Improved micropropagation protocol and enhancement in biomass and chlorophyll content in Stevia rebaudiana (Bert.) Bertoni by using high copper levels in the culture medium

Incorporation of a range of higher concentrations of CuSO4·5H2O in MS medium [Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassay with 240 tobacco tissue cultures. Physiol. Plant 15, 473–497] significantly enhanced direct shoot bud induction and proliferation from cultured leaf and nodal explants taken from mature plants of Stevia rebaudiana. Shoot bud induction medium was supplemented with BAP (2.2 μM) and IAA (2.8 μM). When the concentration of CuSO4·5H2O in the induction medium was raised to 0.5 μM (five times the MS level, i.e. 0.1 μM) there was significant increase in percentage response along with increase in shoot bud number per explant. The shoots were healthy, well developed with dark green broader leaves. There was remarkable increase in total biomass and chlorophyll content at increased (0.5 μM) copper level in the medium. During proliferation stage also presence of high copper levels in the medium favoured increase in shoot bud number per explant.