80 ACTA BOT. CROAT. 77 (1), 2018 Acta Bot. Croat. 77 (1), 80–87, 2018 CODEN: ABCRA 25 DOI: 10.1515/botcro-2017-0012 ISSN 0365-0588 eISSN 1847-8476 In vitro multiplication, micromorphological studies and ex vitro rooting of Hybanthus enneaspermus (L.) F. Muell. – a rare medicinal plant Mahipal S. Shekhawat1*, M. Manokari2 1 Biotechnology Laboratory, Department of Plant Science, M. G. G. A. C. Mahe, Puducherry, India 2 Department of Botany, Kanchi Mamunivar Centre for Postgraduate Studies, Puducherry, India Abstract – Hybanthus enneaspermus is a rare medicinal plant. We defined a protocol for micropropagation, ex vitro rooting of cloned shoots and their acclimatization. Surface-sterilized nodal segments were cultured on Mu- rashige and Skoog (MS) medium with different concentrations of 6-benzylaminopurine (BAP) and kinetin (Kin). Medium supplemented with 1.5 mg L–1 BAP was found optimum for shoot induction from the explants and 6.4±0.69 shoots were regenerated from each node with 97% response. Shoots were further proliferated max- imally (228±10.3 shoots per culture bottle with 7.5±0.43 cm length) on MS medium augmented with 1.0 mg L–1 each of BAP and Kin within 4–5 weeks. The shoots were rooted in vitro on half strength MS medium containing 2.0 mg L–1 indole-3 butyric acid (IBA). The cloned shoots were pulse-treated with 300 mg L–1 of IBA and cul- tured on soilrite® in a greenhouse. About 96% of the IBA-pulsed shoots rooted ex vitro in soilrite®, each shoot producing 12.5±0.54 roots with 5.1±0.62 cm length. The ex vitro rooted plantlets showed a better rate of survival (92%) in a field study than in vitro rooted plantlets (86%). A comparative foliar micromorphological study of H. enneaspermus was conducted to understand the micromorphological changes during plant developmental pro- cesses from in vitro to in vivo conditions in terms of variations in stomata, vein structures and spacing, and trichomes. This is the first report on ex vitro rooting in H. enneaspermus and the protocol can be exploited for conservation and large-scale propagation of this rare and medicinally important plant. Key words: conservation, ex vitro rooting, Hybanthus enneaspermus, micromorphology, micropropagation, rare medicinal plant * Corresponding author, e-mail: smahipal3@gmail.com burning sensations, urinary infections, leucorrhoea, dysuria and sterility (Tripathy et al. 2009). The plant is also valued for its antimicrobial (Retnam and Britto 2007), antiplasmo- dial, antiarthritic (Subramoniam et al. 2013), antimalarial, antirheumatic, emmenagogic, sedative, antispasmodic, an- tiasthmatic, anti-infertility (Nathiya and Selvi 2013), anti- bacterial, anticonvulsant, antidiabetic, antifungal (Arumu- gam et al. 2011), anti-allergic and analgesic, antinociceptive, antioxidant and aphrodisiac properties (Kumar et al. 2013). This plant contains various important phytochemicals like dipeptide alkaloids, aurantiamide acetate, isoarborinol and β- sitosterol, flavonoids, steroids, triterpenes, phenols, tan- nin, glycosides etc. (Krishnamoorthy et al. 2014). There is no commercial cultivation of H. enneaspermus and the plants are collected from wild sources. It is facing genetic threat due to sporadic distribution, poor germina- Introduction Hybanthus enneaspermus (L.) F. Muell. (Formerly Ionid- ium suffruticosum Ging.), belongs to the family Violaceae. It is a rare multipotent herb, endemic to the Deccan Peninsula in India with various invigorating properties (Prakash et al. 1999, Sudeesh 2012). It is a small suffrutescent perennial herb found in India, Sri Lanka, Tropical Asia, Africa and Austra- lia (Anand and Gokulakrishnan 2012). The plant grows up to 15–30 cm in height with many diffused branches (Kirtikar and Basu 1991). H. enneaspermus is traditionally known as Padmavati, Lakshmisheshta, Padmacharini or Purusharathna in India and considered a valuable healing herb in the Indian systems of medicine (Satheeshkumar 2011). It is well docu- mented in the folklore medicine of India for its aphrodisiac and stimulant activity (Awobajo et al. 2009). H. enneasper- mus has therapeutic applications. Moreover, the whole plant is used to treat diarrhea, painful dysentery, and strangury, mailto:smahipal3@gmail.com MICROPROPAGATION OF HYBANTHUS ENNEASPERMUS ACTA BOT. CROAT. 