the use of PPM to Control and Select AMR Endophytes

An excellent alternative to commercial antibiotics is the use of a chemical mixture that is safe for plants in the tissue culture media. Owing to the wide range of microbial species that invade the interior tissues of plants, the use of biocides broadly kills both bacteria and fungi before they spread [35]. PPM is considered a potential replacement for controlling endophytes during the in vitro propagation of N. ciliatus. As evidenced by an increasing number of publications over the past two decades, the development of PPM formulations for controlling tissue culture-associated microbial contaminants has provided tremendous relief for academic and industrial production units engaged in micropropagation and other tissue culture applications [36]. The use of PPM in initiating the in vitro cultivation of diverse woody plants, such as Acacia confusa [37], Dendranthema grandiflora, Betula pendula, and Rhododendron catawbiense [11], has proven to be highly successful. However, the optimal concentration and duration of PPM treatment must be determined for each plant species [35].

Our study demonstrated that soaking explants in 0.3% (v/v) PPM for up to 8 h resulted in the highest survival rate. This finding is partially consistent with the results of Kushnarenko et al. (2022) [35], who reported optimal survival rates with 0.2% PPM. Previous report suggested that incorporating PPM into soaking and inoculation media resulted in significant endophytic eradication and shoot regeneration in Centella asiatica [38, 39]. The reduction in survival rates observed at higher concentrations and longer exposure times aligns with other studies that have reported adverse effects of prolonged PPM treatment on explant viability [40]. The efficacy of lower PPM concentrations was insufficient for endophyte eradication, a result consistent with the findings of other studies [41, 42].

Detection and isolation of PPM tolerant endophytic Bacteria

The detection and identification of PPM-tolerant endophytic bacteria in N. ciliatus underscores their ability to adapt to biocidal agents. Endophytic bacteria are known to possess mechanisms that enable them to tolerate environmental stresses, including exposure to biocides [43]. Our study identified two PPM-tolerant endophytes, Bacillus cereus and Acinetobacter pittii, which were able to survive and proliferate despite the presence of PPM in the culture medium. These findings are in line with previous research by Thomos et al. (2017) [10], who reported the prevalence of diverse PPM-tolerant endophytic bacteria in Papaya carica L. The process by which microorganisms develop antibiotic resistance is strongly related to their enzyme-producing abilities [44].

Enzymatic characterization of endophytic bacteria

Endophytic bacteria play a vital role in plant growth and health, by producing enzymes that aid in nutrient acquisition and breakdown of complex organic compounds in the rhizosphere. These enzymatic capabilities are essential for the release of essential nutrients that support plant growth, development, and protection from pathogens [45, 46]. The enzymatic characterization of B. cereus and A. pittii associated with N. ciliatus offers valuable insights into their metabolic capabilities and functional roles in plant-microbe interactions.

Plant Growth-Promotion attributes of Endophytic Bacteria

The production of indole acetic acid (IAA), phosphate solubilization and stress tolerance by B. cereus and A. pittii proved their potential as plant growth-promoting bacteria (PGPB) and suggest their utility as biofertilizers for N. ciliatus cultivation [47]. IAA is a key phytohormone involved in regulating various aspects of plant growth, including cell elongation, root initiation, and shoot development. The significant production of IAA by B. cereus (28.814 µg) and A. pittii (17.934 µg) highlights their ability to modulate plant hormone levels, promote cell elongation, and stimulate root growth, leading to improved nutrient uptake and overall plant vigor [48]. Furthermore, both B. cereus and A. pittii showed phosphate solubilization potential (17.211 µg and 22.658 µg, respectively), a critical trait that facilitates the mobilization of insoluble forms of phosphate in the soil, making it more accessible to plants and playing a key role in energy transfer, photosynthesis, nucleic acid synthesis, root growth promotion, nutrient uptake, and overall plant performance [49]. Previous studies have confirmed that both B. cereus and A. pittii can produce adequate quantities of IAA [50, 51]. The ability of these endophytes to tolerate stress, including drought and salt stress, further highlights their potential utility as biofertilizers.

