Introduction

There are currently 90 Tylophora species with accepted names, and a multitude of synonyms and species whose nomenclature is being revised (The Plant List 2016). Tylophora indica (Burm. f.) Merr. (syn Tylophora indica var. glabra (Decne.) H. Huber) (The Plant List 2016), of the Asclepiadaceae family and commonly known as Indian ipecacuanha in English, or as Antamul in India (see list of vernacular names in Table 1), is a climbing perennial vine. Kirtikar and Basu (1991) and Schmelzer and Gurib-Fakim (2013) offer a botanical description of this medicinal plant, and indicate that T. indica is a perennial climber that can reach up to 1.5–3 m in length, forms short stocky rhizomes (3–4 mm thick) and fibrous roots. The simple opposite leaves, which can be 2–10 cm long, have margins that can be entire, ovate or orbicular. Many green-yellow flowers (outside) with a purple inside form on an axillary umbel-like cyme, which is the inflorescence. The fruit is 5–10 cm long with many 2–2.5 cm long seeds. The plant is usually propagated by seed collected from plants in the wild and vegetative propagation is poorly explored (Dhandapani and Balu 2002). However, more recently, Mehandru et al. (2014) used stem cuttings of T. indica from field-grown plants for clonal propagation using an aeroponic system and found that 100 % of stem cuttings rooted with 2 g/l indole-3-butyric acid (IBA), performing better than cuttings rooted directly in soil (77 % of cuttings while only 6.65 % of control cuttings rooted in the absence of an auxin).

Table 1 Vernacular names for Tylophora indica
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Tylophora indica has numerous medicinal properties, including antioxidant, antiallergic, anti-angiogenic, antibacterial, anticancer, antifeedant, anti-inflammatory, antimicrobial, antitumor, antiasthmatic, cardioprotective, diuretic, hepatoprotective, and displaying immunomodulatory activity, all of which have been recently reviewed by Shahzad et al. (2016) and will thus not be included in this review. Overexploitation and the lack of organized cultivation strategies underscore the importance of developing biotechnological approaches for the rapid and reproducible in vitro propagation of this medicinal plant species and the stable and improved in vitro and in planta production of its valuable pharmaceuticals that endow it with these widely reported medicinal properties.

Two Tylophora species are on the Red List of the International Union of Conservation of Nature, Tylophora cameroonica N.E.Br., listed as near threatened, and Tylophora urceolata Meve, listed as vulnerable International Union for Conservation of Nature and Natural Resources (IUCN 2015). Other than these two species, currently the use of biotechnology is not a tool for preservation of rare or endangered material, but rather a tool for mass propagation of clonal material, or as a stable base for creating a sterile in vitro milieu to engage in other applied biotechnologies for improvement of medicinal plants such as tissue culture (Yoshimatsu 2008), somatic hybridization (Murch and Saxena 2001), germplasm cryopreservation (Dixit et al. 2004), genetic transformation (Bajaj and Ishimaru 1999; Roychowdhury et al. 2013b), synthetic seed production (Sharma et al. 2013), or bioreactor production of secondary metabolites (Baque et al. 2012). This review highlights the advances that have been made thus far in the tissue culture of Tylophora species, although the analysis in Table 2 reveals that the majority (63/65 studies, or 97 %) of studies have focused on T. indica with only a single study on T. ovata (Lindl.) Hook. ex Steud. (syn. T. ovata var. balansae (Costantin) Tsiang, T. ovata var. brownii (Hayata) Tsiang & P.T. Li, and T. ovata var. ovata), by Jeyachandran and Bastin (2014) and one study on T. subramanii Henry (Murukan et al. 2015).

Table 2 Micropropagation and tissue culture of Tylophora indica, except for Tylophora ovata and Tylophora subramanii (chronological listing)
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Morphogenesis and propagation of Tylophora indica in vitro

The explant is the central unit of plant tissue culture, and its choice depends on seasonal availability or quality of mother plant material, on the experimental objective, and on its responsiveness in vitro. The most popular explant used for the tissue culture of Tylophora species has been leaves (Fig. 1a) from field-grown and micropropagated plants.

