ABSTRACT
The following laboratory exercises use leaves of either chrysanthemum (Dendrathema grandiflora Tzvelev) or orchardgrass (Dactylis glomerata L.) to illustrate some aspects of protoplast technology; however, any number of plants will be suitable for these exercises. If different plants are selected, choose plants with young leaves that are thin and lack substantial pubescence. We have found that, although protoplasts are relatively easy to isolate from leaf tissue of these two species, it is more difcult to regenerate plants from chrysanthemum (Sauvadet et al., 1990) and nearly impossible from orchardgrass. As such, this laboratory is devised only to demonstrate some of the general principles of protoplast methodologies, that is, donor tissue preparation, preculture, tissue digestion, purication, and assessment of purity and viability. We suggest that the laboratory exercise be completed without regard to aseptic conditions since callus and plants will not be regenerated and that some of the complex protoplast culture media discussed in Chapter 26 are not required. The mechanics of the experiment may be completed in 1 day if some students can help out “off and on” for the entire day or if the instructor prepares some of the initial steps of the procedure in advance of the class meeting. The laboratory may also be conducted as a demonstration.