In vivo plant growth-promoting activity

In recent years, the use of endophytes as bioinoculants has gained significant attention for their role in promoting plant growth. Our study demonstrated the in vivo growth-promoting effects of B. cereus and A. pittii on N. ciliatus, indicating their potential as bioinoculants to enhance plant growth and productivity. The differential responses observed in N. ciliatus suggested that microbial interactions play a crucial role in plant growth. B. cereus was particularly effective in enhancing shoot development and overall plant growth, which is consistent with the findings of Joo et al. (2005) [52]. In contrast, A. pittii was more effective in promoting root health, length, and weight. This finding was consistent with the results reported by Kang et al. (2023) [53]. The synergistic effects of the two endophytes resulted in the highest plant growth-promoting attributes of N. ciliatus. Our results are consistent with previous studies that have shown enhanced plant growth when multiple endophytes are used in combination [50].

HPTLC quantification of squalene and stigmasterol

HPTLC quantification of squalene and stigmasterol revealed significant variations in the levels of these compounds in treated N. ciliatus plants. The outcomes unequivocally revealed that the bacterial treatments NCEB1 and NCEB2 individually increased the squalene and stigmasterol content in N. ciliatus plants, with NCEB1 showing a more pronounced effect than NCEB2. Nevertheless, the most significant enhancement was observed with the combined treatment of NCEB1 and NCEB2. This synergism led to a greater increase in the production of these compounds than with either treatment alone.

These findings further highlight the potential of using specific bacterial strains as bioinoculants to maximize the production of valuable phytochemicals in N. ciliatus. Further studies are needed to elucidate the mechanisms underlying this synergistic effect and optimize treatment protocols for commercial applications. This approach could be particularly valuable in enhancing the yield of bioactive compounds in medicinal and agricultural crops.

Conclusion

For the first time, PPM-mediated eradication of endophytes, followed by a rapid and efficient in vitro mass propagation protocol, has been developed to conserve the globally vulnerable medicinal plant, N. ciliatus. This protocol is a potential alternative to conventional propagation as a conservative measure to protect ever-declining medicinal plants. In the future, this protocol could be used to achieve the mass production of hairy roots of target plants and the sustainable production of secondary metabolites of interest.

Furthermore, our study provides valuable insights into the role of endophytic bacteria in promoting plant growth and enhancing secondary metabolite production in N. ciliatus. The synergistic effects observed with combined bacterial treatments suggest a promising avenue for developing effective biofertilizers and bio stimulants to improve crop yields and stress tolerance in sustainable agriculture.

Data availability

All data generated or analyzed during this study are included in this published article. The 16S rRNA gene sequence data that support the findings of this study have been deposited in GenBank (http://www.ncbi.nlm.nih.gov) with the accession numbers [PP237779; PP238070].

Abbreviations

A. pittii :

Acinetobacter pittii

AMR:

Antimicrobial resistant

ANOVA:

Analysis of Variance

B. cereus :

Bacillus cereus

BAP:

Benzyl aminopurine

EtOH:

Ethanol

FeCl3 :

Ferric Chloride (Iron (III) chloride

H2SO4 :

Sulfuric Acid

HgCl2 :

Mercuric Chloride

HPTLC:

High-Performance Thin-Layer Chromatography

IAA:

Indole-3-acetic acid

IBA:

Indole-3-butyric acid

IUCN:

International Union for Conservation of Nature

KH2PO4 :

Potassium Dihydrogen Phosphate

LB medium:

Luria-Bertani medium

MS medium:

Murashige and Skoog

N. ciliatus :

Nilgirianthus ciliatus

NBRIP:

National Botanical Research Institute’s Phosphate Growth Medium

NCEB1:

N. ciliatus endophytic bacteria 1

NCEB2:

N. ciliatus endophytic bacteria 2

PEG:

Polyethylene Glycol

PPM:

Plant Preservative Mixture

RCBD:

Randomized Complete Block Design

SDW:

Sterile Distilled Water

SSP1:

Surface Sterilization Protocol 1

SSP2:

Surface Sterilization Protocol 2

TLC:

Thin-Layer Chromatography

References

  1. Jacoby R, Peukert M, Succurro A, Koprivova A, Kopriva S. The role of soil microorganisms in plant mineral nutrition—current knowledge and future directions. Front Plant Sci. 2017;8:1617.

