Table 1.
Relative abundance of primary metabolites and their associated pathways in shoot base, embryogenic and non-embryogenic calli in B. rotunda.
Table 2.
Estimation of cell density (cell/mm2) in shoot base and embryogenic and non-embryogenic calli in B. rotunda.
Fig 1.
Principal Component Analysis (PCA) plot showing three clusters in callus and explant tissues from B. rotunda (n = 3 biological replicates).
Blue ellipse: embryogenic callus (EC); orange ellipse with green; dry callus (DC) and with purple: watery callus (WC); and red ellipse: shoot base (SB).
Fig 2.
Primary metabolite variables associated with different callus types from B. rotunda (n = 3 biological replicates).
A: Orthogonal Partial Least Square-Discriminant Analysis (OPLS-DA) plot for embryogenic callus (EC) and dry callus (DC); B: OPLS-DA plot for EC and watery callus (WC); C: Blue ellipse in the S plot highlights metabolites associated with EC versus DC with p-value-<0.05, D: Blue ellipse in the S plot highlights metabolites associated with EC versus WC with p-value<0.05, E: OPLS-DA plot for DC and WC; F: S plot showing metabolite comparison between DC and WC.
Fig 3.
Relative abundance of metabolites markers in embryogenic callus and sieved embryogenic cells (n = 3 biological replicates).
EC: Embryogenic callus; EC_S: sieved embryogenic cells. Error bars indicate standard deviation, asterisk represents p-value <0.05 by student T-test.
Fig 4.
Quantitative analysis of five secondary metabolites in B. rotunda callus and explant (n = 3 biological replicates).
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.
Fig 5.
Concentrations (% dry extract) of secondary metabolites in B. rotunda shoot (n = 3 biological replicates).
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.
Fig 6.
Intracellular hormone IAA concentrations (ppb) in dry extracts of B. rotunda callus and explant (n = 3 biological replicates).
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.
Fig 7.
Morphology and histology of B. rotunda explant and callus.
A-D: 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; E-H: 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; I-L: 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; M-P: 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.