Fig. 1
figure 1

The in vitro culture of Tylophora indica. a Direct shoot organogenesis from mature (thick, leathery, and dark green) leaves (from first 10 leaf pairs from the base) after 50 days of culture in Murashige and Skoog (MS) medium supplemented with 2 mg/l BA. Leaf explants were collected from a four-year-old plant maintained inside a shade-net house to avoid direct sunlight and exposed to 60–65 % relative humidity using a misting system. In vitro cultures were incubated inside a growth chamber maintained at 23 ± 2 °C with a 16-h photoperiod and at a photosynthetic photon flux density of approximately 50 µmol m−2 s−1 emitted by cool fluorescent tubes (Philips India Ltd.). b Direct shoot organogenesis from green root segments, excised from 4 to 6-week-old in vitro plant, on MS medium supplemented with 0.5 mg/l IBA, after 8 weeks of culture at 24 ± 1 °C with a 16-h photoperiod. c In vitro root induction from 12-day-old shoot (2.5 cm) derived from (a) on half-strength MS medium fortified with 0.1 mg/l IBA. d Three-month-old hardened plants kept in earthen pots containing a mixture of soil and vermicompost (3:1, v/v) and maintained inside a greenhouse (30 ± 2 °C, 14-h photoperiod, 60–65 % relative humidity). e Ex vitro plant (22 months old) maintained in the field under full sunlight and natural conditions without any additional care. f Flower of ex vitro plant. All photos (unpublished) provided with kind permission of Dr. B. Ghosh (a, c–e), and Dr. D. Roychowdhury (b)

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T. indica has been extensively studied since 1970 when Rao et al. (1970) reported the induction of callus from stem explants which differentiated into roots, shoots and bipolar somatic embryos (SEs). Rao et al. (1970) also demonstrated SE development in a cell suspension culture while the histological basis of morphogenesis was described by Rao and Narayanswami (1972). The morphogenetic potential of dedifferentiated cells of T. indica in vitro was demonstrated using various types of explants, namely leaves (50 % of reports, Table 2; Fig. 1a), internodes or stems, petioles and roots. Micropropagation based on the use of shoot tips or nodal explants involving apical or axillary bud proliferation has been the method of choice in 25 % of published reports (Table 2).

Use of explants from ex situ plants

Leaves from ex situ plants have been the primary source for callus induction and indirect shoot regeneration from dedifferentiated callus, with only few reports on somatic embryogenesis, most of which have not been substantiated by suitable histological analyses.

The earliest report of direct shoot organogenesis from mature leaves of field-grown plants was by Bera and Roy (1993), inducing as many as 304 shoot buds/explant in optimized medium but no histology was performed. Manjula et al. (2000) induced embryogenic callus from mature leaves and subsequent development of SEs, but the conversion frequency to SEs depended on the concentration and combination of indole-3-acetic acid (IAA), 6-benzyladenine (BA) and kinetin. Jayanthi and Mandal (2001) induced SEs from embryogenic callus induced on mature leaf explants from ex situ plants, obtaining 50 plantlets/g of callus in 5 months. Somatic embryogenesis has also been reported from leaf explants by Chandrasekhar et al. (2006) and Sahai et al. (2010a), and from internodes (Thomas 2006).

Faisal and Anis (2003) induced shoots indirectly from callus in 85 % of leaves from field-grown plants, from stem explants (Faisal and Anis 2005) and from petiole explants (Faisal et al. 2005), a similar result being obtained by Verma et al. (2010) leaf, petiole and internode explants. Shoot induction was possible from mature leaf explants (Rathinavel and Sellathurai 2010; Anjum et al. 2014) of young leaves (Kalimuthu and Jeyaraman, 2012). Kaur et al. (2011a) developed a protocol for the induction of shoots from the stems of field-grown plants, and a subsequent protocol for the acclimatization and ex situ establishment of tissue cultured plants (Kaur et al. 2011b). Thomas and Phillip (2005) were the first to provide histological evidence of indirect shoot formation from immature leaves of field-grown plants, noting 100 % regeneration potential by long-term (up to 180 days) callus cultures. Haque and Ghosh (2013) showed a different morphogenic response by young and mature leaves of field-grown plants grown in vitro: while aged leaves formed shoots directly, young leaves first formed nodular meristemoids. The Haque and Ghosh (2013) study is the only publication in which micropropagated plants transferred to field flowered, and 28.5 % of plants produced fruit. The development of a successful protocol for the micropropagation of Tylophora plants (Fig. 1c–e) is a prerequisite for more advanced studies such as genetic transformation.