    Article  PubMed  PubMed Central  Google Scholar 

  2. Verma H, Kumar D, Kumar V, Kumari M, Singh SK, Sharma VK, Droby S, Santoyo G, White JF, Kumar A. 2021. The potential application of endophytes in management of stress from drought and salinity in crop plants. Microorganisms, 9(8), p.1729.

  3. Hardoim PR, van Overbeek LS, Berg G, Pirttilä AM, Compant S, Campisano A, Döring M, Sessitsch A. The Hidden World within plants: ecological and evolutionary considerations for defining functioning of Microbial endophytes. Microbiol Mol Biol Rev. 2015;79:293–320. https://doi.org/10.1128/mmbr.00050-14.

    Article  PubMed  PubMed Central  Google Scholar 

  4. Toppo P, Subba R, Roy K, Mukherjee S, Mathur P. Elucidating the strategies for isolation of endophytic fungi and their functional attributes for the regulation of plant growth and resilience to stress. J Plant Growth Regul. 2023;42(3):1342–63.

    Article  CAS  Google Scholar 

  5. Chouhan S, Agrawal L, Prakash A. Amelioration in traditional farming system by exploring the different plant growth-promoting attributes of endophytes for sustainable agriculture. Arch Microbiol. 2022;204(2):151.

    Article  CAS  PubMed  Google Scholar 

  6. Eid AM, Fouda A, Abdel-Rahman MA, Salem SS, Elsaied A, Oelmüller R, Hijri M, Bhowmik A, Elkelish A, Hassan SED. Harnessing bacterial endophytes for promotion of plant growth and biotechnological applications: an overview. Plants. 2021;10(5):935.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Mishra S, Sahu PK, Agarwal V, Singh N. Exploiting endophytic microbes as micro-factories for plant secondary metabolite production. Appl Microbiol Biotechnol. 2021;105(18):6579–96.

    Article  CAS  PubMed  Google Scholar 

  8. Etesami H, Maheshwari DK. Use of plant growth promoting rhizobacteria (PGPRs) with multiple plant growth promoting traits in stress agriculture: action mechanisms and future prospects. Ecotoxicol Environ Saf. 2018;156:225–46.

    Article  CAS  PubMed  Google Scholar 

  9. Tamošiūnė I, Andriūnaitė E, Vinskienė J, Stanys V, Rugienius R, Baniulis D. Enduring effect of antibiotic timentin treatment on tobacco in vitro shoot growth and microbiome diversity. Plants. 2022;11(6):832.

    Article  PubMed  PubMed Central  Google Scholar 

  10. Thomas P, Agrawal M, Bharathkumar CB. Use of Plant Preservative Mixture™ for establishing in vitro cultures from field plants: experience with papaya reveals several PPM™ tolerant endophytic bacteria. Plant Cell Rep. 2017;36:1717–30. https://doi.org/10.1007/s00299-017-2185-1.

    Article  CAS  PubMed  Google Scholar 

  11. George MW, Tripepi RR. Plant Preservative Mixture™ can affect shoot regeneration from leaf explants of chrysanthemum, European birch, and rhododendron. HortScience. 2001;36:768–9. https://doi.org/10.21273/hortsci.36.4.768.

    Article  Google Scholar 

  12. Khare CP. 2007. Indian Medicinal Plants: An Illustrated Dictionary (Google eBook) 900.

  13. Thomas A, Rajeshkumar S. Effect of Arbuscular Mycorrhizal Fungus and Plant Growth promoting Rhizomicro-Organisms on Productivity of Strobilanthes Ciliatus Nees., an endemic to western ghats. Int J Pharm Sci Invent ISSN. 2014;3:26–9.

    Google Scholar 

  14. George M, Joseph L, Sony S. A review on pharmacological and biological activities of Strobilanthes Ciliatus Nees. World J Pharm Pharm Sci. 2017;6(8):504–12. https://doi.org/10.20959/wjpps20178-9785.

