Proc. Nati. Acad. Sci. USA Vol. 91, pp. 5222-5226, June 1994 Review Genetic instability of plant tissue cultures: Breakdown of normal controls (DNA methylation/genome rearrangements/rearrangement induced premeioticafly/repeat-induced point mutation/heterochromatin) R. L. Phillips*t, S. M. Kaepplert, and P. Olhoft* *Deprtnent of Agronomy and Plant Genetics and Plant Molecular Genetics Institute, University of Minnesota, St. Paul, MN 55108; and *Department of Agronomy, University of Nebraska, Lincoln, NE 68583 ABSTRACT Plants regenerated from However, the frequency of various nated the cell or cells for the tissue cul- relatively undifferentiated callus cultures classes of mutants derived from plant ture. The many levels of genomic modi- possess a vast array of genetic changes. tissue culture is elevated far beyond that fication that already are known and ex- Such variations can result in useful agri- expected in nature. Clearly, tissue cul- pressed as changed genotypes and cultural and horticultural products. For ture processes that involve an undiffer- phenotypes could be potent sources for other purposes, however, variations in entiated callus phase are mutagenic. selection by the plant breeder, and inci- traits other than those of interest may be Variations include, but are not restricted dentally, for theoretical ponderings by undesirable-for example, using cultured to, chromosomal rearrangements and the biologist." cells for genetic engineering. Any steps single-gene mutants (mostly recessive). This article reviews the progress in made toward understanding the basis of DNA methylation changes also have understanding the basis of tissue culture- tissue culture-induced genetic variation been reported in regenerated plants and induced variation. It also explores possi- should be helpful in developing a more their progeny. ble links between tissue culture-induced stable and manipulatable somatic cell sys- In her Nobel lecture, Barbara Mc- variability and mechanisms of sequence tem. This review provides a glimpse at the Clintock (2) said "Some responses to or genomic change. specific kinds of genetic changes encoun- stress are especially significant for illus- Change in tissue culture most likely tered among regenerated plants and their trating how a genome may modify itself occurs by a stress-response mechanism. progeny. Included among these variations when confronted with unfamiliar condi- The relevant mechanism may best be are cytosine methylation alterations of the tions. Changes induced in genomes when described as a programmed loss of cellu- genome. The repeat-induced point muta- cells are removed from their normal lo- lar control. The most commonly ob- tion (RIP) phenomenon, reported for fil- cations and placed in tissue culture sur- served plant tissue culture-imposed amentous fungi, is invoked to provide a roundings are outstanding examples of changes-chromosome rearrangements, framework to consider the origin of vari- this. The establishment of a successful DNA methylation, and mutations-also ation in plant tissue cultures. tissue culture from animal cells, such as are salient features of a phenomenon those of rat or mouse, is accompanied by called repeat-induced point mutation Cells of all living organisms reproduce readily observed genomic restructuring. (RIP, formerly termed rearrangement- with almost exact fidelity and give rise to None of these animal tissue cultures has induced premeiotically) first described in daughter cells of predictable genotype. given rise to a new animal. Thus, the filamentous fungi (3, 4). This review will Errors in the process occur infrequently significance of these changes for the or- describe features of RIP and of tissue due to a remarkable variety of cellular ganism as a whole is not yet directly culture-induced variation. It will also dis- controls that regulate the genome. These testable. The ability to determine this is a cuss processes that might induce the RIP- controls cause expression of genes in a distinct advantage of plant tissue cul- like system in plant tissue culture. developmentally specific manner and fa- tures." cilitate the duplication, recombination, McClintock went on to say "The treat- A Review of Tissue Culture-Induced and distribution of chromosomes. Nor- ment, from isolation ofthe cell or cells of Variation in Plants mal cell behavior is the result of a com- a plant, to callus must inflict on the cells plex cascade of genetic programs that is a succession of traumatic experiences. Mutation induced by plant tissue culture sensitive to disruption by biotic and abi- Resetting of the genome, in these in- has been the subject of numerous scien- otic stresses (1). Although certain forms stances, may not follow the same orderly tific inquiries (reviewed