Efficient plant regeneration from hairy root-derived protoplasts of Hyoscyamus muticus

Successful plant regeneration was achieved for the first time from hairy root-derived protoplasts of Hyoscyamus muticus. High yields (7 × 10⁶ / g fresh weight) of protoplasts were isolated directly from the transformed roots of Hyoscyamus muticus using an enzyme mixture comprising 1 % macerozyme and 2 % cellulase in an osmoticum consisting of 0.2 M CaCl₂ and 0.6 M mannitol. Protoplasts were first cultured in liquid NT/PRO I medium and further on semi-solid NT/PRO II agar medium. The procedure permits highly efficient formation of colonies. The plating efficiency varied from 1-9 %. The small individual colonies regenerated easily into shoots and roots at frequencies of 18 % and 70 %, respectively. The time required for the development of small plantlets from protoplasts was 8-11 weeks. The regenerated plants contained rolB from Ri-T-DNA and exhibited an altered phenotype compared to the control plants.