    Article  CAS  Google Scholar 

  15. Venkatachalapathi S, Ravi S. Antimicrobial activity of Strobilanthes Ciliatus Nees. Indo Am J Pharm Res. 2013;3(4):3124–28.

    Google Scholar 

  16. Nair AK, Chandrasekhar MJ, Shiji Kumar PS. Phytochemcial and pharmacological aspects of strobilanthes ciliatus: a review. Int J Res Ayur Pharma. 2016;7(4):72–7. https://doi.org/10.7897/2277-4343.074137.

    Article  CAS  Google Scholar 

  17. Reneela P, Sripathi SK. Structural characterization of a new compound from Strobilanthes Ciliatus Nees. Nat Prod Indian J. 2011;7:287–90.

    CAS  Google Scholar 

  18. Venkatachalapathi S, Ravi S. Isolation and quantification of lupeol in Strobilanthes Ciliatus Nees by HPTLC method. Int J Pharm Pharm Sci. 2012;4:405–8.

    CAS  Google Scholar 

  19. Jayaraman S, Senthilkumar KL, Jagatheesh K, Arunachalam G. Evaluation of Phytochemical and Biological properties on Strobilanthes Ciliatus Nees. EMCare Cover J PHARMANEST Int J Adv Pharm Sci. 1821;5:1.

    Google Scholar 

  20. IUCN Red List of threatened species [WWW Document]. 2005. Choice Rev. Online. https://doi.org/10.5860/choice.43-2185

  21. Saha D, Ravikumar K, Ved D, Haridasan K. 2015. Nilgirianthus Ciliatus. The IUCN Red list of threatened species 2015: e.T50126629A50131400. https://doi.org/10.2305/IUCN.UK.2015-2.RLTS.T50126629A50131400.en

  22. Rameshkumar R, Largia MJV, Satish L, Shilpha J, Ramesh M. In vitro mass propagation and conservation of Nilgirianthus Ciliatus through nodal explants: a globally endangered, high trade medicinal plant of western ghats. Plant Biosyst. 2017;151:204–11. https://doi.org/10.1080/11263504.2016.1149120.

    Article  Google Scholar 

  23. Duhan P, Bansal P, Rani S. Isolation, identification and characterization of endophytic bacteria from medicinal plant Tinospora cordifolia. South Afr J Bot. 2020;134:43–9. https://doi.org/10.1016/j.sajb.2020.01.047.

    Article  CAS  Google Scholar 

  24. Alsultan W, Vadamalai G, Khairulmazmi A, Saud HM, Al-Sadi AM, Rashed O, Jaaffar AKM, Nasehi A. Isolation, identification and characterization of endophytic bacteria antagonistic to Phytophthora palmivora causing black pod of cocoa in Malaysia. Eur J Plant Pathol. 2019;155:1077–91. https://doi.org/10.1007/s10658-019-01834-8.

    Article  CAS  Google Scholar 

  25. Passari AK, Mishra VK, Gupta VK, Yadav MK, Saikia R, Singh BP. In vitro and in vivo plant growth promoting activities and DNA fingerprinting of antagonistic endophytic actinomycetes associates with medicinal plants. PLoS ONE. 2015;10:e0139468. https://doi.org/10.1371/journal.pone.0139468.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Rajeev M, Sushmitha TJ, Toleti SR, Pandian SK. Culture dependent and independent analysis and appraisal of early-stage biofilm-forming bacterial community composition in the Southern coastal seawater of India. Sci Total Environ. 2019;666:308–20. https://doi.org/10.1016/j.scitotenv.2019.02.171.

    Article  CAS  PubMed  Google Scholar 

  27. Sushmitha TJ, Rajeev M, Toleti SR, Pandian SK. Complete genome sequence of Halomonas boliviensis strain kknpp38, a chlorine-resistant bacterium isolated from the early-stage marine biofilm. Mar Genomics. 2022;62:100890. https://doi.org/10.1016/j.margen.2021.100890.