To induce callus using leaf and stem explants, MS medium supplemented with 2.5–7.5 µM 2,4-D or 2,4,5-T is optimal while shoot organogenesis from this callus can be induced in MS medium supplemented with 5 µM kinetin or BA, or 8 µM thidiazuron (Faisal and Anis 2003, 2005; Thomas and Philip 2005; Fig. 2). Microshoots can be rooted in half-strength MS medium containing 0.5–0.1 µM IBA.

Fig. 2
figure 2

In vitro plant regeneration in and mass propagation of Tylophora indica can be achieved in several ways, based on Table 2

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Use of explants from in vitro plants

All studies that have used in vitro tissue to initiate in vitro cultures have employed leaf and root explants for whole plant regeneration. There are three reports on the use of root explants excised from in vitro plants to develop whole plants. Chaudhury et al. (2004) induced nodular shoot buds from green root segments in the presence of a cytokinin, and embryogenic callus from the same explants in the presence of BA and 2iP (N 6-(2-isopentenyl) adenine), with 42 % of explants converting to SEs. Sahai et al. (2010b) reported direct shoot organogenesis and callus-mediated somatic embryogenesis in green root segments, and provided a detailed histological assessment of shoot development. Nayeem et al. (2014) induced shoots from adventitious roots that developed from leaf explants, either directly or via callus formation. Devendra et al. (2011) claimed to induce SEs from leaf explants but provided no histological evidence.

Use of axillary buds, nodes and shoot tips

Sharma and Chandel (1992) first reported axillary bud proliferation and propagation from nodal stem segments in T. indica in response to cytokinins and auxin in basal medium supplemented with ascorbic acid. Faisal et al. (2007) used 100 mg/l ascorbic acid in addition to auxins and cytokinins to improve shoot number and length from nodes. Axillary bud multiplication and micropropagation were subsequently reported by Gami and Parmar (2010) and Kaushik et al. (2010). Rani and Rana (2010) collected nodal explants during different seasons and found that the frequency of bud break and shoot number/explant were maximum when collected in September–November. Micropropagation from nodal explants was also reported by Mohan et al. (2014) and Patel and Nadgauda (2014). While shoot multiplication using a pre-existing meristem has been the method of choice for clonal propagation of plants in many plant species, in T. indica, the fewest reports of shoot organogenesis involve nodal explants. The rate of multiplication using nodal explants is not mentioned in most publications and needs to be improved. It is also unclear whether a range of endophytic fungi isolated from leaves and stems (Kumar et al. 2011) may impact the effectiveness of organogenesis in vitro.

An optimum number of shoots per nodal explant with an average length of 3–4 cm can be obtained in MS medium supplemented with 2.5 µM BA or kinetin, 0.1 µM NAA, and 50–100 mg/l ascorbic acid after 6 weeks of culture (Faisal et al. 2007). Rooting of these shoots is optimum in half-strength MS medium supplemented with 0.5 µM IBA followed by acclimatization in vermiculite in a growth chamber under a 16-h photoperiod for 4 weeks (Fig. 2).

Protoplast culture and morphogenesis

Mhatre et al. (1984) were the first to report plant regeneration from protoplasts in T. indica from callus induced on stem explants. The yield of viable protoplasts, and induction of shoots, was higher when freshly induced callus was used than from five year old callus that had been regularly subcultured. Thomas (2009) regenerated T. indica plants from mesophyll-protoplast-derived callus that had been induced from the leaves of field-grown plants. Protoplasts have not yet been used to generate hybrids in Tylophora.

Optimal in vitro protocol

Based on the protocols described in Table 2, it is evident that T. indica is an interesting example of a plant species showing morphogenic potential from almost all vegetative parts. However, for the purpose of mass clonal propagation, the most commonly used explant, namely shoot tips or nodes, is not very suitable due to a low rate of multiplication. To maintain the fidelity of the genetic and chemical profile of the parent plant, it is necessary to avoid propagation protocols that involve dedifferentiation of explant cells to friable callus and indirect morphogenesis requiring auxin and/or cytokinin supplementation in the basal medium, which is very prevalent in T. indica. To induce de novo shoots from leaf explants of field-grown plants via the formation of nodular meristemoids, MS medium supplemented with 2–3 mg/l BA or kinetin, together with low levels of IAA (0.2–0.5 mg/l) can be used with subcultures every 4–6 weeks. Similarly, shoot organogenesis can be induced via the formation of nodular meristemoids in green root segments excised from 6 to 8 week old rooted in vitro plants. Thus, induction of nodular meristemoids from leaf explants is a suggested method of choice to establish and in vitro culture and to propagate plants for commercial purposes (Fig. 2).

