Introduction

Solanum khasianum Clarke (Solanaceae), commonly known as Kotahi Begena (Regional Research Laboratory, Jorhat), is a perennial shrub that grows up to a height of 2-3 m. The species has been reported to occur in Khasi, Jaintia, and Naga hills of Assam and Manipur. It also seems to grow widely from different parts of India like North-East, North-West, Southern, and Central India, and it spreads up to Burma and China (Singh and Kaushal 2007).

The members of Solanaceae have attained great significance as an alternative source of several alkaloids. The plant S. khasianum is known for its commercial and medicinal importance. It synthesizes a significant number of alkaloids like solamargine, khasianine, solanine, solasodine and solasonine from various plant parts (Srivastava et al. 2016; Kaunda and Zhang 2019). The important alkaloid solasodine, which has been identified in berries of S. khasianum, has proven to contain antiandrogenic, antibacterial and antifungal activity (Kim et al. 1996). The further pharmacological investigation of the berries has shown to possess anticancer (Rosangkima and Jagetia 2015), anthelmintic and anti-inflammatory properties (Jarald et al. 2008; Kaunda and Zhang 2019). Besides these properties, almost every part of the plant is used for treating several ailments like fever, smallpox, filaria (Ghosh et al. 1994), whooping cough, healing cuts and wounds (Chauhan 1999), trachoma, warts, back pain, male potency, snake bites, toothache, skin infections, bronchitis and rheumatism (Schmelzer et al. 2008).

Conventional plant breeding is time-consuming and very hard to achieve mass propagation. Micropropagation is the most promising method of plant tissue culture for the rapid multiplication and conservation of germplasm. Therefore, the correct choice of PGRs at varying concentrations and combinations is the prime requirement of micropropagation methods. In this regard, many plant regeneration protocols have been established in several species of Solanaceae such as Solanum nigrum (Padmapriya et al. 2011), Solanum americanum (O’Connor-Sanchez et al. 2010), Lycopersicon esculentum (Chaudhry et al. 2010), Solanum melogena (Ray et al. 2011) and Solanum villosum (Iftikhar et al. 2015).

Genetic fidelity screening is important to study the genetic variations in in vitro regenerated plants with the parent plant. The somaclonal variations may occur due to explant type, PGRs, and various subcultures during micropropagation (Lakshmanan et al. 2007). Different kinds of molecular markers such as SCoT and ISSR are available, which can screen total plant genome coding and noncoding sequences and discover the genetic variations (Collard and Mackill, 2009; Thakur et al. 2016). Moreover, genetic homogeneity studies must be analyzed in micropropagated plants, mainly where the industrial application is more. The present investigation has examined the micropropagation responses of S. khasianum in the presence of various plant growth regulators and here we are reporting first-time on rapid micropropagation and SCoT, ISSR based genetic fidelity studies in S. khasianum. Such a regeneration system would enable future studies on genetic transformation and enhancement of potential alkaloids (Solasodine) in S. khasianum.

Materials and methods

Collection of plant material and preparation of explants

The explants were collected from the 1 year old Solanum khasianum plants, maintained in the polyhouse, Biotechnology department, Kakatiya University, Warangal and cleansed thoroughly under running tap water for 10 min. The explants are now initially sterilized by treating with 1% bavistin (a fungicide) solution for 5 min, succeeded by rinsing with autoclaved distilled water for 3 times. Subsequent surface sterilization was achieved with 0.1% w/v HgCl2 for 2 min, followed by sterile distilled water wash for 3 times to eliminate traces of chemical sterilant.

Culture media and conditions

The basal Murashige and Skoog's medium (1962) containing sucrose 3% is used for all the experiments in this study. The solidification of the medium was achieved by 0.8% agar before autoclaving. The medium's pH was adjusted to 5.7 by adding 1 N HCl/1 N NaOH and autoclaved at 121 °C for 15 min. The inoculated cultures were maintained at 25 ± 2 °C with 16 h photoperiod under white fluorescent light and 8 h dark.

Preparation of coconut milk

Coconut water was extracted from fresh coconut and then filtered using a 0.2 µM syringe filter. The filtrate was boiled at 50 °C for 2–3 min and filtered again. The resultant solution was stored at 0–4 °C for future use. The concentration of the coconut milk was made to 10–20% using sterile distilled water.