77 (1), 2018 81 tion of seeds, anthropogenic activities, overgrazing and over exploitation by herbal drug manufacturers (Arunkumar and Jayaraj 2011, Verma and Singh 2011). It has been disappear- ing from the large area of the Western Ghats of India due to widespread cultivation of rubber in the natural habitat of this plant (Joseph et al. 2000). H. enneaspermus is conven- tionally propagated through seeds. The seeds show poor vi- ability and germination in the wild (Arunkumar and Jayaraj 2011). Conventional propagation methods are unable to meet the demand of the pharmaceutical industries and drug research. Therefore, it is necessary to develop a non-conven- tional method for propagation to fulfill the demands of the drug market (Rathore et al. 2008). In vitro propagation meth- ods offer a powerful tool for conservation of germplasm and mass-multiplication of threatened plant species (Murch et al. 2000). They can support the in situ and ex situ conservation of this rare genotype. Since natural propagation is unable to support the demand, in vitro methods could be viable op- tions. Some in vitro work on H. enneaspermus is available in the literature (Arunkumar and Jayaraj 2011, Velayutham et al. 2012, Premkumar et al. 2013, Sudharson et al. 2014). The present work is more effective in terms of number of mul- tiple shoots regenerated per explant. Survival of plantlets in field conditions is the major con- straint in the micropropagation of H. enneaspermus. An ex vitro rooting method could help in better acclimatization which increases the chances of field adaptation of plantlets in the natural environment. Improved rooting and acclima- tization can be achieved simultaneously with ex vitro root- ing of in vitro propagated shoots (Baskaran and Van Staden 2013).This was found to reduce time, labor, energy involved and the cost factor of micropropagated plantlets (Patel et al. 2014). Therefore, the aim of the present study is to establish in vitro methodologies for mass production of this rare plant species using ex vitro rooting and to evaluate the optimum conditions for in vitro development of plantlets. This is the first report on ex vitro rooting of in vitro regenerated shoots in H. enneaspermus. The widespread application of in vitro regeneration tech- nologies is restricted by the difficulties during transfer of plantlets to the field conditions (Pospíšilová et al. 1999). This is due to the sudden change in the culture environment to relatively harsh environments. The ultimate success of micro- propagation depends on successful hardening and field trans- fer of plantlets. The plants micropropagated in a culture vessel are partially heterotrophic; they acquire some developmen- tal changes to make them fully autotrophic after being trans- ferred to the field. The present study also aimed to investi- gate the foliar epidermal micromorphological changes during transfer of plantlets from an in vitro to a field environment. Materials and methods Plant material and surface sterilization Hybanthus enneaspermus was selected from the Coro- mandel Coast (Kanchipuram, Villupuram, Puducherry, Cuddalore, Nagapattinam and Karaikal districts) of India for the present study. Slender young emerging stems were used as the source of explants. The nodal segments (approximate- ly 3.0 cm in length) were harvested from two months old field-grown plant using sterilized surgical scissors. These ex- plants were sterilized with a systemic fungicide (0.1% Bavis- tin; BASF India Ltd., India) and then under laminar air flow bench with 0.1% HgCl2 (w/v) for 4–5 min. The sterilized ex- plants were washed with autoclaved double distilled water 5–6 times to remove the adhered traces of HgCl2. Medium and culture conditions Murashige and Skoog medium (Murashige and Skoog 1962) augmented with 3% sucrose as carbon source and 50 mg L–1 of ascorbic acid and 25 mg L–1 each of arginine, ade- nine sulphate and citric acid were incorporated in the culture medium as additives to initiate the cultures. Culture medi- um was solidified by 0.8% Agar (Hi-Media, India) to sup- port the proper position of the plant material in the medium. The pH of the medium along with plant growth regulators was adjusted to 5.8±0.02 prior to autoclaving. The cultures were maintained at 25±2 °C under a 12 h photoperiod light regime with a light intensity of 40–50 μmol m−2 s−1 photo- synthetic photon flux density (PPFD) implemented by cool white fluorescent lamps (Philips, India). Culture initiation and multiple shoot induction To establish cultures in vitro, stout, green nodal explants were inoculated on MS medium containing different con- centrations of cytokinins (6-benzylaminopurine, BAP; ki- netin, Kin) (Hi-Media, India) ranging from 0.5–3.0 mg L–1 to induce bud break. Cultures showing bud break were further multiplied by subsequent transfer of in vitro regenerated axillary shoot clumps (5–7 shoots) with mother explants by subcultur- ing onto fresh MS medium. The medium was supplement- ed with additives, BAP and Kin (0.1 to 2.0 mg L–1) alone or combinations of optimized concentrations. Subculturing was performed at 4 weekly intervals. In vitro rooting Shoots can be harvested after 3–4 subcultures for root- ing experiments. For root induction under in vitro condi- tions, multiplied shoots longer than 4–5cm were separated individually from shoot clumps and transferred to