In vitro microrhizome production in Decalepis hamiltonii

Microrhizomes were produced from leaf disc derived callus of a threatened endemic medicinal plant Decalepis hamiltonii. Murashige and Skoog (MS) medium supplemented with 2 M 6benzyleaminopurine (BAP) and 6 M 1-napthaleneacetic (NAA) acid was found to be optimum for rapid callus induction and establishment from leaf disc explants. Further differentiation of callus into microrhizome was conquered in MS medium supplemented with 4 M indole-3-butyric acid (IBA) and 8 M NAA. A maximum of 20 microrhizomes in a cluster was produced within 90 days. Yeast extract (0.05%) and polyvinylpyrrolidone (0.05%) further enhanced the microrhizome formation when supplemented along with plant growth regulators (PGRs).


INTRODUCTION
In recent years, there has been a gradual revival of interest in the use of medicinal plants in developed as well as developing countries since herbal medicines have been reported to be safe without side effects (Bahadur et al., 2007).Consequently, several medicinal plants becoming rare, endangered and threatened (RET) categories.Decalepis hamiltonii Wight and Arn.(Asclepiedaceae) is a glabrous extensively climbing shrub growing in moist deciduous forests, scrub jungles of Deccan peninsula and the Western Ghats of India.This is an endemic and endangered medicinal plant of southern peninsula.It prefers to grow along rocky slopes, big rock boulders and rocky crevices and small mounds at an altitude from 300 to 1200 m (Ravikumar and Ved, 2000).Rhizome extract of this plant is reported to have antioxidant, antibacterial, hepatoprotective, antiinflammatory, insecticidal, antidiabetic, anti-atherosclerotic, immunomodulatory and antitumour potentials due to the presence of bioactive principles such as decalepin, salicylaldehyde, vanillin anisaldehyde, 2-hydroxy 4methoxy benzaldehyde and 5,7,4-trihydroxy flavanone 4'o-β-D-glucoside (Srivastava andShivanandappa, 2006, 2010;Harish and Shivanandappa, 2011;Ashalatha et al., 2010;Naveen and Khanum, 2010;Sumalatha et al., 2010;Thangavel et al., 2011).In natural habitat, this species is under severe threat due to poor seed setting and ruthless harvesting by traditional medical practitioners (Ravikumar and Ved, 2000).Among different forms of plant cell and tissue culture systems, specific organ cultures such as root, rhizome and tubers have been reported to hold tremendous potential for in vitro production of uniform, high quality plant based medicines (Tripathi and Tripathi, 2003;Subathra et al., 2006;Majumdar et al., 2010;Poojadevi et al., 2013.Micro-rhizomes have been induced in Curcuma aromatica, Curcuma longa and the efficiency of microrhizome formation was found to be determined by the concentration of cytokinins, sucrose and coconut milk (Naik and Naik, 2006).Production of microrhizomes in vitro has many advantages compared to plantlet production.Besides, the use for extraction of secondary compounds, microrhizomes are feasible to facilitate germ plasm exchange across national borders.Hence, a rapid reproducible protocol for microrhizome production from leaf disc derived callus of D. hamiltonii have been devised in this present study.

Procurement of plant material
Young saplings of D. hamiltonii was collected from Sirumalai hills, Dindigul district, Tamil Nadu, potted and maintained in the department (SPKCES, M. S. University) green house for routine culture work.Identity of the plant was authenticated by Dr. K. Ravikumar, Assistant Director, Repository for Medicinal Plant Resources, FRLHT, Bangalore.Voucher specimen was deposited in departmental herbarium (F.No:144: SPKCESH).Chemicals and plant growth regulators (PGRs) used in this present study were procured from Himedia Laboratories, Mumbai, India.

Surface sterilization of explants
Fresh leaves of D. hamiltonii were collected, thoroughly washed in running tap water and 0.5% (v/v) sodium hypochlorite (NaOCl) with a few drops of "Teepol" for 5 min.This was followed by a thorough wash in running tap water.Then leaves were transferred to sterile laminar air flow cabinet and surface sterilized in 0.05% (w/v) mercuric chloride (HgCl 2 ) solution for 5 min and rinsed thoroughly in sterile distilled water.

