Evaluation of biochemical markers during somatic embryogenesis in Silybum marianum L

Abstract

In present report effects of explants type, basal media and plant growth regulators (PGRs) were tested for induction of indirect somatic embryogenesis in medicinally important plant Silybum marianum L. Leaf, petiole and root explants were exploited in vitro on B5 (Gamborg), SH (Schenk and Hildebrandt) and MS (Murashige and Skoog) media for induction of embryogenic callus followed by somatic embryogenesis. Highest callus induction frequency (76 ± 4.8 %) was recorded when petiole explants of in vitro derived plantlets were cultured on B5 medium supplemented with 1.5 mg l^-1 2,4-dichlorophenoxyacetic acid (2,4_D) in combination with 1.5 mg l^-1 Thidiazuron (TDZ). Induction and multiplication of somatic embryos were observed, when the embryogenic calluses were sub-cultured on to B5 medium containing 0.5 mg l^-1 2,4-D plus 1.5 mg l^-1 TDZ. At this PGRs treatment, 77 % of the cultures responded with 39.1 somatic embryos per callus. Furthermore, MS0 medium was indicated more reponsive for growth and maturation of somatic embryos. Analysis of biochemical markers during various growth phases in somatic embryogenesis revealed that somatic embryos exhibited highest level of total carbohydrate, starch, ascorbic acid and total free amino acids. However, higher protein levels were detected in non-embryogenic callus. Nevertheless, considerable amount of silymarin (4.1 mg g^-1 DW) was detected in somatic embryos than other growth phases. Thus, the present study concluded that biochemical and physiological changes during embryogenesis are influenced by interplay of explants type, basal media and PGRs.

Keywords: Biochemical; Explant; HPLC; Plant growth regulator; Silybum; Somatic embryo.

Fig. 1

Effects of various growth media with or without 1.5 mg l⁻¹ 2,4-D on explants. Data were collected after 4 weeks of culture. Values are the mean ± standard error from three replicates. Column bars sharing the same English letter/s are similar otherwise differ significantly at P < 0.05

Fig. 2

Somatic embryogenesis in Silybum marianum. a Callus formation at cut ends of petiole explants after 1 week of culture (bar = 2 mm). b Embryogenic callus after 2 weeks of culture period (bar = 2 mm). c Induction of somatic embryogenesis,embryonic cells showing isodiametric cells while non-embryonic cells have large vacuole, small starch granules and abundant intercellular spaces (bar = 250 µm)

Fig. 3

Effects of various concentrations and combinations of 2,4-D and TDZ in B5 media on mean number of somatic embryos per embryogenic callus from leaf and petiole explants. Data were collected after 4 weeks of culture. Values are the mean ± standard error from three replicates. Column bars sharing the same English letter/s are similar otherwise differ significantly at P < 0.05

Fig. 4

Effects of various growth media on somatic embryo maturation (%). Data were collected after 2 weeks of culture. EIM Embryo induction medium. Values are the mean ± standard error from three replicates. Column bars sharing the same English letter/s are similar otherwise differ significantly at P < 0.05

Fig. 5

Silymarin content (mg g⁻¹ DW) in in vitro-grown plant samples collected from different growth phases during somatic embryogenesis in Silybum marianum. Values are the mean ± standard error from three replicates. Column bars sharing the same English letter/s are similar otherwise differ significantly at P < 0.05