الفهرس 
Production of Rosmarinic Acid from Callus, Shoot, Cell Suspension and Hairy Root Cultures of Ocimum basilicum L. and Salvia officinalis L. plantsOnly 14 pages are availabe for public view
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Abstract
The present study was carried out with the cooperation between the Faculty of Agriculture, Alexandria University, Department of Floriculture, Ornamental Horticulture and Landscape Gardening and the Tissue Culture Laboratory of the Pharmaceutical Bioproducts Research Department, Genetic Engineering and Biotechnology Research Institute, City of Scientific Research and Technology Applications, New Borg El-Arab, Alexandria, Egypt from the period of 2017 to 2019. Various in vitro cultures such as callus culture, shoot culture and cell suspension culture in addition to the use of some elicitors such as salicylic acid and yeast extract to increase the accumulation of rosmarinic acid and also hairy root cultures for basil Ocimum bsilicum and sage Salvia officinalis plants were prepared .
The world is currently moving towards producing pharmaceutical compounds from their natural sources, ie, plants that contain secondary metabolic compounds such as phenolic and antioxidant compounds, including rosmarinic acid. The increasing demand for rosmarinic acid due to its importance in pharmaceutical, cosmetic industries and food preservation for its antibacterial, antifungal, antiviral effects beside it is also a strong antioxidant. It has been found that the quantity produced by traditional methods does not cover the increasing demand. Therefore, the present study aimed at using plant tissue culture techniques for the production of rosmarinic acid in large quantities and high concentrations from callus, shoots, cell suspension cultures and hairy roots of both basil and sage plants by conducting several research experiments which can be summarized as follows:
Culture establishment of basil and sage plants
- Bail (”Genovase” cultivar) seeds and sage shoot tips cultured after sterilization in basal MS medium + 0.1 g/l myo- inositol for seedling and shoots production and as a source of explants (leaves – lateral buds – internodes) .
The first experiment
Callus cultures production in basil and sage plants
This experiment was carried out to evaluate the effect of explant type (leaf, lateral bud and internode) and growth regulator concentrations on callus production.
In case of basil plant leaf , lateral bud and internode explants cultured on MS medium with different concentrations of 2, 4-D (0.25, 0.5, 1.0 and 2.0 mg / l) and basal MS medium (control) . Two explants cultured for each jar as a replicate. The following data were recorded after four weeks of culture as following:
None of the three tested explants gave a response on basal MS medium (control).
The color and texture of callus was dependent upon the type of explant and 2, 4-D concentration. It was greenish and compact at 0.25mg/l 2, 4 –D and all type of explant, also at 1.5 mg/l 2, 4 –D and leaves. Callus derived from all type of explant grown on 0.5 mg/l ,as well as callus derived from leaves grown on 1mg/l 2,4-D was greenish granular and compact, lateral buds and internodes on 1 and 1.5 mg/l 2,4 –D gave callus light green and semi compact. Callus from 2 mg/l 2, 4-D with all type of
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explant was weak, with white green and compact from leaf explant and yellow friable from lateral bud and internode explants.
Callus fresh weight (g) was analyzed and the data statistical analysis indicated that: there was a significant difference for type of explant, 2, 4-D concentrations and the interaction between them. . The highest significant mean value for callus fresh weight (3.6 g) obtained when leaves cultured on MS + 0.25 mg / l 2,4-D, while the lowest significant mean value of callus fresh weight (0.6 g) resulted on MS + 1.5 mg / l 2,4- D with internode explant .
Callus was sub-cultured on fresh media every month to maintain callus stock. After three subcultures callus was collected and dried in the oven at 44 °C till tissues were completely dried then kept at -20 °C for rosmarinic acid extraction and determination.
For sage plant explants cultured on MS basal (control), and MS medium supplemented with 10 mg/l ascorbic acid, 1mg/l NAA and BA at 1, 3, 5, or 7 mg/l. Two explant cultured for each jar as a replicate .The following data were recorded after 25 days of culture:-
None of the three tested explants gave a response on basal MS medium (control).
Lateral bud was the best explant followed by internode explants and the worst one was leaf explants that were slow in growth or absent in some replicates and often leaves turn to browning stage as result of phenolic compounds secretion.
Lateral bud explants on all callus induction medium compositions formed shoots and callus proliferation was in the base of shoots.
Also, there was a propagules appeared after three or four subcultures from lateral bud explant cultured on MS+1mg/l NAA + 5 mg/l BA or 7mg/l BA, also slightly appeared at 3 mg/l BA.
Dominant callus color was greenish derived from lateral bud and internode explants cultured in all medium compositions except medium sulpplimented with 1mg/l NAA + 7mg/l BA produced light green callus also leaves grown on 1mg/l NAA + 5mg/l BA produced callus greenish. Callus obtained from leaves on 1mg/l NAA + 1mg/l or 3mg/l BA was light green, but callus obtained from leaves cultured on 1mg/l NAA + 7mg/l BA was dark green. Callus was friable from lateral bud and internode explants on 1mg/l NAA +1mg/l BA or leaf explant on 1mg/l NAA + 7mg/l BA. 1mg/l NAA + 3mg/l or 5mg/l BA with lateral bud and leaf explants gave a compact callus. Also, leaf explant on 1mg/l NAA + 1mg/l BA and lateral bud explant on 1mg/l NAA + 7mg/l BA gave a compact callus. Callus was semi compact from internode explant on1mg/l NAA + (3, 5, or 7 mg/l BA).
Callus fresh weight (g) was analyzed and the results showed that: medium compositions and explant type caused significant variance in callus fresh weight while the interaction between them did not cause significant variance in callus fresh weigh. Moreover, MS+ 1mg/l NAA +1 mg/l BA with lateral bud explants gave the highest value of callus fresh weight (1.1 g) and the callus was greenish and friable and some of was compact, while MS + 1mg/l NAA + 7mg/l BA with leaf explants gave the lowest callus fresh weight (0.3 g) and the callus was dark green and friable.
Callus was sub-cultured on fresh media every 25 day to maintain callus stock. After five subcultures callus was collected and dried in the oven at 44 °C till tessues were completely dried then kept at -20 °C for rosmarinic acid extraction and determination.