Protoplast Isolation, Culture, and Regeneration

Protoplasts of Arabidopsis thaliana have been isolated from a variety of explant sources with varying degree of success (1 –3 ), Most workers have faced problems in achieving a high frequency of sustainable division of protoplasts in liquid culture. The problem can be alleviated to a certain extent by embedding mesophyll protoplasts m calcium algmate (4 ). We have developed a technique allowing the culture of protoplasts derived from roots or cell suspensions m llqurd medium and then regeneration to plantlets (2 ). The yield of root-derived protoplasts capable of division and regeneration is considerably higher in comparison to protoplasts obtained from leaf mesophyll cells. The protoplast lsolatton requires a ready supply of root cultures marntamed m auxin containing medium. Alternatively, a high yield of protoplasts, suitable for transient gene expression studies using direct DNA transformatron, can also be obtained from cell suspension cultures, excluding any limitatton of available starting material. Because the methods may have to be tailored to the needs of different experiments, here we describe protocols for the rsolatlon and culture of protoplasts from leaf mesophyll tissue (4 ), as well as from root and cell suspension cultures. These methods are equally effective for the Arabzdop- szs ecotypes Columbia, C24, RLD, and WaSSllJeWSkiJa.