CULTURE OF VEGETATIVE AND FLORAL MERISTEMS IN RYEGRASSES - POTENTIAL TARGETS FOR MICROBALLISTIC TRANSFORMATION

An in vitro culture system allowing the regeneration of fertile plants from isolated vegetative meristems has been established for Lolium mu ltiflorum (Italian ryegrass) and L. perenne (perennial ryegrass). In a ddition, early immature inflorescences before initiation of stamen pri mordia have been isolated and in vitro cultured in L. multiflorum. Dev elopment of flower meristems from cultured early immature inflorescenc es to normal in vitro matured spikelets led to the production of viabl e pollen. Both protocols have been worked out as a basis for microtarg eting-mediated transformation of cells in vegetative and floral merist ems for these important forage grass species. A description based on s canning electron microscopy of different developmental stages of veget ative, transition and floral apices of L. multiflorum allowed the iden tification of potential target meristems appropriate for particle bomb ardment. Plant regeneration from 79% and 99% of the aseptically cultur ed target vegetative meristems, isolated with 1-4 leaf primordia from greenhouse-grown plants, was achieved in Italian and perennial ryegras s, respectively. A stepwise culture system for in vitro development of early immature inflorescences bearing target floral meristems until m aturation of floral organs in 80% of the cases and production of 44% v iable pollen has been optimized for Italian ryegrass. Approximately 10 % of the crosses made with pollen of anthers obtained from in vitro cu ltured early immature inflorescences led to seed setting. Ballistic mi crotargeting to vegetative meristems and to floral meristems of early- staged immature inflorescences allowed for delivery of particles to ce lls in first and second cell layers, and for transient beta-glucuronid ase expression of a rice act1-uidA chimeric gene construct in the micr otargeted cells.