Proc. Nati. Acad. Sci. USAVol. 91, pp. 5222-5226, June 1994

Review

Genetic instability of plant tissue cultures: Breakdown ofnormal 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 ofAgronomy, University of Nebraska, Lincoln, NE 68583

ABSTRACT Plants regenerated fromrelatively undifferentiated callus culturespossess a vast array of genetic changes.Such variations can result in useful agri-cultural and horticultural products. Forother purposes, however, variations intraits other than those of interest may beundesirable-for example, using culturedcells for genetic engineering. Any stepsmade toward understanding the basis oftissue culture-induced genetic variationshould be helpful in developing a morestable and manipulatable somatic cell sys-tem. This review provides a glimpse at thespecific kinds of genetic changes encoun-tered among regenerated plants and theirprogeny. Included among these variationsare cytosine methylation alterations of thegenome. The repeat-induced point muta-tion (RIP) phenomenon, reported for fil-amentous fungi, is invoked to provide aframework to consider the origin of vari-ation in plant tissue cultures.

Cells of all living organisms reproducewith almost exact fidelity and give rise todaughter cells of predictable genotype.Errors in the process occur infrequentlydue to a remarkable variety of cellularcontrols that regulate the genome. Thesecontrols cause expression of genes in adevelopmentally specific manner and fa-cilitate the duplication, recombination,and distribution of chromosomes. Nor-mal cell behavior is the result of a com-plex cascade of genetic programs that issensitive to disruption by biotic and abi-otic stresses (1). Although certain formsof mutagenesis result from a direct sub-stitution, deletion, or insertion of basesequences, genetic changes also can be"self-imposed" by a breakdown of thenormal cellular controls on chromo-somes. If the change deals with largedomains of the genome, a variety of al-terations in chromatin and in gene ex-pression can occur.The past 20 years of research on plant

tissue cultures, regenerated plants, andprogenies of the regenerated plants haverevealed a rich array of culture-inducedgenetic variants. The genetic behavior ofthese variants generally appears similarto that of naturally occurring mutants.

However, the frequency of variousclasses of mutants derived from planttissue culture is elevated far beyond thatexpected in nature. Clearly, tissue cul-ture processes that involve an undiffer-entiated callus phase are mutagenic.Variations include, but are not restrictedto, chromosomal rearrangements andsingle-gene mutants (mostly recessive).DNA methylation changes also havebeen reported in regenerated plants andtheir progeny.

In her Nobel lecture, Barbara Mc-Clintock (2) said "Some responses tostress are especially significant for illus-trating how a genome may modify itselfwhen confronted with unfamiliar condi-tions. Changes induced in genomes whencells are removed from their normal lo-cations and placed in tissue culture sur-roundings are outstanding examples ofthis. The establishment of a successfultissue culture from animal cells, such asthose of rat or mouse, is accompanied byreadily observed genomic restructuring.None of these animal tissue cultures hasgiven rise to a new animal. Thus, thesignificance of these changes for the or-ganism as a whole is not yet directlytestable. The ability to determine this is adistinct advantage of plant tissue cul-tures."McClintock went on to say "The treat-

ment, from isolation of the cell or cells ofa plant, to callus must inflict on the cellsa succession of traumatic experiences.Resetting of the genome, in these in-stances, may not follow the same orderlysequence that occurs under natural con-ditions. Instead, the genome is abnor-mally reprogrammed, or decidedly re-structured. These restructurings can giverise to a wide range of altered phenotypicexpressions. Some of the altered pheno-types are readily observed in the newlyproduced plants themselves. Others ap-pear in their progeny. Their associationwith genomic change remains proble-matic. Other altered phenotypes clearlyreflect genomic restructuring, and vari-ous levels of this have been observed. Itmay be safe to state that no two of thecallus-derived plants are exactly alike,and none is just like the plant that do-

nated the cell or cells for the tissue cul-ture. The many levels of genomic modi-fication that already are known and ex-pressed as changed genotypes andphenotypes could be potent sources forselection by the plant breeder, and inci-dentally, for theoretical ponderings bythe biologist."

