Comparative Analysis of Primary and Secondary Metabolites in Different In Vitro Tissues of Narcissus tazetta var. chinensis

Abstract

Narcissus tazetta var. chinensis is a perennial monocot plant that is well known for its pharmaceutical and ornamental uses. This study aimed to understand the changes in the primary and secondary metabolites in different in vitro tissues of N. tazetta (callus, adventitious root, and shoot) using high-performance liquid chromatography and gas chromatography time-of-flight mass spectrometry. In addition, to optimize the most efficient in vitro culture methods for primary and secondary metabolite production, N. tazetta bulbs were used as explants and cultivated in Murashige and Skoog (MS) medium containing different hormones at various concentrations. In addition, the present study found suitable hormonal concentrations for callus, adventitious root, and shoot induction and analyzed the primary and secondary metabolites. The MS medium supplemented with 1.0 mg L^-1 dicamba, 3.0 mg L^-1 indole-3-butyric acid (IBA), and 3.0 mg L^-1 6-benzylaminopurine (BAP) was the most efficient media for callus, adventitious root, and shoot induction in N. tazetta. The tissue induced in this medium was subjected to primary (amines, amino acids, organic acids, sugars, and sugar alcohols) and secondary metabolite (galantamine and phenolic acids) analysis. The shoots and roots showed the highest amounts of metabolites. This study showed that bulb in vitro culture can be an efficient micropropagation method for N. tazetta and the production of primary and secondary metabolites, offering implications for the mass production of primary and secondary metabolite compounds from N. tazetta tissues generated in vitro.

Figure 1

Establishment of callus, adventitious root, and shoot culture of N. tazetta using meristems in disinfected bulbs. (A) N. tazetta bulb, (B) N. tazetta callus induced on MS agar containing auxin (dicamba 1.0 mg L^–1), (C) N. tazetta adventitious root induced on MS agar containing auxin (IBA 3.0 mg L^–1), and (D) N. tazetta shoot induced on MS agar containing cytokinin (BAP 3.0 mg L^–1). The scale bar indicates 1 cm.

Figure 2

Galantamine content in the callus, roots, and shoot of N. tazetta. The error bar represents the standard deviation of 10 biological replicates.

Figure 3

Heat map of the relative concentrations of hydrophilic metabolites identified from three N. tazetta tissues using HPLC and GC-TOFMS. Three biological replicates were used.

Figure 4

PCA (A) and PLS-DA (B) score and loading plots of the identified metabolites from the three N. tazetta tissues.

Figure 5

Main metabolites that differentiate the three N. tazetta tissues are based on the VIP scores obtained using the PLS-DA model. Colored squares on the right denote the relative concentrations of the three corresponding N. tazetta tissues.

Figure 6

Relationships between the metabolites identified from the three N. tazetta tissues are shown in a correlation matrix; each colored square shows the Pearson’s correlation coefficient for a pair of metabolites; the strength of the red or blue color, as shown on the color scale, represents the correlation coefficient value for each colored box.

Figure 7

Identified metabolites and their pathway impact on three different tissues of N. tazetta. (1) Flavone and flavonol biosynthesis, (2) phenylalanine metabolism, (3) tryptophan metabolism, (4) glycolysis/gluconeogenesis, (5) indole alkaloid metabolism, (6) tropane, piperidine, and pyridine alkaloid biosynthesis, (7) terpenoid backbone biosynthesis, (8) phenylalanine, tyrosine, and tryptophan biosynthesis, (9) nitrogen metabolism, (10) isoquinoline alkaloid biosynthesis, (11) glutathione metabolism, (12) d-amino acid metabolism, (13) thiamine metabolism, (14) lipoic acid metabolism, (15) fatty acid degradation, (16) monobactam biosynthesis, (17) flavonoid biosynthesis, (18) tyrosine metabolism, (19) pantothenate and CoA biosynthesis, (20) fructose and mannose metabolism, (21) propanoate metabolism, (22) biosynthesis of various plant secondary metabolites, (23) sphingolipid metabolism, (24) glucosinolate biosynthesis, (25) pyrimidine metabolism, (26) cyanoamino acid metabolism, (27) ascorbate and aldarate metabolism, (28) amino sugar and nucleotide sugar metabolism, (29) arginine biosynthesis, (30) valine, leucine, and isoleucine degradation, (31) arginine and proline metabolism, (32) glycerophospholipid metabolism, (33) ubiquinone and other terpenoid–quinone biosynthesis, (34) galactose metabolism, (35) glyoxylate and dicarboxylate metabolism, (36) citrate cycle (TCA cycle), (37) beta-alanine metabolism, (38) phenylpropanoid biosynthesis, (39) butanoate metabolism, (40) valine, leucine, and isoleucine biosynthesis, (41) carbon fixation in photosynthetic organisms, (42) inositol phosphate metabolism, (43) cysteine and methionine metabolism, (44) glycerolipid metabolism, (45) pyruvate metabolism, (46) glycine, serine, and threonine metabolism, (47) starch and sucrose metabolism, and (48) alanine, aspartate, and glutamate metabolism.