different strengths of MS media (full MS, ½ MS and ¼th MS) with various concentrations of auxins (indole-3 acetic acid, IAA; indole-3 butyric acid, IBA; α-naphthalene acetic acid, NAA and naphthoxy acetic acid, NOA) (Hi-Media, India) (1.0 to 4.0 mg L–1). The cultures were initially incubated under dif- fused light conditions (20–25 μmol m−2 s−1) for 2–3 days for in vitro root induction and thereafter transferred to an in vi- tro culture environment, and maintained at a light intensity of 40–45 μmol m−2 s−1 PPFD with a 12 h photoperiod per day. Ex vitro rooting of in vitro regenerated shoots Experiments were carried out for ex vitro root induction from in vitro-produced shoots. Basal end (4–6 mm) of in vi- SHEKHAWAT M. S., MANOKARI M. 82 ACTA BOT. CROAT. 77 (1), 2018 tro-raised shoots were treated with different concentrations of auxins (IAA, IBA, NAA and NOA) (50–400 mg L–1) for 5 min and transferred to eco-friendly paper cups containing sterile soilrite® (a combination of perlite with peat moss and exfoliated vermiculite procured from KelPerlite, Bangalore, India) and moistened with one fourth strength of MS basal salts. The cups were kept in the greenhouse for maintenance at 25±2 °C with 80–90% relative humidity (RH). After 4–5 weeks, the rooted plantlets were carefully taken out from the paper cups and transplanted to the nursery polybags in a greenhouse. Acclimatization and field transfer of regenerated plantlets In vitro rooted plantlets were taken out cautiously from the culture tubes and rinsed with distilled water to remove adhered nutrients and agar. They were transferred to auto- claved soilrite® in bottles, moistened with 1/4th strength of MS basal salts and maintained in the greenhouse. The ex vi- tro rooted plantlets were acclimatized in paper cups which were covered with transparent polythene cups to provide enough space for gas exchange. The in vitro rooted plantlets were acclimatized by gradual loosening and then complete- ly removing the transparent cup of the bottles. These plant- lets were subsequently transferred to nursery polybags con- taining soilrite®, garden soil and organic manure (1:1:1) in the greenhouse for further acclimatization process. Plantlets were transferred to the field after 5 weeks of acclimatization. Foliar micromorphological studies of in vitro and field transferred plantlets Experiments were conducted to study foliar micromor- phological developments of veins (vein density and venation pattern), stomata types and density, and trichomes in leaves of plants grown in vitro after 4th subculture in multiplica- tion phase and in those transferred to the field after 6th week. Plants were randomly selected from both the environments. The entire foliar apparatus (leaves) (10 from each stage of plantlets) third to seventh leaves from the base were excised manually for all the experiments. To observe the changes in structure and functioning of developing stomata, epidermal peels were separated manually by the traditional method (Jo- hansen 1940) from the leaves. The leaves were fixed primar- ily in formalin–acetic acid–ethyl alcohol, FAA (1:1:3) and cleared in 70% ethanol (v/v) until the chlorophyll was re- moved (12–24 h), bleached with 5% (w/v) NaOH for 24–48 h, and rinsed three times in distilled water (Sass 1940). The leaves were then stained with 1% safranine (Loba chemie, In- dia) aqueous solution for 4–8 min and rinsed carefully in wa- ter to remove excess stain and then mounted in distilled water and examined under microscope (Labomed iVu 3100, USA). Experimental design, data collection and statistical analysis The experiments were performed with 20 replicates per treatment and repeated thrice. Data were subjected to anal- ysis of variance and the significance of differences was cal- culated by Duncan’s multiple range test using SPSS software (version 16.0). Observations were noted at 4 weeks interval. Results Establishment of cultures and multiplication of cultures in vitro The fresh and light green colored nodal segments re- sponded better than old and dark colored explants. Cultures were initially placed in diffused light (20–25 μmol m−2 s−1) to induce bud breaking, and further transferred to a culture room with a higher light intensity (40–50 μmol m−2 s−1) for proper establishment of culture. The MS medium supple- mented with 1.5 mg L–1 BAP was observed suitable for bud breaking and 97% of the explants responded with 6.4±0.69 shoots from each nodal explants (Fig. 1A, Tab. 1). Maximum Tab. 1. Effect of different concentrations of cytokinins, 6-benzyl- aminopurine (BAP) and kinetin (Kin) on bud breaking from nodal explants of Hybanthus enneaspermus. Significant variation between the concentrations was studied using Duncan’s multiple range test at 0.5% level, SD – standard deviation. Cytokinins (mg L–1) Response (%) Number of