In vitro shoot multiplication of Rumex vesicarius L., and quantification of ascorbic acid and major phenolics from its leaf derived callus

Abstract

An optimised method for enhanced in vitro shoot multiplication of Rumex vesicarius (Polygonaceae)—a branched succulent herb—was achieved. The in vitro seed pre-treatment with 4% urea was able to show 95% seed germination on MS medium within 2 weeks of culturing. In vitro shoot bud induction from shoot tip explants was best in the presence of 2 mg L⁻¹ kinetin on Murashige and Skoog medium (MS medium), wherein 8.3 shoots/explants were observed. Shoot elongation was found to be high (3.75 ± 0.5 cm) in 2 mg L⁻¹ BA comprising medium. The transfer of micro-shoots on to MS medium comprising 1.5 mg L⁻¹ IBA and 1% activated charcoal (w/v) supported efficiently in vitro rooting (2.5–4.0 cm) in 3–4 weeks. Upon hardening, 70% rooted plants survived under greenhouse conditions. Though friable callus was produced on MS medium-containing 2 mg L⁻¹ BA followed by 3 mg L⁻¹ BA, no organogenesis was noticed. The ascorbic acid content of 78.62 ± 0.25 mg 100 g⁻¹ FW was recorded in callus cultures grown on medium supplemented with 2 mg L⁻¹ BA, and it is 1.74-fold more compared to normal ex vitro leaves of the same age. In vitro raised plant leaf showed 1.98-fold more ascorbic acid (89.42 ± 0.18 mg 100 g⁻¹ FW) to that of ex vitro leaves. The total phenolic content was found to be 60 mg in callus as compared to 610 mg (per 100 g GAE FW) of ex vitro leaves. The major phenolic compounds quantified were synergic, chlorogenic, ferulic, and generic acids, respectively. This optimised protocol will facilitate to pursue scale-up studies for in vitro ascorbic acid production and also to further investigate the kinetics of biosynthetic pathway genes involved.