Taxonomic History

The taxonomic history of Decalepis began with the naming of D. hamiltonii by Robert Wight and George Arnott Walker-Arnott in Wight’s “Contributions to the Botany of India” (1834), based on a specimen collected by Wight in Madras, India [11]. Walker-Arnott, who was collaborating with Wight, upon observing what he referred to as “a double row of scales” (referred to in this study as “corona lobes” and “interstaminal nectaries”), placed the new species in its own genus, Decalepis (Greek deca=ten, lepis =scale) [11]. Wight mistakenly referred to the specimen in an earlier, unpublished version of his manuscript as being from the herbarium of the Scottish physician and botanist Francis Hamilton; Walker-Arnott chose to honor Hamilton with the specific epithet hamiltonii despite the fact that the specimen was from the Madras Herbarium and not from Hamilton’s collection [4].

Habit and Habitat

Three species of Decalepis (D. hamiltonii, D. nervosa and D. khasiana) are scandent to climbing twiners, the commonest growth habit among Periplocoideae [13], while two (D. salicifolia and D. arayalpathra) are erect shrubs, a condition that has evolved numerous times within the subfamily and is generally correlated with arid environments or those with seasonal water shortages [15]. Both D. salicifolia and D. arayalpathra are narrow endemics of poor soils in rocky hill slopes and rock fissures in deciduous and evergreen forests, at elevations above 600 m. However, the vining species of Decalepis tend to occupy habitats similar to those of the erect species, particularly the narrow endemic D. nervosa.

Botanical Description

Inota (2006) provided a comparative taxonomic detail of all the five species of Decalepis on the basis of 66 herbarium specimens from BM, CAL, E, K, LM, MO, NY, P and US [16] Figure 1.

Figure 1: Different plant parts of D. hamiltoniiA. Cylindrical tuberous roots excised from a five year old plant.B.Dry follicle with seeds.C.Seeds with silky hairs.D.Fresh seeds.E.Complete plant of D. hamiltonii planted in loamy soil-6 month old.

i. Roots: Three species have clusters of numerous, fleshy, tuberous roots with a sweet, vanilla-like fragrance: D. arayalpathra, D. hamiltonii and D. salicifolia. The tuberous roots of D. arayalpathra and D. salicifolia are moniliform, while those of D. hamiltonii are cylindrical. The roots of D. khasiana are documented as being non-tuberous, and fragrant due to an isomer of vanillin that is also present in the other fragrant-rooted species. No references were found pertaining to the root morphology or chemistry of D. nervosa [4].

ii. Stems: The stems are woody and reddish brown when young, tend to become purplish when older [12]. Lenticels are common on older branches. The stems of D. salicifolia are somewhat succulent, becoming quite wrinkled when dried and the older stems are covered with a grayish wax [4].

Cultivation

For D. hamiltonii, loamy soils are well suited for the growth but soils intermixed with stones encourage the production of long thick roots. The length and thickness of the roots mostly depends on the soils profile rather than the environment [17]. However the information regarding cultivation for other species of Decalepis is not well documented anywhere.

Propagation

All the species of Decalepis can be propagated from seeds as well asby means of stem cuttings. For commercial purpose the first method is preferred. As the direct sowing of seeds in the field does not give good results, seedlings are raised in the nursery before transplanting in the field. Collection of seeds from February-March is preferred to achieve higher viability percentage. Because of short viability, the fresh seeds are to be used. The seeds can be raised either in the nursery beds or in polythene bags. Germination starts after about 2 weeks andmay continue for another week. Three months old seedlings having 4-6 leaves are transplanted at a distance of 60 cm × 60 cm in field. Generally, That sentence will be replaced by this: the crop is cultivated under rain-fed conditions [17]. Seeds of D. hamiltonii were found to germinate soon after early monsoon showers in May on soil floor and rocky areas which are rich in litter and other organic matter. In nature, seed germination rate is 13% out of which seedling establishment rate is 48%. Thus, the overall seedling establishment rate in relation to total seeds sown is only 6% [18]. Anandalakshmi and Prakash [19] have studied the viability and germination of D. hamiltonii seeds under in vitro conditions. They have suggested that the use of moist filter papers was better substrate for germination (83 to 98%) than sand (15 to 28%) after pre-soaking of seeds in hot water (60°C) for 24 hours. Pretreatment study coupled with microscopic studies indicated that about 14% of seeds were found to be hard seeded. Seeds could tolerate desiccation up to 10% moisture content (65% germination) while nil germination was recorded at 5% seed moisture content. The seeds were found to be chilling sensitive, thus showing total mortality at 10°C. Rapid depletion of seed metabolites is indicative of short viability. Based on the storage behaviour, D. hamiltonii seeds could be classified as intermediate seeds. From seed perspective, hard seed coat, need for a well aerated substrate and short viability could be the likely impeding factors for inadequate regeneration of the species in wild. Saini and Giridhar [20] found that exposing seeds to 0.3% H₂O₂ resulted in effective (94%) seed germination in D. hamiltonii. While Vedavathy (2004) reported 100% germination of immature zygoticembryos when placed on MS medium supplemented with GA3 (0.05 ppm), BA (1.0 ppm) and TDZ (1.0 ppm).

