Introduction

Moringa oleifera Lam. (Family: Moringaceae), commonly known as drumstick tree, has been widely used in food, pharmacy, and other economic utilities including bioremediation, water purification, and sustainable agricultural management (Gupta et al 2018). Drumstick tree is considered as a unique plant species that accommodates an exceptionally diverse nutrient and metabolite profile, which is rare to find in other plants (Razis et al 2014). For this reason, the plant has received unprecedented attention as the ‘miracle tree’ that is capable of providing complete nutrition as a natural source of nutraceuticals and food supplements.

Due to the multiple applications, drumstick tree has been overexploited in past decades, causing a potential threat to the long-term existence of its wild types, which requires conservation. Usually, stem cuttings are used for propagation of drumstick tree, but this method reduces the growth and yield of the plant (Islam et al 2005). The tree is also highly susceptible to insect infestations and fungal infections that lead to foliar and floral damage, which deteriorates yield and makes the plant unfit for preparation of any medicinal formulation.

Plant tissue culture is an excellent tool for both conservation and regeneration of infection-free plants. This technique serves as an efficient alternative to the traditional propagation strategies. The method also enables year-round maintenance and availability of plants and plant products to meet the industrial demands, using clonal mass multiplication of plants in a limited space and time (Jain et al 2012; Gupta et al 2018; Hu et al 2019). Micropropagation in drumstick tree has been reported by several researchers using different explants, but the shoot multiplication is limited (Förster et al 2013; Avila-Treviño et al 2017). Direct organogenesis from leaf explants has been reported by Jun-jie et al (2017). Vitrification in the regenerated shoots is a common problem in micropropagation of drumstick tree, and Hassanein et al (2018) reported the use of silver nitrate and salicylic acid in the culture medium for long-term maintenance of healthy shoots without any vitrification. Despite these results, studies to develop an efficient protocol for mass regeneration of genetically identical plants of drumstick tree are still limited. Therefore, in the present study, the effect of different cytokinins and auxins on shoot bud proliferation has been evaluated. Synergism among different combinations of plant growth regulators (PGRs), and their effect on shoot multiplication and elongation, and root induction/formation has also been evaluated. Micropropagated plantlets were subjected to acclimatization and hardening for maximum survival during field transfer. In addition, the regenerated plants were assessed for their clonality using randomly amplified polymorphic DNA (RAPD) markers to deliver maximum reproducibility.

Materials and Methods

Collection of plant material:

Drumstick and nodal segments were collected from Moringa oleifera trees growing in the natural habitat at Jaipur, Rajasthan. Seeds harvested from the pods were decoated prior to any treatment. Decoated seeds and nodes were thoroughly washed under running tap water for 10 min, followed by rinsing with 5% (v/v) Tween®-20 (liquid detergent, HiMedia, Mumbai, India), for 5 min, and washing under running tap water to ensure complete removal of detergent and other contaminants.

Optimization of sterilization method:

The plant is known to be susceptible to various infections of flies, aphids, and other insects; therefore, the explants collected from the wild were subjected to treatment with microbicidal agents prior to surface sterilization in the aseptic conditions. Surface sterilization was done with mercuric chloride (HiMedia) solution (0.1%, w/v) for 2 to 4 min. Explants treated with HgCl2 alone for 2 to 4 min were used as the control. Each treatment step followed thorough washing of the explants with sterile distilled water. Different combinations of microbicidal agents used in the sterilization step are summarized in Table 1.

Table 1. Contamination response of Moringa oleifera under different surface sterilization procedures
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Culture media and growth conditions:

All of the micropropagation experiments in the present study were completed on Murashige and Skoog (MS) culture medium (Murashige and Skoog 1962), which contained 3% (w/v) sucrose (HiMedia) and 0.8% (w/v) plant culture tested agar (HiMedia), and the pH was maintained at 5.8 ± 0.05. All the PGRs used in the study were obtained from Sigma-Aldrich®, Bangaluru, India. Explants for shoot multiplication were inoculated in Erlenmeyer flasks (100 mL, Borosil, Mumbai, India), which contained about 40 mL media, and those for rooting were transferred to boiling tubes (32 × 200 mm, 100 mL, Borosil), which contained 50 mL media in each. The culture media was sterilized by autoclaving at 15 psi, pressure and 121 °C for 20 min.

