Optimization of an efficient micropropagation protocol for Persicaria glabra by the synergistic effect of ancymidol and benzyladenine

Abstract

An efficient in vitro propagation protocol for a medicinal plant, Persicaria glabra (Willd.) M. Gomez, has been established. Nodal explants were cultured in vitro on agar-gelled (0.8%) Murashige and Skoog (MS) medium containing different concentrations of cytokinins, namely N⁶-benzyladenine (BA), 6-(γ,γ-dimethylallylamino)purine (2-iP), or kinetin (KIN). BA (2.5 µM) was found to be the most effective cytokinin for axillary bud proliferation. A growth retardant ancymidol (ANC) was combined with 2.5 µM BA to enhance the multiple shoot induction and ensure the overall health of micro shoots. The nodal explants taken from micro shoots developed in 2.5 µM BA were subcultured on agar-gelled MS medium supplemented with different concentrations of ANC along with 2.5 µM BA. Among the different concentrations tested, 4.0 µM ANC combined with 2.5 µM BA produced a maximum number of multiple shoots (13 shoots per explant). A progressive decline in the shoot height was observed with increasing concentrations of ANC and significantly reduced shoot length (0.5 cm) at a higher concentration (12.0 µM ANC). Reduced leaf area and more chlorophyll were noticed in ANC-treated plants than in BA-alone-treated plants. This facilitated better growth and development of robust micro shoots. Well-developed micro shoots were excised from 4.0 µM ANC along with 2.5 µM BA and subjected to ex vitro rooting experiments using different concentrations of auxins, including α-naphthaleneacetic acid (NAA) or indole-3-butyric acid (IBA). The maximum number of roots (46.33 roots per shoot) and root length (14.53 cm) were observed in 0.5 mM IBA with 89.11% of the response. The rooted plants were successfully acclimatized in field conditions with 100% survival. The genetic fidelity analysis of in vitro–raised plants was tested using inter simple sequence repeat (ISSR) markers, and they showed genetic monomorphism with the mother plant. The present study suggests that adding ANC and BA would enhance the multiple shoots induction and high rate of acclimatization of P. glabra cultures. The addition of ANC resulted in a 2.6-fold increase in shoot number and avoided undesirable elongation of shoots. Healthy shoots, thus, produced in ANC combined with BA-added medium doubled the survival of plants during acclimatization over a BA-alone medium and can be used in many other plant species that have the problem of developing shoots of an unhealthy nature coupled with abnormal internodal elongations.