الفهرس 
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Abstract
MICROPROPAGATION, TRANSPLANTATION AND BIOTRANSFORMATION STUDIES ON DAUCUS GLABER FORSSK. (CAUCALIS GLABER FORSSK.) (FAMILY: APIACEAE)
Daucus glaber Forssk.is a rare, wild, annual herb which is distributed from the Mediterranean area to East Asia. The plant is commonly known as Caspian carrot. This plant seems to be rich in important medicinally active components as volatile oils, phenyl propanoid esters, sesquiterpene lactones and flavonoids which have many biological activities as cytotoxic, antimicrobial and antioxidant activities.
So, the proposed study of this plant presented in this thesis includes:
1- Tissue culture study of Daucus glaber to select the best condition for callus induction.
2- Micropropagation study of the plant through in vitro seed germination, callus induction, organogenesis and acclimatization of whole plant for conservation purposes.
3- Comparative study between the chemical compositions of different parts of the cultivated plants, calli and in vitro regenerated plants, using GLC-MS and HPLC techniques.
4- Screening of the possible biological effects of different plant extracts to reveal biological activities and / or medicinal values.
5- DNA fingerprinting of the plant using RAPD technique.
6- Transformation of the plant using Agrobacterium mediated transformation method to investigate the most appropriate protocol and to study the effect of the inserted gene on the active carotenoids production.
Thus, the present study was performed in two parts. The first part included three chapters (chapter I: Tissue culture and micropropagation; chapter II: GLC/MS and HPLC studies; chapter III: Biological activities). The second part included two chapters (chapter I: DNA fingerprinting using RAPD technique andchapter II: Agrobacterium mediated transformation study).
Part I
Chapter I - Tissue culture, Micropropagation and Transplantation Study of Daucus glaber Forssk.
Protocols for callus induction, somatic embryogenesis and organogenesis from leaf, stem and root explants of the in vitro germinated seeds of Daucus glaber Forssk. were evaluated. Explants were cultured on MS medium supplemented with different concentrations of hormones and hormonal combinations. The study revealed that the best hormonal combinations for callus induction from leaf and stem explants were (TDZ 0.5mg/l + 2,4-D 1mg/l + BAP 0.1mg/l) while the best hormonal combinations for callus induction from root explants were (NAA 1mg/l + BAP 0.1 mg/l). The media with hormonal combinations of (TDZ 0.5mg/l + 2,4-D 1mg/l + BAP 0.1mg/l) and (NAA 1mg/l + BAP 0.1 mg/l) were the best for embryogenesis while multiple shoot were induced from the calli of Daucus glaber by culturing them on MS medium supplemented with (TDZ 0.5mg/l + 2,4-D 1mg/l + BAP 0.1mg/l), averages of 10 shoots were developed per callus. The transplantation trials of the micropropagated plantlets lead to the maintenance of them for about two months in the greenhouse. It can be concluded that the protocols developed are a useful approach to the micropropagation of Daucus glaber species to help in its conservation.
Chapter II - GLC and HPLC Studies of the Contents of Some In vivo and In vitro Tissues of Daucus glaber Forssk.
This study focused on comparing the volatile constituents of different plant parts as well as in vitro induced calli and plants using gas chromatography-mass spectroscopy (GLC/MS), besides screening of three of the major nonvolatile contents previously isolated from the wild plant (angeloyloxylatifolone, glaberinA and talasin A) in different parts of cultivated plant and in vitro induced calli and plants using high performance liquid chromatography (HPLC).
A)The GLC/MS study revealed the composition of the essential oils and ether extracts of fruit, leaf, root and in vitro regenerated plantlets of the cultivated plant of Daucus glaber Forssk. among the essential oils of fruit, leaf and stem of the wild plant:
1- The essential oil of the fruit as well as the leaf showed the presence of the phenyl propanoidmethyl isoeugenol as the major component of the oil. This component has never been detected in the essential oil of the wild plant before.
2- Myristicin is the major essential oil component of the ether extract of the root while it is completely absent from the oil of the fruit and leaf.
3- Just traces of elemicin could be detected in the fruit oil of the cultivated plant, while it is considered a major component of the fruit and stem oil of the wild plant.
4- Sabinene is the major monoterpene hydrocarbon in the oils of cultivated plant, while limonene is considered the major monoterpene hydrocarbon in the oils of wild plant.
5- The ether extract of the root is characterized by the absence of monoterpenes.
6- GC/MSof the ether extract of the root callus, growing on MS media with 1mg/l NAA and 0.1 mg/l BAP, revealed the presence of trans- caryophyllene in a percentage of 0.2%
7- The ether extract of the in vitro regenerated plant contains monoterpene hydrocarbons (2.55%) including α-pinene, sabinene and limonene. sesquiterpene hydrocarbons (2.5%) including trans-caryophylline, α-humulene, β-bisabolene and γ-bisabolene. borneol acetate (0.05%) is the only monoterpene ester in the extract.
