1 Introduction

The Combretaceae is a large family of herbs, shrubs and trees, consisting of nearly 20 genera and 600 species having tropical distribution around the world and centers of diversity in Asia and Africa, and most of them are used for medicinal purpose traditionally (Fyhrquist et al. 2002; de Morais Lima et al. 2012). Combretum species are widely utilized in traditional medicine for diabetes, infections, inflammation, malaria, bleeding, digestive and diarrheal problems (de Morais ima et al. 2012). Combretum ovalifolium Roxb. is a deciduous climber, leaves, elliptic-ovate, apex obtusely accuminate, base cuneate, coriaceous, spikes 1–3 cm, petals oblong, fruits 2–2.5 cm long, obovate, coriaceous, golden yellow with 4-papery wings, four-winged thin seeds (Roy et al. 1992). The presence of fibers with protoplast is a characteristic feature of C. ovalifolium. Prismatic crystals frequently co-occur with the nucleus in the same compartment of the fibers (Rajput and Rao 1999). Seed oil contains 35.4% of palmitic acid, 16.4% myristic acid, 24% of oleic, 17.1% linoleic acids and a small quantity of lauric, stearic, arachidic and behenic acids (Daulatabad and Ankalgi 1983). Leaves of C. ovalifolium are known to have mosquito repellent (Singh and Saratchandra 2005; Modi and Mathad 2016) and insect repellent activity (Singh and Saratchandra 2005) and traditionally utilized for control of insect pest by local farmers (Gupta and Patel 1992). Extracts of C. ovalifolium show larvicidal properties against vector Anopheles stephensi (Krishnappa et al. 2013). Mahida and Mohan (2006) reported the antibacterial activity of C. ovalifolium. Stem bark juice of C. ovalifolium is administered orally as a cure for jaundice (Sanjeev et al. 1997; Vaidyanathan et al. 2013; Nandagoapalan et al. 2016). A decoction of the bark of Albezia procera with the bark of C. ovalifolium and Acacia ferruginea and roots of Blumea eriantha is administered as an antidote for snakebite (Quattrocchi 2012). C. ovalifolium is used as a source of fiber-yielding plant in South Gujarat, India (Gavali and Sharma 2004). A decoction of leaves is used for curing of menstrual problems in Amravati, Maharashtra (Onkar 2016). This is a fiber-yielding plant (Gavali and Sharma 2004) and, therefore, utilized for rope making (Rao et al. 2015). Terminal panicle of C. ovalifolium is crushed and used as local antiseptic and antibiotic (Panda 2002). Combretum sp propagates by seed germination (Onyekwelu 1990; Dalling and Van Staden 1999; Wickens and Gaum 2001). Information about seed germination or any vegetative method of propagation, for C. ovalifolium, is not available. Conventional breeding methods are only limited to the agriculturally important plants, especially plants having a short life cycle. Therefore, such techniques cannot be used for slow growing, long-lived, highly heterozygous and sexually self-incompatible plants (Hammatt 1992). Micropropagation techniques are the popular choice for propagation and conservation of medicinal plants (Nataraj et al. 2016; Kher et al. 2016a; Teixeira da Silva et al. 2016). There have been many studies on the micropropagation of Combretaceae members using nodal explants like Anogeissus sericea var. nummularia King ex. Duthie (Yusuf 2005), Terminalia bellirica Roxb. (Ramesh et al. 2005; Phulwaria et al. 2012a), T. arjuna Roxb. (Pandey et al. 2006), T. catappa Linn (Phulwaria et al. 2012b). The objective of the present research work was propagation of C. ovalifolium through axillary shoot multiplication and in vitro rooting of regenerated plants.

2 Materials and methods

2.1 Plant material and surface sterilization

Nodal explants were collected from Medicinal Plant Garden, Anand Agriculture University, Gujarat, India (Fig. 1a). Nodal explants were carefully washed under running tap water for 5 min and then soaked in 3% Neutral Rankleen detergent (v/v) (RFCL-India) for 10 min followed by 10 min washing in running tap water. Subsequently, the explants were treated with 0.1% mercuric chloride (w/v) solution for 3 min and then rinsed 3–4 times with sterile distilled water (DW).

Fig. 1
figure 1

Axillary shoot multiplication of C. ovalifolium. a C. ovalifolium, b M.S. medium supplemented with 5 mg/l BA + 3 mg/l TIBA + 2 gm/l activated charcoal, c, d callus formation from nodal explant, e MS basal medium, f MS + 5 mg/l TDZ, g MS + 5 mg/l mg/l BA. (Arrow indicates vitrified shoots, circle indicates callus formation)

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2.2 Composition of culture media and culture conditions for in vitro cultures

The culture medium used was Murashige and Skoog (1962): MS medium salts, vitamins with 3% (w/v) sucrose. The pH of all media was adjusted to 5.8 with 0.1 N NaOH or HCl before adding 0.8% (w/v) agar (bacteriological grade; Merck) and autoclaved at 121 °C for 15 min. Cultures were maintained at 25 ± 2 °C under a 16-h photoperiod at a photosynthetic photon flux density of 35 µmol m−2 s−1 provided by cool white fluorescent tubes (36 W Philips).