Callus establishment and microrhizome initiation
Surface sterilized leaves were trimmed into optimum size (0.5 to 1.0 cm).Two discs were aseptically placed per culture vial with sterile Thangavel et al. 1309 MS medium.Culture vials were incubated under complete darkness at 25 ± 2°C.Proliferated callus was sub-cultured in 10 to 15 days interval for further establishment.After establishment, 0.5 g of callus was transferred to liquid MS medium (50 ml) supplemented with indole-3-butyric acid (IBA) and 1-napthaleneacetic (NAA: 2 to 10 µM), coconut milk [5 to 15% (v/v)] and yeast extract [0.05% (w/v)].Suspension cultures were incubated at 24°C in orbital shaker at 100 rpm.Callus was sub-cultured in 15 days interval by filtering the callus mass with several layers of sterile cheese cloth.Partially differentiated microrhizomes were sub-cultured into solid medium for further development.10 replicates were maintained for each media combination, fresh weight of callus was measured 20 days after sub culture.Callus induction efficiency of PGRs in different concentration and combination were evaluated through one way ANOVA.All culture sets were maintained with 10 replicates.Mean values were calculated (Table 1) and interpreted with control.
Significance was taken at the level of P< 0.01.

RESULTS
Among different PGR combinations used, MS medium supplemented with NAA (6 µM) along with BAP (2 µM) exhibited callus proliferation at the maximum level (2.9 ± 0.64 g) after 20 days from sub-culture (Table 1), (Figure 1c).Leaf explants often leached phenolic substances that inhibited the callus growth considerably, but this was suppressed by the addition of 0.05% polyvinylpirrolidone (PVP).Among various levels of sucrose and agar tested, 2 and 1.2% respectively were found to be ideal for rapid callus induction.After establishment, callus was transferred into microrhizome differentiation (liquid) medium, sub-cultured at 15 days interval.In liquid medium, differentiation and development of microrhizomes were observed in a slow rate (Figure 1d and 1e).Hence, the partially developed microrhizome clusters were transferred to solid medium where they grow faster and produced higher number of microrhizomes (Table 2) within 20 days (Figure 1f).Microrhizome differentiation potential was evoked by NAA in combination with IBA, YE and CM (Table 2).Culture medium supplemented with 8 µM NAA + 4 µM IBA + 0.05%YE and 10% CM exhibited maximum efficiency (average: 16 numbers) on microrhizome development and was considered very ideal for producing large number of microrhizomes.During initial stages, microrhizomes were white in color and after 15 days, they turned into pale brown color (Figure 1f).

DISCUSSION
Plant tissue culture techniques have been useful for conserving germplasm of rare and endangered species and considered as an alternative to conventional field gene banks to safeguard against pests and environmental stresses (Dodds, 1991).Tissue culture of medicinal plants holds tremendous potential for in vitro production and exploration of herbal therapeutics.In this  growth regulators to the nutrient medium, cell division, and tissue differentiation could be induced.Callus obtained in this present study was friable in nature (Figure 1c).

Effect of PGRs on callus induction and establishment
Supplementation of auxins at suitable level is a generic requirement for any explant to induce callus.Callus was proliferated initially from the cut surfaces of leaf discs cultured in MS medium supplemented with NAA (Figure 1b).Within 15 days, well proliferated callus covered the entire explant.
Basal medium as well as BAP supplemented medium did not induce callus.In case of medium supplemented with BAP along with NAA callus, the proliferation was rapid.MS medium consist of 6 µM NAA, 2 µM BAP and 0.05% PVP promoted quick induction and rapid proliferation of callus compared to other combinations (Figure 1c).NAA could promote callus induction in several plants where 2,4-D or 2,4,5-T fails (Pawar et al., 2002).Callus induction and organogenesis was successful in several medicinal plants like Petunia inflata, Withania somnifera, Commiphora wightii and Iphigenia indica (Beek and Camper, 1991) from leaf disc explants with the supplementation of NAA in various concentrations.
In this present study, NAA in combination with BAP enhanced callus multiplication at the maximum level (Table 1) and this observation corroborates the previous reports (Giridhar et al., 2004).In D. hamiltonii, in vitro hairy root production was reported with low biomass (Giridhar et al., 2004;Sudha and Seeni, 2001).However, high efficiency of micro-rhizome production is achieved in this present study and it could be a better tool for large scale production of high value secondary metabolites available in the rhizome of this medicinally important plant.

