Plants from protoplasts isolated from a long-term root culture (Super Root) of Lotus corniculatus

In the legume bird's-foot trefoil (Lotus corniculatus L.), a super-growing root (super root) culture has been recently introduced. This system is uniq ue because it allows continuous root cloning, somatic embryogenesis and mas s regeneration of plants under entirely hormone-free culture conditions. He re we report the isolation and culture of protoplasts from this long-term r oot culture (more than three years old) and the regeneration of plants from super root-derived protoplasts. Treatment of pre-cultured super roots with an enzyme mixture containing 4.0 % cellulase and 0.1% pectolyase in 0.25 mol/L mannitol/0.25 mol/L sorbitol plus 0.1 % CaCl2 led to the separation of the root tips from the rest of th e roots and, within four hours, to the isolation of approximately 3.0x10(6) protoplasts per gram root tissue. Protoplasts were released almost entirel y from the separated root-lips and cultured, embedded in small agarose disk s, in a modified KM8P medium supplemented with 0.05 mg/L BAP, 0.1 mg/L 2,4- D and 0.5 mg/L NAA. After five to seven days, the first divisions were observed, and after four weeks micro calli of 0.5 to 1 mm could be recovered. Calli were placed on MS medium containing relatively high concentrations of BAP and NAA (both 1. 5 mg/L) for four weeks, resulting in prolific shoot formation. Elongated sh oots rooted easily on hormone-free medium, and plants could be established. Roots from regenerated plants and roots formed directly on protoplast-deri ved calli were used to establish new root cultures. These newly initiated c ultures expressed all the super root qualities again, including prolific sh oot regeneration upon transfer to light, indicating that the super-growing character is not lost through protoplast isolation and regeneration. The su per root pathway of regeneration, from protoplasts to callus, roots, root c ulture and plants, allows the virtually unlimited mass regeneration of plan ts from root protoplasts. Regenerating super root protoplasts add an import ant component to tissue culture systems for legume/Rhizobium research. Furt hermore, this is the first report on plant regeneration from protoplasts is olated from a true root culture rather than excised roots.