2.3 Effects of cytokinin on axillary shoot multiplication

Surface sterilized nodal explants of 1.5–2.0 cm height with one node (with two axillary meristems) were inoculated with MS medium containing 1.0–5.0 mg/l 6-benzyaminopurine (BA) or kinetin (Kn) or 2-isopentenyladenine (2iP) or thidiazuron (TDZ). Within 15 days of culture, prolific callus formation (Fig. 1c, d) was observed at the cut ends of nodal explants, which inhibited multiplication of shoot (Table 1).

Table 1 Effects of cytokinins and their concentrations on Combretum ovalifolium nodal explants
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2.4 Effects of additives on axillary shoot multiplication

For prevention of callus formation, nodal explants were cultured on MS medium augmented with combination of 5 mg/l BA and 1, 3 and 5 mg/l of 2,3,5-tri-iodobenzoic acid (TIBA) or 5 mg/l BA with 3 mg/l TIBA and 1, 2 and 3 g/l activated charcoal (AC) or 5 mg/l BA with 3 mg/l TIBA and 15, 30, 45 and 60 mg/l adenine sulfate (AdS) Table 4.

2.5 Rooting and acclimatization

In vitro raised shoots of C. ovalifolium were rooted on half-strength MS medium supplemented with various concentrations of auxins (Table 6). Regenerated plantlets of C. ovalifolium with well-developed roots were removed from rooting medium and washed gently under running tap water to remove medium adhering to roots. Subsequently, plantlets were transferred to plastic cups containing cocopeat, soil mixture and sterile sand in 1:1:1 ratio. The transplanted plants were covered with clear transparent plastic bags to retain humidity. Pots containing micropropagated plants of C. ovalifolium were kept in plant tissue culture laboratory and watered with 15 ml of quarter-strength MS medium (quarter-strength of macro- and micronutrients) at 4-day intervals for 15 days. After 15 days, the plastic cover was removed and the uncovered plants were maintained at 25 °C under a 16-h photoperiod and 35 µmol m−2 s−1 in the laboratory for 30 days. After 30 days, plantlets were transferred to the field under natural conditions.

2.6 Statistical analysis

All experiments were conducted with 24 replicates (i.e., explants) per treatment, evaluated after 45 days and evaluated after 45 days. Data were subjected to analysis of variance (ANOVA) to assess treatment differences and interaction, and Duncan’s Multiple Range Test using SPSS-19 for (SPSS Inc., Chicago, IL, USA) calculated the significance of differences.

3 Results and discussion

3.1 Effects of cytokinins on axillary shoot multiplication

Axillary shoot multiplication is the popular method of micropropagation due to the presence of predetermined meristem for shoot formation (Saha et al. 2016; Shekhawat and Manokari 2016). For axillary shoot multiplication, type and concentration of cytkinin play significant roles. Therefore, in this study the response of nodal explants to various concentrations of BA, Kn, TDZ and 2-ip is investigated (Tables 1, 2 and 3). The response of multiple shoot induction varied significantly on MS basal medium with respect to different concentrations of cytokinin type and concentration. Nodal explants exhibited bud breaking within 15 days (Table 1). 66% of nodal explants cultured on basal MS medium exhibited shoot regeneration without any callus formation. 100% regeneration with shoot per node was observed from explants cultured on MS medium augmented with 5 mg/l BA. It was observed that excessive callus formation from the base of a node in all media augmented with cytokinins (Fig. 1c, d, e). Higher concentration (2, 3, 4 and 5 mg/l) of TDZ caused callusing and vitrification of shoots (Fig. 1f). Formation of callus hinders the quality of shoots which are not suitable for further subculture and rooting. Callus formation from nodal explants in cytokinin-enriched MS medium is maybe due to the presence of high concentration of endogenous auxin, which leads to wound healing (Bernabé-Antonio et al. 2012; Ikeuchi et al. 2013). These results indicate that a very high level of BA is necessary for maximal shoot proliferation. Vitrification of shoots due to TDZ has been reported in Pluchea lanceolata (Kher et al. 2014), Aloe polyphylla (Ivanova and Van Staden 2010) and Citrullus lanatus (Vinoth and Ravindhran 2015).