All of the cultures were maintained in growth chambers at 25 ± 1°C with 85% relative humidity, and a 16/8 h photoperiod with an illumination of 25 μmol m−2 s−1 photosynthetic photon flux density.

Establishment of primary cultures:

Decoated seeds were inoculated in 20 replicates of each on one-fourth and half-strength MS medium, both containing 3% sucrose (w/v) followed by incubation in the dark until germination. The optimum medium for in vitro seed germination was identified by recording the number of seeds germinated on both media. The germinated seeds were decapitated and transferred to MS medium supplemented with varying concentrations (0.5 to 5 mg L−1) of different PGRs and incubated in the growth chamber.

Effect of different plant growth regulators:

All the growth hormones used were procured from Duchefa Biochemie, Haarlem, Netherlands. Nodal segments derived from in vitro germinated seeds were inoculated on MS medium supplemented with 6-benzyladenine (BA), Kinetin (Kn), 6-(γ,γ-Dimethylallylamino)purine (2iP), 1-Naphthaleneacetic acid (NAA), indole-3-acetic acid (IAA), and indole-3-butyric acid (IBA) alone, with a concentration range of 0.5 to 5 mg L−1. Combinations of auxins and cytokinins (BA + Kn, BA + IAA, Kn + IAA) were also used for axillary shoot regeneration. Combinations were prepared by varying the concentration of one hormone (0.5 to 5 mg L−1), while keeping the other hormone concentrations constant.

Five flasks with four explants in each were maintained in a culture room, and the number of responding explants, number of shoots per explant, and shoot length (cm) was recorded for every hormone concentration. The observations were made after 4 wk of incubation.

Rooting of in vitro-regenerated shoots:

Healthy elongated shoots (> 2.5 cm) were transferred to half-strength MS basal medium supplemented with IBA and IAA at different concentrations (0.1 and 0.2 mg L−1), either alone or in combinations. Ten replicates for each type of media were maintained in the growth chamber with previously mentioned culture conditions. The number of rooted plants (% response) and number of roots per shoot along with the average root length, shoot length, and number of nodes induced on each rooting medium after 14 and 28 d of incubation was recorded.

Hardening and acclimatization of rooted plantlets:

Rooted plantlets were carefully removed from the culture vessels to ensure there was not any damage to the plantlets. The roots were gently washed with tap water for complete removal of medium remnants, and the plants were potted in small pots containing a mixture of garden soil-organic mixture (Tree House, Jaipur, India) (2:1, w/w). To maintain maximum humidity, the pots were covered with transparent plastic bags and kept for primary hardening in green house. The growth of the plantlets was recorded every week. After 1 mo of primary hardening, the plants were subjected to secondary hardening. Holes in the plastic bags were made at regular intervals, followed by complete removal of bags after 30 d. Plants were monitored for their growth in the secondary hardening, and the number of plants that survived both primary and secondary hardening were recorded.

Data collection and analysis:

The data was statistically analyzed using one-way analysis of variance (ANOVA) to test the significance among the observations recorded. Fisher’s least significant difference (LSD) test was used to identify significantly different means. All the tests were conducted at the 5% level of significance (P < 0.05).

