Augmentation of wedelolactone through in vitro tetraploid induction in Eclipta alba (L.) Hassk.

Abstract

A complete protocol for the in vitro induction of Eclipta alba tetraploids has been optimized to enhance the wedelolactone content, an anti-cancerous compound. The effects of different concentrations of colchicine (0, 0.01, 0.05, 0.1, 0.2 and 0.3%; w/v) along with treatment durations (12, 24, 36 and 48 h) were investigated on shoot tip (ST) and nodal segment (NS). The treated explants were then incubated on Murashige and Skoog (MS) medium having 1.5 mg L−1 N6-benzylaminopurine and 0.5 mg L−1 α-napthalene acetic acid for shoot regeneration and afterward root was induced on 1.0 mg L−1 indole-3-acetic acid enriched ½MS medium. The tetraploids of E. alba were proficiently induced by the treatment of 0.1% colchicine for 24 h. The highest tetraploid induction efficiency was obtained from ST (30.56%) in comparison to the NS (22.22%). Analysis by spectrophotometry and flow cytometry showed that colchicine treated plants contained higher quantity of DNA than diploid plants. Cytological studies demonstrated doubled the chromosome number in tetraploids (2n = 4x = 44) than diploids (2n = 2x = 22). The ploidy level enhancement lead to alteration of other traits, like increased plant height, stem diameter, leaf size, stomatal size and chlorophyll content. As determined through high performance thin-layer chromatography, the ultimate achievement of this technique is the higher accumulation of wedelolactone in tetraploid plants (300.32 µg g−1 dry weight) in evaluation to in vitro diploid (131.31 µg g−1 dry weight) and in vivo diploid mother plants (93.26 µg g−1 dry weight), thus improving the pharmaceutical value of E. alba.