5303
HEBA DESOUKY AHMED KHLIFA
BIOCHEMICAL AND BIOTECHNOLOGICAL STUDIES ON HYPERICUM SINAICUM L. FOR PRODUCTION OF EXPERIMENTAL NATURAL PRODUCT(S) AGAINST BLADDER CANCER
Hypericum sinaicum,Tissue culture , Naphthodianthrones , Hypericin , A. rhizogenesis transformation , SSR finger print , Photodynamic therapy, Antibladder cancer.
Hypericum sinaicum (Hypericeae family) is well known as one of the endemic important Egyptian medicinal plants in Sant Katherin Protectorate South Sinai. Whereas, it contains numerous compounds have biological activities, such as napthodianthrones, including hypericin and pseudohypericin. These compounds are used as bladder antitumor. The aim of this study is in vitro propagation, molecular characterization, active ingredients production and histopathology studies. The best in vitro seed germination of H. Sinaicum seeds was obtained on free-MS medium. In this regarding callus production; the highest value of calli cultures was produced from nodal segment and root explants respectively. The best results of calli production was under dark condition on MS-medium with 3 mg/l of 2,4-D. However, the lowest calli frequency (28 and 20 %) and fresh weights (1.4 and 1.0 g/ Jar) were estimated with nodal and root explants on MS medium with 0.25 mg/l 2,4-D + 0.25 mg/l TDZ under light condition. Also the maximum value of growth rates 0.33 and 0.18 (gm/day) were recorded at the end of the 3rd week of cultivation for nodal and root explants, respectively. Regarding, direct/ indirect shoot regeneration; the maximum number of direct shootlets regeneration was 24 shootlets/ explant from nodal segment on MS medium supplemented with 0.1 mg/l NAA plus 1mg/l BA at light condition. Likewise, the maximum number of indirect shootlets regeneration 14 shootlets/callus was recorded with the same previous explant on MS medium with 0.5 mg/l TDZ and 0.05 mg/l NAA under light and darkness condition. While in vegetative growth dynamic the highest shoot growth rate (0.273 gm/day) was recorded for shoots derived from nodal segment at the 3rd week of cultivation. In addition to, H. sinaicum was successfully regenerated after preservation and cryopreservation treatment. Concerning, Agrobacterium rhizogenesis transformation experiment two protocol were investigated. In 1st protocol, A4 pgfp strain was gave the best transgenic hairy roots formation with leaf, stem and root explants of H. sinaicum. While in 2nd protocol found that LBA1334pgfp strain gave the highest shoot elongation (8.82 cm), root elongation (6.58 cm), branches shoots (2.5 shootlets), and branches roots (10.9 rootlets) values after 3 weeks of incubation. Regarding molecular characterization SSR finger print(s) was carried out and applying total polymorphism (100%) of in vitro derived plants compare to in vitro intact plant when using Hyp-1 primer. In state of chemical analysis it was found that, the highest value of hypericin contents 93.952 and 90.0395 µg/g dry weight were estimated in shootlets regeneration or calli induction from nodal segments on MS containing 2mM of algae mixture (diatomas) as biotic elicitors by using HPLC. Ultimately, the high dose of H. sinaicum crude extract (10 mg/kg daily) for 2 months can be used for prevention from bladder cancer at early stage.
2017
Ph.d
Sadat City
Agriculture