In vitro Callus Induction and Preliminary Phytochemical Studies of Cissampelos pareira L

The present investigation was aimed to evaluate the effect of growth regulators on induction of callus from leaf segments of Cissampelos pareira. The surface sterilized young leaves were inoculated on Murashige and Skoog (MS) medium fortified with different concentration and combination of growth regulators for induction of callus. The phytochemical constituents were analyzed in the different solvent extracts using standard methods. Maximum callus induction was observed on MS medium supplemented with 2 mg/L of NAA or 2, 4-D alone and in combinations of NAA (2 mg/L) + BAP (1 mg/L) and NAA (2 mg/L) + KIN (0.5 mg/L). The preliminary phytochemical screening of leaf and leaf derived callus revealed the presence of flavonoids, alkaloids, phenols, terpenoids, coumarins etc. The developed protocol is useful for large scale production of callus from leaf explants which may be useful for isolation of important bioactive compounds for the treatment of various diseases.


INTRODUCTION
Medicinal plants are widely used in traditional system of medicine and even in modern medicine [1,2]. Medicinal plants have enormous potential to synthesize aromatic substances like phenols or their oxygen substituted derivatives as secondary metabolites that serve as plant defense mechanisms against microorganisms, insects and herbivores. Medicinally active constituents of plant tissues are extracted with a broad spectrum of organic solvents of choice through standard procedures [3]. The extracts obtained in liquid, semi-solid, powder form are complex mixtures of metabolites like alkaloids, glycosides, terpenoids, flavonoids, lignin and others [4].
Cissampelos pareira L. (C. pareira) is an important medicinal plant, belongs to the family Menispermaceae. It is commonly called as Patha or Laghupatha in Indian traditional medicine [5]. There are 37 plant species which are distributed worldwide come under this genus, but only one species occurs in India [6]. The plant is commonly found in orchards, hedges, parks and gardens mainly on moist soil. It is distributed throughout the tropical and subtropical India. The plant has various medicinal properties and is used for dyspepsia, indigestion, flatulence, abdominal pain, diarrhea, dysentery, blood disorders, cough, asthma, bronchitis, skin disorders, leprosy, migraine, leucorrhoea, gonorrhea, burns, wounds, astringent, antispasmodic, piles, urogenital affections and scabies [7].
This medicinal plant is over exploited for its wider range of phytoconstituents and diverse applications. Micropropagation of plants through tissue culture is an alternate method to prevent the loss of important medicinal plants from the natural habitat. Therefore, the present study aims to develop the efficient protocol for callus induction from leaf explants and to screen the phytoconstituents present in different solvent extracts of leaf and leaf derived callus of C. pareira.

Collection of plant material and sterilization
Plants were collected in and around Jnanabharathi campus and maintained at the Department of Botany in the greenhouse condition. The leaves segments (0.5 to 1 cm 2 ) were surface sterilized with 0.1% bavastin fungicide for 20 mins, then washed thoroughly with tap water followed by liquid detergent (1-2 drops of teepol) and kept under running tap water to remove the traces of detergent followed by three to four rinses with distilled water. Finally, the explants were sterilized with 0.1% mercuric chloride for 3 min and rinsed 3-4 times with sterile distilled water.

Culture condition
The leaf explants were transferred into culture bottles on MS medium supplemented with 3% sucrose and was solidified with 0.8% agar (Plant tissue culture grade, Himedia) with different concentrations (0.5-4 mg/L) of auxins (2, 4-D, NAA, IAA and IBA) and in combination with cytokinin (BAP, KIN) for callus induction. The pH of the media was adjusted to 5.6-5.8, then autoclaved at 121 º C for 20 mins. The cultures were incubated at 25±2 º C under 16 hrs photoperiod with light intensity (3000-4000 Lux) provided by white florescent bulbs.

Preparation for plant extracts
Leaf and leaf derived callus were collected and finely powdered. 10 gms of the powdered material was extracted with different solvents (methanol, ethanol, chloroform, aqueous, hexane and petroleum ether) by soaking the material for 72 hrs. The extracts were filtered through Whatman No. 1 filter paper. The procedure was repeated for another two cycles to ensure complete extraction of phytochemical compounds. The filtrates were lyophilized and stored at 4º C until further analysis.

Statistical analysis
The data were expressed as Mean ± SE. All the experiments were repeated thrice and 15 replicates for each experiment. The data were analyzed statistically by one-way analysis of variance followed by Turkey's HSD test using SPSS software. Probability values P < 0.05 were considered significant.

Effect of auxins
Auxins

Effect of cytokinins
There was no callus induction from leaf explants of C. pareira inoculated on MS medium supplemented with different concentrations of cytokinins such as 6-Benzylaminopurine (BAP) and 6-Furfurylaminopurine (KIN) ranging from (0.5-4 mg/L).

Effect of auxins and cytokinins
Synergistic effect of auxins and cytokinins on leaf explants of C. pareira on callus induction was studied.  [20]. Sugandha also observed the maximum callus induction from leaf explants of Adansonia digitata on MS medium fortified with BAP (3 mg/L) + NAA (1 mg/L) [21].

Phytochemical screening
Plants have enormous potential to synthesize secondary metabolites and play an important role in plant defense mechanism against prey, microorganisms, insect, herbivores and stress as well as interspecies protection. These secondary metabolites have been used as a drug from the time immemorial, hence screening of phytochemicals serve as the initial steps in predicting the potential active compounds in the plant extracts [22]. The preliminary phytochemical screening of leaf and callus extract of C. pareira revealed the presence of major phytochemicals like alkaloids, flavonoids, phenols, tannins, saponins, steroids, terpenoids, glycosides, carbohydrates, reducing sugars and volatile oils. Gums and resins were found to be absent in leaf and leaf derived callus. Ethanol and methanolic extracts of leaf and callus extracts were proven to be better solvents for the extraction of major phytochemical compared to other solvents ( Table  2). Arumugam et al. reported that maximum phytochemicals were extracted in methanolic extract of leaf and leaf derived callus of Centella asiatica [23]. Whereas, Jhonson et al. found ethanol to be better solvent for extraction of phytochemicals in Baliospermum montanum [24]. IV. CONCLUSION The present study was aimed to develop the effective protocol to induce callus from leaf segments through in vitro propagation of medicinally important plant. The preliminary phytochemical screening of leaf and leaf derived callus showed the presence of alkaloids, phenols, flavonoids, steroids, tannins, terpenoids and volatile oils. This study will provide an efficient protocol for rapid induction of callus for the production of bioactive compounds. There will be possibilities of making plant cell factories for the enhanced production of bioactive compounds, which may prevent the exploitation of natural plant resources.