In vitro callus induction of Spilanthes calva DC [Spilanthes acmella auct. non L,.] (Maha Akmella)

Abstract

Spilanthes calva DC. (Maha Akrnella) is a valuable medicinal plant belongs to Family Asteraceae. Itis widely used in indigenous medicine to treat toothache in most of the Asian countries. Not only it hasanesthetic properties, but also contain secondary metabolites, with the insecticidal properties, whichcould be used as potential bio insecticide. This is an annual plant, which grows to a height about 30ern. After flowering mother plant is dried off. Four to six weeks later seeds are germinated and newseedl ings are produced. Viabil ity of seeds loses with in short period of time. Even though seeds aregerminated percentage of germination is low (about 30%). Rooting of cuttings is also not possible.This is a limitation in using this valuable medicinal plant for commercial production. Therefore it isvery important to develop a protocol for mass propagation through tissue culture and establishing cellcultures will be useful for large-scale chemical extraction in industrial purposes.Leaf discs were used as explant for callus initiation. In order to identify the suitable maturity stage forcallus initiation, leaves were harvested at different maturity stages i.e first, second and third fullyopened leaf.Leaves were washed with Dettol" soap and soaked in a solution of Teepol" for 5 minutes. After thatleaves were washed with running tap water for 45 minutes, In order to surface sterilize. Leaves werewashed with 10% Clorox ™ (5.25% Sodium hypochlorite v/v) for 5 minutes and then with 70% alcoholfor 30 seconds each followed by three successive washings in sterile distilled water. These operationswere carried out inside the laminar airflow cabinet before inoculation. Basal media tested for the study were full strength MS (Murashige and Skoog, 1962) medium and Y2 MS (both macro andmicronutrients) medium. Media were supplemented with different concentrations (1.0 mgl' - 3.0mgl") of BAP and 2,4-0. Cultures were incubated under complete dark at 25± I °C in the growthroom.Study conducted by Haw and Keng (2003) on the same species produced multiple shoots from axillarybud explants without inducing callus in MS medium supplemented with 2.0 mgl.:' BAP. In the presentstudy, callusing was observed within 5 days of incubation in full strength MS medium supplementedwith BAP and 2AO. It took longer period to initiate callus when both macro and micro nutrients in thebasa l rned ium was lowered to ha If and the amount of callus produced was also very low even after 6thweek of incubation. In order to observe the time taken to produce maximum amount callus freshwe iuht was measured after 2".1,4th and 6tltweek of incubation. It was observed that maximum amountofc~llus was produced within 4 weeks in all explant types tested with a maximum of 0.88 g:': 0.23 inleaf discs obtained from first fully opened leaf.In order to determine the best growth regulator combination for callus initiation, calli fresh weightswere measured after fourth week of incubation in different growth regulator combinations tested.Highest amount of calli were in MS medium in the presence of2.25 mgl' BAP and 1.0 mgl' 2.4-0.Fragile calli, which were transulant and mucilaginous in nature were observed within 15 days ofincubation, which could lead to cell suspension cultures.