Introduction

The genus Mahonia (family: Berberidaceae) widely distributed in East and Southeast Asia, Western North America, Central America, and Western South America. In context to the Himalayan region the genus is reported from Nepal, India, Bhutan, China, and Vietnam at an altitude between 1,000–2,400 m. s. l. [1]. Mahonia has about 70 species worldwide [2], out of which 13 have been reported from Indian Himalaya [3] and four species (viz. M. acantfolia, M. borealis, M. nepalensis, M. jaunsarensis) from Uttarakhand [4], one of these M. jaunsarensis is endemic to Uttarakhand [4, 5]. Among the berberidaceae plants, Mahonia jaunsarensis Ahrendt is the most valuable woody perennial shrub. The species is endemic, found particularly in the Chakrata region of Dehradun (West Himalayan) and is called “khoru” locally. Only a very small number of populations of this plant grow naturally in oak banj forest at an altitude of 2,300 to 4,300 m. s. l. Local people use this plant for treating fevers, cough, asthma by using its edible fruit, root and stem bark. The species reported to possess antioxidant activity [6] and have diverse medicinal uses, thus requires adequate populations in nature. The natural habitat of M. jaunsarensis is diminishing due to increasing anthropogenic activities, long period of dormancy, fungal and insect attack, indiscriminate collection, deteriorating natural ecosystem, etc. Although, Bisht et al. [7] standardized a protocol for vegetative propagation of the species using stem cuttings but unable to produce enough number of plants and required longer response time. Over the years, plant tissue culture has emerged as one of the most effective alternate method for large-scale production, maintaining genetic uniformity and secondary metabolite production [8, 9]. As a result of this, many rare and endangered species have been propagated in the last few decades [10]. Successful in vitro studies have been performed on other species of genus Mahonia, namely M. leschenaultia [11] and M. soft carees [12], however, in vitro propagation protocol for M. jaunsarensis along with alkaloid studies of in vitro raised plants have not been conducted yet. Therefore, the present study aimed to (i) develop an effective in vitro regeneration protocol, (ii) analyse the secondary metabolites in in vitro raised and wild plant leaves, and in vitro developed callus, and (iii) evaluate the antioxidant and antimutagenic activity of both wild-grown and in-vitro regenerated plants.

Materials and Methods

Sample Collection

Ripened fruits of M. jaunsarensis were collected from the natural habitat of Chakrata, Dehradun, Uttarakhand (N 30°45′5.76″; E77°52′10.56″; altitude: 2200 m. s. l.) and the voucher specimens were deposited at the Botanical Survey of India, Dehradun, Uttarakhand (Acc. No.56). All the experiment conducted at G. B Pant National Institute of Himalayan Environment (NIHE), Kosi-Katarmal, Almora, Uttarakhand (India).

Explant Preparation

Briefly, the seeds were rinsed with running tap water (2–3 times) then washed by distilled water (2-3times). After that, 3–5 drops of surfactant (Tween 20; Hi-Media, Mumbai, India) were added and washed thorughly. Treatment with fungicide (Bavistin) solution at (0.1% 0.5%, 0.01%) was applied for 10, 20, 30 min, followed by shaking in a magnetic stirrer. Thereafter, seeds were treated with different concentrations (0.1%, 0.5%, 0.01%) of mercuric chloride (HgCl2) for 5 min, 10 min, and 15 min under laminar airflow. Finally, seeds were washed with sterile double distilled water for 4–6 times.

Effect of media

To determine the best culture medium, seeds were inoculated (four seeds per flask) on Murashige and Skoog media (MS) (Murashige and Skoog, 1962) as well as on Woody Plant media (WP) (Lloyd & McCown, 1981). The pH of the medium was adjusted to 5.8 using 1 N NaOH and HCl and the medium was supplemented with 3% (w/v) sucrose and solidified with agar (0.8% w/v) (Hi-media, Mumbai, India). The prepared media was dispensed into the culture tubes and autoclaved at 15 psi for 20 min at 121 °C. Inoculated culture tubes were kept in culture room conditions (25 ± 20 °C) for the 16-h photoperiod under cool-white fluorescent light.

