Factors Affecting In Vitro Culture of Asphodelus aestivus l. and Secondary Metabolites Production

Abstract

Asphodelus aestivus L. (Liliaceae), Arabic name “Gysalan” is a geophytes plant distributed all over the Mediterranean region. It has been used in traditional (folk) medicine to treat skin disease, jaundice, and psoriasis. Asphodelus aestivus propagation by conventional methods is problematic, since the seed show low germination percentage, and also show high sensitivity against light, humidity, and salt concentration. Therefore using plant biotechnology techniques offer an excellent alternative to propagate and study A. aestivus. In the present study, the main objective was to implement plant in vitro culture to propagate Asphodelus aestivus, and then assess secondary metabolites content in different parts of the plant. To test in vitro seed germination, seeds were cultured on water-agar medium, full strength or half strength MS medium. Seeds show slow and low germination rates (10%). Seeds pretreatment with 1.0 mg/L GA3 increased germination percentage to 43% on half strength MS medium. More rapid establishment of motherstock plants was achieved using sterilized nodes excised from the plant tuberous roots. Highest number of shoot (8 shoot/flask) was achieved on MS medium with 4.0 mg/L BA, 0.4 mg/L IAA and 1.0 g/L PVP. In vitro shoot proliferation was tested by culturing shoots on MS medium with IAA at 0.0 or 0.5 mg/L in combination with BA, and 2-ip at the levels 0.0, 0.5, 1.0, 2.0 or 4.0 mg/L. MS medium supplemented with 0.5 mg/L IAA, 1.0 mg/L BA and 1.0 mg/L 2-ip gave the highest number of shoots (3.5 ± 0.56) and leaves (18.63 ± 2.36). For in vitro rooting, shoots were cultured on MS medium supplemented with 1.0, 2.0, and 3.0 mg/L of IAA, NAA, or IBA. Results showed that MS medium with 2.0 mg/L NAA show the highest rooting percentage (93.8%) with a mean root number of (8.75 ± 1.66), and the longest roots was observed on MS medium with 1.0 mg/L NAA with a mean of (3.69 ± 0.76). Callus was induced from immature seed embryos and from newly developed roots under dark condition. The highest percentage of callus induction was obtained from seed embryos cultured on MS medium with 2.0 mg/L BA, and 1.0 mg/L 2,4-D. Thin layer chromatography iv (TLC) analysis between ex vitro and in vitro growing roots revealed several and different biochemical compounds in the root extract. This implies that growing A. aestivus under in vitro techniques resulted in different biochemical content of root extract. Key words: Asphodelus aestivus, In vitro propagation, Callus culture, Secondary metabolites, Traditional medicine.