Harvesting

The roots of D. hamiltonii come to maturity in about 12-14 months after planting depending upon the soil and climatic conditions. The roots can easily be harvested by digging around the plant leaving the central core of the root and stem for regeneration. Some plants could be used to harvest roots up to 4-5 years, as vigorous regeneration and growth resumed even with a single left over root. Each root is 5-10 cm in diameter and 4-10 roots regenerate from the rootstock. A 2-3 year old plant produces 15-20 kg of roots, while one year old plant produces 1-2 kg of roots. Roots are harvested during summer months mostly by the Yanadi tribe of Chittoor district and it is the main source of income to them until the agricultural works resumes.

Chemical constituents

The roots of D. hamiltonii have been reported to contain aldehyde, inositols, saponins, amyrins, lupeols [21,22]. Morphologically as well as chemically the plant resembles African liana called Mondia whitei. Both have similar ethno botanical uses and the presence 2-hydroxy-4-methoxy benzaldehyde (2H4MB), an isomer of vanillin is reported from the volatile oils (96%) [23]. Further GC-MS analysis of this oil showed, along with the major component, the presence of benzaldehyde (0.017%), salicylaldehyde (0.018%), methyl salicylate (0.044%), benzyl alcohol (0.016%), 2-phenylethyl alcohol (0.081%), ethyl salicylate (0.038%), p-anisaldehyde (0.01%), and vanillin (0.45%) in minor quantities which are biologically significant [23]. Hydro-distillation of D. hamiltonii roots yielded an essential oil (0.33% v/w) that contained 2H4MB (37.45%), 2-hydoxybenzaldehyde (31.01%), 4-O-methylresorcylaldehyde (9.12%) and benzyl alcohol (3.16%) as major constituents, with aromatic aldehydes constituting the main fraction of its roots’ essential oil [24]. Thangadurai et al. [25] isolated two other compounds from the root extract that potentially account for its insecticidal and anti-microbial properties: α-atlantone (2.06% v/w of oil), commonly used to control household insects and nontoxic to mammals, and β-pinene (2.01%), which is used as an insecticide and disinfectant. Whereas, Zhang et al. [26] isolated four new pregnane glycosides (Stelmatocryptonoside A, B, C and D) from the stems of D. khasiana; and four species of sariva (D. hamiltonii, Cryptolepis buchananii, Ichnocarpus frutescens and H. indicus) were shown to have potent anti-inflammatory properties [27].

A gas chromatographic (GC) procedure was developed by Nagarajan and Rao [28] for the assay of 2H4MB in both fresh and dried roots of D. hamiltonii from different origins. They used benzyl butyrate as an internal standard. Among the methods tried, steam hydrodistillation was found most suitable for extraction of the volatile oils. The quantity of this aromatic compound varied from 0.03 to 0.54% (Nagarajan and Rao 2003). In addition 4-hydroxyisopthalic acid, 14-aminiotetradecanoic acid, 4-(1-hydroxy-1-methylethyl)-1-methyl- 1,2-cylohexane diol, 2-(hydroxymethyl)-3-ethoxybenzaldehyde, 2,4,8-trihydroxybicycle (3,2,1) octan-3-one, bis-2,3,4,6-galloyl-α/β- D-glucopyranoside, bornerol and ellagic acid have been identified in D. hamiltonii root [29,30].