Effect of PGRs

The best growth hormone was determined by inoculating seeds in different medium supplemented with gibberellic acid (GA3; 0.5–10 µM) and N6-benzylaminopurine (BA; 0.5–10 µM) (Table 1). After 30 days, leaves of newly germinated seedlings were used as explant for callus induction.

Table 1 Effect of different concentrations of BA and GA3 on in vitro seed germination of M. jaunsarensis
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Callus induction

Callus initiation was achieved by removing young leaves from in vitro produced seedlings and cut into pieces (1 × 1 cm2) before being cultured in MS basal medium supplemented with 3% (w/v) sucrose and 0.8% (w/v) agar containing N6-benzylaminopurine (BA; 0.5 µM), α-naphthalene acetic acid (NAA; 0.1–4 µM) with various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D; 0.1—4 µM) either alone or in combination of BA (0.5 µM). After 30 days of culture, data on callus induction was recorded.

Shoot regeneration and proliferation

For the shoot regeneration callus were transferred on MS medium supplemented with different concentrations and concentrations of plant growth regulators including thidiazuron (TDZ; 0.25–1 µM), N6-benzylaminopurine (BA; 0, 2.0, 4.0, 6.0 and 8.0 µM), α-naphthalene acetic acid (NAA; 0.5 µM) and indole-3-acetic acid (IAA; 0.5 µM). Data on the percentage of responsive calli, shoot number and shoot length was recorded after eight weeks of culture.

Root induction

Well-developed microshoots (> 5 cm long) were transferred to half MS media supplemented with different concentrations of indole-3-butyric acid (IBA; 0.01–0.1), indole-3-acetic acid (IAA; 0.01–0.1), and α-naphthalene acetic acid (NAA; 0.01–0.1 µM). The plantlets were removed from the culture flask after six weeks of culture for data recording.

Acclimatization

Acclimatization of the well-rooted healthy plantlets was performed after 14 weeks. Roots were removed carefully from culture bottles and washed thoroughly with water to remove the traces of gelling agent (agar). Then, they were transferred into plastic pots (15 cm height and 12 cm diameter). Different substrate types viz. vermiculite, perlite, sand, farmyard manure and garden soil and their combinations were used to identify the optimal conditions for acclimatization of in-vitro raised plantlets. The five substrate combinations namely sand + farmyard manure (1:1), garden soil + farmyard manure (1:1), vermiculite + garden soil + farmyard manure (1:1:1), vermiculite + sand + farmyard manure (1:1:1) were used. All these substrate combinations were sterilized before transplanting the plants, which were then covered with perforated plastic bags (Fig. 3 (i, j, k) and Fig. 3 (i and j) to maintain humidity. Initially, these plantlets were acclimatized in culture room conditions (25 ± 5 °C, 16/8 light/dark photoperiod) and after four weeks perforated polybags were removed and plants were placed inside glass houses for further growth and development.

Phytochemical and antioxidant activity

The accumulation of phytochemicals and antioxidant activities in different in vitro raised and wild plant leaves, and in vitro raised callus, were determined following standard methodologies [13].

Alkaloid quantification

The amount of berberine and palmatine were measured with High Performance Liquid Chromatography (Allaince Waters e2695, Waters, Milford, USA) equipped with photo diode array (PDA 2998) detector as described by Belwal et et al. [14]. In the separation, mobile phase consisted of acetonitrile (A) and 0.13% potassium dihydrogen phosphate with a pH of 2.5 (B) in a 50:50 ratio at 1.0 ml/min for a total run time of 20 min on a SPHERISORB C18 reverse phase column. In both cases, the detection wavelength was set to 345 nm for both alkaloids. Using berberine and palmatine concentrations ranging from 10 to 50 mg/l, standard curves were created for developing linear regression equations, and their authenticity was assessed using regression coefficients (R2). Chemicals were identified by their retention time (Rt) and quantified by their area under the curve (AUC). The results were expressed in milligram per gram of dry weight (mg/g DW).