Secondary metabolite production in vitro

Benjamin and Mulchandani (1973) first reported secondary metabolite production in T. indica in vitro callus induced from stem segments and roots from in vitro germinated seedlings. The callus did not form any phenanthroindolizidine alkaloids even after feeding precursors, but phytosterols were detected. Benjamin et al. (1979) further investigated alkaloid synthesis in callus cultures and in vitro regenerated plants: while alkaloids were not detected in callus, the alkaloid profile of tissue culture-derived flowering plants were similar to field-grown plants. Jha et al. (2005) investigated the potential of differentiated or morphogenic root-derived callus cultures and plants regenerated from root segments of T. indica. The level of tylophorine—a phenanthroindolizidine alkaloid and the main alkaloid in T. indica—in nodular meristemoid and friable embryogenic cultures and in plantlets regenerated in vitro prior to and after transfer to the field was assessed by HPLC. Tylophorine was detected in all in vitro cultures, in the shoots and roots of in vitro plantlets as well as in the leaves, stems and roots of one-year-old plants after transfer to the field. Friable embryogenic cultures had double the tylophorine content when the culture period was extended from 4 to 12 weeks, and 12-week-old tissue cultured plantlets had a 21-fold higher tylophorine content than 4-week-old plants. The tylophorine content of one-year-old micropropagated plants growing in the field and wild plants was comparable. Kaur et al. (2011a) detected 71–80 µg/ml of tylophorine in tissue culture-derived plantlets, confirmed by Kaur et al. (2011b) study (80 µg/ml). Kaur et al. (2011b) also found that suspension cultures and callus produced 28.3 and 24.5 µg/ml of tylophorine, respectively. Soni et al. (2015a), using precursor feeding of 2 mg/l tyrosine, induced 27.7, ~12.5, ~9.5, and ~4.5 µg/ml of tylophorine in in vitro-derived plantlets, shoots, callus and mother plants, respectively.

Molecular verification of somaclonal variation

Molecular markers have not been extensively used in Tylophora biotechnology. While two studies (Jayanthi and Mandal 2001; Haque and Ghosh 2013) showed no variation (i.e., polymorphism) in random amplified polymorphic DNA (RAPD) banding between mother plants and in vitro regenerants, Chaturvedi and Chowdhary (2012) reported 37.5 % polymorphism while Pathak et al. (2013) showed 62.1 % polymorphism.

Genetic transformation, hairy root production, secondary metabolites and bioreactors

Tissue culture forms an important structural basis for genetic transformation studies. Several studies reporting on the development of transgenic T. indica exist. The first (Chaudhuri et al. 2005) documents the induction of hairy roots in excised leaf and stem explants infected with Agrobacterium rhizogenes strain A4. In that study, as many as 60 % of inoculated shoots formed hairy roots with different transformed root clones (Fig. 3a) that accumulated tylophorine at 0.16–0.29 mg in roots/Petri dish and 1.03–1.29 mg/g root dry weight. The transformed roots could be successfully cultured in liquid medium, forming higher biomass, yield and tylophorine content than in solid medium. This is a prerequisite for scale-up studies. The secretion of tylophorine in liquid root culture medium was a significant finding for large-scale production using bioreactors.

