Introduction

Ranunculus wallichianus Wight & Arnn belonging to the family of Ranunculaceae is a rare and endemic medicinal plant of the Western Ghats, India, and is commonly called as Wallich Buttercup (Mathew 1996; Kumar 2004). The genus Ranunculus has more than 600 species throughout the world (Tamura 1993, 1995). In Asian countries, Ranunculus species are traditionally used to treat fever, rheumatism and asthma (Iqbal et al. 2011; Aslam et al. 2012) as well as pharmacological properties as antifungal (Mares 1987), antibacterial, anticancer, and antioxidant agents (Cao et al. 1992; Yin et al. 2008; Kaya et al. 2010; Akkol et al. 2012).

Being an endemic and over-used plant, R. wallichianus needs to be conserved and micropropagation is an effective tool to accomplish this (Fay 1992; Pathnaik and Chand 1996). Based on the previous literature, tissue culture studies have been reported in the R. asiaticus (Meynet and Duclos 1990; Pugliesi et al. 1992; Beruto and Debergh 1992, 2004; Beruto et al. 1996, 1999) and R. lyallii (Bicknell et al. 1996), although no in vitro culture studies of R. wallichianus have not been reported. The aim of the present study was to develop a protocol for producing R. wallichianus in large quantities from nodal explants by adding various concentrations of coconut water (CW) to the medium along with cytokinins to produce more shoots by micropropagation.

Materials and methods

Collection of plants and surface sterilization

The young and mature plants were collected from the Pampar Shola of Western Ghats, Tamilnadu, India. The explants were washed with running tap water for 30 min, followed by immersion in 1% (w/v) Teepol solution (soap solution) (Reckitt Benckiser Pvt. Ltd., Himachal Pradesh, India) containing 5% Bavistin (fungicide) (Crystal Crop protection Pvt. Ltd., New Delhi, India) for 3 min. Explants were washed with distilled water six times and then with double-distilled water six times before surface sterilization with 0.1% (w/v) HgCl2 (Hi-Media, Mumbai, India) for 3 min under aseptic conditions in a laminar air flow chamber (Atlantis Applications Engg. Pvt. Ltd., New Delhi, India). Finally explants were washed with sterile double distilled water five times to remove the traces of HgCl2 (Dubey 1999).

Culture media and conditions

Murashige and Skoog (MS; Murashige and Skoog 1962) medium containing 3% (w/v) sucrose (Hi-Media, India) and 0.8% (w/v) agar (Hi-Media, India) was used for the experiments. The pH of the medium was adjusted to 5.7 using 0.1 N NaOH (Hi-Media) and 0.1 N HCl (Spectrum Reagents and Chemicals Pvt. Ltd., Cochin, India) and autoclaved at 15 psi/121 °C for 15 min.

Extraction of coconut water

CW was extracted from the mature fruit (Ghandi Market, Tiruchirappalli, India) by drilling holes through two of the micropyles under the sterile laminar air flow chamber (Nasib et al. 2008). Then the CW was passed through 0.45-μm sterile filters and heated at 80 to 100 °C for 10 min with continuous stirring to precipitate proteins, fats, and other materials. The precipitate was removed by filtration and the filtrate was stored at – 20 °C for future use (George 1993).

Multiple shoot induction

Nodal explants were cultured on half-strength MS medium supplemented with 1.0 to 5.0 mg L−1 BA, KIN, or TDZ (Hi-Media) either individually or in combination with 5%, 10%, or 20% CW. The cultures were maintained at 25 ± 2 °C with 16 h photoperiod (50 μmol m−2 s−1 Photon Flux Density provided by white cool fluorescent tube light) and 8 h darkness and sub cultured at 10-day intervals. The number of shoots per explants and shoot length were recorded after 30 d of culture.

In vitro rooting and acclimatization of regenerated plants

In vitro propagated shoots exceeding 5 cm in length were excised and transferred to half-strength MS medium containing 250 mg L−1 activated charcoal with 0.5 to 3.0 mg L−1 indole-3-butyric acid (IBA) or 0.5 to 3.0 mg L−1 indole-3-acetic acid (IAA) for in vitro rooting. The percentage of rooting, mean number of roots per shoot, and mean shoot length was recorded after 2 wk transfer to rooting medium. Well-rooted plantlets were transferred to pots containing a mixture of sterilized red soil (TNB Analytical Laboratory Pvt. Ltd. Tiruchirappalli, India), vermiculite (Vermicompost yard, St. Joseph’s College, Tiruchirappalli, India), and coconut husk (Agaram Trading Company, Tiruchirappalli, India) (1:1:1 v/v) combination for ex vitro acclimatization. The potted shoots were fertilized with quarter strength of M.S. liquid medium for 1 wk. After 4 wk, plantlets were transferred to pots containing garden soil and maintained in a greenhouse and eventually transferred to the field.

Statistical analysis

Each experiment was repeated three times with 15 replicates, and the results were recorded after 6 wk culture. The mean number of shoots, shoot length, mean number of roots, and root length with standard error was calculated with Sigma plot statistical software version 17.0 (SPSS Inc., Chicago, IL). The treatment means were compared using Duncan’s multiple range test (DMRT) at 5% probability level (Gomez and Gomez 1976).

