Culture of Vegetative and Floral Meristems in Ryegrasses: Potential Targets for Microballistic Transformation

Abstract

Summary An in vitro culture system allowing the regeneration of fertile plants from isolated vegetative meristerns has been established for Lolium multiflorum (Italian ryegrass) and L. perenne (perennial ryegrass). In addition, early immature inflorescences before initiation of stamen primordia have been isolated and in vitro cultured in L. multiflorum . Development of flower meristems from cultured early immature inflorescences to normal in vitro matured spikelets led to the production of viable pollen. Both protocols have been worked out as a basis for microtargeting-mediated transformation of cells in vegetative and floral meristems for these important forage grass species. A description based on scanning electron microscopy of different developmental stages of vegetative, transition and floral apices of L. multiflorum allowed the identification of potential target meristems appropriate for particle bombardment. Plant regeneration from 79 % and 99 % of the aseptically cultured target vegetative meristems, isolated with 1–4 leaf primordia from greenhouse-grown plants, was achieved in Italian and perennial ryegrass, respectively. A stepwise culture system for in vitro development of early immature inflorescences bearing target floral meristems until maturation of floral organs in 80 % of the cases and production of 44 % viable pollen has been optimized for Italian ryegrass. Approximately 10 % of the crosses made with pollen of anthers obtained from in vitro cultured early immature inflorescences led to seed setting. Ballistic microtargeting to vegetative meristems and to floral meristems of early-staged immature inflorescences allowed for delivery of particles to cells in first and second cell layers, and for transient β-glucuronidase expression of a rice act1-uidA chimeric gene construct in the microtargeted cells.