Traditional uses

Four of the five species of Decalepis (all except D. nervosa) are utilized in tribal and traditional Indian and Chinese medicine for the treatment of a wide range of ailments including those of the digestive system, lungs and circulatory system [31-33,26]. The principal organs used are the tuberous roots, which are fragrant and sweet tasting (fragrant but non-tuberous in D. khasiana), with a vanilla-like taste and odor [34,33,28,35]. D. hamiltonii, the type species, is the most widespread and utilized species in the genus.

Medicinal Importance

Numerous recent investigations have tested the medicinal properties of the different species of Decalepis supported the veracity of various traditional ethnomedicinal claims. As compared to other species, D. hamiltonii has been much evaluated for its medicinal properties as below;

i. Anti fungal activity: The antifungal activity of aqueous extract of D. hamiltonii was evaluated at different concentrations by poisoned food technique against 8 species of Fusarium, 10 species of Aspergillus, 3 species of Penicillium, 2 species of Drechslera and Alternaria alternata. [43]. Mohana et al. [44] found that the antifungal active principle is a phenolic compound, 2H4MB. In vitro antifungal activity assay of different concentrations of 2H4MB isolated from D. hamiltonii against 6 important seed-borne fungal pathogens, namely, A. alternata, Drechslera tetramera, Fusarium oxysporum, Fusarium proliferatum, Pyricularia oryzae and Trichoconis padwickii isolated from paddy seeds revealed that, the compound 2H4MB showed significant antifungal activity. Among the fungi tested, F. proliferatum showed the highest inhibitory activity, whereas P. oryzae showed least inhibitory activity [44].

ii. Antibacterial activity: Thangavel et al. [45] studied antibacterial potential of crude petroleum ether extract of leaf callus tissue of D. hamiltonii against 5 bacterial pathogens using agar well diffusion method. Among the tested bacterial strains, inhibitory activity of the callus extract was minimum against Klebsiella pneumoniae (5 mm) and followed by Proteus vulgaris (6 mm). Maximum inhibitory activity was observed against Salmonella typhi (11 mm). From this observation it was evident that phytochemical principles which are responsible for the curative (antibacterial) activity of D. hamiltonii could have a constant expression pattern in a specialized set of cells even after their rapid division. Later on Devi and Latha [46] also reported antibacterial activity of various root extracts of D. hamiltonii against E. coli, K. pneumoniae, S. typhi, Proteus mirabilis, Vibrio cholerae, Shigella sonnie, Serritias species, S. aureus and Bacillus subtilis by disc diffusion method.

iii. Insecticidal activity Several studies have been conducted to evaluate the insecticidal activity of D. hamiltonii against 3 coleopteran stored product pests,viz., rice weevil, the lesser grain borer and red-rust flour beetle [24,47].Further, the residual deposits of 2H4MB has been assayed for contact toxicity on rice weevil (Sitophilus oryzae), Rhyzopartha dominica and Tribolium castaneum. Thus, the aromatic chemicals of D. hamiltonii have been established with insecticidal and pesticidal properties.

