The use of protoplasts for genetic research.

Protoplasts isolated from cells of higher plants have provided a new, much heralded, experimental material for cell biologists and physiologists (1, 2). Though the potential of plant protoplasts as a system for genetic research has often been stressed, very little has been written about the kinds of unique genetic questions that can be resolved by the use of protoplasts. This paper describes the attributes of plant protoplasts relevant to their use in genetic experimentation, and reviews recent work from the author's laboratory using protoplasts in a diverse array of experimental contexts. For a discussion of the method involved in protoplast isolation, manipulation, and culture, several recent review articles may be consulted (2-4). Protoplasts have several characteristics that make them a unique material for the study of the genetics of higher eukaryotes. (i) It is possible to generate large, homogeneous populations of protoplasts of known genetic composition from single plants. Protoplasts of any genotype, be it haploid or diploid, mutant or aneuploid, can be easily obtained. This property is in contrast to single cells derived from in vitro cell cultures, that vary markedly in their genetic composition and physiological state. (ii) Protoplasts are devoid of the normal plant cell wall, and can be treated experimentally much as cultured mammalian cells. The absence of a wall permits protoplasts to fuse with one another, and allows the uptake of externally supplied particles or objects. (iii) Protoplasts can be cultured with high efficiency. It is common to have plating efficiencies of 50% or over with protoplasts, while this is a rare event with single cells derived from in vitro cell cultures. (iv) Protoplasts of some plant species are capable of regenerating into entire organisms. This attribute makes it possible to genetically analyze the mechanisms and processes of development. Other plant cell cultures also accomplish this feat. However, the possibility of combining regeneration to whole plants with the ease of working with haploid single cells makes plant protoplasts an unequaled material for genetically dissecting and analyzing biological processes. Perhaps these features are best understood by considering examples of the kinds of genetic experiments that have successfully used protoplasts. All of the work described below has been made possible or easier by the availability of protoplasts. Protoplasts used in the following experiments were derived from leaf mesophyll cells and were subsequently cultured according to the methods described by Nagata and Takebe (5).