Growth regulators, DNA content and anatomy in vitro-cultivated Curcuma longa seedlings

Abstract

Curcuma longa L., from the Zingiberaceae family, generally reproduces through its rhizomes, which are also utilized for therapeutic purposes because they are rich in terpenoids. Its conventional propagation has low efficiency due to the small number of seedlings and their contamination by pathogens. Therefore, this study aimed to evaluate the influence of growth regulators on the development of in vitro-cultivated C. longa as well as to determine their influence on DNA content and foliar anatomy. Shoots were inoculated in MS culture medium with the addition of 30 g/L of sucrose and 6.5 g/L of agar, and a pH adjusted to 5.8. Two assays were built to study the multiplication and rooting phases of growth. The first assay evaluated the influence of eight concentrations of cytokinins and auxins on the multiplication phase. Leaf samples were analyzed for DNA content through flow cytometry, utilizing two reference standards, green pea, and tomato. Characteristics of leaf anatomy were also measured in four time periods. The second assay analyzed the influence of six auxin concentrations on the rooting phase. The first assay showed that the root systems grew more in treatment 3 (4.44 μM benzylaminopurine [BAP], 0.46 μM kinetin [KIN]) and reached greater dry mass in T8 (8.88 μM BAP,  0.92 μM KIN, 2.16 μM naphthalene acetic acid [NAA]). The largest fresh matter of the main shoot was found in T2 (4.44 μM BAP). The estimated DNA content varied depending on the presence of supplemental growth regulators, from 2.38 to 2.77 pg, and was greater in T4 (4.44 μM BAP, 1.08 μM NAA) and T5 (4.44 μM BAP, 0.46 μM KIN, 1.08 μM NAA). Results from the latter two treatments were not significantly different. Estimates of DNA content were precise, as indicated by coefficients of variation that were much lower than 5%. The results also showed quantitative variation of evaluated anatomical traits. In general, there was a thin epidermis layer with rectangular cells, followed by parenchyma with octahedral cells and differentiated xylem and phloem. In leaf parenchyma, the presence of idioblasts containing phenolic compounds was observed in all growth stages. In the rooting phase, the supplementary auxins affected the dry matter of the aerial part and roots. The highest averages were observed in treatments with 2.0 μM NAA.

Keywords: Turmeric, micropropagation, flow cytometry, vegetal anatomy