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2004 sivb congress symposium proceedings ““thinking outside the cell””: optimized chemodiversity in protoplast-derived lines of st. john's wort (hypericum perforatum l) Plant

PAN, Z. G., LIU, C. Z., MURCH, S. J., SAXENA, P. K.
In vitro cellular & developmental biology 2005 v.41 no.3 pp. 226-231
Hypericum perforatum, antioxidant activity, benzyladenine, callus, cell division, culture media, genetic variation, genotype, germplasm, glucose, hypocotyls, naphthaleneacetic acid, organogenesis, phytopharmaceuticals, plantlets, protoplasts, salts, seedlings, shoots, somatic hybridization, thidiazuron
A procedure for protoplast isolation and plant regeneration of St. John's wort has been developed to utilize cell-to-cell variability for optimum production of valuable medicinal compounds. Calluses, induced from hypocotyl segments of St. John's wort seedlings, were used for protoplast isolation, induction of sustained cell division, and ultimately, plant regeneration. Callus-isolated protoplasts at a density of 2.0××10⁵ per ml were embedded in 0.6%% Na-alginate blocks and cultured in a medium containing modified Murashige and Skoog (MS) salts, 2.5 μμM 6-benzylaminopurine (BA), 5.0 μμM αα-naphthaleneacetic acid (NAA), and 0.5 mol l⁻⁻¹ glucose. Protoplast-derived colonies formed compact calluses when transferred onto 0.35%% gellan gum-solidified MS medium supplemented with 2.5 μμM BA and 2.5 μμM NAA. Shoot organogenesis from the protoplast-derived callus was induced on MS medium supplemented with 5 μμM thidiazuron. Complete plantlets were obtained from the regenerated shoots on MS basal medium. A greater than 3-fold variation of antioxidant activity was observed among the protoplast-derived plantlets and chemically distinct germplasm lines were selected on the basis of phytochemical profiles. The protoplast to plant regeneration protocol developed in this study provides the foundation for development of novel genotypes with potential expansion of the genetic diversity through somatic hybridization, and organelle transplantation.