Effect of modified sucrose and agar concentrations
Establishment of callus was successful only when the sucrose and agar concentrations were altered.In general, this plant stores high quantity of starch in all vegetative organs.Reducing the normal concentration of sucrose from 3 to 2% induced regenerative callus and the callus survived a longer time.Reduced level of sucrose in culture medium has been reported in medicinal plants like Ficus lyrata and Solanum melongena (Mukherjee et al., 1991).
Increasing the concentration of agar (1.0 to 1.2%) enhanced the survival and curtailed hyperhydricity based suppression during callus establishment.This could be attributed to the natural conditions where this plant is exposed to xeric habitats.Thus, creating a near natural environment facilitated growth of callus and differentiation of microrhizome significantly.Such a similar observation was made earlier in Solanum surattense by Pawar et al. (2002).

Effect of PGRs on microrhizome differentiation
Specialized organ cultures such as root, rhizome and tubers will provide an efficient means of biomass production due to fast growth and stable metabolite production.Callus mass was established and transformed into microrhizome in suspension and later on solid medium.During the course of such differentiation, profound influence of exogenous plant growth regulators was observed.As the bioactive principles of this plant are produced in rhizome, the in vitro rhizome production would be of much commercial interest and value.Microrhizome was differentiated at the maximum level in MS medium supplemented with IBA (4 µM), NAA (8 µM), yeast extract (0.05%) and coconut milk (10%).
This combination induced only four to seven microrhizomes in 90 days in suspension culture under agitated condition (Figure 1d).Rate of differentiation, further growth and development of microrhizome was successful only after transfer into solid medium containing similar hormone and adjuvant combinations (Figure 1f).Each cluster had a maximum of 20 microrhizomes and an average of 16 on solid medium.Callus mediated microrhizome was also produced from Hemidesmus indicus (Sreekumar et al., 1998), Bunium persicum (Grewal, 1996) and Solanum tuberosum (Piao et al., 2003) with various hormone supplementations.Microrhizomes have also been developed in vitro from various rhizomatous medicinal plants such as Zingiber officinale (Sharma and Singh, 1995), Curcuma aromatic (Nayak, 2000) and Curcuma longa (Sunitibala et al., 2001).In all these reports, supplementation of PGRs such as NAA and IBA (2 to 15 μM), adjuvants like coconut milk, maleic hydrazide, activated charcoal and yeast extract were found to be crucial for microrhizome development.Photoperiod was reported to play a key role in induction of storage organs such as rhizome and tuber in vitro (Jean and Cappadocia, 1991).In the present study, cultures incubated in 16 h photoperiod produced more number of microrhizomes.Under 16 h light regime, large number of tubers have been produced in Dioscorea abysinica and Dioscorea alata, whereas continuous dark conditions hindered the tuberization process.Compared to other forms of cell aggregates, microtubers, microrhizomes and adventitious tuberous roots could serve as better alternative for large scale extraction of secondary metabolites (Jean and Cappadocia, 1991;Xie et al., 2000).Accordingly, the protocol devised in this present study will find an immense application in pharmaceutical industries for extraction and formulation of herbal products.
this present study, a rapid, reproducible protocol has been devised for the development of microrhizome from leaf disc derived callus of D. hamiltonii.Plant growth regulators were amended at different concentrations and combinations.However, the specific combination of the medium resulted in different extent of callus induction and differentiation of microrhizome.With the stimulus of endogenous or by addition of exogenous

Table 2 .
Microrhizome development from leaf disc derived callus.