Anti-mutagenic activity

Plasmid (pBR322) DNA was tested for DNA damage activity based on the method of Singh et al. [15]. The plasmid DNA (2 µL, 180 ng) was damaged under UV radiation for 15 min in the presence of H2O2 (3.5%). Indicators like conversion of the plasmid supercoiled (S) DNA to the open-circular (OC) or linear (L) is marked as a sign of DNA damage. The extent of DNA damage and preventive effect of the test samples were analysed on agarose gel (1.0%) in 1X TAE buffer run over 2 h at 50 Volt at room temperature. The band density was calculated under the gel documentation system (UVI pro Platinum ver. 12.9, UK). The following equation was used to calculate the supercoiled DNA intensity:

$$(S\;(\%)\;=\;\lbrack S\;band\;density)\;\backslash \;(density\;of\;(S\;+\;L\;+\;OC)\rbrack)\;\times\;100.$$

The recovery of supercoiled DNA was also calculated with reference to positive control as relative supercoiled DNA (%) = s (%) of test sample/s (%) of positive control.

Data Analysis

During seed germination, seedlings were monitored regularly, and if the radicle was visually identified, seeds were considered as germinated. The following formulas were used to calculate percentage of germination (GP) and mean germination time (MGT) [16]

$$\mathrm G\mathrm e\mathrm r\mathrm m\mathrm i\mathrm n\mathrm a\mathrm t\mathrm i\mathrm o\mathrm n\;\mathrm p\mathrm e\mathrm r\mathrm c\mathrm e\mathrm n\mathrm t\mathrm a\mathrm g\mathrm e\;\left(\mathrm{GP}\right)=\mathrm{GN}/\mathrm{SN}\times100$$
$$\mathrm M\mathrm e\mathrm a\mathrm n\;\mathrm g\mathrm e\mathrm r\mathrm m\mathrm i\mathrm n\mathrm a\mathrm t\mathrm i\mathrm o\mathrm n\;\mathrm t\mathrm i\mathrm m\mathrm e\;\left(\mathrm{MGT}\right)=\left({\mathrm N}_1{\mathrm T}_1+{\mathrm N}_2{\mathrm T}_2\dots\dots..\mathrm{NnTn}\right)\mathrm{GN}$$

where, GN – total number of seeds germinated in 40 days, SN – total number of seeds tested, Sn – number of seed germinated each day of observation, N- days for germination, N1T1- number of seed germinated at time T1, SL – mean seedling length (mm). The observed data on various parameters was analysed using IBM SPSS Statistics V21.0 software.

Results and Discussion

Sterilization and Explant Establishment

Disinfected seeds were treated with (0.1%, 0.5%, 0.01% of HgCl2 and Bavistin) for different time durations (5 min, 10 min, and 15 min for HgCl2) and (10 min, 20 min, and 30 min) for Bavistin. Among the various pre-treatments used, 0.1% HgCl2 for 5 min produced the highest (70%) aseptic cultures (Fig. 1). In addition, 0.1% Bavistin on applying for 30 min resulted maximum aseptic cultures (70.00%). Similar findings were obtained in Berberis asiatica, where 0.1% HgCl2 for 10 min and Bavistin for 30 min gave best response [17]. Likewise, Brijwal et al. [18] reported application of 0.7% Bavistin and 0.1% HgCl2 for 20 min as best treatment for seed sterilization in Berberis aristata, Pandey et al. [19] reported 0.5% Bavistin for 30 min and 0.1% HgCl2 for 12 min as best seed sterilization treatment for Berberis chitria, and Dhar et al. [20] sterilized seeds of Berberis lycium with 0.5% Bavistin for 10 min and 0.1% HgCl2 for 2 min. Apart from seeds the nodes of Mahonia leschenaultia were surface sterilized with 0.1% HgCl2 for 5–10 min [11]. Besides, other woody plant species, such as Cnidoscolus aconitifolius [21] and Atriplex taltalensis [22] were also reported best sterilized with 0.1% HgCl2 for 5 min and 10 min respectively.

