Tissue culture-based selection of high rosmarinic acid producing clones of rosemary (Rosmarinus officinalis L. ) using Pseudomonas strain F

Natural phenolic antioxidants from genetically heterogeneous plants are becoming important for prevention of lipid oxidation in food. To produce uniform and enhanced levels of antioxidants we have developed elite clonal lines of rosemary using plant tissue culture techniques. A cytokinin (benzyladenine)-induced adventitious shoot multiplication was developed for rosemary. Optimum shoot multiplication occurred in Murashige and Skoog medium containing 1 mg/l benzyladenine and 2 mM proline. About 3-10 adventitious shoots were induced from each explant originating from a single heterozygous seed in 45-60 days and was designated as a single clonal line. Individual shoots of each clonal line were then transferred to hormone free medium to regenerate whole plants. Using this approach several clonal lines with each originating from a single heterozygous seed were isolated from a heterogeneous seed lot. It is well established that microbial elicitors can stimulate plant secondary metabolites. Based on this concept, we inoculated several shoots of each clonal line of rosemary with a novel Pseudomonas sp. to stimulate synthesis of total phenolics and rosmarinic acid. Total phenolics and rosmarinic acid content of each clonal line were measured after 25 and 60 days to specifically select fast growing and microbial elicitor-induced high rosmarinic acid-containing clonal lines. Using this strategy 5 classes of clonal lines were isolated: low RA containing clonal line--R-1; medium RA--R-7 and R-12; high RA--R-16 and R-35; Pseudomonas mediated inhibition of RA--R-8; (v) high RA with growth inhibition--R-15. Selection of such genetically uniform phenotypes offer potential for production of phenolic antioxidants from rosemary with consistent quality. Genetically uniform clonal lines can also be used to understand the regulation of biosynthetic pathways associated with phenolic antioxidants in response to microbial elicitors.