Mesophyll protoplasts were produced from clones of two cultivars of Medicago sativa, ''Rangelander'' and ''Regen S''. Protoplasts from the ''Regen S'' clone generally gave rise to calli while those from the ''Rangelander'' clone would undergo direct embryogenesis. Effects of plant growth conditions, donor tissue pretreatment and protoplast culture conditions on mesophyll protoplast production and subsequent development patterns were investigated. The major factor determining whether or not mesophyll protoplasts would be produced from either of the clones was the pretreatment in water of shoots excised from the donor plants. Pretreatments in water containing growth regulators did not alter protoplast production or development in the ''Regen S'' clone. Pretreatment of the ''Rangelander'' clone shoots whith abscisic acid or naphthaleneacetic acid was slightly beneficial to embryo production while pretreatment with benzylaminopurine was detrimental. Altered leaf morphology induced by growth condition changes did not affect mesophyll protoplast production or subsequent development patterns when shoots were pretreated in water. Culture of protoplasts in liquid droplets or solid agar medium increased low density protoplast survival and subsequent embryo production in the ''Rangelander'' clone.
