Improvements in Protoplast Isolation Protocol and Regeneration of Different Cabbage (Brassica oleracea var. capitata L.) Cultivars

Protoplasts are a versatile tool in plant biotechnology since they can be used for basic biological studies as well as for breeding strategies based on genome editing. An efficient protoplast isolation protocol is essential for conducting protoplast-based studies. To optimize the protoplast isolation protocol in cabbage (Brassica oleracea var. capitata L.), different enzyme solutions were tested for the isolation of leaf mesophyll protoplasts. In our experiments, the combination of 0.5% Cellulase Onozuka RS and 0.1% Macerozyme R-10 showed the best result. The optimized protocol proved suitable for the isolation of protoplasts from five different cabbage cultivars with yields ranging from 2.38 to 4.63 x 10(6) protoplasts/g fresh weight (fw) and a viability of 93% or more. After three weeks in culture, protoplasts from all of the tested cultivars formed micro-calli, but further callus growth and shoot regeneration depended strongly on the genotype and regeneration protocol used. For shoot formation, 1 mg/L BAP in combination with auxin 0.2 mg/L NAA showed the best results with a regeneration of 23.5%. The results obtained will contribute to the development of different applications of cabbage protoplasts and facilitate the breeding process of this important horticultural crop.

Automated summary

Through our experiments, we found that a combination of 0.5% Cellulase Onozuka RS and 0.1% Macerozyme R-10 yielded the best results in isolating protoplasts from cabbage leaves. After three weeks in culture, protoplasts from all tested cabbage cultivars formed micro-calli, but further callus growth and shoot regeneration varied depending on the genotype and regeneration protocol used. The best regeneration results, with a rate of 23.5%, were obtained using a medium supplemented with 1 mg/L BAP and 0.2 mg/L NAA. These findings will contribute to the development of various applications for cabbage protoplasts and facilitate the breeding process of this important horticultural crop.