shoots (mean ± SD) Shoot length (cm) (mean ± SD) 0.00 0 0.0±0.00a 0.00±0.00a BAP 0.50 32 2.3±1.06abc 3.12±0.31bcd 1.00 56 4.2±0.28bcd 3.40±0.18cd 1.50 97 6.4±0.69d 5.60±0.49e 2.00 76 4.9±1.02cd 4.16±0.38de 2.50 50 4.1±0.57bcd 3.28±0.33cd 3.00 37 2.3±1.09abc 2.06±0.12bc Kin 0.50 26 2.1±0.54abc 1.32±0.43ab 1.00 41 2.9±0.76bc 2.07±0.64bc 1.50 68 3.0±0.43ab 2.78±0.21bcd 2.00 59 3.6±0.47bc 3.08±0.30bcd 2.50 50 2.0±0.53ab 3.12±0.36bcd 3.00 40 2.1±0.49ab 2.17±0.17bc Fig. 1. Micropropagation of Hybanthus enneaspermus: induction of shoots from the nodal segments with 6-benzylaminopurine (A); multiplication of shoots on MS medium (B); in vitro rooting of the excised shoots on MS medium with indole-3 butyric acid (C); ex vitro rooted plantlets (D), scale bars = 2 cm. MICROPROPAGATION OF HYBANTHUS ENNEASPERMUS ACTA BOT. CROAT. 77 (1), 2018 83 Tab. 2. Effect of different concentrations and combinations of cy- tokinins 6-benzylaminopurine (BAP), kinetin (Kin), and combina- tion of BAP + Kin on shoot multiplication of Hybanthus enneasper- mus. Significant variation between the concentrations was studied using Duncan’s multiple range test at 0.5% level, SD – standard deviation. Cytokinins (mg L–1) Number of shoots (mean±SD) Shoot length (cm) (mean±SD) 0.00 0.0±0.00a 0.00±0.00a BAP 0.10 14±0.10ab 4.0±0.54b 0.50 25±0.93abc 6.4±0.32hi 1.00 42±1.51d 6.6±0.23i 1.50 40±7.37d 5.3±0.17ef 2.00 34±6.24cd 6.4±0.62hi Kin 0.10 19±8.91bc 5.0±0.44de 0.50 22±6.74bc 4.3±0.33bc 1.00 36±5.13cd 4.6±0.63cd 1.50 29±7.11abc 5.1±1.70def 2.00 20±8.83bc 4.9±0.54de BAP + Kin 0.10 98±8.76e 5.6±1.30fg 0.50 172±9.27g 5.9±1.05gh 1.00 228±10.3h 7.5±0.43j 1.50 121±10.1f 6.1±0.79ghi 2.00 93±9.22e 5.1±0.43def Ex vitro rooting of in vitro produced shoots The basal end of in vitro-produced micro-shoots was treated (5 min) with root-inducing growth regulators and transferred to a greenhouse environment. The lower part (4– 6 mm) of in vitro-regenerated shoots evaluated with 300 mg L–1 IBA exhibited about 96% rooting (Tab. 4). A maximum of 12.5±0.54 roots per shoot with 5.10±0.62 cm length was observed within 4 weeks (Fig. 1D). Poorer rooting than with IBA was recorded with all the concentrations of IAA, NAA, and NOA in this study. Acclimatization and field transfer of regenerated plantlets In vitro and ex vitro rooted plants were acclimatized effi- ciently in a greenhouse (Figs. 2A–2C). A profusely branched root system was observed in ex vitro rooted plantlets during transfer to the field. It resembled the conventional root sys- tem obtained under natural conditions. The hardened plant- lets were successfully transferred to the field with 92% sur- vival rate (Fig. 2D) but the survival rate of in vitro rooted plantlets was only 86%. Micromorphological studies of micropropagated plantlets The plants developed under in vitro conditions possessed normal leaves with hairs and denticulate margins. The mid- Tab. 3. Effect of auxins indole-3 acetic acid (IAA), indole-3 butyric acid (IBA), α-naphthalene acid acid (NAA), and naphthoxy acetic acid (NOA) on in vitro root induction, number and length of roots on half strength MS medium. Significant variation between the concentrations was studied using Duncan’s multiple range test at 0.5% level, SD – standard deviation. Auxins (mg L–1) Response (%) Number of roots (mean±SD) Length of root (cm) (mean±SD) 0.00 0 0.00±0.00a 0.0±0.00a IAA 1.0 26 12.9±0.16d 2.98±0.83h 2.0 40 16.6±10.75h 3.00±0.38h 3.0 34 16.0±1.56gh 2.35±0.33f 4.0 31 14.8±0.40f 2.05±0.20de IBA 1.0 63 18.5±3.43i 2.15±0.26e 2.0 98 25.7±3.90k 6.21±0.78k 3.0 71 21.0±2.37j 4.18±0.39j 4.0 59 13.7±1.70e 3.24±0.49i NAA 1.0 29 10.5±0.80b 1.78±0.13c 2.0 33 13.6±0.75de 2.90±0.23h 3.0 49 15.9±0.23gh 1.90±0.91cd 4.0 32 11.5±0.10c 1.95±0.26cd NOA 1.0 30 11.9±1.43c 2.71±0.83g 2.0 42 13.6±1.90de 3.32±0.38i 3.0 54 15.3±2.37fg 2.93±0.33h 4.0 50 10.5±1.20b 1.43±0.20b response of the explants was observed on MS medium aug- mented with BAP rather than with Kin in present study. The maximum number of shoots (228±10.3 shoots with 7.5±0.43 cm length) was regenerated on a subculture of the in vitro regenerated shoot clumps on MS medium fortified with 1.0 mg L–1 each of BAP and Kin within 4–5 weeks (Tab. 2). The shoot number and shoot length was increased by repetitive subculturing up to 3–4 passages onto fresh me- dium (Fig. 1B). Six fresh shoots from a single explant trans- ferred to fresh medium yielded a maximum of 228 shoots (228/6=38) within 4–5 weeks. The rate of shoot multiplica- tion increased more than 35 fold in 4–5 weeks. Cream col- ored callus was observed from the basal part of the cultures if the medium was augmented with auxins (IAA and IBA) along with cytokinins. In vitro rooting of the shoots Among the different strengths of MS medium and aux- ins experimented for in vitro root induction, ½ strength MS medium supplemented with 2.0 mg L–1 IBA was observed best for in vitro rooting (Fig. 1C). Maximum number of roots (25.7±3.90) with highest length (6.21±0.78 cm) were record- ed with this medium combination (Tab. 3). Poor response with smaller root number and root length was reported on media fortified with IAA, NAA and NOA. SHEKHAWAT M. S., MANOKARI M. 84 ACTA BOT. CROAT. 