iv. Anti-oxidant activity: Free radicals are implicated in the etiology of several diseases, such as cancer, antherosclerosis, diabetes, neurodegenerative disorder and aging. Antioxidants prevent the damage to macromolecules and cells by interfering with the free radicals. Natural products containing antioxidants from plants are believed to modulate oxidative stress and to prevent or delay degenerative disorders [48]. Harish et al. [29] characterized 6 pure compounds with strong antioxidant properties from the methanolic extract of D. hamiltonii roots by using MS, 1H NMR, 13C NMR and two-dimensional NMR spectroscopic techniques and including a novel antioxidant molecule, bis-2,3,4,6-galloyl-α/β D glucopyranoside (aptly named decalepin) from D. hamiltonii root extracts. Srivastava et al. [29] also reported antioxidant potential of the methanolic and aqueous extracts of D. hamiltonii roots. Murthy et al. [49] found that antioxidant activity was associated with 2H4MB. They subjected different parts of aromatic roots viz., whole tuber, peel, tuber without peel and medullary portion. Maximum antioxidant activity was found with peel extract followed by medullary Portion [49]. Later on, Srivastava et al. [50] isolated another antioxidant compound from the aqueous extract of the roots of D. hamiltonii and identified it as ellagic acid (EA), based on NMR and MS studies. Anti-typhoid activity Kumuda et al. [51] studied in vitro anti-typhoid activity of various roots extracts of D. hamiltonii. The powdered roots were extracted with various solvents viz., petroleum ether, chloroform, and ethyl acetate by Soxhlet extraction method and the extracts were tested on typhoid causing organisms. Among these, extracts of petroleum ether and chloroform showed a significant activity against S. Typhi (ATCC 14028), S. paratyphi A and S. paratyphi B respectively on comparison with Ciprofloxacin [51].

v. Ulcer preventive activity Srikanta [30] reported a pectic polysaccharide from D. hamiltonii roots containing a sulphonamide group and phenolics as an effective antiulcer compound in vivo. They envisaged a multi-potent role for this phenolic polysaccharide in the upregulation of mucin, antioxidant levels, modulation of oxidative status, inhibition of H⁺, K⁺-ATPase activity against swim and ethanol stress induced ulcers in experimental animal models, in addition to its ability to inhibit Helicobacter pylori. Roots containing phenolics at 0.12 g GAE/g, prevented stress-induced gastric ulcers in animal models by 80-85%. Histopathological analysis revealed protection to the disrupted gastric mucosal layer and epithelial glands. SRPP also inhibited H⁺, K⁺-ATPase in vitro, at an IC50 of 77 μg/mL as opposed to that of 19.3 μg/mL of Lansoprazole and H. pylori growth at Minimum Inhibitory Concentration (MIC) of 150 μg/mL. In addition, free radical scavenging (IC50-40 μg/mL) and reducing power (3200 U/g) activities were also observed. They also concluded that SRPP is non-toxic as opposed to other known antiulcer drugs, and therefore may be employed as a potential alternative for ulcer management.

vi. Anti-diabetic activity: Sumalatha et al. [52] evaluated the anti-diabetic activity of extracts of the D. hamiltonii in alloxan induced diabetic rats. The fall of blood glucose levels after administration of aqueous, methanol and petroleum ether extracts at a dose of 200 mg/ kg body weight were found to be 69.43, 62.04, and 49.61%, respectively, after 4 hours of oral administration. At the same dose the acute oral administration of aqueous extract showed significant decrease of blood glucose loaded normoglycemic rats. In vitro glucose uptake studies suggest that D. hamiltonii root extracts has direct insulin like effect which can enhance the peripheral utilization of glucose by rat hemi diaphragm.

vii. Antitumor activity: Cancer is a major aliment that affects several organs and chemothaerapy is widely practiced to treat the disease. Chemotherapy with or without radiation, although effective for many cancers, is accompanied by severe side effects due to the toxicity of the anticancer drugs. Several anticancer drugs are derived from plants such as taxol, cycolphosphamide, vincristine, vinblastineand there is great interest in the search for newer and safer drugs from plants. In this regard, Zarei and Shivanandappa [53] have shown the antitumor effect of D. hamiltonii root extract (DHA) against Ehrlich Ascites Tumor (EAT) cells in mice and compared its effect with that of the anticancer drug, cyclophosphamide (CP). Treatment of EAT cell bearing mice with aqueous extract of D. hamiltonii (50 mg/kg and 100 mg/kg b.w.) showed a significant reduction in ascites tumor volume, tumor cell counts and increased median survival time (MST). EAT cells induced alterations in hematological profile and the serum marker enzymes aspartate abd alanine transaminases (AST, ALT), alkine phosphatase (ALP) and lactate dehyrogenase (LDH) were restored by DHA treatment. The antitumor effect of DHA was comparable to that of the anticancer drug cyclophosphamide suggesting the anticancer potential of the root extract of D. hamiltoniii [53].

Table 1: In vitro shoot regeneraion using different explants of D. hamiltonii (chronological order).