    Article  CAS  PubMed  Google Scholar 

  28. Khanam B, Chandra R. Optimization of surface sterilization process of selected dye-yielding plants for isolation of bacterial endophytes. Appl Biotechnol Sustain Dev. 2017;45–50. https://doi.org/10.1007/978-981-10-5538-6_7.

  29. Stevens LH, Tom JY, Mendes O, van der Zouwen PS, van der Wolf JM. Chemical Disinfection of Potato Cutting Machinery to avoid dissemination of Clavibacter Sepedonicus. Potato Res. 2022;65:21–9. https://doi.org/10.1007/s11540-021-09506-z.

    Article  CAS  Google Scholar 

  30. Ray SS, Ali N. Biotic contamination and Possible Ways of Sterilization: a review with reference to Bamboo Micropropagation. Brazilian Arch Biol Technol. 2017;60. https://doi.org/10.1590/1678-4324-2016160485.

  31. Kumar S, Sehrawat SK, Malik A, Yashveer S, Kumar D, Kumar P. Optimizing Surface sterilization for Micropropagation of Gerbera jamesonii: a study on Explant Survival and Contamination Control. Plant Cell Biotechnol Mol Biol. 2023;83–8. https://doi.org/10.56557/pcbmb/2023/v24i3-48345.

  32. Landa F, Chia YC, Ombokou R, Aziz ZA. Effective surface sterilization method using plant preservatives mixture and shoot multiplication of Clinacanthus nutans. Int J Curr Microbiol Appl Sci. 2020;9:3240–51. https://doi.org/10.20546/ijcmas.2020.903.371.

    Article  CAS  Google Scholar 

  33. Fang JY, Hsu YR. Molecular identification and antibiotic control of endophytic bacterial contaminants from micropropagated Aglaonema cultures. Plant Cell Tissue Organ Cult. 2012;110:53–62. https://doi.org/10.1007/s11240.

    Article  CAS  Google Scholar 

  34. Volk GM, Bonnart R, de Oliveira ACA, Henk AD. Minimizing the deleterious effects of endophytes in plant shoot tip cryopreservation. Appl Plant Sci. 2022;10:e11489. https://doi.org/10.1002/aps3.11489.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Kushnarenko S, Aralbayeva M, Rymkhanova N, Reed BM. Initiation pretreatment with Plant Preservative Mixture™ increases the percentage of aseptic walnut shoots. Vitr Cell Dev Biol - Plant. 2022;58:964–71. https://doi.org/10.1007/s11627-022-10279-4.

    Article  CAS  Google Scholar 

  36. Romadanova NV, Tolegen AB, Kushnarenko SV, Zholdybayeva EV, Bettoni J.C. Effect of Plant Preservative MixtureTM on Endophytic Bacteria Eradication from in Vitro-Grown Apple shoots. Plants. 2022;11:2624. https://doi.org/10.3390/plants11192624.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Ho WJ, Huang YK, Huang WW, Huang YC, Chung JP. Effective in vitro culture using dormant bud of nodal sections from a mature Acacia tree. Vitr Cell Dev Biol - Plant. 2022;58:437–46. https://doi.org/10.1007/s11627-021-10235-8.

    Article  CAS  Google Scholar 

  38. Moghaddam SS, Jaafar HB, Aziz MA, Ibrahim R, Rahmat AB, Philip E. Optimization of an efficient semi-solid culture protocol for sterilization and plant regeneration of centella asiatica (L.) as a medicinal herb. Molecules. 2011;16:8981–91. https://doi.org/10.3390/molecules16118981.

    Article  CAS  PubMed  Google Scholar 

  39. Joshee N, Biswas BK, Yadav AK. Somatic embryogenesis and plant development in Centella asiatica L., a highly prized medicinal plant of the tropics. HortScience. 2007;42:633–7. https://doi.org/10.21273/hortsci.42.3.633.

    Article  CAS  Google Scholar 

  40. Rihan HZ, Al-Issawi M, Al-swedi F, Fuller MP. The effect of using PPM (plant preservative mixture) on the development of cauliflower microshoots and the quality of artificial seed produced. Sci Hortic (Amsterdam). 2012;141:47–52. https://doi.org/10.1016/j.scienta.2012.03.018.