Clonal fidelity analysis using RAPD markers: 

The genetic similarity among the regenerated plantlets was assessed using RAPD markers. DNA was extracted from 16 randomly selected micropropagated plantlets by the cetyl trimethylammonium bromide (CTAB) method (Doyle 1990). A total of 15 random primers belonging to T and F Operon® series (Operon Technologies, Alameda, CA), were tested to ascertain the clonal fidelity of the plantlets. The polymerase chain reaction (PCR) was carried out in a total reaction volume of 25 μL, which contained the DNA sample, 2.5 μL of 10X Taq Buffer B, 200 μM dNTPS each, 2.5 mM MgCl2, 0.75 U of Taq polymerase (Bangalore Genei, Bangaluru, India), 0.8 μM primer, and deionized water (Merck Millipore, Bangaluru, India). The PCR cycle was performed in C1000 Touch™ Thermal Cycler (Bio-Rad, Dalkeith, UK), and the amplification conditions were as follows: initial denaturation at 95°C for 5 min followed by 35 cycles of 95°C for 60 s, 37°C for 45 s, and 72°C for 45 s, with a final extension at 72 °C for 5 min. The amplicons were separated using 1.2% (w/v) agarose (Himedia), with horizontal gel electrophoresis (Bangalore Genei). The gel was stained with ethidium bromide (Himedia) and photographed using the Gel Documentation System (Gel Doc XR+ Imager, Bio-Rad).

Results and Discussions

Effect of sterilization methods:

Different treatments were used to sterilize the decoated seeds, from which T5 was found to be the most effective disinfection method compared to the other treatments (Table 1). The combination of Bavistin and Streptocycline significantly reduced the contamination from the decoated seeds. Pre-treatment with fungicide and antibiotic reduced microbial load from the explant and enhanced the efficiency of HgCl2. The seeds remain in direct contact with environment, which increases the susceptibility to microbial attack, and removal of the seed coats prior to surface sterilization significantly reduced the endophytic infection. Decoating the seeds for complete removal of contaminants was also reported in Moringa by Avila-Treviño et al (2017) and Hassanein et al (2018).

Nodal segments excised from field-grown trees were divided in two groups, (A) mature, and (B) immature nodal segments. Out of all treatment regimes, T10 reduced the contamination significantly in ‘group A,’ and T15 and T16 aided in significant reduction of contamination (< 20%) in ‘group B’ (Table 1). Although the contamination was significantly reduced in nodal segments, the prolonged treatment from disinfectants caused browning followed by death of explant tissues, which in turn resulted in reduced bud break. Förster et al (2013) also reported lower survival rates and response of nodes after surface sterilization. Therefore, due to lower survival rates (< 40%) of disinfected nodes, in vitro-germinated seedlings were used as the explant source for the present study.

Primary culture:

Primary cultures were established using in vitro germination of the decoated seeds. The decoated seeds cultured on one-fourth strength MS basal medium that contained 3% (w/v) sucrose showed the maximum germination efficiency of 89.13%. The seed germination percentage was greatly reduced (58.26%), on half-strength MS basal medium and 3% (w/v) sucrose. In previous studies, a germination frequency as high as 84% was reported on MS basal medium and filter paper beds (Stephenson and Fahey 2004; Steinitz et al 2009; Avila-Treviño et al 2017).

Germinated seedlings were inoculated on MS medium supplemented with different PGRs. Apart from cotyledonary node elongation, multiple shoot proliferation from the decapitated seeds inoculated on MS medium supplemented with different concentrations of cytokinins and auxins was also observed (Table 2, Fig. 1a, b). Shoot multiplication from decapitated seeds of Moringa was also reported previously, with a maximum regeneration of six shoots (Steinitz et al 2009), which affirmed the tendency of Moringa seeds to regenerate multiple shoot after decapitation. Shoot morphogenesis from decapitated seedlings can occur either from meristems in the axil of the cotyledonary node or through de novo apical shoot meristems. Similar conclusions were also drawn by Avila-Treviño et al (2017), after the apical shoot was removed from the seedlings of drumstick.

Table 2. Effect of different PGRs on Moringa oleifera Lam. multiple shoot morphogenesis from decapitated seeds
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Figure 1.
figure 1

Multiple shoot proliferation/regeneration from decapitated Moringa oleifera Lam. seeds on Murashige and Skoog (MS) medium supplemented with (a) 6-benzyladenine (1 mg L−1), (b) 1-Naphthaleneacetic acid (1 mg L−1), (c) Shoot bud proliferation from cultured nodal segments on MS + BA (1 mg L−1), (d) Shoot bud proliferation through callus on MS + BA (3 mg L−1), (e) Shoot elongation on MS + Kinetin (3 mg L−1), and (f) Multiple shoots from nodal segments on MS + Indole-3-acetic acid (1 mg L−1).