This article reviews the progress inunderstanding the basis of tissue culture-induced variation. It also explores possi-ble links between tissue culture-inducedvariability and mechanisms of sequenceor genomic change.Change in tissue culture most likely

occurs by a stress-response mechanism.The relevant mechanism may best bedescribed as a programmed loss of cellu-lar control. The most commonly ob-served plant tissue culture-imposedchanges-chromosome rearrangements,DNA methylation, and mutations-alsoare salient features of a phenomenoncalled repeat-induced point mutation(RIP, formerly termed rearrangement-induced premeiotically) first described infilamentous fungi (3, 4). This review willdescribe features of RIP and of tissueculture-induced variation. It will also dis-cuss processes that might induce the RIP-like system in plant tissue culture.

A Review of Tissue Culture-InducedVariation in Plants

Mutation induced by plant tissue culturehas been the subject of numerous scien-tific inquiries (reviewed in refs. 5-17).Two general concepts have emergedfrom these studies. (i) Tissue culture-induced mutation has been detected in allspecies studied. Rarely have individualgenotypes within species been identifiedwhich show no mutation, and in thosecases they most likely resulted from sam-pling small numbers of individuals orfrom scoring for only one or a few typesof mutation. Trends toward low mutationrates within species are usually corre-

Abbreviation: RIP, repeat-induced point mu-tation.tTo whom reprint requests should be ad-dressed.

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lated with their "culturability." Cellgrowth and regenerability indicate lowlevels of cell stress, levels which exper-imentally are reflected by relativelylower mutation rates. (ii) Tissue culture-induced mutation is a general increase inmost types of mutation rather than in afew specific types. Tissue culture-induced variation has usually been basedon phenotypic differences in regeneratedplants and their progeny. However, ge-nomic changes appear to be the basis forthe phenotypic alterations.

Phenotypic changes found in regener-ated plants and their progeny are moststrikingly observed as qualitative mu-tants which have major phenotypic ef-fects (Table 1). The mutants have phe-notypes similar to those previously ob-served in the respective species, such aschlorophyll deficiencies, dwarfs, and de-fective seeds. In some cases, allelismwith previously characterized mutantshas been proven. The qualitative mutantsare generally inherited as single Mende-lian factors and are most frequently re-cessive alleles. The relative frequencyand gene action of the different types ofqualitative mutations are consistent withstudies using mutagenic agents that in-duce mutations at random sites such asethyl methanesulfonate. Several unstableor nontransmissible mutations have beenobserved, indicating that transposable el-ements and/or epigenetic modificationmay be the basis of some tissue culture-induced mutations (ref. 18; C. L. Arm-strong and R.L.P., unpublished observa-tion). Transposable element activity hasbeen detected in the progeny of regener-ated plants (19, 20), but a direct associ-ation between an active transposable el-ement and a phenotypic mutant has notyet been demonstrated. Quantitative traitvariation has also been observed by sev-eral researchers (21-24). While this typeof variant is somewhat more subtle than

single-gene phenotypic mutants, it arisesat least as frequently.

Specific genetic changes associatedwith particular tissue culture-inducedphenotypic mutants have been elucidatedonly in rare cases (25, 26). However, avariety of mutation types have been char-acterized which most likely are responsi-ble for the observed phenotypic variation.These changes include cytological aberra-tions which are primarily the result ofchromosome breakage, single basechanges, changes in the copy number ofrepeated sequences, and alterations inDNA methylation pattern. The genomicchanges represent a wide array of differ-ent alterations. The thread connectingthese diverse mutations is that all could bethe result ofa disruption ofnormal cellularcontrols. Pardue (1) has hypothesized thatgenomic stability is not the default statebut is the result of a rather finely tunedsystem ofchecks and balances. The tissueculture environment may cause a generaldisruption of cellular controls, leading tothe numerous genomic changes present intissue culture regenerants. There aremany examples of observed genomicchanges to the cellular process which maybe functioning abnormally.Chromosome aberrations are fre-

quently found in plants regenerated fromtissue culture. These aberrations, whichare the result of chromosome breakageevents, have been most well character-ized in maize and oat (12) but have alsobeen observed in other species. Translo-cations, inversions, deletions, and dupli-cations have all been detected. Thebreakpoints of the various aberrationsgenerally have been found either be-tween distal heterochromatin and thecentromere or within centric heterochro-matin. Late replication of heterochro-matic blocks followed by chromosomebridges and breakage has been hypothe-sized as the mechanism explaining thelocation of the breakpoints. Normal cell

Table 1. Types of single-gene visible mutants segregating in progeny of regeneratedmaize plants