in refs. 5-17). of mutagenesis result from a direct sub- sequence that occurs under natural con- Two general concepts have emerged stitution, deletion, or insertion of base ditions. Instead, the genome is abnor- from these studies. (i) Tissue culture- sequences, genetic changes also can be mally reprogrammed, or decidedly re- induced mutation has been detected in all "self-imposed" by a breakdown of the structured. These restructurings can give species studied. Rarely have individual normal cellular controls on chromo- rise to a wide range ofaltered phenotypic genotypes within species been identified somes. If the change deals with large expressions. Some of the altered pheno- which show no mutation, and in those domains of the genome, a variety of al- types are readily observed in the newly cases they most likely resulted from sam- terations in chromatin and in gene ex- produced plants themselves. Others ap- pling small numbers of individuals or pression can occur. pear in their progeny. Their association from scoring for only one or a few types The past 20 years of research on plant with genomic change remains proble- ofmutation. Trends toward low mutation tissue cultures, regenerated plants, and matic. Other altered phenotypes clearly rates within species are usually corre- progenies of the regenerated plants have reflect genomic restructuring, and vari- revealed a rich array of culture-induced ous levels of this have been observed. It Abbreviation: RIP, repeat-induced point mu- genetic variants. The genetic behavior of may be safe to state that no two of the tation. these variants generally appears similar callus-derived plants are exactly alike, tTo whom reprint requests should be ad- to that of naturally occurring mutants. and none is just like the plant that do- dressed. 5222 Downloaded by guest on September 24, 2021 Rmveview: Phillips et al. Proc. Natl. Acad. Sci. USA 91 (1994) 5223 lated with their "culturability." Cell single-gene phenotypic mutants, it arises cycle controls, which prevent cell divi- growth and regenerability indicate low at least as frequently. sion before the completion of DNA rep- levels of cell stress, levels which exper- Specific genetic changes associated lication, are presumed to be disrupted by imentally are reflected by relatively with particular tissue culture-induced tissue culture, resulting in chromosome lower mutation rates. (ii) Tissue culture- phenotypic mutants have been elucidated breakage. Chromosome breakage with- induced mutation is a general increase in only in rare cases (25, 26). However, a out the reunion ofbroken fragments leads most types of mutation rather than in a variety ofmutation types have been char- to deletions of chromosome segments; few specific types. Tissue culture- acterized which most likely are responsi- chromosome breakage followed by the induced variation has usually been based ble for the observed phenotypic variation. reunion of broken ends leads to translo- on phenotypic differences in regenerated These changes include cytological aberra- cations, inversions, duplications, and de- plants and their progeny. However, ge- tions which are primarily the result of letions. chromosome breakage, single base Chromosome breakage could be in- nomic changes appear to be the basis for altered levels of DNA the phenotypic alterations. changes, changes in the copy number of duced by methyl- repeated sequences, and alterations in ation. Heterochromatinization of chro- Phenotypic changes found in regener- matin has been associated with increased ated plants and their progeny are most DNA methylation pattern. The genomic changes represent a wide array of differ- methylation. Nucleosomes of mouse strikingly observed as qualitative mu- chromatin have been fractionated and tants which have major phenotypic ef- ent alterations. The thread connecting these diverse mutations is that all could be probed with antibodies against 5-methyl- fects (Table 1). The mutants have phe- cytosine (27). More methylcytosine is notypes similar to those previously ob- the result ofa disruption ofnormal cellular associated with isolated nucleosomes served in the respective species, such as controls. Pardue (1) has hypothesized that is not the default state that contain histone H1. Because H1 is chlorophyll deficiencies, dwarfs, and de- genomic stability involved in chromosome condensation, fective seeds. In some cases, allelism but is the result of a rather finely tuned increased methylcytosine conceivably with previously characterized mutants system ofchecks and balances. The tissue culture environment may cause a general could affect the rate of DNA replication. has been proven. The qualitative mutants disruption of cellular controls, leading to This delayed replication could