Protoplasts were isolated from the primary leaves of lettuce (Lactuca sativa L.) seedlings 10 days after in vitro germination. The leaves were stripped and incubated in an enzyme mixture consisting of 1.2% Cellulase R-10 and 0.3% Macerozyme R-10 in cell and protoplast washing solution (CPW) overnight. The average protoplast yield was 8.25 x 106 protoplasts per g of fresh leaf tissue. When protoplasts were cultured at a density of 3.0 × 105 protoplasts/mL in agarose solid KM8P/KM8 medium, first and second divisions were observed in the protoplasts within a week. Protoplast-derived microcolonies formed after 4 weeks of culture, and visible colonies were present after 3 months of culture. Protoplast-derived microcalli were transferred to Murashige and Skoog medium supplemented with 2.0 mg/L kinetin and 0.1 mg/L NAA and incubated in the light for 3 weeks. They grew into callus, which then regenerated into plants after 7 weeks of culture. The regenerated plants grew as apparently normal flowering fertile plants.

Abstract
Introduction
Materials and Methods
    1. Plant material and protoplast isolation
    2. Protoplast culture
    3. Plant regeneration
Results and Discussion
References

• Bo-Hwa Son(Researcher, Division of Life Science, Gyeongsang National University)
• Chul-Gyoo Park(Lecturer, Department of Science Education, Chinju National University of Education)
• Dhruba Khakurel(Graduate student, Department of Biology, Gyeongsang National University)
• Hae-Jun Hwang(Assistant manager, Department of Water Industry Demonstration, Korea Environment Corporation)
• Tea-Won Kim(Researcher, Gyeongsangnam-do Agricultural Research & Extension Services, Medicinal Resources Research Institute)
• Byeong-Gyun Jeon(Professor, Department of Biology Education, Gyeongsang National University)
• Sung-Ho Lee(Professor, Division of Life Science, & Institute of Agriculture and Life Science, Gyeongsang National University) Corresponding author