Morphological and biochemical alterations during in vitro microrhizome formation of Curcuma caesia Roxb

Curcuma caesia Roxb., a critically endangered herb in the Zingiberaceae family, can be conserved through microrhizomes, which are easily transported, germinate like seeds, and are independent of seasonal variations. The current investigation attempts to induce microrhizomes of this endangered herb for conservation purpose using high concentration of sucrose. To encourage the establishment of microrhizomes, six-month-old cultures of C. caesia were transferred to Murashige and Skoog supplemented with containing 8 mg L⁻¹ benzyladenine, 8 mg L⁻¹ kinetin, 100 mg L⁻¹ citric acid, 200 mg L⁻¹ adenine sulphate, and 2 mg L⁻¹ indole-3-acetic acid (standard medium). For this, standard medium was examined with sucrose concentrations of 3%, 6%, 9%, and 12%. The standard medium with 9% sucrose showed the highest rate of microrhizome formation (now referred as microrhizome production medium, MPM). During acclimatization, the survival rate of microrhizomes exceeded 90%. The physiology behind the microrhizome formation was also evaluated using enzymatic and non-enzymatic tests on days 0, 30, and 60 after inoculation. Superoxide dismutase activity, an enzymatic defence molecule, and total soluble sugar and ascorbate content, a non-enzymatic defence molecule, both increased in the MPM microrhizomes relative to the control [shoot multiplication medium (standard medium with 3% sucrose) at day 0]. Further, protein, 2-thiobarbituric acid reactive substances, and hydrogen peroxide content also increased. The biochemical results proved that 9% sucrose in MPM induces osmotic stress which eventually led to the formation of C. caesia microrhizomes, an in vitro storage organ.