Table 2 Two-way ANOVA analysis showing the influence of type and concentration of cytokinins on number of shoots (Table 1)
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Table 3 Two-way ANOVA analysis showing the influence of type and concentration of cytokinins on shoot length (Table 1)
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3.2 Effects of additives on axillary shoot multiplication

Anti-auxin 2,3,5 tri-iodobenzoic acid (TIBA), a well-known polar auxin transport inhibitor, adenines sulfate (AdS) and activated charcoal were used to study effects on shoot multiplication from nodal explants of C. ovalifolium (Table 4). 100% axillary shoot multiplication was observed in all concentrations and combinations of plant growth regulators (Table 4). Excellent shoot formation was observed within 30 days on MS medium augmented with 5 mg/l BA, 3 mg/l TIBA and 30 mg/l AdS (Fig. 2b). Best results were observed on the same medium (7.5 shoots/node with average 4.7 cm shoot length); it was noted that when cultures are kept for 45 days, it showed root formation from nodal regions (Fig. 2b). Application of AdS and AC enhances shoot multiplication and shoot length (Fig. 1b; Tables 4, 5). The Increase in concentration of AdS from 15 mg/l to 30 mg/l increased shoot multiplication and shoot length while beyond 30 mg/l did not show any further increase in shoot number and length. Application of AdS for axillary shoot multiplication of other Combretaceae plants are reported for, e.g., 50 mg/l for Terminalia arjuna (Choudhary et al. 2015), 25 mg/l for Terminalia catappa (Phulwaria et al. 2012b) and Anogeissus latifolia (Shekhawat et al. 2000). TIBA controls the polar transport of auxin from cell to cell by adjusting the efflux carrier for auxin (Li et al. 2012). Bhau and Wakhlu (2001) reported that TIBA decreases the accumulation of auxin by suppressing the transport of endogenous auxins in the callus cells. As in the present study, Lall et al. (2005) found that TIBA promoted axillary shoot multiplication and reduced callus formation in Alnus glutinosa. The inclusion of TIBA in the culture media promoted axillary bud break and reduced callus in Capsicum sp (Christopher and Rajam 1994), Alnus glutinosa (Lall et al. 2005), Cucumis sativus (Shukla et al. 2014) and Clerodendrum phlomidis (Kher et al. 2016b).

Table 4 Effects of various combinations of BA: 6-benzyladenine with 2,3,5-tri-iodobenzoic acid (TIBA), activated charcoal (AC) and adenine sulfate (AdS) on Combretum ovalifolium nodal explants cultured on MS medium
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Fig. 2
figure 2

Axillary shoot multiplication of C. ovalifolium. a MS + 5 mg/l BA + 3 mg/l TIBA + 30 mg/l AdS photographed after 30 days, b MS + 5 mg/l BA + 3 mg/l TIBA + 30 mg/l AdS photographed after 45 days, c ½ MS + 2 mg/l IBA, d PGR free ½ MS medium, e Acclimatized micropropagated plant after 60 days in natural condition

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Table 5 One-way ANOVA of Effects if BA, TIBA, AC and Ads on nodal explants of C. ovalifolium cultured on MS medium (Table 4)
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3.3 Rooting and acclimatization

Rooting and acclimatization of in vitro raised shoots is a crucial step of micropropagation of any plants (Hazarika 2003; Osório et al. 2013). For root induction, individual microshoots were transferred to half-strength MS medium augmented with different concentrations of auxins (IAA, NAA and IBA). In vitro rooting (100%) was observed in MS medium augmented with auxins as well as on auxin free half-strength MS medium (Table 6; Fig. 2d). Two-way ANOVA of auxin type and concentration had no significant effect on number of root per shoot (Table 7) and root length (Table 8). Formation of roots on auxin-free medium is a possible indication of the presence of endogenous auxin. Best results were obtained on half-strength MS medium augmented with 2 mg/l IBA (6.3 roots/shoot with average root length of 5.3 cm). The effectiveness of IBA over other auxin for in vitro rooting is reported for other Combretaceae members such as Quisqualis indica (Mandal 2013). In the present research work, 85% of plants successfully survived (Fig. 2e) under natural conditions.

Table 6 Effects of various concentrations of auxins on in vitro rooting of Combretum ovalifolium
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Table 7 Two-way ANOVA analysis showing the influence of type and concentration of auxins on in vitro rooting (Table 6, number of roots/shoot)
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Table 8 Two-way ANOVA analysis showing the influence of type and concentration of auxins on in vitro rooting (Table 6, root length)
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4 Conclusion

In conclusion, the present work describes a regeneration method for C. ovalifolium from the nodal explants. Micropropagation of C. ovalifolium was achieved by supplementing MS medium with 3 mg/l TIBA, an inhibitor of auxin transport plus 5 mg/l BA with 30 mg/l AdS for shoot multiplication, and 2 mg/l IBA for rooting of regenerated shoots. This protocol could be viable option for large-scale propagation of other Combretum species.