B)The qualitative and quantitative HPLC analysis showed the following:
1- All in vivo tissues (leaf, fruit and root) containangeloyloxylatifolone.
2- Angeloyloxylatifolone can be detected in the leaf calli those grew on MS medium with 1mg/l NAA and 0.1 mg/l BAP and MS with 0.5 mg/l TDZ, 1 mg/l 2, 4 D and 0.1 mg/l BAP, besides the extract of the in vitro regenerated plants.
3- Talasin A can only be detected in the n-hexane fraction of the leaf in a percentage of 6.7%.
4- The phenyl propanoid trimester,glaberin A, could be detected in the n-hexane fraction of the leaf and root in a percentage of 0.2 and 0.13% respectively.
5- Talasin A and glaberin A cannot be detected in any of the extracts of the in vitro tissues.
6- The use of mobile phase in a concentration of (55 water: 40 methanol: 5 acetonitrile) is more preferable than (60 water : 40 methanol) as it gives more sharp peaks and also good separation of the tested components.
7- The n-hexane fraction of the fruit showed the highest percentage of angeloyloxylatifolone (15.55%) while the extract of in vitro regenerated plants showed the lowest percentage 0.01%.
Chapter III:Some Biological Activities forIn vivo and In vitro Tissues of Daucus glaber Forssk.
A-Cytotoxicity evaluation of some in vivo and in vitro extracts of Daucus glaber Forssk. wascarried out against Hep-G2 cells using cell viability assay.
The phenylpropanoid ester, angeloyloxylatifolone had the strongest cytotoxic activity against HEp-2 cell from the isolated compound under the investigation (IC50 3.67), followed by glaberin A (IC50 6.7), while the sesquiterpene lactone, talasin A showed the least activity (IC50 ˃50). All the tested extracts showed strong cytotoxic activities against Hep-G2 cells in comparison to doxorubicin standard. The volatile oil of the fruit showed the greatest activity followed by the ether extract of root and the alcoholic extract of leaf.
B-The anti-microbial activity of the different plant extracts were tested against Aspergillus fumigatus and Geotricum candidum as fungi, Streptococcus Pneumoniae and Bacillus subtilis as Gram positive bacteria and Pseudomonas aeruginosa, Escherichia coli as Gram negative bacteria. Ampicillin and Gentamicin were used as positive control against Gram positive and Gram negative bacteria, respectively. Amphotericin B was used as positive control for fungi.The total alcoholic extracts and ether extracts of fruit, leaf, root and in vitro regeneratedplant as well as the volatile oilsof fruit and leaf of Daucus glaber Forssk.had a significant antifungal and antibacterial effect against the tested microorganisms except for Candida albicans and Pseudomonas aeruginosa. Ether extract of theroot of Daucus glaber Forssk. had the least activity against susceptible microorganisms, while the ether extract of the leaf has the maximum activity against all tested microorganisms.
C-No signs of acute toxicity or mortality were observed for all tested extracts within doses from 10 mg/kg upto 5000 mg/kg of body weight. We can conclude that the aerial part’s alcoholic extract and the volatile oils of leaves and fruits are practically non-toxic.
Part II
Chapter I: DNA fingerprinting using RAPD technique:
The RAPD banding patterns of Daucus glaber Forssk. were performed using 10primers (A1, A4, A6, A7, A8, A9, A10, A11, A12 and A13)which were able to amplify the DNA from Daucus glaber Forssk. via PCR. The number of well-defined and major bands for a single primer ranged from 2 to 5. The maximum number of well-defined or major bands was ob¬served with primer A8 and A12 (5 bands each).This technique suggests the use of primers A8 and A12 for the selective discrimination of Daucus glaber Forssk.
Chapter II: Agrobacterium Mediated Transformation in Daucus glaber Forssk.
In this study the cyanobacterial gene PSY (encodes for Phytoene synthase) was cloned into suitable plant expression vector (pTRA-K) in order to transfer the PSY activity into Daucus glaber Forssk. The PSY gene was PCR amplified using Synechococcus elongatus (PCC6803) as template and PSY gene specific primers flanked by NcoI and XbaI recognition sequences. The resulted PSY PCR product was further cloned into pTRA-K vector. The resulted pTRA-K-PSY plasmid DNA was used to transform Daucus glaber Forssk. via Agrobacterium tumefaciens mediated plant transformation. The resulted explants after these transformation events were selected on medium with Kanamycin. Presence of PSY gene in transformed calli was confirmed using PCR. The effect of the inserted gene on accumulation of carotenoids in the callus was performed by comparing the UV absorption of the extracted carotenoids in transformed and non-transformed calli at different time intervals (30, 50 and 70 days) at 460 nm. The insertion of PSY in Daucus glaber Forssk. significantly increased the accumulation of carotenoids (provitamin A).