الفهرس 
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Abstract
This study was carried out in Laboratory of Genetics Department, Faculty of agriculture, Ain Shams University and Plant Tissue Culture unit belonging to Genetic Resources Department, in Desert Research Center, to introduce the new species Plectranthus barbatuse (Coleus forsckoli) belonging to family: Lamiaceae to the Egyptian flora for its medicinal importance due to presence of forskolin the active ingredient of Plectranthus barbatuse.
Part 1: Micropropagation experiment
This part is concerned with the effect of cytokinin on the propagation of Plectranthus barbatus by in vitro micropropagation to give a large number of plantlets by using stem node section.
1. In multiplication stage, the axillary shoots per explant reached the highest significant value (6.19) on MS medium contained 2mg/L TDZ followed by 1.5mg/L TDZ at the value (5.72) when compared with the control value (3.03). Less concentrations of TDZ showed decrease in the mean number of axillary shoots per explant.
2. The highest value of mean length was highly significant (6.44 cm) on MS medium contained 1mg/L Kin followed by 0.5mg/L Kin (5.20 cm), when compared with the control value (4.81 cm).
3. The root formation and elongation did not respond in the control to form roots. The different concentrations of NAA and IBA as auxins induced root induction, the mean number of roots per explant of reached the highest significant value (30.00 ) on 1/2 MS medium contained 0.5mg/L IBA followed by 2mg/L IBA (25.67),4. The highest length of roots mean was 3.93 cm on 1/2 MS medium contained 0.5mg/L IBA followed by 1.5mg/L IBA 2.88cm.
5. Length of shoots mean (cm) recorded the highly significant value with 11.8 cm on 1/2 MS medium containing 2mg/l NAA followed by 1.5mg/L NAA with 11.4 cm.
6. It is noticed that when the concentration of NAA decreased, the length number of shoots mean decreased.
7. The survived acclimatized plants were 93% after two weeks from transferring rooted plantlets of Plectranthus barbatuse.
8. After a month from transferring the rooted plantlets, the percentage of surviving was 83%. In rooting stage, the plants were transferred from the closed artificial environment to the open environment
Part 2.Production of forskolin by callus culture
This stage aimed at the production of forskolin on large scale using callus culture with different concentrations of 2,4-D auxine through five subcultures.
1. This part was inducted on both callus of leaves and stems node explants with auxin and gave a negative result with MS medium without any auxin (Control). The induced callus from leaf explant was a yellowish friable callus.
2. The fresh weight mean of leaf callus at the third subcultures of Plectranthus barbatuse recorded the highest value (20.19g/jar) and the highest growth rate (16.44 g/jar) on MS medium contained 2mg/L 2,4-D with 9.7% callus induction.
3. The concentration of 2mg/L 2,4-D at the third subculture gave the best callus induction with leaf explant, the growth rate increased from the first subculture and reached the maximum at the third subculture, then return to decrease again at the fourth and fifth subcultures.4. The number of stem node callus mean at the third subcultures on MS medium contained 0.5mg/L 2,4-D of Plectranthus barbatuse recorded the highest value (27.73 g/jar) and the highest growth rate (23.53 g/jar) with 10.6% callus induction.
5. The concentration 0.5mg/L 2,4-D at the third subculture gave the best callus induction with stem node explant and the growth rate increased from the first subculture and reached the maximum at the third subculture, then return to decrease again at the fourth and fifth subcultures
Part 3: Extraction and quantitative estimation of forskolin by HPLC
1. The highest amount of forskolin content in leaf callus on MS medium contained 2mg/L 2,4-D of Plectranthus barbatuse recorded the highest value 467.76 mg/g in the fifth subculture. Followed by 0.5 mg/L 2,4-D with value 377.97mg/g in the fifth subculture compared with the control.
2. The highest amount of forskolin content in stem callus on MS medium contained 0.5mg/L 2,4-D of Plectranthus barbatuse recorded the highest value 751.96 mg/g in the fifth subculture.
3. The third subculture gave the best callus weight for both leaf and stem node with 2mg/L and 0.5mg/L 2,4-D. So that the elicitor feeding was added in the third subculture with the previous concentrations for leaf and stem node.
4. The highest amount of forskolin content in leaf callus on MS medium contained 2mg/L 2,4-D +45g/L sucrose 277.83 mg/g.
Followed by 2mg/L 2,4-D +0.2g/L casein hydrolysate with value 158.38 mg/g compared with the control.
5. The highest amount of forskolin content in stem callus on MS medium contained 0.5mg/L 2,4-D +0.2g/L casein hydrolysate of Plectranthus barbatuse recorded 81.62 mg/g compared with the control.6. The highest amount of forskolin content was obtained in leaf callus with 2mg/L 2,4-D in the fifth subculture. While, forskolin content in stem callus with 0.5mg/L 2,4-D in the fifth subculture gave the highest percentage of effective substance.
Part4: Relative expression analysis using quantitative real time RT-PCR (qPCR).
1. The relative fold change in gene expression of Cytochrome P450 genes in both leaf and stem callus with casein hydrolysate elicitor was measured on it by using three primers for three genes
)Cytochrome P450c4, Cytochrome P450c6 and Cytochrome P450c7(.
2. Both Cytochrome P450c4 and Cytochrome P450c6 expression increased (up-regulated) in stem callus on MS medium with 0.5mg/L 2,4-D +0.2g/L casein hydrolysate.
3. In leaf callus on MS medium with 2mg/L 2,4-D +0.2g/L casein hydrolysate, the expression of these two genes decreased (down-regulated).
4. Cytochrome P450c7 gene expression increased (up-regulated) in leaf callus on MS medium contained 2mg/L 2,4-D +0.2g/L casein hydrolysate, while decreased (down-regulated) in stem callus on MS medium contained 0.5mg/L 2,4-D +0.2g/L casein hydrolysate.