Chlorophyll deficienciesVirescentPale greenLuteusStriatelojapAlbinoYellow greenZebra stripeUnstable albino

Necrotic leavesLower leaf necrosisNecrotic leaf (lethal)Necrotic leaf spots

StatureDwarfSemi-dwarf

Seed characteristicsDefective kernelShrunkenSugaryViviparous

Leaf morphologyCrinklyWiltedAdherent (epidermal cell fusions)

Reproductive structuresMale sterilityRamosa tasselAnother color

cycle controls, which prevent cell divi-sion before the completion of DNA rep-lication, are presumed to be disrupted bytissue culture, resulting in chromosomebreakage. Chromosome breakage with-out the reunion ofbroken fragments leadsto deletions of chromosome segments;chromosome breakage followed by thereunion of broken ends leads to translo-cations, inversions, duplications, and de-letions.Chromosome breakage could be in-

duced by altered levels of DNA methyl-ation. Heterochromatinization of chro-matin has been associated with increasedmethylation. Nucleosomes of mousechromatin have been fractionated andprobed with antibodies against 5-methyl-cytosine (27). More methylcytosine isassociated with isolated nucleosomesthat contain histone H1. Because H1 isinvolved in chromosome condensation,increased methylcytosine conceivablycould affect the rate ofDNA replication.This delayed replication could causeanaphase bridges, chromosome break-age, and rearrangements. The type ofrearrangement would depend on thechromosome location of the heterochro-matin, whether homologues or hetero-logues are involved, and the ploidy level(28). Alternatively, chromosome break-age could be caused by decreases inmethylation. Decreases in methylationhave been implicated in the nondisjunc-tion of chromosomes in both rye (ref. 29;methylation decreased by exposure to5-azacytidine) and Neurospora (ref. 30;methylation decreased because ofmutantmethyltransferase). According to thesemechanisms, maintenance ofDNA meth-ylation rather than DNA replicationcould be the cellular process which hasgone awry and given rise to chromosomebreakage events.

Single base-pair changes have been de-tected in the progeny of tissue cultureregenerants and have been shown to bethe basis of two independent, tissue cul-ture-induced alcohol dehydrogenase mu-tants in maize (25, 26). More often, theoccurrence of single base-pair changeshas been inferred based on the presence ofrestriction fragment length polymor-phisms detected by specific-sequenceprobes (31, 32). Single base-pair changescould theoretically result by two mecha-nisms: (i) deamination of methylated cy-tosine, resulting in a C -* T or G -- Atransition following mismatch repair; and(ii) loss of precision of the DNA replica-tion/repair machinery, resulting in transi-tion or transversion types of base-pairchanges. Both ofthese mechanisms couldbe due to a reduction in the normal con-trols which maintain a standard level ofsequence integity. It is possible that se-quence changes involving a small numberof contiguous nucleotides could also bethe footprint left as a result of excision of

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a transposable element. The frequency ofelement activity in maize does not cur-rently support transposable-element exci-sion as a major effector of contiguousbase-pair changes, although the range oftypes ofelements tested for and the extentof the testing have certainly not beenexhaustive enough to exclude the possi-bility.Tandemly repeated sequences show

increased levels of instability in tissueculture with "clonal" regenerants con-taining variable copy numbers (33, 34).Variability in the copy number of tan-demly repeated sequences is a conse-quence of various cellular stresses andhas been found to be the basis of severalhuman genetic diseases (35). It is notunexpected, therefore, that repeated-sequence variation has been detectedamong tissue culture regenerants andthat this variation may be responsible forsome of the observed phenotypic vari-ability. Copy-number variability is mostlikely effected by mitotic recombination.Either interchromatid unequal crossingover or intrachromatid exchange of in-verted repeats could result in the loss orgain of genetic information.DNA methylation patterns are fre-

quently altered by the tissue culture envi-ronment (17, 28, 31, 32, 36-40). Exami-nation ofregenerated plants or their prog-eny with methylation-sensitive restrictionenzymes has revealed both hyper- andhypomethylation, depending on the re-port. Several kinds of probes have beenemployed, including those detecting sin-gle-copy and repeated sequences, as wellas cDNAs for known expressed genes andrandom Pst I genomic sequences. Alter-ations in methylation in plant tissue cul-tures do not appear to be restricted tospecific kinds ofDNA sequences.

