Temporal variations in metabolite profiles at different growth phases during somatic embryogenesis of Silybum marianum L.

Silybum marianum, commonly known as Milk thistle, is a popular herbal supplement used for the treatment of jaundice and liver cirrhosis worldwide. Here we established methods for somatic embryogenesis and comparative metabolite profiling of the different growth phases during embryogenesis in S. marianum. Highest embryogenic potential was observed for calli previously derived from petiole explants on Schenk and Hildebrandt medium containing 2.5 mg l−1 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l−1 N6-benzyladenine (BA). Somatic embryos (SE) were induced when embryogenic calli with pre-embryoid masses (PEMs) were subcultured on same media as used for induction of embryogenic callus. Highest number of somatic embryos (46 somatic embryo per callus) was observed at 1.5 mg l−1 2,4-D and 1.5 mg l−1 BA, however ½ strength MS medium showed optimal response for maturation followed by germination of somatic embryos at 1.5 mg l−1 GA3. Metabolite profiles from developmental stages of non-embryogenic callus (NEC), PEMs, SE and embryos germinating into intact plantlets (GSE) were obtained using Electro spray ionization mass spectrometry ESI/MS. Principal component analysis (PCA) was carried out to identify key metabolites in different growth phases during somatic embryogenesis. The loading scatter plots enabled the detection of several bin masses responsible for separating samples from different growth stages. Based on the values of % total ions count and average intensity of selected bins in all biological samples, putatively known metabolites were obtained from in-house bin program. Amino acids associated with various biosynthetic pathways like arginine, asparagine and serine were abundantly detected in GSE, while they were detected at decreased intensities in NEC. However, tryptophan was measured with increased signals in SE when compared to other growth phases. Glucose, fructose and fructose-6-phosphate were mostly accumulated in NEC; however they were detected with lowest intensities in GSE. Moreover, sucrose and significant secondary metabolites like cinnamic acid, kaempferol, quercetin, myricetin, linolenic acid, and 5-enolpyruvyl-shikimate-3-phosphate were found at higher amount in SE when compared to other embryogenic phases.