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Effect of cytokinins on shoot bud multiplication: 

Nodal segments derived from in vitro regenerated shoots were inoculated on MS medium fortified with various concentrations (0.5 to 5 mg L−1) of BA, Kn, and 2iP. Multiple shoot morphogenesis was observed on all the concentrations of BA and Kn except at 5 mg L−1 Kn, whereas, there was no significant shoot multiplication on any of the concentrations of 2iP (Table 3).

Table 3. Effect on axillary bud proliferation from nodal segments of Moringa oleifera Lam. cultured on Murashige and Skoog (MS) medium supplemented with different cytokinins and auxins
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Shoot multiplication from nodal segments followed swelling and callusing at the cut end at lower concentrations of BA (0.5, 1, and 2 mg L−1) (Fig. 1c), while at high BA concentrations (3 and 5 mg L−1), prominent shoot proliferation through callus was observed (Fig. 1d). The maximum response (88.33 ± 7.26%) was observed on MS + 0.5 mg L−1 BA and declined with increased concentrations of the hormone. At higher BA levels (3 mg L−1), indirect organogenesis was observed with a maximum of 17.62 ± 2.35 shoot buds per explant, but the shoot buds were not able to elongate. Regeneration of dwarf shoots (< 1 cm) from callus can be attributed to the differential signal transduction of the endogenous growth hormone(s) and due to the presence of massive intervening callus (Rasool et al 2013). Direct regeneration of multiple shoots using nodal explants in drumstick trees has been reported by several researchers (Islam et al 2005), but indirect organogenesis using BA has been addressed for the first time in the present study.

Nodal segments cultured on MS + Kn (0.5 to 5 mg L−1) showed both shoot multiplication and elongation (Fig. 1e) with a lower shoot count (Table 3). The nonsignificant regeneration pattern indicated differential signaling of BA and Kn during morphogenesis. Use of Kn alone for shoot multiplication was not preferred by earlier researchers in medicinal herbs and trees including drumstick (Takayama and Misawa 1982; Jain et al 2009; Jun-jie et al 2017) due to its lower regeneration potential. The effect of another cytokinin (2iP) was also studied on the nodal explants and 2iP did not show any significant shoot proliferation or elongation, compared to BA and Kn (Table 3).

Although a range of responses was observed with each of the cytokinins used alone, none of the hormone regimes resulted in optimum shoot growth and development, and different combinations of cytokinins (BA (0.5 to 5 mg L−1) + Kn (0.5 to 5 mg L−1)) were also tested.

Effect of auxins on shoot bud regeneration: 

Explants cultured on MS medium supplemented with IAA (0.5 to 5 mg L−1) showed two types of responses: (i) formation of new shoot buds from the node and (ii) elongation of the shoots regenerated from node (Fig. 1f). The shoots regenerated on IAA were vitrified and responded well after subculture on the same medium. The maximum number of shoots (11.5 ± 1.32) was obtained on 2 mg L−1 IAA (Table 3), with a little or reduced callus at the base. Higher IAA concentration (5 mg L−1) facilitated elongation of shoots with an average shoot length of 9.27 ± 1.32 cm and 9.67 ± 1.28 for the average number of nodes per shoot (Table 3).

The average shoot length at lower concentrations of IAA (0.5 to 2 mg L−1) was comparable with that recorded at the same concentration of Kn (Table 3), but at higher IAA concentrations (3 to 5 mg L−1), shoot length was significantly higher than the respective counterparts using MS + Kn (3 to 5 mg L−1). In contrast, the average number of nodes per explant was higher at lower concentrations of Kn (0.5 to 1 mg L−1) than IAA, which can be attributed to the cumulative effect of both endogenous and supplemented levels of auxin in the explant (Rani and Raina 2000).