Results and discussion

In vitro multiple shoot induction

Multiple shoots were produced from nodal explants cultured on half-strength MS medium supplemented with 1 to 5 mg L−1 BA, KIN or TDZ (Table 1). Of these cytokinins, 2 mg L−1 TDZ produced maximum rate (78%) of shoots, with an average of 4.2 ± 0.28 shoots per explant (Table 1, Fig. 1b). Several studies reported high frequency shoot regeneration with medium containing TDZ (Huetteman and Preece 1993; Tiwari et al. 2001; Faisal et al. 2005; Sujatha and Ranjitha Kumari 2007; Guo et al. 2011, 2012; Cheruvathur et al. 2013; Fatima et al. 2015; Vaidya et al. 2016). On the other hand, 3.0 mg L−1 BA and KIN produced shoots with 60% (3.4 ± 0.21 shoots per explant) (Fig. 1a) and 45% (2.1 ± 0.21 shoots per explant) efficiency, respectively. The highest average of shoot length was 3.4 ± 0.21 cm with 2 mg L−1 TDZ after 6 wk of culture. With 3 mg L−1 BA and KIN treatments, optimum average shoot length was 3.3 ± 0.13 and 2.5 ± 0.11 cm, respectively (Table 1). Meynet and Duclos (1990) reported similar results for direct regeneration of R. asiaticus on half-strength MS media supplemented with 2.2 μM BA in combination with 0.45 μM 2,4-D. Adventitious shoots of R. asiaticus were achieved by Pugliesi et al. (1992) using MS medium supplemented with a combination of 4.6 μM KIN and 5.4 μM NAA. The effective shoot proliferation in R. lyallii was demonstrated using half-strength MS basal medium supplemented with 0.2 mg L−1 BAP (Bicknell et al. 1996).

Table 1. Effect of cytokinins on micropropagation of Ranunculus wallichianus Wight & Arnn using nodal explants cultured on half-strength Murashige and Skoog medium for 6 wk
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Fig. 1
figure 1

In vitro shoots regeneration from nodal explants of Ranunculus wallichianus Wight & Arnn. (a) Shoot initiation on half-strength Murashige and Skoog (MS) medium supplemented with 3.0 mg L−1 of 6-benzyladenine (BA). (b) Shoot induction on half-strength MS medium containing 2.0 mg L−1 of thidiazuron (TDZ). (c) Shoot induction on half-strength MS medium with 4.0-mg L−1 kinetin (KIN) and 10% of coconut water (CW). (d) Multiple shoot induction on half-strength MS medium with 4.0 mg L−1 of BA and 10% of CW. (e) Multiple shoot initiation on half-strength MS medium with 3.0 mg L−1 of TDZ and 10% of CW after 2 wk. (f) Shoot multiplication on half-strength MS medium with 3.0 mg L−1 of TDZ and 10% of CW after 4 wk (scale bar 5 mm)

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The effect of 5%, 10%, and 20% CW in combination with cytokinins on multiple shoot induction from nodal explants is shown in Table 1. Ninety-five percent of nodal explants cultured on medium containing 3 mg L−1 TDZ and 10% CW produced multiple shoots and averaged 6.7 ± 0.26 shoots per explant with an average shoot length of 6.3 ± 0.21 cm (Table 1: Fig. 1e, f). Roy and Banerjee (2002) documented that the combination of coconut water with various types of plant growth regulators enhances plant growth and development. Agampodi and Jayawardena (2009) and Tan et al. (2011) reported that plant growth regulators used in the plant tissue culture are naturally present in CW. The combination of CW and synthetic auxins are known to be more promotive of shoot multiplications in tissue culture (Loc et al. 2005). When utilizing BA, the maximum number of shoots (5.9 ± 0.25 shoots/explants with 5.2 ± 0.21 cm mean shoot length) was produced from nodal explants cultured on MS medium containing 4 mg L−1 BA and 10% CW (Table 1; Fig. 1d), while KIN was much less effective for shoot induction (Table 1; Fig. 1c). The number of shoots per explant and the shoot length were inhibited at higher concentrations of CW (20%). Al-Khayri (2010) demonstrated that somatic embryogenesis of date palm (Phoenix dactylifera L.) was inhibited on media supplemented with 20% of CW. The morphological features and growth of Calanthe hybrids were decreased at high concentrations of CW (Baque et al. 2011). Our results suggest that 10% of CW with 3.0 mg L−1 TDZ is the optimal for rapid shoot production from nodal explants of Ranunculus wallichianus. A comparison of efficiency of hormones indicates that TDZ is superior to BA and KIN (Table 1; Fig. 1e, f).

In vitro rooting and acclimatization

Root initiation from well-elongated in vitro microshoots was observed after 7 d of culture on rooting medium and plantlets suitable for transplantation could be produced in 20 d. The high frequency (94% response) of root induction (4.8 ± 0.23 roots per shoot with 3.7 ± 0.18 cm root length) was noted on half-strength MS media containing 2.5-mg L−1 IBA with 250-mg L−1 activated charcoal (Table 2; Fig. 2a). A recent investigation by Choudhary et al. (2020) reported similar results on root induction in Farsetia macrantha with 2.0-mg L−1 IBA and 100-mg L−1 activated charcoal. For root induction, IBA with activated charcoal were also reported for Hagenia abyssinica (Feyissa et al. 2005) and Cassia angustifolia (Siddique and Anis 2007). In the present study, IBA was found to be more effective than IAA in terms of percent rooting and mean number of roots per shoot. After the sufficient root and shoot development, plantlets were successfully transferred to the field with a 93% survival rate (Fig. 2b).

Table 2. Effect of auxins and 250 mg L−1 activated charcoal on root induction from in vitro raised shoots of Ranunculus wallichianus Wight & Arnn on half-strength Murashige and Skoog medium after 4 wk culture
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Fig. 2
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In vitro root induction and acclimatization of Ranunculus wallichianus Wight & Arnn. (a) Root induction on half-strength Murashige and Skoog (MS) medium with 2.5 mg L−1 of indole-3-butyric acid and 250 mg L−1 of activated charcoal after 20 d. (Scale bar 1 cm) (b) Hardened plantlet transferred to plastic pots for survival (scale bar 2 cm)

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