In summary, the types ofgenomic vari-ation observed in tissue culture are di-verse. The parallels between the varia-tion induced by tissue culture and theseemingly unrelated RIP phenomenonare intriguing. These parallels may holdthe key to further understanding thiscomplex process.

Applying the RIP Hypothesis to PlantTissue Culture

Selker (42) reported that a DNA se-quence transformed into Neurospora ledto the mass methylation of the cytosinesspecifically within the limits of the se-quence of both the introduced copy andthe endogenous copy. The cytosinemethylation was indiscriminant in thesense that any cytosine could be methyl-ated, not just those present as CpG di-nucleotides or CpNpG trinucleotides(where N could be any other base) as isthe usual case. Point mutations occurfrequently in the newly methylated se-quences as the result of deamination of

the methylated cytosines, thus the termrepeat-induced point mutation (RIP).Neurospora has a rather low level ofDNA methylation but it is not knownwhether this is relevant to the RIP phe-nomenon in this species. The frequencyof RIP was higher for cases where theintroduced sequence was linked to theendogenous sequence. This observationindicated that RIP might depend on thepairing or matching of like sequences.From an evolutionary viewpoint, it hasbeen speculated (49) that such a processmakes the sequences divergent in orderto be less likely to recombine and thusleads to rearrangements.The phenomenon of RIP originally was

shown to occur in the premeiotic stage inthe dikaryotic ascogenous hyphae ofNeu-rospora before the nuclei fused to give riseto the zygote (42). Later, Pandit andRusso (43) found reversible gene inacti-vation in somatic tissue, which suggeststhe possibility ofRIP occurring in somaticplant tissue cultures. Because of the oc-currence of these events in other than thepremeiotic cell, the term repeat-inducedpoint mutation was preferred. In Ascobo-lus, the induced methylation also is coex-tensive with the length of the duplications(44), but the process is reversible, mean-ing that the repeat-induced methylatedcytosines are replaced by unmethylatedcytosines instead ofremaining methylatedor being deaminated to form thymine.Although both methylation and mutationsare observed in plant tissue cultures, wewill use the term RIP because mutationsare observed at high frequencies.A mechanism similar to RIP is cer-

tainly present in plants (reviewed in ref.45). Quiescent duplicated sequences,such as the transposable element Ac, arepresent throughout the genome of maize.Paramutation of the R locus has beenshown in specific maize crosses. Cosup-pression of transgenes is common withmethylation and altered gene expressionfound in both the transgenic duplicate aswell as the original sequence (see ref. 46for a recent review on cosuppression).While these examples suggest a compar-ison and methylation of duplicated se-quences, characterization of ensuingbase-pair changes is not as thorough inplants as in Neurospora.

Regenerants from plant tissue culturecontain a high frequency of mutations,many methylation changes, and chromo-some structure abnormalities. It is thefrequency and types of mutations thatlead us to speculate that a RIP-like mech-anism is involved in tissue culture-induced mutagenesis. However, a num-ber of issues suggest that the process ofmutagenesis in tissue culture is not ex-actly like RIP as described in Neurospora.The first difference is that the plant ge-nome is littered with duplications includ-ing disperse and tandem repeats of both

high and low copy sequences. Recentevidence from restriction fragment lengthpolymorphism (47, 48) indicates that themaize genome has a high degree of dupli-cation. Over 70% of the single-copy riceprobes that were hybridized to maize ge-nomic DNA detected duplications. Thesesequences are usually not susceptible tothe RIP process in normal plants, thusindicating that the plant genome hasreached a state of equilibrium. Basechanges due to methylation can occur.Evidence in mammals (49) and in plants(50) supports the idea that cytosines havebeen converted to thymines over time andthat C -+ T transitions occur much morefrequently than other base-pair changes inmethylated regions. Spontaneous deami-nation of 5-methylcytosines is the likelyexplanation for these changes (51, 52).However, the rate ofmutation is very lowrelative to that found in tissue culture,suggesting that the potent RIP mechanismis not operating. Why then is the RIPmechanism induced by tissue culture andnot in normal plants?A second difference between tissue cul-

ture-induced variation and RIP in Neuro-spora is that methylation and sequencechanges in Neurospora are specific to anarea homologous to an introduced se-quence, whereas methylation changes andmutations found in tissue culture regener-ants are found frequently at sequencesscattered throughout the genome of thesame regenerated plant. This fact arguesfor induction of the RIP process by agenome-wide mechanism rather than byspecific sequence or chromosomal dupli-cations which do occur in culture (14).What genome-level process could initiatesuch wide-ranging changes?