Fig. 3
figure 3

Spontaneous regeneration of Ri-plant from hairy root culture of Tylophora indica. a Ri-transformed root culture on MS medium (bar 1.3 cm). b Spontaneously regenerating Ri-transformed callus cultured under 16-h photoperiod on MS medium showing somatic embryogenesis (bar 0.5 cm). c Single germinated somatic embryo on MS medium (bar 0.3 cm). d Ri-transformed plant

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Spontaneous regeneration of plants from Ri (root-inducing)-transformed roots in plant growth regulator-free basal medium (Fig. 3b, c) was reported by Chaudhuri et al. (2006). The Ri-transformed T. indica plants had 160–280 % higher tylophorine content than untransformed plants, and an equivalent 350–510 % higher biomass (Chaudhuri et al. 2006). The same group (Roychowdhury et al. 2013a, 2015a, b) then assessed the morphological and genetic stability of long-term (4–6 years) in vitro hairy root cultures and plants derived from transgenic hairy roots (Fig. 3d). Among the most notable morphological variations observed were shorter shoots with more nodes and leaves/plant, both in in vitro plantlets and in one-year-old greenhouse-grown plants (Roychowdhury et al. 2013a). Despite this variation, no genetic variation (RAPD profiles) was detected (Roychowdhury et al. 2015a). The rolA, rolB, rolC, and rolD genes were stably inserted (Fig. 4), as confirmed by RT-PCR, in all clones and tylophorine content, as confirmed by HPTLC, was almost two-fold higher than in non-transformed plants (Roychowdhury et al. 2013a, 2015b).

Fig. 4
figure 4

Molecular confirmation of integration of the rolA, rolB, rolC, and rolD genes at the transcription level. The β-actin gene served as the internal control (see primers and additional evidence of integration in Roychowdhury et al. 2015a, b) using RT-PCR of Ri-transformed Tylophora indica plant derived using procedures explained in Figs. 1 and 2 (Chaudhury et al. 2006 protocol; Table 2). Lane 1 molecular markers (100-bp plus DNA ladder); lane 2 positive control (pLJ1, which carries TL-DNA); lane 3 negative control (genomic DNA from non-transformed plant); lanes 4–13 amplified cDNAs of Ri-transformed plants lines. Unpublished photos

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The morphogenic potential of transformed hairy root cultures was not affected by the presence of rol genes of the Ri plasmid and plants regenerated both via direct (less common) and indirect (more common) organogenesis as well as via callus-mediated somatic embryogenesis (Chaudhuri et al. 2006; Roychowdhuri et al. 2015b). In these studies, since Ri-transformed plants showed enhanced tylophorine production, and since such high tylophorine-containing plantlets could be stably micropropagated as non-transformed plants, this technique can be commercialized for the production of T. indica secondary metabolites. A protocol for the induction of Ri-transformed roots, regeneration of plants via direct organogenesis and indirect somatic embryogenesis in T. indica, does not require exogenous supplementation of phytohormones at any stage thereby ensuring the genetic stability of Ri-transformed plants.

Conclusions and future perspectives

The development of an economically viable scale-up culture system using transformed root cultures is a pre-required for the large-scale production of tylophorine (Roychowdhury et al. 2013b), although there are several other means of establishing in vitro cultures (Fig. 2). Hairy roots serve as a continuous source of target metabolites of parent plants due to their genetic stability, and ability for rapid growth in plant growth regulator-free liquid medium in bioreactors (Stiles and Liu 2013; Mehrotra et al. 2015), allowing for hairy root-mediated biotransformation (Banerjee et al. 2012). These technologies would allow for the use of T. indica hairy roots to further improve the biosynthetic potential of superior clones. Studies on the factors affecting the biosynthesis of T. indica alkaloids and other secondary metabolites need to be conducted using transgenic cultures via exogenous and endogenous elicitation (Chaudhuri et al. 2009; Ramirej-Estrada et al. 2016) and biotransformation (Banerjee et al. 2012).

The following topics still need to be explored in Tylophora in vitro biotechnology: anther culture (e.g., Teixeira da Silva et al. 2015), the use of thin cell layers technology (Teixeira da Silva and Dobránszki 2013, 2015), in vitro flower induction (e.g., Teixeira da Silva et al. 2014), or CO2 enrichment for increasing biomass (e.g., Norikane et al. 2013). More recently, an aqueous extract of T. indica leaves was used to synthesize silver nanoparticles (Oke et al. 2015), but that procedure would need to be improved to make it more economically viable than for tylophorine production. The use of these detailed protocols and advice may also be useful for the in vitro propagation of other Tylophora species, such as T. ovata (Jeyachandran and Bastin 2014) and T. subramanii (Murukan et al. 2015).

Author contribution statement

Both authors contributed equally to all aspects of the development and writing of this review.