Fig. 1
figure 1

Effect of HgCl2 and Bavistin concentrations and treatment duration on explant (seed) establishment of M. jaunsarensis

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Effect of Media on In Vitro Seed Germination in M. Jaunsarensis

Germination was observed in all tested media, but their germination rates were different (Fig. 2). The present study found that MS medium yielded the best seed germination response among the used media. In MS medium, with a minimum average time (24.31 ± 0.09 ds) the maximum percentage of germination (80.00 ± 5.77 %) and average plant height (2.55 ± 0.08 cm). While the seedling height in WP medium was comparatively lower than MS medium, the seedling germination percent was (73.33 ± 3.33 %) with an average germination time of (25.37 ± 0.27 ds) and an average seedling height of (1.99 ± 0.15 cm). Similary, Pandey et al. [19] reported maximum (78.89%) seed germination in WP as compared to MS (50%) in Berberis chitria. However, Sharma et al. [23] observed best seed germination (65%) in MS medium in B. chitria. The maximum shoot multiplication was also observed in WP medium for Berberis aristata [18]. Moreover, the best in vitro propagation results for B. asiatica were obtained in MS medium by Bisht et al. [17] and Dobhal et al. [24]. MS medium was also found to be the most effective in germination of seeds in Berberis lycium [20]. According to the studies, MS is the preferred medium for number of species, including woody ones. However, tissue culture of such woody species as M. leschenaultia, and Mahonia ‘Soft Caress’ has also been accomplished with other nutrient formulations. In M. leschenaultia, Radha et al. [11] used MS, WP and Schenk and Hildebrandt (SH) medium, and found the best shoot multiplication in SH medium. Similarly for Mahonia ‘Soft Caress’ Rounsaville et al. [12] tested Gamborg B5 (B5), Quoirin and Lepoivre (QL), and MS medium and reported best shoot multiplication in B5 medium. Other woody plant species such as Shorea robusta [25], Quercus serrata [26], Zanthoxylum armatum [27] and Salix acmophylla [28] also propagated in vitro using WP medium.

Fig. 2
figure 2

Effect of media on in vitro seed germination in M. jaunsarensis

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Effect of Plant Growth Regulators on Seed Germination

In vitro seed germination response of M. jaunsarensis under different treatment was studied. The radical emergence was observed in 12–15 days of inoculation and complete growth of cotyledons was seen between 30 to 35 days. After ten weeks of inoculation seeds of M. jaunsarensis showed significantly (p < 0.05) higher germination percentage (83.33 ± 3.33 %) in MS medium supplemented with BA (1 µM) with the least germination time (23.45 ± 0.18 ds) (Table 1). However, in control condition, the lowest germination percentage (46.66 ± 3.33 %) with an average time of (28.44 ± 0.27 ds) was observed. Similarly, Sharma et al. [23] reported a maximum (65.46%) seed germination in B. chitria using BA (6 mg/l), Bisht et al. [17] also observed higher (50%) germination in B. asiatica when treated with BA (1 μM). Similarly, Belwal et al. [16] studied seed germination response of B. aristata and B. jaeshkeana under six treatments (viz. GA3, salicylic acid, thiourea, vermiwash, and cow urine) and found that vermiwash and GA3 produced the best seed germination responses. Gibberellic acid treatment promotes the synthesis and production of hydrolases, especially amylase, which breaks down starch into maltose (a chain of two glucose molecules), thereafter, maltose breaks down into glucose, which is used for the growth of plumule and radicle hence, result in the germination of seeds [28, 29]. In addition, it has been well documented that BA independently serves as a better cytokinin for shoot induction [30,31,32].

Callus Induction from Leaf

In order to induce callus from leaf explants, different concentrations of cytokinin and auxin were used individually or in combination (Table 2). Explants were unable to form callus when cultured on MS medium without auxin or cytokinin (control). After seven-week incubation period the formation of compact callus was observed in plant growth regulator containing media. It was observed that the highest percentage of compact green callus formation (76.66 ± 3.33%) occurred in MS medium supplemented with 2,4-D + BA (1.0 + 0.5 µM), followed by (70.00 ± 0.00%) in MS medium supplemented with 2, 4-D (0.1 µM) (Fig. 3, b, c, d). Callusing percentage was observed low (20.00 ± 0.00 %) for the medium containing NAA + BA (3.0 + 0.5 µM) and NAA + BA (4.0 + 0.5 µM) and produced black compact textured callus (Fig. 3, f). In all treatments, a decline in callus induction was observed with an increase in concentration of PGRs (viz. 2,4-D, BA and NAA) from 2 µM to 4 µM. The combination of 2,4-D and cytokinins known to improve callus regeneration in both monocots and dicots Mehta et al. [33] Bisht et al. [17]. The results of our study are in line with Bisht et al. [17], Rawat et al. [34] and Santos et al. [35] who observed that cytokinin and auxin synergistically induce better callus growth in Berberis asiatica, Aconitum violaceum, and Piper permucronatum.