    Article  CAS  Google Scholar 

  41. Karjadi AK, Gunaeni N. 2021. Effect of added PPM on Murashige and Skoog media for shallot meristematic proliferation. In IOP Conference Series: Earth and Environmental Science (Vol. 752, No. 1, p. 012010). IOP Publishing.

  42. da Silva Ledo A, Leite do Amaral A, Jenderek M, Harrison M, Manter MK, D. Sterilization procedures and plant preservative mixture on in vitro establishment of Miscanthus sinensis Andersson. Plant Cell Cult Micropropag. 2020;15:27–32. https://doi.org/10.46526/pccm.2019.v15i2.139.

    Article  Google Scholar 

  43. Compant S, Clément C, Sessitsch A. Plant growth-promoting bacteria in the rhizo- and endosphere of plants: their role, colonization, mechanisms involved and prospects for utilization. Soil Biol Biochem. 2010. https://doi.org/10.1016/j.soilbio.2009.11.024.

    Article  Google Scholar 

  44. Uddin TM, Chakraborty AJ, Khusro A, Zidan BRM, Mitra S, Emran T, Bin, Dhama K, Ripon MKH, Gajdács M, Sahibzada MUK, Hossain MJ, Koirala N. Antibiotic resistance in microbes: history, mechanisms, therapeutic strategies and future prospects. J Infect Public Health. 2021;14:1750–66. https://doi.org/10.1016/j.jiph.2021.10.020.

    Article  PubMed  Google Scholar 

  45. Kandel SL, Joubert PM, Doty SL. Bacterial endophyte colonization and distribution within plants. Microorganisms. 2017;5:77. https://doi.org/10.3390/microorganisms5040077.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Sopalun K, Iamtham S. Isolation and screening of extracellular enzymatic activity of endophytic fungi isolated from Thai orchids. South Afr J Bot. 2020;134:273–9. https://doi.org/10.1016/j.sajb.2020.02.005.

    Article  CAS  Google Scholar 

  47. Mehmood A, Hussain A, Irshad M, Hamayun M, Iqbal A, Khan N. In vitro production of IAA by endophytic fungus aspergillus awamori and its growth promoting activities in Zea mays. Symbiosis. 2019;77:225–35. https://doi.org/10.1007/s13199-018-0583-y.

    Article  CAS  Google Scholar 

  48. Ferreira SdaC, Nakasone AK, do Nascimento SMC, de Oliveira DA, Siqueira AS, Cunha EFM, de Castro GLS, de Souza CRB. Isolation and characterization of cassava root endophytic bacteria with the ability to promote plant growth and control the in vitro and in vivo growth of Phytopythium Sp. Physiol Mol Plant Pathol. 2021;116:101709. https://doi.org/10.1016/j.pmpp.2021.101709.

    Article  CAS  Google Scholar 

  49. Mei C, Chretien RL, Amaradasa BS, He Y, Turner A, Lowman S. Characterization of phosphate solubilizing bacterial endophytes and plant growth promotion in vitro and in greenhouse. Microorganisms. 2021;9:1935. https://doi.org/10.3390/microorganisms9091935.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Faria PSA, Marques VdeO, Selari PJRG, Martins PF, Silva FG, Sales JdeF. Multifunctional potential of endophytic bacteria from Anacardium Othonianum Rizzini in promoting in vitro and ex vitro plant growth. Microbiol Res. 2021;242:126600. https://doi.org/10.1016/j.micres.2020.126600.

    Article  CAS  PubMed  Google Scholar 

  51. Umapathi M, Chandrasekhar CN, Senthil A, Kalaiselvi T, Santhi R, Ravikesavan R. Isolation, characterization and plant growth-promoting effects of sorghum [Sorghum bicolor (L.) moench] root-associated rhizobacteria and their potential role in drought mitigation. Arch Microbiol. 2022;204(6):354.