Profuse callusing followed by gradual senescence of the explants cultured on MS medium supplemented with NAA (0.5 to 5 mg L−1) was observed after 2 wk of culture. MS medium containing 1 mg L−1 NAA induced tuberous roots along with callusing from the cut end of the nodal explant. These nodes did not show any bud break and did not survive for long. Formation of tuberous roots could be attributed as a metabolic consequence in presence of NAA, as no other auxin used in this study led to tuberization of roots. In vitro root tuberization has also been reported in Trichosanthes kirilowii cultured on half-strength MS medium that contained 2.7 μM NAA and 5% (w/v) sucrose (Zhao et al 2018).

Effect of different combinations of PGRs:

No PGR in MS medium alone was found to be suitable for development of healthy shoots. Therefore, combinations of cytokinin with other cytokinins and auxin were used to attain optimum development of regenerated shoots. The effect of three combinations (BA + IAA, Kn + IAA, and BA + Kn) on axillary bud proliferation, growth, and development was studied further.

A total of 45 combinations with varying concentrations of cytokinins (BA and Kn) and IAA and 10 combinations of BA and Kn were tested for axillary bud proliferation (Table 3). Callus formation at the base of the nodal segments followed by shoot proliferation was noted on all combinations of BA and IAA (Table 3). The percentage of responding explants decreased with increasing concentrations of BA and IAA. Shoot count also decreased with increased IAA concentrations, while the BA concentration was kept constant. High shoot count (10) was observed on MS medium supplemented with BA (3 mg L−1) and a lower concentration of IAA (0.5 mg L−1) (Table 3), whereas the maximum shoot length (2.4 ± 0.2 cm) was observed with 0.5 mg L−1 of BA and IAA (3 mg L−1 and 5 mg L−1) (Table 3, Fig. 2a). Callus formation also accelerated at increased concentrations of BA and IAA. As previously discussed, the maximum shoot count and shoot length was attained at higher BA and IAA concentrations, when used alone, respectively. When used in combination, both shoot proliferation and elongation was attained, which can be attributed to the synergistic effect of BA and IAA. Synergism between BA and IAA was greatly affected by the varied concentrations of BA and declined with the increasing BA concentrations (1 to 5 mg L−1), irrespective of the IAA counterpart (Fig. 2b–c), and MS + BA (0.5 mg L−1) + IAA (0.5 to 5 mg L−1) was the most effective (Table 3).

Figure 2.
figure 2

Response of Moringa oleifera Lam. nodal segments cultured on Murashige and Skoog medium supplemented with different combinations of plant growth regulators: (a) 6-benzyladenine (BA 0.5 mg L−1) + Indole-3-acetic acid (IAA 1 mg L−1); (b) BA (1 mg L−1) + IAA (0.5 mg L−1); (c) BA (3 mg L−1) + IAA (1 mg L−1); (d) BA (0.5 mg L−1) + Kinetin (Kn 5 mg L−1); (e) BA (1 mg L−1) + Kn (5 mg L−1); and (f) IAA (1 mg L−1) + Kn (1 mg L−1).

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Similarly, combinations of Kn and IAA resulted only in shoot elongation, with the maximum shoot length of 8 ± 0.6 cm on MS + Kn (0.5 mg L−1) + IAA (3 mg L−1), and the maximum average number of nodes per shoot (8.5 ± 0.3) on MS + Kn (3 mg L−1) + IAA (3 mg L−1) (Table 3). The average shoot length and nodes per shoot increased significantly with the higher concentration of IAA than that of Kn. In addition, higher concentrations of IAA (2 to 5 mg L−1) resulted in the formation of callus at the base of the nodal segments, whereas swelling with negligible callusing at the base was found in those cultured on MS that contained lower concentrations of IAA (0.5 to 1 mg L−1) and Kn (0.5 to 5 mg L−1). Explants cultured on MS + IAA (2 mg L−1) + Kn (3 mg L−1) resulted in the formation of multiple axillary buds at the nodes of the elongated shoots. It was also found that the explants showed significant shoot elongation along with little shoot bud proliferation on MS medium augmented with IAA or Kn alone, and when used in combination, their cumulative effect resulted only in shoot elongation (Fig. 2f).