77 (1), 2018 rib was fairly prominent projecting equally on both the sides, but bluntly conical on the adaxial side and hemispherical on the abaxial side. Veins and vein-islets were fewer in in vi- tro than in field transferred plantlets (Figs. 3A and 3B). The vein density and distinct vein-islets were increased during the hardening period, and became distinct, rhomboidal and rectangular in shape after field transfer of the plantlets. The stomata were more frequent in the inter-coastal ar- eas than in the coastal areas, facing all directions with ir- regular distribution. The stomatal frequency was greater in the in vitro environment than in the field transferred plants with anisocytic stomata predominating, but these were non- functional as they were always in open condition (Figs. 3C and 3D). Anisocytic (cruciferous), paracytic (rubiaceous), diacytic (caryophyllaceous), anomocytic (rununculaceous), anisotricytic, isotricytic, tetracytic, staurocytic, desmocyt- ic and pericytic stomata were observed in the in vitro pro- duced leaves. Anomocytic and pericytic stomata were oc- casionally observed in these leaves. The field-transferred plants possessed the aforementioned stomatal types except for the anomocytic and pericytic. Anisocytic and paracytic stomata were prominent but diacytic and desmocytic sto- mata were rare in this plant. Trichomes were simple, unicel- lular or uniseriate emerging from the epidermis. Under in vitro conditions, the trichomes were unicellular, less frequent and underdeveloped but these were fully developed in field transferred plants after 6 weeks (Fig. 3E). The uniseriate and unicellular hairs were frequent but the bicellular and tricel- lular hairs were occasionally observed after plantlets were transferred to the field (Fig. 3F). Adaxial surface possessed numerous shaggy trichomes, and the trichome density was found maximum in field transferred plantlets compared to Fig. 2. Hardening of Hybanthus enneaspermus plantlets in the greenhouse (A-C), and in vitro raised plantlets under field condi- tions (D). Fig. 3. Micromorphological studies of Hybanthus enneaspermus: venation pattern in leaves of in vitro shoots (A); and field plant (B); stomatal pattern in leaves of in vitro shoots (C), and field plant (D); and trichomes in leaves of in vitro shoots (E), and field plant (F). Tissues were stained with 1% safranine aqueous solution. Scale bars = 100 µm. Tab. 4. Effect of auxins indole-3 acetic acid (IAA), indole-3 butyric acid (IBA), α-naphthalene acid acid (NAA), and naphthoxy acetic acid (NOA) on ex vitro root induction from in vitro raised shoots. Significant variation between the concentrations was studied using Duncan’s multiple range test at 0.5% level, SD – standard deviation. Auxins (mg L–1) Response (%) Number of roots (mean±SD) Length of root (cm) (mean± SD) 0.00 2 0.43±0.13a 1.21±0.32b IAA 50 30 0.45±0.30a 1.19±0.54b 100 45 0.62±0.11e 2.56±0.36h 200 51 1.93±0.15f 4.01±0.44m 300 50 3.22±0.32f 3.92±0.19l 400 43 2.07±0.28cd 2.05±0.30g IBA 50 35 0.96±0.21c 3.15±0.73k 100 42 2.78±0.59h 3.84±0.61l 200 56 4.89±0.41i 4.10±0.49n 300 96 12.5±0.54l 5.10±0.62p 400 84 5.83±0.49j 4.25±0.53o NAA 50 23 0.39±0.33a 1.03±0.69a 100 35 0.73±0.21b 1.41±0.74d 200 47 2.73±0.19h 2.05±0.21g 300 44 6.21±0.48l 3.91±0.28l 400 39 6.10±0.30k 3.01±0.35j NOA 50 33 0.11±0.45c 1.33±0.30c 100 47 0.29±0.29d 1.49±0.49e 200 56 2.39±0.16g 1.90±0.24f 300 41 3.26±0.72h 2.94±0.92j 400 38 2.30±0.64d 2.70±0.62i MICROPROPAGATION OF HYBANTHUS ENNEASPERMUS ACTA BOT. CROAT. 77 (1), 2018 85 the in vitro grown leaves. Mucilaginous cells were also ob- served in field transferred plants but these were totally absent in the in vitro grown plantlets. Discussion The success of tissue culture experiments basically de- pends on the selection of starting material. The mature ex- plants responded later than the fresh and light green col- ored nodal segments under diffused light conditions in the present experiment. BAP induced more shoots on MS me- dium than Kin. Similar results were reported by many re- searchers recently in a number of plant species (Panwar et al. 2012, Premkumar et al. 2013, Rathore et al. 2013a, Sud- harson et al. 2014). Shoot multiplication was achieved by re- petitive transfer of mother explants with regenerated shoots onto fresh medium and by subculturing of freshly regener- ated shoots isolated from the mother explants. This approach of shoot multiplication has been used in several plant spe- cies (Rai et al. 2010, Patel et al. 