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Hany M. El-Naggar, Paul E. Read, and Ayed Al-Abdallat

Phenylalanine ammonia-lyase (PAL) enzyme is the most extensively studied enzyme in the phenylpropanoid pathway. Studies on the biosynthesis of rosmarinic acid (RA) showed that the PAL enzyme catalyzes the initial step of the phenylpropanoid pathway. The increase in RA content in plant tissues in vitro coincided with the increase in PAL activity. The aim of this study was to investigate the activity of the gene responsible for the production of the PAL enzyme in the five rosemary genotypes; this will give more understanding about the accumulation of rosmarinic acid in the five rosemary genotypes. The genotypes were Majorca, Rosmarinus officinalis, Pine Scented, Madeline Hill and APR. Northern blot hybridization between the PAL gene primer and the five genotypes' cDNA showed bands at 300 bp in all the five genotypes for the PAL gene. The expression of the PAL gene was high in genotypes Majorca, Rosmarinus officinalis, and Madeline Hill, while the expression was low in genotypes Pine Scented and APR. It was expected that the genotypes having the highest PAL gene expression will produce the highest amount of RA, but the highest genotype in PAL gene expression Madeline Hill had the lowest RA production in their leaves. This could occur due to the tissue specific regulation inside plant tissues. Inside the callus tissues, where the specific tissue regulation no longer exists, the RA was produced in repetitively large amounts in genotypes with high PAL gene expression.

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Hany M. El-Naggar, Paul E. Read, and Susan L. Cuppett

Rosemary Rosmarinus officinalis is a member of the Lamiaceae. Rosmarinic acid (RA) is a very strong antioxidant produced in the chloroplast, and used to protect plant tissues against oxidative stress. A number of investigations showed that the sucrose concentration in the callus growing medium greatly influenced the production of secondary metabolites of the phenylpropanoid pathway such as RA. The aim of this study was to test the effect of elevated sucrose concentrations (2%, 3%, 4%, 5%, and 6% sucrose) and the effect of light and dark treatments on the production of RA in the callus of five different genotypes. The genotypes were Majorca, Rosmarinus officinalis, Pine Scented, Madeline Hill, and APR. It was found that the dark treatment produces more RA than the light treatment in all genotypes, and in all sucrose concentrations. The RA concentration increased with increasing the sucrose concentration from 2%—reaching the highest concentration at 4% and 5% in most genotypes. The RA concentration declined again at 6% sucrose in all genotypes. We concluded that for the extraction of RA from rosemary callus it is preferred to be produced in the dark—this will save energy and will produce more RA than the light treatment. Also it is preferred to use sucrose concentration at 4% for genotypes Rosmarinus officinalis, Pine Scented, and APR; and 3% sucrose for genotype Madeline Hill in the dark condition. While for the light condition, it is preferred to use 5% sucrose with genotypes Majorca, Rosmarinus officinalis, Pine Scented, and Madeline Hill; and 4% sucrose for genotype APR.

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Hany M. El Naggar, Paul E. Read, and Susan L. Cuppett

Rosemary (Rosmarinus officinalis) belongs to the Lamiaceae family, and is native to the Mediterranean and one of the most important medicinal herbs containing antioxidants in its leaves. One of the most important antioxidants is rosmarinic acid (RA). The aim of this study was to test the concentration of (RA) and chlorophyll content in leaves and callus of five successive subcultures of five different genotypes of rosemary. They were: 1) `Majorca'; 2) Rosmarinusofficinalis; 3) `Pine Scented'; 4) `Madeline Hill', and 5) APR. It was found that the highest concentration of RA in leaves was in `Pine Scented', while the lowest concentration was for APR and `Madeline Hill'. However, in the callus the highest RA concentration was for Rosmarinusofficinalis in the second subculture and `Madeline Hill' in the third subculture, while the lowest RA concentration was for `Majorca', `Pine Scented', and APR. The RA concentration in callus declined after the second and the third subculture for Rosmarinusofficinalis and `Madeline Hill', respectively. We concluded that it is preferred to use `Pine Scented' for RA extraction from the leaves while for RA extraction from callus it is better to use Rosmarinusofficinalis in the second subculture or `Madeline Hill' in the third subculture.