<p>The response to <em>in vitro</em> tissue culture of five commercial annual ryegrass cultivars, namely ‘Magnun’, ‘Bargala’, ‘Tetragold’, ‘Hercules’ and ‘Maximus’, was evaluated. Morphogenic callus induction and plant regeneration capacity were assessed in shoot apices cultured on six media formulations that included Murashige and Skoog medium supplemented with casein hydrolyzate and different concentrations of 2,4 phenoxyacetic acid (2,4-D; 1 or 2 mg L^-1), benzylaminopurine (BA; 2 or 4 mg L^-1) and adenine (40 or 80 mg L^-1). Two morphological kind of calli were observed: one of them friable and regenerable and the other watery without regeneration capacity. The highest morphogenic callus formation (37 % in ‘Tetragold’ and ‘Hercules’ varieties <em>vs.</em> 58 % and 65 % in ‘Maximus’ and ‘Magnun’, respectively) was achieved using 2D4B80A medium (2 mg L^-1 2,4-D, 4 mg L^-1 BA and 80 mg L^-1 adenine). On the other hand, the highest number of regenerated plants per g^-1 fresh weight (FW) of morphogenic callus was 48.71 for ‘Magnum’ shoot apices cultured on 2D4B40A medium (2 mg L^-1 2,4-D, 4 mg L^-1 BA and 40 mg L^-1 adenine). Regenerated plants grew in soil under greenhouse conditions reached maturity and subsequently produced seeds. The results obtained in this study confirm the suitability of using the shoot tips of <em>Lolium multiflorum</em> as starting material when cultured on the formulation media developed in this study.</p>