Elicitation of secondary metabolites in cotyledonary callus of

Elicitation of secondary metabolites in cotyledonary callus of Ziziphus spina-christi and measurement of antioxidant activity in different lines using FRAP method Wattan Basheer Supervised by: Dr. Rami Arafh

Introduction • Ziziphus spina-christi , (Rhamnaceae) is a tropical evergreen tree from Sudanese origin. known as Christ's Thorn and its Arabic name is "Sidder“ • In Palestine, it is found in the Jordan Valley and the Dead Sea area. • It is known in traditional medicine for treatment of several illnesses like dermatitis, diuretic, eye inflammation, stomachache, rheumatoid arthritis and has anti-diabetic activity • The plant is rich in pharmaceutical compounds including alkaloids, glycosides, flavonoids, and volatile oils.

Introduction • Due to the increasing demand on Ziziphus spina-christi in traditional medicine, there is a need for sufficient plant material to satisfy the market needs • Alternative methods are used for the propagation and culture of Z. spina-christi including seed and stem cutting propagation. • In vitro propagation method offer a powerful alternative for production of large amount of plants • Plant tissue culture also offers an effective alternative for utilization of explants that are not possible to be produced with conventional methods like root culture and cells from cotyledons.

Introduction Advantages of in vitro plant culture • In the micropropagation method, only a small amount of tissue is required to regenerate millions of clonal plants in a year • Some plant parts such as cotyledons and hypocotyls which cannot be found in nature are utilized • In vitro stock can be quickly proliferated as it is season independent • Enables long term storage of valuable germplasm

Introduction Advantages of in vitro plant culture • The production of huge amount of plants away from conventional methods (by seeds or rooted cuttings) • Pest free plants are quickly multiplied for various agricultural uses • Production of secondary metabolites which is difficult to be synthetically produced or by intermediate agents like bacteria

Aim of the study q Examine chemical factors and test their effect on secondary metabolites production in cotolydonary callus q Explore antioxidant properties of callus extract

Objectives of the study q Examine the effect of media type and chemical elicitation on secondary metabolites production in cotolydonary callus q Explore antioxidant activity with FRAP method

Methodology : 1 2 3 • Establishment of callus from cotyledon • Extraction of different callus cell line and compare the UV absorbance of different extracts • Measure antioxidant potential of callus extract using FRAP assay

Methodology : Establishment of callus from cotyledonary tissue 1 - The dried fruits of Ziziphus spina-christi (L. ) Desf. were collected in October 2012 from Jericho city then seeds were isolated manually 2 - Seeds were germinated in vitro on PGR-free water-agar media

Methodology : 3 - Cotyledons were excised and used for callus induction on MS, B 5, Mcc or QL media with 1. 3 mg/l BA + 0. 3 mg/l NAA MS QL B 5 MCC

Methodology : Extraction of different callus cell line and compare the UV absorbance of different extracts 5 - Aqua extract of callus obtained from each media

Methodology : 6 - Ultraviolet–visible spectroscopy used for secondary metabolites detection 7 - FRAP assay used to measure the antioxidant power of three media calli “MS, QL, B 5”.

Results : 1 -The preliminary results indicate significant differences in the crude secondary metabolites extracted when using MS, QL and B 5 media B 5 MS QL

Results : 2 - The antioxidant power of the three extract B 5, QL, MS also differ with an absorbance value (0. 189), (0. 327) and (0. 380) respectively at 593 nm Using FRAP assay.

Discussion • The change in the amount of secondary metabolites produced from different media lines indicate that the micro and macro nutrients influenced the growth and affected the type of biochemical's produced. • The difference in the amounts of these compounds was clear in changing the oxidation power of different media depending on the secondary metabolites they produce.

Discussion • Calli grown on B 5 has the highest antioxidant power this correlate with a peak observed at 333 nm only in the extract from B 5 media B 5 QL MS

Future work : • Characterization of the compound absorbed at 359 nm using NMR. • Use HPLC to separate the produced compound. • Different other chemical and electrical stress will be used to enhance other secondary metabolites.

THANKS