2014, Shekhawat and Mano- kari 2016). The higher rate of shoot multiplication during repeated transfer may be due to inhibition of apical domi- nance which stimulates the basal dormant meristematic cells to produce young shoots (Phulwaria et al. 2013). A maxi- mum of 228 shoots were induced per culture vessel within 4–5 weeks in this study. Premkumar et al. (2013) induced the most (52.3) shoots, when the regenerated shoots were subcultured on MS medium containing IAA along with Kin and BAP. Contrary to this report, callus formation was ob- served when the medium was supplemented with IAA and IBA along with BAP and Kin in the present study. Sudhar- son et al. (2014) reported maximum of 11.8 shoots, when the cultures of H. enneaspermus were inoculated on MS medium supplemented with 2.0 mg L–1 BAP. Maximal 90 shoots were reported in this plant by Velayutham et al. (2012) from the callus cultures on MS medium augmented with BAP and Kin. This supports our findings where the most shoots were regenerated on BAP and Kin, but our results were far better than the earlier reports in multiple shoot formation. The shoots were rooted maximally on half strength MS medium augmented with IBA. The half strength MS salts and sucrose in medium was appropriate for in vitro root- ing and supports many authors’ findings in different plant species (Rai et al. 2010, Premkumar et al. 2013, Patel et al. 2014). We report more roots (25.7±3.90) per shoot in this study than were found in earlier works on H. enneaspermus. Maximum 2.8 roots per shoot was reported by Prakash et al. (1999), 5–8 roots by Velayutham et al. (2012) and 21.3 roots by Premkumar et al. (2013) in this plant species. The superiority of IBA over other auxins for root induction has been recognized by several researchers in a number of plants (Barreto and Nookaraju 2007, Rai et al. 2010, Rathore et al. 2013b). Plants rooted under ex vitro environment were bet- ter suited to natural conditions and reported easy to hard- en (Yan et al. 2010). It has been reported that ex vitro root- ed plants are better suited to tolerate environmental stresses (Pospíšilová et al. 1999, Tiwari et al. 2002, Shekhawat et al. 2015a). About 96% shoots were rooted with IBA with maxi- mum 12.5 roots per shoot in this report. IBA is more effec- tive than NAA and NOA in ex vitro root induction in many plant species, and applied economically worldwide (Debergh et al. 1992, Yan et al. 2010, Ranaweeraa et al. 2013, Shekha- wat et al. 2015b). This is the first report on ex vitro rooting of in vitro regenerated shoots in H. enneaspermus with maxi- mum rate of survival under natural conditions. The rooted plantlets were hardened in greenhouse with development of profusely branched root system in ex vitro rooted plantlets. About 92% ex vitro rooted and 86% in vitro rooted plantlets survived in the field conditions. Ex vitro rooting reduced the time, energy of production of plantlets and mortality during hardening and field transfer. Normal flowering and fruiting was observed in the field transferred plantlets. These micromorphological studies of micropropagated plantlets were performed to understand the developmental changes in the leaves of plantlets, when they were transferred to field conditions. The stomata were present on both surfac- es of the leaf but the frequency was less on the adaxial sur- face (Narayanaswamy et al. 2006, Retnam and Britto 2007) therefore, the abaxial surface was further considered for the study. The specific artificial conditions in vitro are respon- sible for the structural changes occurring in micropropagat- ed plantlets. The lesser stomatal density under field condi- tions may help to check the rate of transpiration and prevent water loss (Singh et al. 2003). Transitional types of stomata between anisocytic and paracytic are also present in H. en- neaspermus (Inamdar 1969). Anisotricytic and isotricytic stomata could be the transitional form between anisocyt- ic and paracytic types of stomata. Unicellular, less frequent and underdeveloped trichomes were observed under in vitro conditions but fully developed trichomes were reported in field-transferred plants. The mucilaginous cells were not ob- served with the in vitro leaves but found in field-transferred plants. Our findings are supported by the results of various researchers (Chandra et al. 2010, Rathore et al. 2013b, Lodha et al. 2015). Understanding the changes in foliar micromor- phology of in vitro grown and hardened plantlets could be useful in improvement of in vitro clonal propagation proto- cols and for large scale production of plants. Conclusion An efficient in vitro propagation protocol has been de- veloped using various plant growth regulators for successful conservation of this rare plant species. Excellent rate of shoot multiplication was achieved in vitro. Ex vitro rooting has been successfully demonstrated in H. enneaspermus, which could save time, labor and energy in production of plantlets. The hardened plantlets were successfully transferred to the field with a 92% survival rate. The results of foliar micromor- phological studies could help in understanding the response of plantlets under field conditions. The data could contribute significantly to meeting the market demand for this multipo- tent healing herb and conservation of this valuable genotype through biotechnological interventions. SHEKHAWAT M. S., MANOKARI M. 86 ACTA BOT. CROAT. 