Morphology and histology of <i>B</i>. <i>rotunda</i> explant and callus.

<p><b>A-D</b>: morphology of samples; A: cross section of 1 cm x 1 cm shoot base tissue; B: friable pale yellowish callus; C: compact, dense and dry callus, D: spongy and wet callus; <b>E-H</b>: SEM images (100x magnification); E: regular-shaped and -sized cells with arrows showing the presence of starch; F: regular-shaped cells with fibrils; G: rounded, compact cells; H: elongated and irregular-shaped cells; <b>I-L</b>: morphology of each sample viewed under fluorescent microscopy with diphenylboric acid 2-aminoethylester (DPBA) stain (100x magnification); I: fluorescent yellowish-green lining of cell membrane, J: fluorescent greenish-blue spots observed with yellow lining of cell membrane; K: fluorescent greenish blue spots observed with yellow lining of cell membrane; L: yellowish lining of cell membrane; <b>M-P</b>: morphology of each sample viewed under light microscopy with Periodic acid Schiff (PAS) stain (100x magnification); E: organized and compact cells with presence of vascular bundles (VB) and purplish-red starch granules; F: presence of dark blue clusters indicates active cell division and purplish-red starch granules; G: presence of dark blue clusters indicates active cell division and red-purplish starch granules; H: irregular-shaped and -sized cells without starch granules. SB: shoot base; EC: embryogenic callus; DC: dry callus; WC: watery callus.</p

Principal Component Analysis (PCA) plot showing three clusters in callus and explant tissues from <i>B</i>. <i>rotunda</i> (n = 3 biological replicates).

<p>Blue ellipse: embryogenic callus (EC); orange ellipse with green; dry callus (DC) and with purple: watery callus (WC); and red ellipse: shoot base (SB).</p

Relative abundance of metabolites markers in embryogenic callus and sieved embryogenic cells (n = 3 biological replicates).

<p>EC: Embryogenic callus; EC_S: sieved embryogenic cells. Error bars indicate standard deviation, asterisk represents p-value <0.05 by student T-test.</p

Concentrations (% dry extract) of secondary metabolites in <i>B</i>. <i>rotunda</i> shoot (n = 3 biological replicates).

<p>Error bars indicate standard deviation; RH: rhizome; SB: shoot base; T1: region of shoot 1–5 cm distal from the shoot base; T2: region of shoot 6–10 cm distal from the shoot base.</p

Quantitative analysis of five secondary metabolites in <i>B</i>. <i>rotunda</i> callus and explant (n = 3 biological replicates).

<p>A: values expressed in percent dry weight. B: values expressed in percent wet weight. Red: shoot base (SB); blue: embryogenic callus (EC); green: dry callus (DC); purple: watery callus (WC). Error bars indicate standard deviation and different letters represent significant differences for each metabolites at 95% confidence level by Tukey’s test.</p

Amino Acid and Secondary Metabolite Production in Embryogenic and Non-Embryogenic Callus of Fingerroot Ginger (<i>Boesenbergia rotunda</i>)

<div><p>Interest in the medicinal properties of secondary metabolites of <i>Boesenbergia rotunda</i> (fingerroot ginger) has led to investigations into tissue culture of this plant. In this study, we profiled its primary and secondary metabolites, as well as hormones of embryogenic and non-embryogenic (dry and watery) callus and shoot base, Ultra Performance Liquid Chromatography-Mass Spectrometry together with histological characterization. Metabolite profiling showed relatively higher levels of glutamine, arginine and lysine in embryogenic callus than in dry and watery calli, while shoot base tissue showed an intermediate level of primary metabolites. For the five secondary metabolites analyzed (ie. panduratin, pinocembrin, pinostrobin, cardamonin and alpinetin), shoot base had the highest concentrations, followed by watery, dry and embryogenic calli. Furthermore, intracellular auxin levels were found to decrease from dry to watery calli, followed by shoot base and finally embryogenic calli. Our morphological observations showed the presence of fibrils on the cell surface of embryogenic callus while diphenylboric acid 2-aminoethylester staining indicated the presence of flavonoids in both dry and embryogenic calli. Periodic acid-Schiff staining showed that shoot base and dry and embryogenic calli contained starch reserves while none were found in watery callus. This study identified several primary metabolites that could be used as markers of embryogenic cells in <i>B</i>. <i>rotunda</i>, while secondary metabolite analysis indicated that biosynthesis pathways of these important metabolites may not be active in callus and embryogenic tissue.</p></div

Intracellular hormone IAA concentrations (ppb) in dry extracts of <i>B</i>. <i>rotunda</i> callus and explant (n = 3 biological replicates).

<p>Red: shoot base (SB); blue: embryogenic callus (EC); green: dry callus (DC); purple: watery callus (WC). Error bars indicate standard deviation and different letters represent significant difference at 95% confidence level by Tukey’s test.</p