Stigmasterol production from Abutilon indicum cell suspension cultures
Stigmasterol production from Abutilon indicum cell suspension cultures
Abutilon indicum, a therapeutically valuable shrub can act as a continuous source of stigmasterol, accredited with pharmacological significance. In this study, the content of stigmasterol when analyzed in both in vivo and in vitro plants was found to be 13.89 ± 1.43 and 20.50 ± 2.34 μg/gFW, respectively. The callus obtained from the in vitro plants of A. indicum was found to contain 10.78 ± 0.19 μg/gFW of stigmasterol and was used for initiation of suspension cultures. In comparison to the calli, suspension cultures of A. indicum accumulated considerable amounts of stigmasterol (16.08 ± 1.92 μg/gFW) on the 12th day, i.e., end of log phase. The suspensions on further elicitation with cadmium chloride have shown a significant increase (2.59-fold) in the amount of stigmasterol compared to the initial calli, reaching 41.73 ± 3.77 μg/gFW. Thus, cell suspensions of A. indicum offer a unique advantage for large-scale production of stigmasterol under in vitro conditions, by retaining its natural essence and safety in human consumption.
Materials and methods
Mature seeds of A. indicum collected from greenhouse – grown plants were thoroughly washed and treated with concentrated sulphuric acid for 10 min to soften the tough seed coat. The seeds were then surface sterilized with 0.1% mercuri chloride for 5 min followed by thorough washing (8-10 rinses)with sterile distilled water. They were then left to imbibe(48 h) before transfer to semi-solid (0.8% agar) MS media(Murashige and Skoog 1962) supplemented with gibberellic acid (GA3) at a concentration of 1 mg/L. The seeds were incubated under standard culture controlled conditions.The nodal explants from in vitro germinated seedlings of A. indicum were cultured on MS media supplemented with different concentrations (0.125-1.00 mg/L) of cytokinins, viz. kinetin (Kn), zeatin (Z), thidiazuron (TDZ), benzylaminopurine (BAP), for multiple shoot induction. The basal callus obtained was transferred to MS semi-solid media with a combination of cytokinins (Kn, BAP and TDZ) to initiate embryogenesis under standard culture conditions. The somatic embryo formation was recorded after a culture period of 6-8 weeks.
Callus induction
Explants obtained from leaves, stems (nodal and intermodal sections) and roots of in vitro germinated seedlings were transferred to MS semi-solid media fortified with different combinations of auxins [2,4–dichlorophenoxyacetic acid (2,4-D), α-naphthaleneacetic acid (NAA)] and cytokinins (BAP; Kn) in the range of 0.25-1 mg/L, for callus initiation. The obtained callus was periodically sub-cultured for further proliferation under standard culture conditions.
Growth pattern of A. indicum suspensions
Friable callus (3 g) obtained from the leaf explants was transferred to conical flasks (100 mL volume) containing 30 mL of MS liquid media (sterilized by autoclaving at 121 °C under a pressure of 15 psi for 20 min) supplemented with 2,4-D, Kn and NAA at a concentration of 1 mg/L each; and incubated on an orbital shaker, under standard culture conditions (25 ± 2 °C temperature; 40–50 μmol m2/s illumination;16/8h (light/dark) photoperiod; 120 rpm aeration). The increase in biomass was assessed along with stigmasterol content by harvesting the suspensions at regular intervals, i.e., every 4 days for a period of 28 days, until the culture reached its decline phase. The biomass and stigmasterol content in the suspensions are expressed as gram fresh weight (gFW) and microgram per gram fresh weight (μg/gFW), respectively.
Elicitor treatment
An abiotic elicitor, Cadmium Chloride (CdCl2), was used for eliciting stigmasterol production in the suspension cultures of A. indicum. The elicitor was prepared as a stock solution (1 M) in distilled water and filter sterilized (0.22 μm membrane filters). CdCl2 was added aseptically to the suspensions in the range of 0.5-4 mM, on the 11th day of growth studies. Stigmasterol production in combination with biomass was monitored at an interval of 24 h for three consecutive days (24, 48 and 72 h).
Cell viability
The A. indicum cell suspensions were analysed for cell viability preceding the addition of elicitors by selective labelling of cells with 75 μg/mL fluorescein diacetate and the obtained results are expressed in terms of gram fresh weight (gFW).
Stigmasterol quantification
The amount of stigmasterol was quantified in various samples i.e., non-elicited and CdCl2 elicited samples, by a modified HPLC method. The stigmasterol present in the samples was quantified by comparing the obtained HPLC data with that of standard stigmasterol
Conclusions
The naturally occurring phytosterol-stigmasterol considered as an equivalent of cholesterol in plants can reduce cholesterol absorption thereby lowering the cholesterol levels by 8-10%. A. indicum suspension cultures proved to be a sustainable platform for production of stigmasterol, which has achieved a further 2.59-fold increase by employing the abiotic elicitor-CdCl2. The suspensions hold promise in becoming a continuous source of the naturally occurring steroidal sapogenin-stigmasterol that can combat hyperlipidemia involved in obesity and cardiovascular health similar to diosgenin, alliin, glycyrrhizin, among others.
Reference:
Rao, K., Chodisetti, B., Gandi, S., Giri, A. and Kavi Kishor, P.B., 2022. Cadmium chloride elicitation of Abutilon indicum cell suspension cultures for enhanced stigmasterol production. Plant Biosystems-An International Journal Dealing with all Aspects of Plant Biology, 156(3), pp.613-618.