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In vitro shoot regeneration (direct or indirect) and rootinduction

D. hamiltonii: For the first time, George et al. [62] studied in vitro growth and development of multiple shoots from leaf calli of D. hamiltonii. They used “Response Surface Methodology” (RSM) to evaluate the effect of BA, NAA and sucrose on multiple shoot induction and to develop an efficient micropropagation system. The optimum combination for maximum shoot regeneration (4.4 shoots) was 1.9 mg L-1 BA and 0.05 mg L-1 NAA. Shoot number exhibited a positive linear relationship with BA concentration and was maximum at the highest levels of BA, while sucrose had no significant effect. However, maximum shoot length was observed when sucrose concentration was maximum (3%) in the optimal medium. Bais et al. [63] used axillary bud for micropropagation and achieved maximum shoot number (12.8 shoots per culture), shoot length (5.8 cm) and number of leaves (16.4) on MS medium supplemented with BA (2.0 mg L-1) and NAA (0.5 mg L-1) after 30 days of incubation. Similarly, Anitha and Pullaiah [64] used nodal segments and reported maximum of 4 shoots per explant on MS medium supplemented with 17.76 μM BA and 0.53 μM NAA. Reddy et al. [57] studied the effect of triacontanol (TRIA) on shoot multiplication of in vitro derived shoot tips. Among different concentrations (2-20 μg L-1) of TRIA used, 20 μg L-1 TRIA was found to be most effective for shoot proliferation. While Giridhar et al. [65] found that the addition of phenylacetice acid (PAA) had a significant effect on shoot multiplication through nodal segments. The maximum number of shoots per culture (6.4 shoots) was produced on MS medium supplemented with 31.08 μM BA and 14.68 μM PAA, while the longest shoot length (4.5 cm) and nodes were obtained on MS medium supplemented with 22.2 μM BA and 14.68 μM PAA. Shoot subcultured on MS medium supplemented with 22.2 μM BA and 14.68 μM PAA elongated along with secondary shoot formation.

Similarly Gururaj et al. [66] found that phloroglucinol (PG) had synergistic effect on shoot multiplication though nodal segments when added with BA and GA3. Maximum number of shoots per culture was observed on MS medium containing 1.1 μM BA, 5.8 μM GA3 and 800 μM PG. Subculturing of the shoots onto MS medium containing optimum concentration of BA (5.6 μM), PG (200 μM) and TRIA (0.11 μM) produced elongated shoots along with secondary shoot formation. Later on, Giridhar et al. [67] compared the influence of different cytokinins (2-iP, BA, Kn, TDZ and zea) on shoot multiplication through shoot tips. Maximum number of multiple shoots (6.5) was noticed on MS medium supplemented with 4.9 μM 2-iP. Further elongation of shoots and adventitious shoot formation was obtained on MS medium with 2.5 μM 2-iP and 0.3 μM GA3 (Table 1).

Giridhar et al. [60] reported the improvement of 2H4MB of micropropgated plants under TRIA treatment. The microppropgated plants were raised from nodal explants on MS medium supplemented with 2.0 mg/l and 0.5 mg/l NAA in stage I and MS medium supplemented with 2.0 mg/l IBA in stage II by following the method reported earlier by Bais et al. [63]. They were then hardened in a green house for 2 months. These micropraogated plants of 12-15 cm shoot length were used in experiments. They observed improved plant growth and yield of tubers with enhanced flavor compound, by using TRIA soil drenching. The administration of TRIA (0.228 μM) soil drenching twice by giving second treatment with a gap of 60 days after initial treatment, showed fast sign of growth and good yield of tubes with enhanced flavor content. The GC-MS profile of 2H4MB in tubers showed significant improvement over controls (1.5 times more), which could be useful for various commercial application.

Giridhar et al. [68] analysed the production of 2H4MB in roots of tissue culture raised and acclimatized plants of D. hamiltonii. They used axillary buds excised from field grown plants to initiate multiple shoots on MS medium supplemented with 2 mg/l BA and 0.5 mg/l IAA. Profuse rooting was achieved when actively growing shoots were cultured on MS medium supplemented with 1.0 mg/l IBA. Regenerated plants were grown successfully in the plains, in contrast to wild growth in high altitudes and rocky crevices of hilly regions. Roots of different sizes from one-year-old tissue culture raised field grown plants had the same profile of 2H4MB as that of wild plants. A maximum of 0.14% and 0.12% 2H4MB was produced in roots of one year old tissue culture derived plants and greenhouse grown plants respectively.

