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.

This study aimed to develop an environmentally friendly horticultural substrate that promotes the growth of organic onion(Allium cepa L.) seedlings. Four substrates were prepared by mixing different ratios of peatmoss, cocopeat, perlite, vermiculite, and zeolite. Their pH and electrical conductivities ranged from 5.12 to 5.60 and from 0.07 to 0.08 dS/m, respectively. Beneficial microorganisms, molasses, sesame oil cake, and sulfur were added to one substrate combination, which was named “environmentally friendly horticultural substrate” (EFHS). The chemical properties of the EFHS were analyzed and compared with a commercial organic horticultural substrate (OHS) and a commercial general horticultural substrate (GHS). The organic matter and inorganic ion (nitrogen, potassium, calcium, zinc, and sulfur) contents in the EFHS were higher than those in the OHS and GHS. The germination rates of onion seeds in the EFHS were higher than in the OHS and GHS. The mean number of leaves, sheath diameter per seedling, and weight of 30-day-old seedlings grown on the EFHS were greater than those of seedlings grown on the OHS and GHS. The length of the seedlings grown on the EFHS was comparable to that of the seedlings grown on the OHS and greater than that of the seedlings grown on the GHS. Overall, the growth parameters of onion seedlings grown on the EFHS were better than those of seedlings grown on the OHS and GHS, suggesting that the EFHS may be used as an organic horticultural substrate for growing organic onion seedlings.