Callus induction

For callus induction, MS medium amended with BAP (0.5- 2.5 mg l−1) alone or in combination with varying concentrations of IAA/IBA/2,4-D were used to determine the morphogenic response of leaf, petiole, and nodal explants. The data pertaining to the texture and proliferation of callus, mean fresh weight (mg), and dry weight of callus (mg) was recorded after 4 weeks of culture.

Multiple shoot regeneration

For multiple shoot induction, a small piece of friable callus subcultured on MS medium amended with varying concentrations of TDZ alone or in combination with IAA/IBA was used to compare the regeneration capacity of different explants. Statistics concerning the % of regeneration, number of shoots regenerated from callus, and length were recorded after 8 weeks of culture.

In vitro rooting

The individual shoots were dissociated from the cluster of multiple shoots and shifted onto MS medium augmented with varying concentrations of IAA/IBA/NAA for in vitro rooting. At the end of the 4th week, the data regarding root number and root length were collected and recorded.

Hardening and acclimatization

The complete plantlets were taken away gently from the medium and cleaned under running tap water to eliminate the adhered media components and shifted to pots filled with sterile soil and sand in 2:1 proportion. After that, the pots were covered with polythene bags to retain the relative humidity and irrigated every alternate day with half-strength MS solution for 3 weeks. Later plants were acclimatized under greenhouse conditions.

DNA Isolation and genetic fidelity analysis by SCoT, ISSR markers

For genetic fidelity analysis, whole plant genomic DNA was extracted from 500 mg leaf samples of ten randomly selected in vitro propagated acclimatized plants and mother plant of S. khasianum by using the CTAB method (Doyle and Doyle 1987). The purity of isolated plant DNA was analyzed by agarose gel electrophoresis (0.8%). For analysis of genetic homogeneity, ten each type of SCoT and ISSR markers were used (Tables 4, 5). The total volume of 50 µl PCR reaction was carried out containing plant DNA (100 ng/µl), 10 p mole primer, and 2X master mix (GCC Biotech, India). The PCR technique was performed in a thermal cycler (Eppendorf, Germany) with the conditions of 95 °C initial denaturation for 5 min and followed by 35 cycles with 95 °C denaturation for 30 s and primer annealing for 45 s at 52 °C for SCoT, 50 °C for ISSR and extension for 2 min at 72 °C with a final extension at 72 °C for 10 min and the temperature was lowered down to 4 °C. The amplified PCR samples were separated by agarose gel electrophoresis (1%) using TAE (1X) buffer with 50 V for 2 h and 30 min. The gel was imaged using a gel documentation system (Bio-Rad, USA). The sizes of the amplified PCR bands were calculated by the 1 kb DNA marker (Thermo Scientific, USA). All the amplifications were repeated thrice with molecular markers (SCoT, ISSR) to check the banding pattern's consistency.

Statistical analysis

The entire experiments were conducted thrice with 10 replicates. The collected data was analyzed statistically by ANOVA (One-way). The significant difference between the sample means was detected by DMRT (Duncan’s Multiple Range Test) at 5% level of significance (p < 0.05) using SPSS software.

Results and discussion

Callus induction and its proliferation

In the present investigation, the explants cultured on MS medium augmented with various BAP concentrations or combined with auxins like IAA/IBA/2,4-D achieved the only callus and failed to induce multiple shoots. Among all the explants, leaf explants promoted an optimal callus induction with a mean fresh and dry weight (220.1 ± 0.43 mg and 10.9 ± 0.43 mg) on MS medium amended with BAP (1.5 mg l−1) and 2,4-D (1.5 mg l−1) (Fig. 1b). Whereas the petiole explants exhibited a maximum fresh and dry weight (182.3 ± 0.47 mg and 9.2 ± 0.53 mg) on MS medium supplemented with BAP (1.0 mg l−1) and 2,4-D (1.0 mg l−1), followed by nodal explants (175.9 ± 0.37 mg and 8.9 ± 0.48 mg) on BAP (1.5 mg l−1) and 2,4-D (1.5 mg l−1) (Table 1). Similar findings were reported in Citrullus colocynthis (Savitha et al. 2010), Trachyspermum ammi (Fazeli-Nasab 2018), Solanum trilobatum (Pendli et al. 2019), and Gynura procumbens (Nurokhman et al. 2019), whereas in Momordica cymbalaria, MS medium augmented with 2,4-D alone induced maximum amount of callus (Chaitanya et al. 2020). The explants have shown various texture of callus on different PGR’s tested. All the explants cultured on the MS medium supplemented with BAP and BAP + IAA developed green compact callus, whereas white nodular callus was developed on MS medium augmented with BAP + IBA and white friable callus on BAP + 2,4-D.