In contrast to the effect of BA + IAA and BA + Kn on shoot morphogenesis from nodal segments, combinatorial effects of Kn and IAA led to single shoot elongation, and the average length of the shoots obtained was significantly higher than those found on MS + IAA or Kn, which suggested synergy between Kn and IAA at all the various combinations.

When both cytokinins, BA and Kn, were used in combination, profuse shoot multiplication along with elongation at lower concentrations of BA was found (Fig. 2d–e). The shoot count was found to increase significantly on MS medium supplemented with 1 mg L−1 BA and varying Kn concentrations (0.5 to 5 mg L−1), with a maximum shoot count of 17.36 ± 2.32 on MS + BA (1 mg L−1) + Kn (1 mg L−1). MS medium augmented with BA (0.5 mg L−1) + Kn (0.5 to 5 mg L−1) resulted in both shoot bud proliferation and elongation from the cultured nodal segments, while at higher BA concentrations, there was no significant shoot elongation (Table 3). These results suggested synergistic effects of both the hormones in combination that results in a cumulative response on the shoot multiplication and elongation, based on their individual effects when used alone.

These observations were similar to the results observed with combination of BA and IAA. In both scenarios, the concentration of BA governed the synergy in combination. It can also be deduced from the results that MS medium containing BA (0.5 mg L−1), along with Kn (0.5 to 5 mg L−1) or IAA (0.5 to 2 mg L−1), can be further optimized to obtain the maximum synergy for shoot morphogenesis from nodal segments.

The synergy between cytokinin and auxin has been widely reported for efficient shoot regeneration in various plants including Sarcostemma brevistigma (Thomas and Shankar 2009), Artemisia amygdalina (Rasool et al 2013), and Lilium (Takayama and Misawa 1982). Combinations of cytokinins and auxins for shoot proliferation and elongation from drumstick tree explants was recently reported (Shaaban and Maher 2016; Jun-jie et al 2017), but their synergistic effect has not been studied in detail. This effect can be attributed to the interaction between the hormones due to differences in their signaling pathways. Another explanation could be the ratio of endogenous and exogenous hormone levels, and the minimum threshold levels required for optimum proliferation and elongation (Takayama and Misawa 1982; Rasool et al 2013). In addition, BA has different physiological effects than Kn, due to different physiochemical properties. These differences have been identified as the potential reasons for the synergism between different hormone combinations at different concentrations (Takayama and Misawa 1982).

Effect of auxins on rooting of regenerated shoots:

Healthy and elongated shoots (> 3 cm) were transferred to half-strength MS medium fortified with or without auxins. For rooting, IAA (0.1, 0.2 mg L−1) and IBA (0.1, 0.2 mg L−1), alone or in combination, were used in MS medium. Rooting initiated within 2 wk of culture from the shoots maintained on both with and without auxins. A 100% rooting response was observed from the shoots cultured on half-strength MS + IBA or IAA (0.1 mg L−1) + IBA (0.1 mg L−1) (Table 4). The percentage of shoots developing roots reduced on IAA (0.1 mg L−1) + IBA (0.1 mg L−1) (Table 4). The maximum number of roots (14.1 ± 2.9) developed from the shoots inoculated on half-strength MS supplemented with IBA (0.2 mg L−1) and followed by IAA (0.1 mg L−1) (12.8 ± 0.7) (Table 4). In contrast, some studies reported the maximum number of roots (4) and 100% rooting efficiency on MS medium without any PGR (Islam et al 2005; Förster et al 2013). The longest roots with an average root length of 7.3 ± 0.8 cm developed on half-strength MS + IBA (0.1 mg L−1). The length of roots formed on other types of rooting media ranged from 4.4 to 6.4 cm, which indicated that there was no significant effect of the media on the length of the roots formed. Slight callus formation was observed when IAA and IBA were used alone, while direct root initiation was observed on IAA + IBA (Fig. 3a–b). The internodal length was also observed to be significantly reduced on MS + IAA (0.1 or 0.2 mg L−1) + IBA (0.1 or 0.2 mg L−1), which indicated the presence of potential synergy between IAA and IBA (Table 4). Saini et al (2012) also reported that combinations of IAA and IBA induced the longest and maximum number of roots per shoot in drumstick tree.