The success of in vitro regeneration relies on an efficient rooting in regenerated shoots and their subsequent acclimatization which is often problematic in some plant species. Bais et al. [63] investigated in vitro rooting of D. hamiltonii by using silver nitrate (AgNO₃). They found that medium comprising of MS salts with IAA (2.88 μM), induced poor rooting and root emergence did not occur until after 25 days. The resultant roots were stunted. However, the addition of 40 μM AgNO₃ improved root initiation and elongation. The promotive effects of AgNO₃ on rooting may result from inhibition of ethylene action. It was noticed that the addition of ethephon to the rooting medium, excessive callusing was observed in explants in all the treatments. Addition of 40 μM AgNO₃ to ethephon containing medium resulted in improvement in root initiation and elongation. Ethylene production was monitored in all the treatments with IAA/ AgNO₃/ethephon and it was observed that the treatment with IAA (2.88 μM) alone showed a greater increase in ethylene production when compared with AgNO₃, ethephon and their combinations.

For efficient rooting of in vitro-derived microshoots by using phloroglucinol (PG), activated charcoal and CoCl₂ was investigated by Reddy et al. [58]. Transfer of in vitro-derived shoots to MS medium supplemented with 8.8 μM IBA and 1.43 μM IAA resulted in root induction. IBA was found to be a good root-promoting agent, dipping of explants in 4.4 μM IBA for 30 min and subsequent inoculation on MS basal medium was also beneficial for root induction Reddy et al. [58]. While, Anitha and Pullaiah [64] reported a maximum of 2 to 3 roots on MS medium containing 16.11 μM NAA. Whereas Reddy et al. [57] found that TRIA at 5 and 10 μg played an effective role in root induction in D. hamiltonii. Giridhar et al. [65] found that the shoots rooted on medium containing 9.8 μM IBA showed 80-90% survival rate under field conditions. While Gururaj et al. [66] found best rooting on 5.38 μM NAA and 400 μM PG containing MS medium. The efficient root formation was achieved when the elongated shoots of D. hamiltonii inoculated on medium supplemented with various phenolic compounds along with 9.8 μM IBA within 5-6 wk [65]. They found that PG and salicylic acid interaction with IBA stimulated maximum in vitro rooting of shoots [Table 2].

Table 2: Effect of different treatments on in vitro root induction of microshoots of D. hamiltonii (chronological order).

D. arayalpathra: There are only two reports for the micropropagation of D. arayalpathra in the year 2005. Gangaprasad et al. [59] cultured single node from young top shoots on MS medium supplemented with 0.1 to 5 mg/l BA. All the concentrations of BA induced single axillary shoots of varying length. However, MS medium supplemented with 0.1 mg/l BA supported rapid growth and produced the longest shoots (6.8 cm) in 60 days. For further multiplication, the nodes and shoot tips from in vitro derived shoots were re-cultured on MS medium fortified with 0.5 mg/l BA which produced 8 cm long shoot having 5-7 nodes in 30 days period. Further, the top microshoot cuttings (3-5 cm) with 2-3 nodes, subcultured onto MS medium supplemented with 1.5 mg/l IAA, produced an average of 6.3 roots in 30 days period. The rooted plants after hardening were reintroduced into their natural habitat at Kallar reserve forest, Thiruvananthapuram. After 2 years, 84% survival of reintroduced plants was recorded.