Fig. 1
figure 1

Callus induction and multiple shoot regeneration with various explants and PGRs in S. khasianum. a, e, i Initiation of friable callus from leaf, petiole and nodal explants on BAP and auxin fortified medium, b, f, j induction of complete white friable callus from leaf, petiole and nodal explants, c, d initiation and regeneration of maximum frequency of multiple shoots from leaf explants after 8 weeks of culture on MS medium with TDZ (1.5 mg l−1) and IBA (1.5 mg l−1), g, h initiation and regeneration of maximum frequency of multiple shoots from petiole explants after 8 weeks of culture on TDZ (1.5 mg l−1) and IBA (1.5 mg l−1) medium, k, l initiation and regeneration of maximum frequency of multiple shoots from nodal explants after 8 weeks of culture on TDZ (1.5 mg l−1) and IBA (1.5 mg l−1) medium, m root induction on MS medium supplemented with IBA (1.5 mg l−1) and measurement of shoot and root length, n hardening of plantlets

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Table 1 Effect of BAP alone and in combination with IAA/IBA/2,4-D on callus induction from leaf, petiole, and node explants of Solanum khasianum Clarke
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The other BAP combinations and with IAA/IBA tested showed less response than BAP and 2,4-D combination. MS medium augmented with BAP (1.5 mg l−1) and IBA (1.5 mg l−1) induced callus with a maximal mean fresh and dry weight (219.4 ± 0.61 mg and 10.7 ± 0.42 mg) in petiole explants (Fig. 1f), followed by leaf explants (216.2 ± 0.80 mg and 10.6 ± 0.37 mg) and nodal explants (212.2 ± 0.41 mg and 10.5 ± 0.54 mg) (Fig. 1j; Table 1). But in contrast, BAP and IBA combination induced multiple shoots in Cicer arietinum (Sadhu et al. 2020).

On MS medium augmented with BAP (1.0 mg l−1) and IAA (1.0 mg l−1), optimal mean fresh and dry weight of callus (214.1 ± 0.43 mg and 10.5 ± 0.34 mg), (213.6 ± 0.60 mg and 10.5 ± 0.26 mg) and (192.2 ± 0.62 mg and 9.7 ± 0.36 mg) was observed in the petiole, leaf and nodal explants (Table 1). Shah et al. (2015) also reported high callus induction on BAP and IAA in S. lycopersicum. On MS medium amended with BAP (1.5 mg l−1) induced an optimum fresh and dry weight of callus (214 ± 0.36 mg and 10.7 ± 0.30 mg) and (194.8 ± 0.80 mg and 9.8 ± 0.51 mg) in leaf and nodal explants, whereas the petiole explants exhibited optimal weight of callus (209.8 ± 0.41 mg and 10.4 ± 0.47 mg) at 1.0 mg l−1 BAP (Table 1). The supplementation of coconut milk (10% or 20%) to the MS medium either alone or in combination with BAP resulted in browning of callus or ceased tissue growth. A similar result of decreased growth parameters was observed on media supplemented with coconut milk (Baque et al. 2011; Souza et al. 2013). In contrast, MS medium fortified with BAP and Coconut milk induced green compact callus and later developed shoots in S. trilobatum (Alagumanian et al. 2004). The amount of callus produced on MS medium amended with BAP was comparatively lower when compared to BAP in combination with auxins IAA/IBA/2,4-D, which indicates the synergistic effect of BAP and auxins.

