Rapid plant regeneration in industrially important Curcuma zedoaria revealing genetic and biochemical fidelity of the regenerants

Abstract

The present investigation was carried out to establish an efficient and reproducible micropropagation protocol for the production of morphologically, genetically and chemically uniform plants of Curcuma zedoaria. Axillary bud explants of C. zedoaria were inoculated into MS basal medium supplemented with various combinations and concentrations of 6-benzyladenine (2.2–22.2 µM, BA), kinetin (2.3–23.2 µM, Kin), indole-3-acetic acid (2.9–11.4 µM, IAA), α-naphthalene acetic acid (2.7–10.2 µM, NAA) and adenine sulphate (33.9–203.6 µM, Ads). Almost 95% of rhizome buds sprouted on MS medium supplemented with 13.3 μM BA, 5.7 μM IAA and 63.9 μM Ads giving rise to an average of 12.89\u2009±\u20090.02 shoots within 6 weeks. However, the maximum number of roots (25.8\u2009±\u20090.07 roots per explant) was obtained on half strength MS medium supplemented with 7.4 µM of IBA after 4 weeks of inoculation. Morphological characteristics were similar in both conventionally propagated and micropropagated plants. Additionally, genetic homogeneity of in vitro plants was further confirmed through ISSR and flow cytometry analysis. A total of 27 ISSR primers were screened, out of which 13 ISSR primers generated 58 monomorphic and reproducible bands thereby confirming the genetic uniformity of obtained plants. The mean 2C DNA content of the mother plant (2.96 pg) was similar to that of in vitro derived plants (3.07 pg). Gas chromatography-mass spectrometry (GC–MS) analysis showed similarity in the qualitative profile of chemical constituents of essential oil and high-performance liquid chromatography analysis revealed no significant differences in curcumin content in the tissue culture regenerants and mother plants of C. zedoaria. Therefore, the present micropropagation protocol could be effectively employed to generate true to type plantlets of C. zedoaria.