Finally, the methylation changes foundamong tissue culture regenerants are notalways the result of large increases in thefrequency of methylated cytosine as isfound in Neurospora. Our results indi-cate that hypomethylation is the rule, butwe recognize that other researchers (37),even those using the same inbred maizeline, find hypermethylation as well ashypomethylation. Why this differenceexists is not clear. IfRIP causes a generalincrease in methylation, why would prog-eny of regenerated maize plants usuallyhave less methylation when specific se-quences are compared between controls(plants from kernels borne on the sameear from which embryos were explantedfor culture initiation) and regeneratedplants? The explanation might lie in theRIP mechanism (Fig. 1). Methylated cy-tosines are deaminated and are thereforeconverted to thymines. This creates mis-matched T-G base pairs. According toBrown and Jirieny (53), working withsimian cells, the thymine is often (92%)repaired to a cytosine; note that thisprocess replaces methylated cytosineswith unmethylated cytosines. About 4%

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Cytosinemethylation

CmC GG Deamination CT GG -< Repair of >G GmCC GGmCC Mismatch to G:C

No repair (2%) (90%)I

Replication

CTGGGACC(Point mutation)

CCGGGGCC (cytosine-

CC GG Replication methylation lost)GGmCC A GG'CC

CC GG

Repair ofmismatch to A:T (8%)

GGmCC

CC GGI

MaintenanceMethylation4

G GmCCCmC GG(No change)

CT GGGAmCC

Replication

CTGGGACC(Point mutation)

MaintenanceMethylation

4G GmCCC-C GG(No change)

GAmCCCT GG(Point mutation)*

FIG. 1. Potential creation of mutations and non-methylated cytosines in plant tissue cultures by RIP. *, The methylated cytosine will notbe maintained due to loss of CG symmetry.

mismatches stay as such until replica-tion, which then results in a transitionmutation. In the remaining 4%, the gua-nine is repaired to an adenine, againresulting in a mutation.The duplicated sequences in Neuro-

spora become riddled with C -+ T transi-

tions following the extensive cytosinemethylation. Since mismatches are usu-ally repaired to the original base, thisimplies that an even greater frequency ofthe TUG mismatches were repaired back toC-G pairs. In maize, with a higher level ofmethylcytosines, RIP would be expectedto produce a higher frequency of methyl-cytosine cytosine changes. This highfrequency of change could explain whymaize regenerants homozygous for newDNA methylation patterns occur at suchhigh frequency; 17% of the DNA methyl-ation changes are apparently homozygousin the original regenerant (17). Dependingon the number of CmCGG sites repre-sented in the probed region (using Hpa II

and Msp I isoschizomers), a relativelyhigh frequency of altered sites might bepossible, leading to higher than expectedhomozygosity frequencies.

Induction of Tissue Culture-ImposedMutagenesis and the RIP Mechanism

A RIP mechanism might be induced intissue culture in three ways: (i) duplica-tions occurring in tissue culture couldinitiate the process, (ii) an agent in thetissue culture medium could substantiallyincrease the general level of sequencemethylation with ensuing changes follow-ing a RIP-like process, or (iii) the genomicbalance which inhibits RIP in normalplants could be disrupted. The secondmechanism would better be described asmethylation-induced mutation rather than

repeat-induced mutation, since compari-son of repeats is not directly involved.Does the evidence support the fre-

quency of newly generated duplicationsneeded to subsequently observe such ahigh frequency of methylation alterationsand apparent point mutations? The evi-dence for duplications is mostly fromcytological analysis of meiotic tissue.Duplications are usually recognized asheteromorphic bivalents at diakinesis ormetaphase I or the occurrence ofa buckleat pachynema in heterozygotes. In bothcases, distinction between duplicationsand deletions is difficult. Many of theaberrations are probably duplications, asjudged from the absence of the degree ofsterility expected from deletions. Al-though heteromorphic pairs are detectedat an appreciable frequency (14), cyto-logical analysis most likely reveals only asmall fraction of the total. The occur-rence of a multitude of cytologically un-detectable rearrangements is likely.Thus, a sufficient number of duplicationscould be generated in tissue cultures toinitiate a substantial amount ofRIP. Mostof these duplications might be smallenough to have normal transmission and,therefore, possibly be subject to RIP insubsequent generations.Although an argument can be made for