Effect of PGR’s on multiple shoot induction

The friable callus obtained on MS medium fortified with BAP (0.5- 2.5 mg l−1) and auxins (0.5- 2.5 mg l−1) was excised into small pieces and subcultured onto MS medium augmented with varying concentrations of TDZ alone or in combination with IAA/IBA showed a synergistic effect and successfully induced multiple shoots within 3 weeks (Fig. 1c, 1g, and 1k). Among the explants tested, leaf explants were predominant over petiole and nodal explants for multiple shoot induction. Among the different concentrations and combinations of TDZ tested, TDZ (1.5 mg l−1) with IBA (1.5 mg l−1) was considered the most efficient for multiple shoot induction in all the explants.

The leaf explants cultured on MS medium amended with TDZ (1.5 mg l−1) and IBA (1.5 mg l−1) showed the optimal percentage of response (92.73%) with maximal mean shoot number (53.5 ± 0.47) and shoot length (11.2 ± 0.53 cm) after 8 weeks of culture (Fig. 1d, 1m; Fig. 2; Table 2). Followed by petiole and nodal explants with an optimal percentage of response (85.76% and 78.50%) with mean shoot number (20.1 ± 0.49 and 15.1 ± 0.43) and shoot length (8.6 ± 0.26 cm and 8.4 ± 0.47 cm) on the same media (Fig. 1h,1l; Table 2). By raising or lowering the optimal concentration of TDZ alone or combined with IAA/IBA, showed the reduction of multiple shoot induction in all the explants. The combination of TDZ and IBA was also found to be effective in different species like Ficus carica (Kim et al. 2007), Lythrum salicaria (Turker et al. 2009), Alocasia indica (Rong et al. 2011), Lycopersicon esculentum (Vinoth et al. 2012), and Malus niedzwetzkyana (Jin et al. 2014; Kazemi et al. 2019).

Fig. 2
figure 2

Effect of TDZ alone and with IAA and IBA on shoot length of multiple shoots in leaf, petiole and nodal explants

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Table 2 Effect of TDZ alone and in combination with IAA/IBA on multiple shoot induction from callus derived from leaf, petiole, and nodal explants of Solanum khasianum Clarke
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The explants cultured on MS medium amended with different concentrations of TDZ and IAA achieved lower response than explants cultured on TDZ and IBA. Among the various concentrations of TDZ and IAA tested, TDZ (1.0 mg l−1) and IAA (1.0 mg l−1) were considered to be effective in leaf explants for induction of multiple shoots, whereas, in petiole and nodal explants, TDZ (1.5 mg l−1) and IAA (1.5 mg l−1) showed to the best combination. Therefore the optimal percentage of response (87.43 ± 0.14, 78.50 ± 0.12, and 74.96 ± 0.14) and mean shoot number (33.8 ± 0.56, 15.2 ± 0.56 and 13.1 ± 0.57) was obtained in leaf, petiole, and nodal explants on TDZ and IAA (Table 2). The similar result were reported in Capsicum annuum (Kasula et al. 2008), Woodfordia fruticosa (Bulle et al. 2012), and Citrullus colocynthis (Dasari et al. 2015), where TDZ + IAA showed maximum regeneration of multiple shoots.

The MS medium augmented with TDZ alone failed to regenerate multiple shoots in petiole and node explants, whereas only leaf explants developed multiple shoots. The highest percentage of response (73.33 ± 0.05) and number of multiple shoots (23.2 ± 0.61) was achieved on TDZ (1.5 mg l−1) (Table 2). TDZ alone was also found to be effective in Solanum aethiopicum and Solanum macrocarpon (Gisbert et al. 2006), Solanum melongena (Mallaya et al. 2013), Vigna subterranea (Silue et al. 2016), Bienertia sinuspersici (Northmore et al. 2016), Solanum nigrum (Afrasiab et al. 2017) and Solanum erianthum (Sarkar and Banerjee 2020).

Thidiazuron, substituted phenyl urea, is a potent cytokinin for the regeneration of multiple shoots in several plants (Sajid and Aftab 2009; Gharari et al. 2019; Matand et al. 2020; Savitikadi et al. 2020). TDZ is also showed to have better efficiency than other cytokinins like BA or Kinetin, even at low concentrations (Bhattacharyya et al. 2016).