    Article  CAS  PubMed  Google Scholar 

  52. Joo GJ, Kim YM, Kim JT, Rhee IK, Kim JH, Lee IJ. Gibberellins-producing rhizobacteria increase endogenous gibberellins content and promote growth of red peppers. J Microbiol. 2005;43:510–5.

    CAS  PubMed  Google Scholar 

  53. Kang SM, Hoque MIU, Woo JI, Lee IJ. Mitigation of salinity stress on soybean seedlings using Indole Acetic Acid-Producing Acinetobacter pittii YNA40. Agric. 2023;13:1021. https://doi.org/10.3390/agriculture13051021.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The authors gratefully acknowledge the Board of Research in Nuclear Sciences (BRNS), Department of Atomic Energy (DAE), Government of India for providing financial support to carry out this work (54/14/05/2020-BRNS dated 11.09.2020). The authors also thank RUSA 2.0 [F.24-51/2014 - U, Policy (TN Multi-Gen), Dept of Edn, GOI]. The authors are grateful to Dr. T. J. Sushmitha and Prof. S. Karutha Pandian, Department of Biotechnology, Alagappa University for their help rendered for the identification and molecular characterization of PPMTM-resistant bacteria. JR extended his sincere gratitude to Prof. S. Karutha Pandian, Department of Biotechnology, Alagappa University for vetting the manuscript.

Author information

Authors and Affiliations

  1. Department of Biotechnology, Alagappa University, Science Campus, Karaikudi, Tamil Nadu, 630003, India

    Jeevan P. S. Ram, Pavan Kumar K & Ramesh Manikandan

  2. Blaustein Center for Scientific Cooperation Postdoctoral Fellow @ French Associates Institute for Agriculture and Biotechnology of Drylands, The Jacob Blaustein Institutes for Desert Research, Ben-Gurion University of the Negev, Negev, Israel

    Rameshkumar Ramakrishnan

  3. Nuclear Agriculture and Biotechnology Division, Department of Atomic Energy, Bhabha Atomic Research Centre, Anushaktinagar, Mumbai, 400094, India

    Sudhir Singh

  4. Homi Bhabha National Institute, Anushaktinagar-400094, Mumbai, India

    Sudhir Singh

  5. N. Rama Varier Ayurveda Foundation and AVN Ayurveda Formulation Private Limited, 175- A, Vilachery Main Road- 625004, Madurai, Tamilnadu, India

    Anitha Kumari R. & Suresh Govindan

  6. Department of Soil Science and Agricultural Chemistry, Anbil Dharmalingam Agricultural College and Research Institute (ADAC&RI), TNAU, Madurai, Tamil Nadu, India

    Rathika Selvaraj

Authors
  1. Jeevan P. S. Ram
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Rameshkumar Ramakrishnan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Pavan Kumar K
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Sudhir Singh
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Anitha Kumari R.
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Suresh Govindan
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Rathika Selvaraj
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Ramesh Manikandan
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

MR and JR envisioned and designed the experiments. JR performed the experiments. JR and PK analyzed the results. JR wrote the manuscript. MR, SS, RR, and RS revised the manuscript. AK and SG helped in HPTLC analysis, and the final manuscript has been reviewed and approved by all authors. MR approved and uploaded the final version of this manuscript.

Corresponding author

Correspondence to Ramesh Manikandan.

Ethics declarations

Ethics approval and consent to participate

‘Not applicable’.

Consent for publication

‘Not applicable’.

Conflict of interest

The authors declare that they have no conflicts of interest.

Data access statement

All relevant data are within the paper and its Supporting Information files.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Material 1

Supplementary Material 2

Supplementary Material 3

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ram, J.P.S., Ramakrishnan, R., K, P.K. et al. Phyto-safe in vitro regeneration and harnessing antimicrobial-resistant endophytes as bioinoculants for enhanced growth and secondary metabolites yield in Nilgirianthus ciliatus. BMC Plant Biol 24, 872 (2024). https://doi.org/10.1186/s12870-024-05582-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s12870-024-05582-8

Keywords

BMC Plant Biology

ISSN: 1471-2229

Contact us