Table 2 Effect of different PGRs concentrations on callus induction from in vitro germinated M. jaunsarensis seedling leaves
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Fig. 3
figure 3

In vitro propagation of Mahonia jaunsarensis. (a,b,c) in vitro seed germination in 25 days; (d) callus induction after seven weeks; (e, f) shoot induction after 5 weeks; (g) root induction after 6 weeks; (h) well rooted plantlet ready for hardening; (i) in vitro raised plant after transfer to cups (kept inside culture room covered with transparent polybags to maintain humidity); (j and k) plants transferred to potting mixture; (l) well-developed in vitro grown plant in glasshouse condition after 120 days of transfer to nursery bags

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Plant Regeneration Through Callus

The healthy callus taken after 6 weeks of culture and transferred to the shoot regeneration medium. Different PGRs exhibited different responses and data related to leaf number, shoot length, and shoot number were observed for shoot regeneration (Table 3). Among the various treatment, MS medium containing TDZ (0.75 µM) resulted significantly (p < 0.05) higher shoot number (3.06 ± 0.03), however, best average shoot length (3.37 ± 0.13 cm) and average leaf number (2.87 ± 0.23) were recorded in combination of BA + NAA (6 + 0.5 µM) (Fig. 3, d) Whereas, lowest average number of shoots (0.70 ± 0.00), average shoot length (0.11 ± 0.01 cm) and average leaf number (0.22 ± 0.06) were observed in BA + IAA (2.0 + 0.5 µM). The species grown in vitro responded well to TDZ (0.75 µM) and was considered the most effective medium for promoting shoot initiation. Application of TDZ has been reported effective in growing endangered dicotyledonous plants from the Himalayas, including recalcitrant and woody plant species [36,37,38]. However, for shoot length and shoot multiplication, TDZ was not found to be the best cytokinin since it inhibited growth. Similarly, in Mahonia 'Soft Caress', the shoot length significantly decreased after applying TDZ. Many medicinal plants have demonstrated a synergistic effect of hormones on shoot length and shoot multiplication in Dendrocalamus latiflorus [36], Zanthoxylum armatum [37], Bambusa tulda Roxb and Dendrocalamus stocksii Munro [39].

Table 3 Effect of different concentrations and combination of PGRs on indirect organogenesis in M. jaunsarensis
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In Vitro Rooting

The rooting response of micro shoots under different auxin (IAA, IBA and NAA) treatments responded differently. The significantly (p < 0.05) higher rooting percentage (56.66 ± 3.33%), average root number (2.53 ± 0.13), average root length (3.33 ± 0.11 cm) were recorded in IBA (0.01 µM) treatments, while, minimum rooting (23.33 ± 3.33%), average root number (0.29 ± 0.10), average root length (0.13 ± 0.02 cm) were observed in NAA (0.01 µM) treatments (Table 4). The in vitro rooting of M. jaunsarensis shoots are shown in Fig. 3, g &h. In Mahonia ‘Soft Caress’ microcuttings treated with IBA (8 µM) and kept in dark showed the highest rooting percentage (37%) [12]. In Mahonia leschenaultia Radha et al. [11] also found the best rooting (78%) responce in IBA (1 mg/l). Similar results were observed for Berberis chitriya with (100%) rooting in IBA (100 µM) [19] and (71%) rooting for B. aristata in IBA (50 µM) [18]. However, Bisht et al. [17] reported higher rooting percentage (70%) in IAA (0.05 µM) for B. asiatica. Likewise, Sharma et al. [23] found that the combination of NAA (1.0 mg/l), IAA (0.5 mg/l), and IBA (0.5 mg/l) produced the maximum rooting (76%) in B. chitriya. Several studies elsewhere also reported IBA as the most suitable auxin for rooting in a wide variety of plant species [40, 41].