Rooting and acclimatization

The in vitro developed shoots were rooted efficiently on the MS medium amended with auxins like IAA, IBA, and NAA (0.5 mg l−1 to 2.5 mg l−1). The rooting efficiency varied significantly among the three auxins tested. The cultured shoots induced root formation after 2 weeks of culture. Among all the auxins tested, IBA was superior to IAA and NAA. The optimum number of roots (12.8 ± 0.36) per shoot was observed on MS medium amended with IBA (1.5 mg l−1), subsequently followed by NAA (1.0 mg l−1) with 7.9 ± 0.52 mean number of roots, whereas negligible rooting response (1.9 ± 0.32) was achieved on MS medium fortified with IAA (1.0 mg l−1) (Table 3). Kannan et al. (2006) also reported similar results in Solanum nigrum with 0.5 mg/L IBA. IBA also induced in vitro rooting in different plants (Hussain et al. 2018; Vemula et al. 2019; Jogam et al. 2020). The high frequency of in vitro rooting with IBA is due to its fine structure, stability, and easy translocation to tissues (Hussain et al. 2018). The well-rooted complete plantlets were acclimatized with a high survival rate of 80% (Fig. 1n).

Table 3 Effect of various auxins on the induction of rooting in S. khasianum Clarke
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SCoT and ISSR molecular markers based on genetic fidelity studies

Genetic variations are common in in vitro raised plantlets. So, molecular marker-based PCR studies are preferable to study the variations among micropropagated plants with the mother plant. In the present study, SCoT and ISSR molecular markers are used to check the somaclonal variations. The ten SCoT primers amplified 50 scorable amplicons with the approximate range from 400 to 3000 bp size (Table 4; Fig. 3a). The average of 5 PCR bands per one SCoT primer was amplified. The ten ISSR primers amplified 40 scorable amplicons with an approximate range of 300 to 3100 bp size (Table 5; Fig. 3b). The average of 4 PCR bands per one ISSR primer was amplified. All the molecular markers amplified PCR products were found to be monomorphic.

Table 4 List of SCoT primers used and their response on the assessment of genetic fidelity studies in S. khasianum Clarke
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Fig. 3
figure 3

a SCoT markers based genetic fidelity analysis of S. khasianum amplified with S4 primer. Lane M: 1 kb marker, Lane MP: PCR amplification of mother plant DNA, Lane 1–10: PCR amplification of in vitro regenerated plants DNA. b ISSR markers based genetic fidelity analysis of S. khasianum amplified with I8 primers. Lane M: 1 kb marker, Lane MP: PCR amplification of mother plant DNA, Lane 1–10: PCR amplification of in vitro regenerated plants DNA

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Table 5 List of ISSR molecular markers used and their response on the assessment of genetic fidelity studies in S. khasianum Clarke
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SCoT and ISSR are most advanced and have high nucleotide length compared with RAPD markers (Goyal et al. 2015). These markers have a high annealing temperature to result in better reproducibility without nonspecific amplification (Bornet and Branchard 2011). Screening of genetic homogeneity among micropropagated plants by two molecular markers is always preferable to reconfirm the genetic variations (Rohela et al. 2019). ISSR and SCoT markers are constant, low cost, reproducible, examining plants' total genome randomly (Rohela et al. 2020). Similar Genetic fidelity studies of in vitro regenerants using SCoT molecular marker was reported in Pisum sativum (Ajithan et al. 2019), Helicteres isora (Muthukumar et al. 2020), and Cicer arietinum (Sadhu et al. 2020) and ISSR molecular marker-based genetic fidelity among micropropagated plants were reported in Eleusine coracana (Babu et al. 2018), Morus spp (Rohela et al. 2018), and Flemingia macrophylla (Sirikonda et al. 2020).

Conclusion

The micropropagation technique is the best-employed method for the regeneration of S. khasianum. Among all the three explants (leaf, petiole, and node) used, leaf explants showed maximum multiple shoot induction (53.5 ± 0.47) on TDZ (1.5 mg l−1) with IBA (1.5 mg l−1) from friable callus. The variations in the regeneration frequencies are due to the type of explants and their physiological conditions. The regenerated plantlets showed no somaclonal variations when assessed by ISSR and SCoT markers. In the present investigation, we have developed an efficient micropropagation method for mass propagation in S. khasianum plants without any genetic variability with biotechnological tools.