In another study of Sudha et al. [54] it was suggested that nodal explants of greenhouse-raised plants were more desirable than cotyledonary nodal explants of aseptic seedlings of D. arayalpathra. The basal nodes (73%) of 12-16-week-old greenhouse-grown plants cultured in MS medium containing 12.96 μM BA, 2.48 μM 2-iP and 2.68 μM NAA formed 16-17 cm long unbranched robust solitary shoots in 8 weeks. Cotyledonary nodal explants cultured in the same medium showed multiple shoot formation and axillary branching. But the shoots were thin, fragile and not suitable for mass propagation. Single nodes of a solitary shoot subcultured on MS medium containing 2.22 μM BA and 0.24 μM 2-ip together produced 9.8 nodes from 18.0 cm long shoots within 5-6 weeks. The basal nodes of the shoots so formed were repeatedly subcultured to increase the stock of propagules while 2.5-3.0 cm terminal cuttings were used for rooting. The best root induction (68%) and survival (86%) was achieved on half-strength MS medium supplemented with 1.07 μM NAA. Fieldestablished plants showed uniform growth and phenotypic similarity to parental stock [54].

Somatic Embryogenesis: As compared to the other species of Decalepis, somatic embryogenesis is only reported in D. hamiltonii through leaf culture by Giridhar et al. [69]. Nodular embryogenic callus was developed from the cut end of the explants on MS medium containing 2, 4-D and BA. On subsequent transfer of explants with primary callus on to the MS medium containing zea (13.68 μM) and BA (10.65 μM) resulted in the differentiation of somatic embryos directly from nodular tissue. The maturation of embryos took place on the same medium. The efficiency of somatic embryogenesis may enhanced when the embyrogenic calli with somatic embryos were subcultured onto MS basal medium supplemented with 4.56 μM zea and 10.65 μM BA. The mature embryos developed into complete plantlets on growth regulator-free MS medium.

Hydrogel Encapsulation: Encapsulation technology represents a new and powerful tool in the plant nursery field as well as in approaches to germplasm conservation and exchange of plant materials between laboratories [70]. Previously, synthetic seed or artificial seed (or synseed), described as ‘‘artificially encapsulated somatic embryos (bipolar structure) which can be used for sowing under in vitro or ex vitro conditions’’. But due to low success and high cost of somatic embryo production, shoot tips, nodes, bulbs or other meristematic tissue (unipolar structures) that can produce a whole plant may also be encapsulated which are also considered as synthetic seeds [71]. A wide range of woody plants has been re-grown from encapsulated shoot tips or nodes [72,73].

An efficient protocol has also been developed for short-term storage and conservation of D. hamiltonii using encapsulated nodal segments [74]. Only juvenile nodal segments, excised from aseptic seedlings were used for encapsulation. It was found that the encapsulation significantly affected by the concentrations of sodium alginate (Na-alginate) and calcium chloride (CaCl₂·2H₂O). A gelling matrix of 4% Na-alginate and 100 mM CaCl₂·2H₂O was most suitable for the production of ideal Ca-alginate beads. Maximum shoot regrowth (77.00%) was recorded on MS basal medium supplemented with 5.0 μM BA, 0.5 μM IAA and 30.0 μM adenine-sulphate (ADS) after 6 weeks of culture. Microshoots, recovered from encapsulated nodal segments (capsule) were best rooted on half-strength MS medium containing 2.5 μM NAA. Capsules of D. hamiltonii could be stored up to 8 weeks at low temperature (4°C) [75].

Being small in size the capsule, therefore can provide an effective tool for storage and exchange of this endemic and endangered plant species, potentially overcoming many of the difficulties associated with long-distance transport of plant germplasm. However, in this protocol rooting could not be possible in a single step on re-growth media which calls for further refinement in the established protocolin such a way that rooting could be achieved in a single step (on regrowth-medium).

Similar to the somatic embryogenesis, exploitation of encapsulation technique for the conservation of other species of Decalepis is still needed for their successful reintroduction in natural habitat.

Normal root culture

D. hamiltonii: Giridhar et al. [60] studied the production of 2H4MB by normal root cultures of D. hamiltonii. The flavour compound was extracted with dichloromethane, evaporated to dryness and dissolved in ethanol for qualitative (TLC) and quantitative (GCMS) analysis. Maximum root biomass and the maximum content of flavour compound (40 μgg-1 dry weight) were recorded after 45 days of growth on Murashige and Skoog (MS) medium containing 1.0 mgL-1α-naphthaleneacetic acid (NAA).