duplications in tissue cultures inducingthe RIP process, agents in the mediumalso might be the leading cause. LoSchi-avo et al. (54) have shown that a commoncomponent of the plant tissue culturemedium, the hormone 2,4-diphenoxyace-tic acid (2,4-D), causes a dramatic eleva-tion of cytosine methylation in plant tis-sue cultures. An increase in 2,4-D con-centration in carrot (Daucus carota)suspension cultures from 0.5 to 2 pg/mlraises the percent 5-methylcytosine from

16% to 40% in only 5 days; 2 pg/ml is acommonly used concentration of2,4-D inmonocot tissue cultures. Other auxinssuch as 1-naphthaleneacetic acid and in-doleacetic acid also caused increases inDNA methylation. This known effect of2,4-D makes it a possible inducer of RIPin plant tissue cultures. However, theextrapolation of this effect to culturesother than carrot has not been shown.Preliminary analysis of the amount ofmethylation in maize tissue culture foundsimilar levels in non-cultured plants com-pared with callus cultures (P.O., unpub-lished data). More research is needed onthe cytosine methylation effects of hor-mones other than 2,4-D, other mediacomponents, and other culture systems.Hormones may effect tissue culture

variation by causing a general increase inmethylation in most monocot cultures.However, hormones found in the tissueculture medium, such as 2,4-D, may actby a quite different mechanism. The her-bicidal mode ofaction of2,4-D is not wellunderstood but apparently involves sub-stantial general increases in transcription(55). The events leading to the increasedtranscription may alter the chromatinstructure. These alterations could disruptthe stability of the genome and result inthe comparison of repeated sequences,an event which does not normally occur.Among the features of RIP is the con-

tinuing mutation over cell cycles andgenerations. In Neurospora, the degreeof mutation is a function of the number ofcell cycles. If RIP is occurring in planttissue cultures, the commonly observedculture age effect might be expected. Theolder plant tissue cultures would haveundergone more cell divisions and per-haps more RIP. This is consistent with

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the increasing mutation frequency ob-served as culture age increases.D. P. Cummings (personal communi-

cation) observed cases of maize tasselssectored for anther color in sixth-generation lines derived from regener-ated plants. The genes for anther colorshould have been homozygous, since re-generation was from an inbred line. Ap-pearance of variants at later generationswould be expected on the basis of RIP.The process occurs until a certain point isreached which has been postulated todepend on the ability of the duplicatedsequences to still recognize each other.Those duplicated sequences which arenot linked take longer in Neurospora toundergo RIP, presumably because of thelower frequency of sequence pairing ormatching. A sequence duplicated at anunlinked position lost l1O% of the G-Cpairs in one generation, whereas a closelylinked duplicated sequence lost abouthalfofthe G-C pairs after two generations(56). Thus, it is possible that new muta-tions could be produced several genera-tions removed from the regeneratedplant. In Neurospora, duplications canundergo further RIP even after six gen-erations (41). The proportion of progenyshowing RIP decreased with generations.Again, linked duplications diverged morethan independent ones. Interestingly,many of the duplicated sequences inmaize are unlinked; perhaps a compari-son of linked and unlinked repeats inmaize regenerants would shed light onthe importance of RIP in tissue culture-induced variation in this species.

Summary

Specific genomic alterations associatedwith tissue culture variation have beenwell characterized, but the mechanismleading to these changes is not well un-derstood. There are numerous parallelsbetween RIP in Neurospora and tissueculture-induced variation in plants. How-ever, differences in the two mechanismsalso exist. Understanding why differ-ences and similarities exist in these twosystems should ultimately lead to a betterunderstanding of tissue culture-inducedvariation. It seems likely that a preexist-ing mechanism for genomic change, suchas RIP, could be the effector of tissueculture-induced mutagenesis. Under-standing the mechanism of mutation willlead to a better understanding of (i) ge-nomic change in response to stress, (ii)factors contributing to genomic stability,and (iii) methods to control variationamong tissue culture regenerants.

This is paper no. 21,094, Scientific JournalSeries, Minnesota Agricultural ExperimentStation. This work was partly supported byU.S. Department of Agriculture Grant 91-37301-6376.

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