Protoplast isolation, colony formation and shoot regeneration from Lolium perenne

Abstract

Lolium perenne (ryegrass) is the most widely sown pasture grass in New Zealand providing forage for millions of sheep and cattle. Genetic improvement of Lolium species through conventional breeding has been particularly difficult because of its outbreeding and heterozygous nature. Biotechnology techniques that are not based on genetic modification technology, such as protoplast fusion, could help in the breeding of L. perenne as it enables the introgression of DNA (and associated desirable traits) from other species of plants that are otherwise unobtainable by conventional means. We intend to adapt this technology for L. perenne and report on our progress with the isolation of protoplasts, micro colony and callus formation, and shoot regeneration. Friable callus was induced from L. perenne seeds and used for isolation of protoplasts. After testing several combinations of enzyme mixtures at different molarities, a high yield of protoplasts (1.1 × 107 g-1 FW) with a viability of 82% has been obtained. Callus colonies were formed from protoplasts cultured on a nitrocellulose membrane over a feeder layer. Plating efficiencies of 0.1-0.2% were obtained. Regeneration of plantlets from these colonies were obtained on Linsmaier and Skoog medium supplemented with growth hormones. All the plants regenerated to date have an albino appearance.