D. arayalpathra: Sudha and Seeni [35] reported for fast-growing normal root culture of D. arayalpathra from leaf and inter nodal explants of in vitro-raised shoot cultures in MS medium containing 2.5 mg/l BA, 0.5 mg/l 2-iP and 0.5 mg/l NAA. Shoot cultures were maintained on MS agar medium supplemented with 0.5 mg/l BA and 0.05 mg/l 2-iP or and 0.05 mg/l NAA and sub-cultured at 5 weeks interval. Leaf explants incubated in total darkness on half-strength MS medium supplemented with 0.5 mg/l IBA and 0.2 mg/l NAA favoured induction of roots (5.820 g). A 100 mg fresh root tissue cultured in 80 ml half-strength MS liquid medium supplemented with 0.2 mg/l IBA and 0.1 mg/l NAA, under continuous agitation (80 rpm), yielded 3.433 g fresh and 0.734 g dry weight of roots. Roots grown in this optimal medium produced maximum content of 2H4MB (0.16%) after 6 weeks of culture. The root cultures were maintained up to the 7th passage without decline of growth [35].

Hairy root culture

The normal root culture system was slow growing with a short life-span, as mentioned in an extensive review [76]. Advances in plant cell and tissue culture, combined with improvement in genetic engineering, specifically transformation technology has opened new avenues for high volume production of pharmaceuticals, nutraceuticals, and other beneficial substances [77]. Transgenic hairy root cultures have revolutionized the role of plant tissue culture in secondary metabolite production from the wild and commercial medicinal plants. Hairy root culture is an attractive experimental system, as they are long-term aseptic root clones, genetically stable with growth are comparable to those of the fastest-growing cell suspension culture [75]. This approach has been successfully applied for D. hamiltonii and D. arayalpathra as below;

D. hamiltonii: Samydurai et al. [78] reported Agrobacterium rhizogenes mediated hairy root culture and genetic transformation of D. hamiltonii. They have reported the total dry matter content of 2H4MB by hairy root culture of D. hamiltonii from internode and roots explants inoculated with A. rhizogenes. The addition of acetosyringone to basal MS liquid medium fortified with NAA and IBA considerably enhanced the hairy root production. PCR analysis of Ri plasmid on transformed internode and root tissues showed DNA amplification at 780 bp amplified in internode, root derived hairy roots. The highest biomass of dry matter content (25.36 and 24.12 g) was observed in liquid MS medium containing 0.3 mg/L NAA, cocultivated with A. rhizogenes on the internode and root explants. Afterthat the internode and root explants were cultured on MS medium supplemented with 0.3 mg/L IBA thereby exhibited improved biomass content of dry matter 23.56 and 22.91 g respectively. The dry matter content of biomass was extracted with 1 mg/ml methanol to measure the total content accumulation of 2H4MB in hairy roots cultures by using HPLC [77].

D. arayalpathra: Sudha et al. [79] studied A. rhizogenes mediated transformation of D. arayalpathra by infecting juvenile hypocotyl and cotyledon explants with different strains, including A4, MTCC 532, TR105 and LBA 5402. Hypocotyl explants induced hairy roots at a higher frequency (53.2 ± 0.3 %) than cotyledons (32.1 ± 0.2 %) when infected with the most virulent strain TR105. The explants co-cultivated 48 h in half-strength salts and vitamins of MS basal medium (half-MS medium) induced hairy roots either directly from the wounds or follwed by the formation of gall like structures. Irrespective of the explants, the strain MTCC 532 induced callus alone. The root initials on the galls proliferated vigorously and elongated more rapidly when they were segmented and subcultured on half-MS medium than the proliferation and elongation of directly emerged roots. The established hairy roots showed intermittent gall formation which was the active sites for hairy roots induction. The molecular evidence of rol A and rol C gene integration was confirmed by PCR amplification and southern blot hybridization. Growth of the hairy roots was undertaken by measuring root growth unit after culturing root tips in half-MS medium and determined fresh weight/dry weight/conductivity during time-course study in shake flask cultures. The maximum biomass and accumulation of the root specific compound, 2H4MB (0.22 % dry weight), was recorded at the 6th week of growth, which was more than that observed in normal root cultures (0.16 % dry weight) [78].