Improved plant regeneration from cell suspensions of commercial cultivars, breeding- and inbred lines of perennial ryegrass (Lolium perenne L.)

The impact of 16 different culture media compositions on the establishment of regenerable cell suspensions from chopped mature embryos of two inbred lines (L6, L31) of Lolium perenne L. was analysed in a multifactorial design. The replacement of sucrose by maltose, dicamba by 2,4-D and the stepwise increase of the BAP concentration in the culture initiation medium significantly improved the regeneration frequency of the cell suspensions. Significant interactions of genotype x carbohydrate source and genotype x auxin source were observed. The best genotype (L6) produced on the optimised culture medium on average 488 green shoots per gram suspension freshweight. Rapid establishment of fine regenerable suspensions was demonstrated by passing small cell colonies through a 500 mu m mesh four weeks after suspension culture initiation and their subsequent regeneration to plants. Regeneration potential was maintained in most of the suspension cultures over a 33 weeks culture period. The optimised culture medium allowed the establishment of regenerable eel suspensions from four commercial cultivars and four breeding lines with an average regeneration frequency from 36 green shoots per gram suspension freshweight in cv. Aurora up to 388 green shoots per suspension freshweight in cv. Limes. Regenerated plants produced germinable seeds. Since cell suspensions are the preferred target for gene transfer into perennial ryegrass our improved culture protocol has the potential to support elevated transformation frequencies and extension of the ztechnology to recalcitrant cultivars or genotypes.