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.

Growth Regulators Affect the Growth and Biochemical Activity of Curcuma longa Plants Grown in vitro

“…In addition, there was an increase in the number of leaves (13.0) and the number of roots (7.0), which were double in number than those obtained the M2 medium (Table 3). We emphasize that this culture medium was also shown to be efficient for the development of explants in the studies by Antoniazzi et al (2016) and Ferrari et al (2016).…”

“…Among the growth regulators, cytokinins and auxins are the most frequently used plant hormones in the in vitro culture medium, both of which may influence both plant morphology and physiological responses, as reported by Antoniazzi et al (2016). These authors showed, during their trials with C. longa, that the addition of auxins and cytokinins to the Murashige and Skoog (MS) medium increases the shoot fresh mass, as well as the number and dry mass of the roots.…”

“…The different tested compositions of the culture media are shown in Table 1. All the culture media were supplemented with 6.5 g L -1 of agar (Kasvi ® ), and the pH was standardized to 5.8 (Antoniazzi et al, 2016;Ferrari et al, 2016).…”

“…The use of 6-benzylaminopurine (BAP) and kinetin (KIN) along with α-naphthaleneacetic acid (NAA) was tested in this in vitro assay. Concentrations of the growth regulators were established based on the results of Antoniazzi et al (2016). The sterilized explants were inoculated into 350-mL transparent glass vials, containing MS culture medium supplemented with 30 g L -1 of sucrose, 6.5 g L -1 of agar (Kasvi ® ), and pH adjusted to 5.8.…”

Growth and Anatomical Alterations in Leaves of Popcorn Induced by Abiotic Stresses

“…To remove excess paraplast, slides with the fixed tissue were then placed in a water bath containing butyl acetate to be dehydrated in butyl series ethanol. Slides were stained with safrablau and a blend of astra blue and safranin (9:1, v/v) (Bukatsch, 1972) followed by modification according to the method of Antoniazzi et al (2016). Finally, glass varnish was applied to ensure adherence of the coverslip (Paiva, 2006).…”