PROTOPLAST CULTURE AND GENERATION OF TRANSGENIC PLANTS IN RED FESCUE (FESTUCA-RUBRA L)

Embryogenic callus cultures, derived from mature embryos, were initiat ed for three cultivars of red fescue (Festuca rubra L.). Morphogenic s uspension cultures were established for different genotypes of red fes cue cvs. 'Roland' and 'Gondolin', 5 months after their initiation. Sus pension cultures of red fescue cv. 'Roland' showed the capacity to reg enerate green plants efficiently over a period of 14 months. Similar b ehavior, concerning growth and plant regeneration, was observed for em bryogenic cell suspensions when thawed and re-established after cryopr eservation for their long-term storage. Protoplasts isolated from thes e highly morphogenic suspensions and cultured in agarose beads using n urse cells formed microcalli with 2 x 10(-3) overall plating efficienc y. More than 85% of the protoplast derived microcalli grew further and allowed for regeneration of green plantlets in vitro. Forty represent ative plants from protoplasts were established in soil and grown under greenhouse conditions. To develop transgenic red fescue plants, exper iments using direct gene transfer to protoplasts, polyethylene glycol treatment and a chimeric phosphinotricin acetyltransferase gene driven by rice actin 1 5' sequences were performed. Upon selection with 50 m g/l phosphinotricin, resistant clones were obtained with 10(-6) overal l transformation frequency and several transgenic plants were recovere d and grown in soil. Stable integration of the transgene in the genome of plants regenerated from resistant callus clones was shown by South ern hybridization analysis. Expression of the transgene in mature plan ts was demonstrated by phosphinotricin-herbicide spraying.