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

Abstract

Summary In the legume bird's-foot trefoil ( Lotus corniculatus L.), a super-growing root (super root) culture has been recently introduced. This system is unique because it allows continuous root cloning, somatic embryogenesis and mass regeneration of plants under entirely hormone-free culture conditions. Here we report the isolation and culture of protoplasts from this long-term root 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 % CaCl 2 led to the separation of the root tips from the rest of the roots and, within four hours, to the isolation of approximately 3.0 × 10 6 protoplasts per gram root tissue. Protoplasts were released almost entirely from the separated root-tips and cultured, embedded in small agarose disks, 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 shoots rooted easily on hormone-free medium, and plants could be established. Roots from regenerated plants and roots formed directly on protoplast-derived calli were used to establish new root cultures. These newly initiated cultures expressed all the super root qualities again, including prolific shoot regeneration upon transfer to light, indicating that the super-growing character is not lost through protoplast isolation and regeneration. The super root pathway of regeneration, from protoplasts to callus, roots, root culture and plants, allows the virtually unlimited mass regeneration of plants from root protoplasts. Regenerating super root protoplasts add an important component to tissue culture systems for legume/ Rhizobium research. Furthermore, this is the first report on plant regeneration from protoplasts isolated from a true root culture rather than excised roots.