Table 4 Effect of different auxin concentrations on M. jaunsarensis root induction of in vitro raised shoots
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Acclimatization

Well-developed in vitro rooted plants of M. jaunsarensis were transferred to different potting mixtures for further growth and development (Fig. 3, i, j& k). The highest survival percentage (55%) was observed in potting mixture containing vermiculite, garden soil and farmyard (1:1:1), whereas minimum (30%) survival percentage was observed in potting mixture containing sand and farmyard manure (1:1) (Table 5).

Table 5 Effect of different fertilizers on the survival of M. jaunsarensis
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In Vitro Phytochemical production

In vitro raised leaves of M. jaunsaresis (15-week-old) had significantly higher total phenol (36.65 ± 0.45 mg GAE/g FW), tannin (10.35 ± 0.15 mg TAE/g FW) content, and ABTS (19.45 ± 0.75 mM AAE/100 g FW), FRAP activity (3.63 ± 0.11 mM AAE/100 g FW) activities. However, DPPH (8.90 ± 0.20 mM AAE/100 g FW) activity was significantly lower in in vitro raised plant leaves as compared to wild plant leaves (Table 6). Although, flavonoid content was observed similar in both in vitro (2.22 ± 0.05 mg QE/g FW) and wild (2.01 ± 0.05 mg QE/g FW) leaf samples. The HPLC results indicate that the in vitro raised plant leaves have more alkaloid ontent [viz. berberine (3.54 ± 0.00 mg/g FW) and palmatine (0.58 ± 0.00 mg/g FW)] then wild plant leaves. In the present study, in vitro-regenerated plant samples showed higher phytochemical, phenolic, tannin, and flavonoid content, which can be corelated to the exogenous supply of different PGRs in in vitro condition. The PGRs are probable precursors for the conversion of phytochemicals into active forms during stress or adverse environmental conditions, where they enhance phytochemicals [42]. Besides, the phytochemical contents may vary depending on factors such as the climate, soil texture, habitat, aspect, and slope, etc. [17]. A similar finding were observed in Habenaria edgeworthii [43], Justicia gendarussa [44], Atropa acuminata [45], Berberis asiatica [17], where in vitro raised plants had higher phenolic content than wild plants.

Table 6 Phytochemical, antioxidant activity of in vitro leaves and wild leaves extracts of M. jaunsarensis
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Anti-Mutagenic Activity

In vitro raised plant leaf extract of Mahonia jaunsarensis showed considerable anti-mutagenic activity. The damaged pBR322 plasmid DNA when treated with extracts of in vitro raised plant leaves at concentration of 10 µg/ μL, showed 39.66% recovery of damaged plasmid DNA (Fig. 4). Further, the efficaceae of the plant extract was compared with the standard marker antioxidant compounds (i.e., gallic acid) at a concentration of 4 µg/μL and mother plant extract at a concentration of 10 µg/ μL, which showed recovery percent of 64.84% and 61.80% respectively. Overall, the standard marker compounds, gallic acid showed maximum anti-mutagenic activity followed by mother plant leaf extract (Fig. 4), while tissue culture gown plant leaf extract showed comparatively low activity. Several Himalayan medicinal plants have also been studied for DNA damage protection activity, including Ashtvarga species (Habenariya edgeworthi, H.intermedia, Malaxcis acuminata, M. mucifera, Polyganatum cirrifollium, P. verticillatum, Rosceoa procera and Lillum polyphyllum [46], Quercus serrata [26], Origanum vulgare [13] and B. asiatica [17] The antimutagenic properties of the species can be correlated to it’s polyphenolic content and antioxidant properties.

Fig. 4
figure 4

Anti-mutagenic activity of different extracts of M. jaunsarensis. (+C) Positive control; (-C) Negative control; (A) Gallic acid (4μg/ μl; 64.84% Recovery percent), (B) Mother plant leaf extract (10μg/ μl; 61.80% Recovery percent), (C) In vitro growing plant leaf extract (10μg/ μl; 39.66% Recovery percent)

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Conclusion

This study is the first report on in vitro propagation of M. jaunsarensis which can be used to conserve this endemic species by facilitating its mass propagation and transplantation into the natural environment, also replicated in other Himalayan species of the genus Mahonia and Berberis. Further, study revealed that, M. jaunsarensis possess a wide array of alkaloids and have shown good antioxidant and antimutagenic activities which highlights it's potential as a vital source of antioxidant polyphenolics.