Table 4. Rooting response of Moringa oleifera Lam. regenerated shoots on half-strength Murashige and Skoog media supplemented with different combinations of auxins
Full size table
Figure 3.
figure 3

Rooting response of in vitro regenerated Moringa oleifera Lam. shoots on half-strength Murashige and Skoog medium supplemented with (a) Indole-3-acetic acid (IAA 0.1 mg L−1) + Indole-3-butyric acid (IBA 0.2 mg L−1), (b) IAA (0.1 mg L−1), and

(c) pot transferred plants after 1 mo.

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Response of rooted plantlets to hardening and acclimatization Transfer of rooted plants to small pots containing a mixture of garden soil-organic mixture (2:1, w/w) for primary hardening was done by carefully removing the media remnants with minimal damage to the roots. The plants were successfully established during primary hardening in the greenhouse. About 80% of the plants survived and were transferred to a natural environment for secondary hardening. About 60% of the plants were able to establish well in secondary hardening. All the established micropropagated plants were healthy and followed a uniform growth pattern (Fig. 3c).

RAPD analysis:

Out of 15 random primers (OPT 1–11 and OPF 16, 18–20) that were used, eight (OPT 1, 5, 6, 7, 11, and OPF 16, 19, 20) showed significant amplification with distinct and reproducible bands. A total of 480 amplicons of length ranging from 400 to 900 bp, with an average of 3.75 bands per primer set, was produced. Among all the primers that resulted in amplification, OPT 6 and OPF 20 showed the maximum reproducibility in the banding pattern (Fig. 4a–b). The banding pattern for a set of random primers was found to be identical, which confirmed clonality of all of the micropropagated plants. In a similar report, genetic stability of the micropropagated plants of drumstick tree was demonstrated using randomly amplified microsatellite polymorphism (RAMP) markers (Avila-Treviño et al 2017), which indicated that shoot proliferation is an efficient method for clonal propagation of M. oleifera with minimal risk of somaclonal variation. Molecular markers, RAPD, and inter-simple sequence repeats (ISSR), in particular, have been widely used to demonstrate the clonal fidelity of in vitro raised plantlets and validate their true-to-type nature (Arora et al 2004; Jain et al 2011; Behera et al 2019).

Figure 4.
figure 4

Agarose gel electrophoresis of randomly amplified polymorphic DNA (RAPD) fragments showing banding pattern amplified by (a) OPT-6 primer and (b) OPF-20 primer. M = Molecular marker and C = Control.

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Conclusions

The synergistic effect of different combinations of PGRs on axillary multiplication from nodal explants of an economically and medicinally important tree species M. oleifera has been reported in the present investigation. The study also reports an efficient and reproducible micropropagation protocol for drumstick, with a maximum number of shoots per explant (17.62 ± 2.35), using axillary bud proliferation for the first time. The clonality of the regenerated plants was also ascertained through RAPD analysis. The species is prone to the infections that could affect the yield. Therefore, this protocol can be used for ex situ conservation of wild types and scaling-up to regenerate hundreds of plantlets to meet the huge market demand. This micropropagation study can be extended to study the effect of various biotic and abiotic stresses on biosynthesis of industrially important metabolites and gum exudates. In addition, strategies will be developed for enhanced secretion of gum and biosynthesis of important metabolites in in vitro raised cultures of M. oleifera.