77 (1), 2018 Acknowledgements The authors are grateful to the University Grants Com- mission, New Delhi, Government of India and the Depart- ment of Science, Technology and Environment, Government of Puducherry for providing financial support to their labo- ratory as Major Research Project and Grant–In-Aid Scheme respectively. References Anand, T., Gokulakrishnan, K., 2012: Phytochemical analysis of Hybanthus enneaspermus using UV, FTIR and GC-MS. IOSR Journal of Pharmacy 2, 520–524. Arumugam, N., Sasikumar, K., Malipeddi, H., Sekar, M., 2011: Antifungal activity of Hybanthus enneaspermus on wet clothes. International Journal of Research in Ayurveda and Pharmacy 2, 1184–1185. Arunkumar, B. S., Jayaraj, M., 2011: Rapid In vitro callogenesis and phytochemical screening of leaf and leaf callus of Ionidi- um suffruticosum, Ging. – A seasonal multipotent medicinal herb. World Journal of Agricultural Sciences 7, 55–61. Awobajo, F. O., Olatunji-Bello, I. I., Adegoke, O. A., Odugbemi, T. O., 2009: Phytochemical and antimicrobial screening of Hy- banthus enneaspermus and Paquentina nigricense. Recent Re- search in Science and Technology 1, 159–160. Barreto, M. S., Nookaraju, A., 2007: Effect of auxin types on in vitro and ex vitro rooting and acclimatization of grapevine as influenced by substrates. Indian Journal of Horticulture 64, 5–11. Baskaran, P., Van Staden, J., 2013: Rapid in vitro micropropaga- tion of Agapanthus praecox. South African Journal of Botany 86, 46–50. Chandra, S., Bandopadhyay, R., Kumar, V., Chandra, R., 2010: Ac- climatization of tissue cultured plantlets: from laboratory to land. Biotechnology Letters 32, 1199–1205. Debergh, P., Aitken-Christie, J., Cohen, D., Grout, B., Arnold von, S., Zimmerman, R., Ziv, M., 1992: Reconsideration of the term vitrification as used in micropropagation. Plant Cell Tissue and Organ Culture 30, 135–140. Inamdar, J. A., 1969: Epidermal structure and development of sto- mata in vegetative and floral organs of Hybanthus enneasper- mus (Linn.) F. Muell. Biologia Plantarum 11, 248–255. Johansen, D. A., 1940: Plant microtechnique. McGraw Hill Co., New York. Joseph, T. S.,. Skaria, B. P., Sajithakumari, 2000: Disappearing me- dicinal plant resources of Kottayam district of Kerala State. In- dian Journal of Areca Nut, Spices and Medicinal Plants 2, 79–81. Kirtikar, K. R., Basu, B. D., 1991: Indian medicinal plants. Vol I. Periodical Experts Book Agency, Delhi, India. Krishnamoorthy, B. S., Nattuthurai, Logeshwari, R., Dhaslima, N., Syedali, H., Fathima, I., 2014: Phytochemical study of Hyban- thus enneaspermus (Linn.) F. Muell. Journal of Pharmacogno- sy and Phytochemistry 3, 6–7. Kumar, S. B., Vijaya Kumar, J., Selvaraj, R., 2013: Aphrodisiac ac- tivity of Cycas circinalis and Ionidium suffruticosum Ging. on male wister albino rat. Asian Journal of Pharmaceutical and Clinical Research 6, 215–217. Lodha, D., Pate, A., Shekhawat, N. S., 2015: A high-frequency in vitro multiplication, micromorphological studies and ex vitro rooting of Cadaba fruticosa (L.) Druce (Bahuguni): a multi- purpose endangered medicinal shrub. Physiology and Mo- lecular Biology of Plants 21, 407. Murashige, T., Skoog, F., 1962: A revised medium for rapid growth and bioassays with tobacco cultures. Physiologiae Plantarum 15, 473–497. Murch, S. J., Choffe, K. L., Victor, J. M. R., Slimmon, T. Y., Raj, K., Saxena, P. K., 2000: Thiazuron-induced plant regeneration from hypocotyls cultures of St. John’s wort (Hypericum perfo- ratum L. cv. Anthos). Plant Cell Reports 19, 576–581. Narayanaswamy, V. B., Kumar, D. C., Setty, M. M., Shirwaikar, A., 2006: Histological and physico-chemical evaluation of Hy- banthus enneaspermus (L.) F. Muell. Natural Products Science 12, 104–108. Nathiya, S., Selvi, S. R., 2013: Anti-infertility effect of Hybanthus enneaspermus on endosulfan induced toxicity in male rats. International Journal of Medicine and Biosciences 2, 28–32. Panwar, D., Ram, K., Harish, Shekhawat, N. S., 2012: In vitro prop- agation of Eulophia nuda Lindl. – an endangered orchid. Sci- entia Horticulturae 139, 46–52. Patel, A. K., Phulwaria, M., Rai, M. K., Gupta, A. K., Shekhawat, S., Shekhawat, N. S., 2014: In vitro propagation and ex vitro rooting of Caralluma edulis (Edgew.) Benth. & Hook. f.: an en- demic and endangered edible plant species of the Thar Desert. Scientia Horticulturae 165, 175–180. Phulwaria, M., Rai, M. K., Patel, A. K., Kataria, V., Shekhawat, N. S., 2013: A genetically stable rooting protocol for propagat- ing a threatened medicinal plant Celastrus paniculatus. AoB Plants 5, pls054. Pospíšilová, J., Ticha, I., Kadleck, R., Haisel, D., Plzakova, S., 1999: Acclimatization of micropropagated plants to ex vitro condi- tions. Biologia Plantarum 42, 481–497. Prakash, E., Sha Valli, K. P. S., Sairam, R. P., Rao, K. R., 1999: Re- generation of plants from seed-derived callus of Hybanthus enneaspermus L. Muell., a rare ethnobotanical herb. Plant Cell Reports 18, 873–878. Premkumar, G., Arumugam N., Muthuramkumar, S., Varathara- ju, G., Rajarathinam, K., 2013: Improved micropropagation in Hybanthus enneaspermus L. Muell. American Journal of Plant Sciences 4, 1169–1172. Rai, M. K., Asthana, P., Jaiswal, V. S., Jaiswal, U., 2010: Biotechno- logical advances in guava (Psidium guajava L.). Recent devel- opments and prospects for further research. Trees Structure and Function 24, 1–12. Ranaweeraa, K.K., Gunasekarab, M. T. K., Eeswara, J. P. 2013: Ex vitro rooting: a low cost micropropagation technique for tea (Camellia sinensis (L.) O. Kuntz) hybrids. Scientia Horticul- turae 155, 8–14. Rathore, M. S., Dagla, H. R., Singh, M., Shekhawat, N. S., 2008: Rational development of in vitro methods for conservation, propagation and characterization of Caralluma edulis. World Journal of Agricultural Science 4, 121–124. Rathore, M. S., Rathore, M. S., Shekhawat, N. S., 2013a: Ex vivo im- plications of phyto-hormones on various in vitro responses in Leptadenia reticulata (Retz.) Wight. and Arn. – an endangered plant. Environmental and Experimental Botany 86, 86–93. Rathore, N. S., Rathore, N., Shekhawat, N. S., 2013b: In vitro prop- agation and micromorphological studies of Cleome gynandra: a C4 model plant closely related to Arabidopsis thaliana. Acta Physiologiae Plantarum 9, 2691–2698. Retnam, R. K., Britto, A. J. de, 2007: Antimicrobial activity of a medicinal plant Hybanthus enneaspermus (L.) F. Muell. Natu- ral Product Radiance 6, 366–368. Sass, J. E., 1940: Elements of botanical microtechnique. McFraw- Hill Book Co. New York and London. Satheeshkumar, D., Kottai, M. A., Manavalan, R., 2011: Antioxi- dant potential of various extracts from whole plant of Ionidi- um suffruticosum (Ging). Research Journal of Pharmaceutical, Biological and Chemical Sciences 2, 286–293. MICROPROPAGATION OF HYBANTHUS ENNEASPERMUS ACTA BOT. CROAT. 77 (1), 2018 87 Shekhawat, M. S., Kannan, N., Manokari, M., 2015a: In vitro prop- agation of traditional medicinal and dye yielding plant Morin- da coreia Buch. -Ham. South African Journal of Botany 100, 43–50. Shekhawat, M. S., Kannan, N., Manokari, M., Ravindran, C. P., 2015b: Enhanced micropropagation protocol of Morinda citrifolia L. through nodal explants. Journal of Applied Re- search on Medicinal and Aromatic Plants 2, 174–181. Shekhawat, M. S., Manokari, M., 2016: In vitro propagation, mi- cromorphological studies and ex vitro rooting of Alternan- thera philoxeroides (Mart.) Griseb.: an important aquatic plant. Aquaculture International 1, 423–435. Singh, I. P., Parthasarathy, V. A., Handiqu, P. J., 2003: Comparative growth of micropropagated plantlets and seedlings of citrus varieties. Agrotropica 15, 9–16. Subramoniam, A., Madhavachandran, V., Gangaprasad, V., 2013: Medicinal plants in the treatment of arthritis. Annals of Phytomedicine 2, 3–36. Sudeesh, S., 2012: Ethnomedicinal plants used by Malyaraya tribes on Vannapuram village in Idukki, Kerala, India. Indian Jour- nal of Science and Technology 1, 7–11. Sudharson, S., Anbazhagan, M., Balachandran, B., Arumugam, K., 2014: Effect of BAP on in vitro propagation of Hybanthus enneaspermus (L.) F. Muell., an important medicinal plant. International Journal of Current Microbiology and Applied Sciences 3, 397–402. Tiwari, S. K., Tiwari, K. P., Siril, E. A., 2002: An improved micro- propagation protocol for teak. Plant Cell Tissue and Organ Culture 71, 1–6. Tripathy, S., Sahoo, S. P., Pradhan, D., Sahoo, S., Satapathy, D. K., 2009: Evaluation of anti-arthritic potential of Hybanthus en- neaspermus. African Journal of Pharmacy and Pharmacol- ogy 3, 611–614. Velayutham, P., Karthi, C., Nalini, P., Jahirhussain, G., 2012: In vi- tro regeneration and mass propagation of Hybanthus ennea- spermus (L.) F. Muell. from the stem explants through cal- lus culture. Journal of Agricultural Technology 8, 1119–1128. Verma, S., Singh. S. P., 2011: Current and future status of herbal medicines. Vet World 1, 347–350. Yan, H., Liang, C., Yang, L., Li. Y., 2010: In vitro and ex vitro root- ing of Sratia grosvenorii – a traditional medicinal plant. Acta Physiologiae Plantarum 32, 115–120.